Immunization of Volunteers with Salmonella enterica Serovar Typhi Strain Ty21a Elicits the Oligoclonal Expansion of CD8+ T Cells with Predom
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感染与免疫杂志 2005年第6期
Center for Vaccine Development, Department of Pediatrics, University of Maryland School of Medicine, Baltimore, Maryland
ABSTRACT
CD8+ T cells are likely to play an important role in host defense against Salmonella enterica serovar Typhi by several effector mechanisms, including lysis of infected cells (cytotoxicity) and gamma interferon (IFN-) secretion. In an effort to better understand these responses, we studied the T-cell receptor (TCR) repertoire of serovar Typhi-specific CD8+ T cells in humans. To this end, we determined the TCR beta chain (V) usage of CD8+ T cells from three volunteers orally immunized with Ty21a typhoid vaccine by flow cytometry using a panel of monoclonal antibodies. Although TCR V usage varied among volunteers, we identified oligoclonal V subset expansions in individual volunteers (V 2, 5.1, 8, 17, and 22 in volunteer 1; V 1, 2, 5.1, 14, 17, and 22 in volunteer 2; and V 3, 8, 14, and 16 in volunteer 3). These subsets were antigen specific, as shown by cytotoxicity and IFN- secretion assays on V sorted cells and on T-cell clones derived from these volunteers. Moreover, eight-color flow cytometric analysis showed that these clones exhibited a T effector memory phenotype (i.e., CCR7– CD27– CD45RO+ CD62L–) and coexpressed gut homing molecules (e.g., high levels of integrin 47, intermediate levels of CCR9, and low levels of CD103). In conclusion, our results show that long-term T-cell responses to serovar Typhi in Ty21a vaccinees are oligoclonal, involving multiple TCR V families. Moreover, these serovar Typhi-specific CD8+ T cells bearing defined V specificities are phenotypically and functionally consistent with T effector memory cells with preferential gut homing potential.
INTRODUCTION
Typhoid fever remains an important public health priority, particularly in developing countries, with an estimated 16 million new cases annually and 600,000 deaths (17, 21). The appearance of antibiotic-resistant strains of Salmonella enterica serovar Typhi, the causative agent of typhoid fever, has added new urgency for the development of improved typhoid vaccines. New-generation attenuated serovar Typhi vaccine strains have the potential to become the preferred public health tool to immunize against typhoid fever because of their ability to elicit long-lasting protective systemic and mucosal immune responses. In addition, significant efforts have been focused in recent years on the use of attenuated serovar Typhi vaccines as carriers of foreign antigens to deliver foreign genes because of their ability to gain access to dendritic cells (51, 52) and deliver antigens or DNA coding for the antigens (12, 31, 32). These characteristics make the use of Salmonella live vectors particularly attractive for mass immunization programs. Moreover, live attenuated serovar Typhi vaccines are expected to have relatively low manufacturing costs compared to many other types of vaccines.
CD8+ T cells might play an important role in host defense against serovar Typhi by several effector mechanisms, including lysis of infected cells and gamma interferon (IFN-) secretion (35, 36, 44, 45). These CD8+ T cells recognize antigenic peptides bound to self-major histocompatibility complex via T-cell antigen receptor (TCR) (26, 48) on the surface of infected cells. The TCR on most peripheral T cells is a heterodimeric molecule, composed of (V) and (V) chains (7). Functionally, V has been associated with the antigen recognition process, and specific recognition of peptide results in the clonal expansion of a subset of V antigen-specific T cells (7, 26). Thus, direct examination of TCR V specificities has been used to identify and track oligoclonal antigen-specific T-cell responses during infection and after immunization (1, 14, 30). Approximately 60 TCR V gene segments are known in humans (30). TCR V usage has been studied in various systems, and either broad or restricted patterns have been identified in a number of areas, including autoimmunity (29, 53), response to viral (10, 25, 40, 42) or bacterial (8, 11, 16, 27) antigens, and tumor immunity (22).
The induction of strong, long-lived immunologic T- and B-cell memory responses is of paramount importance in the development of effective vaccines. Thus, in the present study, we analyzed the TCR V gene usage of specific CD8+ T cells from healthy adults 5 to 40 months after oral immunization with serovar Typhi strain Ty21a, the only licensed attenuated typhoid vaccine, to gain further insights into the clonality and characteristics of T-cell responses to serovar Typhi. Although the responses were oligoclonal, we observed that CD8+ T cells from several V families participated in the anti-serovar Typhi response. Moreover, we studied the induction of memory T-cell subsets and the homing potential of these cells. The specificity of the expanded cell subsets was demonstrated by functional assays on V sorted cells and on clones. Moreover, we observed that V serovar Typhi-specific CD8+ T cells express surface molecules associated with a T memory effector phenotype with gut homing potential.
MATERIALS AND METHODS
Subjects. Three healthy volunteers, 32 to 41 years old, recruited from the Baltimore-Washington area and University of Maryland at Baltimore campus, participated in this study. Volunteers were vaccinated following the routine immunization schedule for the Ty21a typhoid vaccine, i.e., four spaced doses of 2 x 109 to 6 x 109 CFU of Ty21a at an interval of 48 h between doses (20, 35). Volunteers were screened for good health by medical history, physical examination, normal laboratory tests, including blood counts, and the absence of antibiotic treatment at the times of leukapheresis. The purpose of this study was explained to volunteers, and they gave informed, signed consent. Cells from volunteers 1, 2, and 3 were collected 5, 25, and 40 months after immunization, respectively, to perform a time course analysis of the immune responses to serovar Typhi. Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation (35).
Target and stimulator cells. Blasts were established from PBMC isolated from Ty21a vaccinees following standard procedures (35, 36, 44) and maintained in RPMI 1640 (Gibco, Grand Island, New York) supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, 50 μg/ml gentamicin, 2 mM L-glutamine, 2.5 mM sodium pyruvate, 10 mM HEPES buffer, 1% nonessential amino acids, and 10% heat-inactivated fetal bovine serum (R10) plus 20 IU/ml recombinant human interleukin-2 (rhIL-2) (Boehringer GmbH, Mannheim, Germany). Blasts were infected by incubating in RPMI (without antibiotics) for 3 h at 37°C with wild-type serovar Typhi strain ISP1820 (35, 36). In some experiments, the following day, blasts were -irradiated (4,000 rads) and used as stimulators. For cytotoxic T lymphocyte (CTL) assays, infected and noninfected cells were labeled with 200 μCi of sodium chromate (51Cr) (Amersham Pharmacia Biotech, Piscataway, New Jersey) for 1 h at 37°C, washed, and used as targets.
To confirm that targets were infected with serovar Typhi, cells were stained with anti-CSA-1-fluorescein isothiocyanate (FITC) (KPL, Gaithersburg, MD) and analyzed by flow cytometry (35, 36).
Effector cells. For cytotoxic assays, both ex vivo and in vitro-expanded PBMC from immunized volunteers were used as effectors. In vitro-expanded effectors were obtained using a modification of a previously described technique (35, 36). Briefly, PBMC were restimulated weekly with irradiated autologous serovar Typhi-infected blasts at an effector-to-stimulator cell ratio of 7:1 for 3 weeks in R10 containing 20 IU/ml of rhIL-2. Aliquots of these effector cells were frozen weekly before each new restimulation. The expression of surface molecules on these cell lines was determined by flow cytometry. To this end, effectors were stained with a panel of anti-TCR V monoclonal antibodies (MAbs) (TCR V Repertoire kit, IO Test Beta Mark; Beckman-Coulter, Miami, FL) and combinations of MAbs to CD3 (clone UCHT1), CD4 (clone IM0704), CD25 (clone B1.49.9), CD28 (clone IM1236), HLA-DR (clone H279) (Beckman-Coulter), CD8 (clone SK1), CD56 (clone B159), CD38 (clone HB7), CD45RO (clone UCHL-1), and CD62L (clone Dreg-56) (BD Pharmingen, San Diego, CA). MAbs or the corresponding isotype controls conjugated to the following fluorochromes were used in these studies: FITC, phycoerythrin (PE), energy-coupled dye (ECD) (PE-Texas Red conjugate), peridinin chlorophyll protein (PerCP), PE-Cy5 conjugate, PE-Cy7 conjugate, allophycocyanin (APC), and APC-Cy7 conjugate or biotin (followed by streptavidin-Pacific blue [Molecular Probes, Eugene, Oregon]). The TCR V panel used in these studies included MAbs that recognize 24 different V specificities (i.e., V 5.3, V 7.1, V 3, V 9, V 17, V 16, V 13.1, V 13.6, V 8, V 18, V 5.1, V 20, V 5.2, V 2, V 12, V 23, V 1, V 21.3, V 11, V 22, V 14, V 13.2, V 4, and V 7.2) (see Table 1 for more details), which cover 60% of the known specificities (47) (Table 1). Samples were run in a MoFlo flow cytometer/cell sorter system (Dako-Cytomation, Fort Collins, CO) and analyzed using the WinList flow cytometry software analysis package (Verity Software House, Topsham, ME).
For IFN- enzyme-linked immunospot (ELISPOT) assays, both ex vivo and in vitro-expanded PBMC (as described above) were also used as effectors.
T-cell cloning. T cells from volunteers 1 and 3 were expanded for one cycle in the presence of serovar Typhi-infected autologous blasts and cloned by limiting dilution and polyclonal stimulation with 2.5 x 105 irradiated allogeneic blasts and 2.5 x 105 K562/CD32/4-1 BBL cells (kindly provided by M. Mauer and C. June, University of Pennsylvania, Philadelphia, PA) prepulsed with both anti-CD3 (clone OKT3; ATCC, Rockville, MD) and anti-CD28 (clone 9.3; BD Pharmingen) MAbs (0.5 μg/ml each) (24). These cell lines and clones were cultured in R10 supplemented with 60 IU/ml of rhIL-2 for 10 days and expanded by multiple cycles of in vitro restimulation every 10 days with allogeneic blasts and K562/CD32/4-1 BBL cells at a 2:1 ratio of T cells:stimulator cells. The expression of surface homing and/or effector memory molecules was assessed in these T-cell clones by eight-color flow cytometry (MoFlo). The following MAbs (coupled to the indicated fluorochromes) were used: integrin 47 (clone ACT-1) was kindly provided by W. Newman, Leukosite, Cambridge, MA, and conjugated to Alexa 488 using an Alexa 488 labeling kit (Molecular Probes); CD27-PE (clone M-T271); CD62L-PE-Cy5 (clone Dreg-56); CCR7-PE-Cy7 (clone 3D12) and CD8-APC-Cy7 (clone Sk1) (BD Pharmingen); CD45RO-ECD (clone UCHL-1) and CD103-biotin (clone 2G5) (Beckman-Coulter); and CCR9-APC (clone 112509; R&D Systems, Minneapolis, MN). Alexa 405-streptavidin was purchased from Molecular Probes.
Cytotoxicity (chromium release test). As previously described in detail, cytotoxicity against uninfected or serovar Typhi-infected targets was determined by using a 4-h 51Cr release assay (34-36, 44). Specific cytotoxicity mediated by effector cells was calculated by substracting the lysis of uninfected targets from the lysis of serovar Typhi-infected targets. Lysis of noninfected targets was generally less than 10%. The cutoff for positive responses in CTL assays was defined as >10% specific cytotoxicity (34-36, 44).
IFN- ELISPOT assays. The human IFN- ELISPOT assay was carried out as previously described (34-36). Briefly, PBMC from immunized volunteers were incubated with uninfected or serovar Typhi-infected stimulator cells. Effector cells cultured without stimulator cells or with CD3/CD28 beads (0.6 μl/ml; Dynal) were used as negative and positive controls, respectively. After 16 h, cultures were assayed for IFN- by ELISPOT. Anti-IFN- (5 μg/ml, clone 2G1; Endogen, Woburn, MA) and biotinylated anti-IFN- (2 μg/ml, clone B133.5; Endogen) MAbs were used as capture and detection reagents, respectively. Complexes were detected by incubation with avidin-peroxidase conjugate (Sigma), and TrueBlue reagent (KPL) was used as a substrate. Each sample was tested in triplicate. Spots were enumerated using a stereomicroscope. Net frequencies were calculated as previously described (34-36). The cutoff for ELISPOT assays was established as the frequency of IFN- spot-forming cells/106 PBMC in cocultures of effectors with noninfected targets plus 3 standard errors (SE).
Statistical analysis. All tests were performed using SigmaStat software (version 3.0; SPSS Science software products, Chicago, IL). P values that were <0.05 were considered significant.
RESULTS
Generation of serovar Typhi-specific polyclonal cell lines for V analysis. Our initial approach to evaluating the V repertoire of specific T cells elicited in humans following immunization with the attenuated Ty21a typhoid vaccine involved the enrichment of serovar Typhi-specific T cells by repeated cycles of in vitro restimulation with serovar Typhi-infected polyclonal autologous blasts. We have previously reported the use of serovar Typhi-specific T-cell lines following one cycle of in vitro stimulation with serovar Typhi-infected autologous polyclonal blasts to demonstrate specific cytotoxic activity and IFN- production in clinical trials of attenuated serovar Typhi oral live vector vaccines (34-36). In the present studies, serovar Typhi-specific polyclonal cell lines from three Ty21a typhoid vaccinees generated following one cycle of in vitro stimulation were further expanded by weekly restimulation with irradiated autologous serovar Typhi-infected blasts for up to 3 weeks before specificity and TCR V repertoire analysis. Aliquots of these effector cells were frozen weekly before each new restimulation, and the phenotype of these cell lines was determined. The specificity of these cell lines was evaluated by measuring their ability to secrete IFN- and to kill serovar Typhi-infected targets.
Secretion of IFN- by effector cells in response to stimulation with serovar Typhi-infected targets was measured by ELISPOT in ex vivo PBMC and in polyclonal cell lines after one, two, and three cycles of in vitro restimulation. Marked increases in the frequency of IFN--secreting cells in response to stimulation with serovar Typhi-infected targets were observed. The largest increase of IFN- production occurred after one cycle of in vitro restimulation. Similarities in the levels of IFN- production following two and three cycles of in vitro restimulation suggest a stabilization of these responses with continued in vitro stimulation. Compared with preimmunization levels without in vitro restimulation, postimmunization PBMC without in vitro restimulation showed a 3-fold increase, while polyclonal cell lines showed an average 35-fold increase in serovar Typhi-responsive cells (63-, 24-, and 19-fold after one, two, and three cycles of in vitro restimulation, respectively) (Fig. 1). This corresponds to 0.3 to 1% of the total cell population (2,853 to 9,640 spots/106 cells in these polyclonal cell lines).
51Cr release assays and correlation with IFN--secreting effector T-cell populations. Cytolytic function of the polyclonally expanded cell lines was measured by standard 51Cr release assays. After 3 weeks of in vitro restimulation with autologous serovar Typhi-infected targets, we observed that these cell lines maintain high levels of CTL activity against serovar Typhi-infected targets. As shown in Fig. 2, similar results were obtained in volunteers 1 and 2. Consistent with our previous results, ex vivo PBMC from both volunteers were unable to lyse autologous blasts infected with serovar Typhi (35, 36). However, both volunteers showed increased levels of cytotoxic activity after in vitro restimulation. Although CTL activity is at best semiquantitative, it is important to note that, as observed with IFN- production, the highest increase of the cytotoxicity levels occurred after one in vitro restimulation followed by stabilization after two and three cycles of in vitro restimulation (e.g., volunteer 2 exhibited 33, 17, and 17% of specific lysis at an effector-to-target cell [E:T] ratio of 3:1 after one, two, and three in vitro restimulation cycles, respectively). Moreover, as we have previously described (35), when we compared the results obtained by ELISPOT with those obtained by 51Cr release assays, a positive correlation (R2 = 0.910, P < 0.046) was observed between the frequencies of serovar Typhi antigen-responsive cells, as evidenced by IFN- production and the cytotoxic activity of these cell lines at E:T ratios of 3:1 (data not shown).
Taken together, the results from IFN- ELISPOT and CTL assays clearly show that the polyclonal cell lines remain functional after repeated cycles of specific antigenic stimulation.
Characterization of the effector population. We next investigated which cell populations in PBMC from immunized volunteers were preferentially expanded after several cycles of in vitro restimulation with serovar Typhi-infected autologous blasts. In agreement with previous results using a similar system (34-36), a major expansion in CD8+ T-cell populations, as determined by flow cytometry, occurred during in vitro restimulations. The percentage of CD8+ T-cell populations increased progressively after each additional in vitro restimulation (e.g., 26, 44, and 63% after one, two, and three cycles of in vitro restimulation, respectively). These increases were accompanied by concomitant decreases in the percentage of CD4+ cells at the same time points (28, 13, and 9% after one, two, and three cycles of in vitro restimulation, respectively) (Fig. 3). No changes were observed in the percentage of NK (CD56+) or T cells bearing / T-cell receptors (Fig. 3).
To further characterize the composition of polyclonal cell lines after in vitro restimulation, the proportion of cells expressing molecules involved in T-cell activation and those associated with memory effector T cells in populations following three cycles of in vitro expansion were compared to those present in PBMC without in vitro restimulation. To this end, PBMC from Ty21a vaccinees ex vivo or cocultured with stimulators for up to 3 weeks were stained with MAbs to CD3, CD4, CD8, CD56, CD25, CD28, CD38, CD45RO, CD62L, and HLA-DR and analyzed by multicolor flow cytometry. The results are shown in Table 2. Of the various CD3+ CD8+ cell subpopulations expressing CD45RO+, a T-cell memory marker, only the percentages of CD38+ and HLA-DR+ differed significantly between ex vivo and in vitro-restimulated PBMC. Five-color flow cytometric analysis showed that in vitro-expanded CD3+ CD8+ CD45RO+ T cells contained a greater proportion of cells bearing activation molecules (HLA-DR, 97%; CD38+, 39%) than did the initial ex vivo population (HLA-DR, 24%; CD38+, 9%). Smaller increases were also observed in the percentages of CD3+ CD4+ CD45RO+ populations coexpressing HLA-DR and CD38 after each cycle of restimulation (Table 2). Of note is that no major differences were observed in the expression of CD62L and CD28 in either cell population (Table 2).
Taken together, the results from the functional assays and the analysis of surface expression of activation/memory molecules suggest that activated CD8+ T cells coexpressing memory markers remain functional after several rounds of in vitro stimulation and are therefore appropriate for studying the TCR V repertoire induced by immunization of volunteers with the Ty21a typhoid vaccine.
Flow cytometric V analysis in polyclonal cell lines. Following functional validation and characterization of the polyclonal cell lines, we next evaluated the TCR V repertoire of these populations. First, we examined whether CD8+ T cells with a broad TCR V repertoire or a limited selected repertoire are involved in the host's serovar Typhi response after administration of the Ty21a typhoid vaccine. To this end, we studied the TCR V usage of these cell lines by flow cytometry using a panel of 24 MAbs which cover 60% of the known specificities (47).
Five-color cytofluorometric analysis was performed to determine whether CD4+ or CD8+ T cells of defined V were preferentially expanded by in vitro restimulation. Changes above the mean plus 3 SE of the ex vivo effector cells (controls) were defined as being increased. Although TCR V diversity varied greatly from volunteer to volunteer, we observed the oligoclonal expansion of particular V subsets in each individual (V 2, 5.1, 8, 17, and 22 in volunteer 1; V 1, 2, 5.1, 14, 17, and 22 in volunteer 2; and V 3, 8, 14, and 16 in volunteer 3). As shown in Fig. 4, volunteer 1 exhibited a marked increase in the percentage of CD8+ T cells expressing TCR V 17 after three in vitro restimulations (26.4%) compared to postimmunization levels without in vitro restimulation (3.0%), whereas CD4+ V 17+ cells did not change significantly over time (5.8, 5.4, 5.4, and 5.8% ex vivo and after one, two, and three restimulations, respectively) (data not shown). In volunteer 2, a major increase in the percentage of CD8+ T cells expressing TCR V 2 was found even after a single cycle of in vitro restimulation (14.3%), and it continued to rise after three successive cycles of in vitro restimulation (20.5%), compared to postimmunization levels without in vitro restimulation (4.1%) (Fig. 4). CD4+ V 2+ cells did not change significantly over time (4.2, 6.3, 5.3, and 6.5% ex vivo and after one, two, and three restimulations, respectively) (data not shown). Taken together, the most prominent changes were observed in the V 2, 3, 5.1, 14, 16, and 17 families. Increases in V 2, V 5.1, and V 17 were observed in both volunteer 1 and volunteer 2. Of note is that although in volunteer 3 the V 2 and V 17 changes did not reach the mean percentage plus 3 SE of ex vivo effector cells used as a cutoff level of positivity, increases of 5 to 18 times over the values observed ex vivo without restimulation were observed after the first and second restimulations (i.e., for V 2, 0.1, 1.43, and 1.85% and for V 17, 0.19, 1.02, and 2.43% of positive cells were observed ex vivo, after one cycle, and after two cycles, respectively).
It is important to note that except for the overrepresentation of V 13.2 in volunteer 2 and V 12 in volunteer 3, the reported distributions of V usage observed in ex vivo effectors (controls) are comparable to the V usage previously reported for healthy controls (Table 1) (47). Moreover, the sum average of the percentages of all V specificities in ex vivo CD8+ PBMC after immunization was 69% (volunteer 1, 72%; volunteer 2, 72%; and volunteer 3, 61%), further supporting the contention that the V usage in these volunteers is similar to that previously reported for healthy individuals (60% of CD8+ PBMC) (Table 1) (47).
We next evaluated whether, following the remarkable expansion of CD8+ TCR V 2 T cells, they remain functional in mediating CTL activity against serovar Typhi-infected cells. To this end, all three in vitro-restimulated postimmunization PBMC aliquots from volunteer 2 were pooled and surface-stained for CD8, CD56, CD3, V 2, V 5.2, and V 12 and the TCR V 2+ CD8+ CD56– CD3+ and TCR V 2– CD8+ CD56– CD3+ populations sorted (Fig. 5A). The CTL activity of sorted as well as unsorted populations was measured by 51Cr release assays against serovar Typhi-infected autologous blasts. More than 10% specific lysis was observed in both sorted and unsorted PBMC at an E:T ratio of 3:1 (Fig. 5B). These results demonstrate that both TCR V 2+ CD3+ CD8+ and TCR V 2– CD3+ CD8+ populations are functional and specific for serovar Typhi-infected targets. Moreover, these results also indicate that TCR V specificities other than V 2 are also able to mediate CTL activity. Taken together, these observations support the notion that more than one TCR V family is involved in the host's response to serovar Typhi after vaccination with the Ty21a typhoid vaccine.
TCR V repertoire and expression of molecules involved in homing and effector and memory function in T-cell clones specific for serovar Typhi antigens. To confirm the V usage of T cells elicited by immunization, as well as to identify additional TCR V specificities that could be involved in specific responses to serovar Typhi, T-cell clones were derived from PBMC from volunteers 2 and 3. T-cell clones were derived by limiting dilution, as described in Materials and Methods, and established cell lines were examined for TCR V usage, a procedure that also helped to confirm the clonality of the populations. The functional activity of these T-cell clones was examined by specific cytotoxicity and frequency of IFN--secreting cells against serovar Typhi-infected targets by 51Cr release and IFN- ELISPOT assays, respectively. Figure 6 summarizes the IFN- production, cytotoxicity, and TCR V usage of eight CD8+ T-cell clones. In ELISPOT assays, two CD4+ T-cell clones (TP1-24 and TS1-24) were used as negative controls. The clones that either killed infected targets or produced IFN- were considered responders. Among the six responder CD8+ T-cell clones (TP1-6, TP1-16, TP1-38, TP1-39, TP1-40, and TS1-27), one clone expressed V 7.2, one expressed V 3, and two expressed V 16. For two clones (TP1-16 and TP1-38), no reactivity was observed with any of the anti-TCR V MAbs in our panel. This is likely the result of the lack of a MAb for the appropriate V specificity in the panel, which in the case of volunteer 3 identified 61% of the TCR V repertoire. Of note is that while some T-cell clones (e.g., TP1-38 and TS1-27) exhibited both CTL and IFN- production capabilities, other clones (e.g., TP1-16) were only either cytotoxic or able to produce IFN-, suggesting heterogeneity in the effector function of different T-cell subpopulations following immunization with serovar Typhi vaccines.
It is widely recognized that oral immunization induces effector memory cells carrying distinct homing receptors (e.g., integrin 47, CCR9, and CD103) that might endow the effector cells with the capability to migrate preferentially to the gut (13, 18, 33, 39). Thus, it was important to assess whether these molecules are present on the T-cell clones from volunteers immunized orally with the Ty21a typhoid vaccine described above. Moreover, it was also important to determine whether these T-cell clones exhibit an effector memory phenotype. To this end, clones were stained with MAbs to CD8, CD27, CCR7, CD45RO, CD62L, 47, CCR9, and CD103 and analyzed by eight-color flow cytometry. Despite the observed functional and TCR V usage differences among these clones, there were remarkable similarities in their phenotype and expression of homing molecules. All serovar Typhi-specific T-cell clones were found to be CCR7– CD27– CD45RO+ CD62Llow, i.e., exhibit an effector memory phenotype (9, 37). Moreover, clones expressed high levels of integrin 47 (>92%), moderate levels of CCR9 (8 to 35%), and low levels of CD103 (1 to 8%) (Fig. 7). Taken together, these results confirm an oligoclonal expansion of V serovar Typhi-specific CD8+ T cells, which have a phenotype consistent with T effector memory cells with preferential gut homing potential.
DISCUSSION
In the present study, we evaluated the TCR V specificities of CD8+ T cells from three healthy adults orally immunized with the attenuated serovar Typhi Ty21a strain typhoid vaccine. PBMC from Ty21a typhoid vaccinees were expanded by weekly restimulation with irradiated autologous serovar Typhi-infected blasts for up to 3 weeks and used as effector cells. We found that CD8+ T cells from several V families participated in the anti-serovar Typhi response. Although TCR V usage varied from one volunteer to another, our results are consistent with an oligoclonal V subset expansion in each volunteer (V 2, 5.1, 8, 17, and 22 in volunteer 1; V 1, 2, 5.1, 14, 17, and 22 in volunteer 2; and V 3, 8, 14, and 16 in volunteer 3). Moreover, we observed that these subsets were antigen specific, as shown by functional assays on cells sorted based on their V expression and on T-cell clones. Furthermore, we show that V serovar Typhi-specific CD8+ T cells exhibit a phenotype consistent with effector memory cells with gut homing potential.
Restricted T-cell V repertoires have been shown in infections with bacteria, including Streptococcus (27, 46), Staphylococcus (8, 38), Mycobacterium tuberculosis (11, 28), Listeria (15), and Shigella (16). Although the TCR V repertoire observed with these pathogens was somewhat diverse, TCR V 2 and V 3 usage appears to be favored. The observation in our studies with volunteers immunized with serovar Typhi that V 2 and V 3 specificities were preferentially used appears to support this notion. Interestingly, a study with Shigella (16), a pathogen that shares 80% homology with serovar Typhi, showed a pattern of TCR V usage surprisingly similar to that observed in our expanded T-cell lines. Despite the fact that in this study a limited panel composed of just nine MAbs to different TCR V specificities was used (compared to the 24 V specificities evaluated in our study), the increases in TCR V 2, V 3, V 5.1, V 13.6, and V 17 reported were remarkably similar to the ones observed in our study (TCR V 1, V 2, V 3, V 5.1, V 8, V 14, V 16, V 17, and V 22). Taken together, these observations support the notion that it is likely that T-cell responses against some, if not most, bacteria might be oligoclonal in nature and use a relatively restricted set of TCR V specificities.
The aim of the studies presented in this paper was to analyze the TCR V usage in healthy adults 5 to 40 months after oral immunization with serovar Typhi strain Ty21a to evaluate the persistence of serovar Typhi-specific responses over time. Because the postimmunization specimens were collected long after the T-cell memory contraction phase took place, it is not surprising that we did not observe differences when comparing the ex vivo TCR V usage before and after immunization in two of the volunteers (volunteers 1 and 2) (data not shown). To overcome this difficulty, we evaluated the V repertoire of specific T cells after repeated cycles of in vitro restimulation with serovar Typhi-infected polyclonal autologous blasts. Although this procedure does not allow us to estimate their frequency in vivo, available evidence with other gram-negative bacteria suggests that it should not have negatively affected the identification of at least some of the different specificities elicited by immunization with serovar Typhi. In this context, although some reports studying virus-specific CTL populations showed that in vitro expansion of lymphocytes can introduce a significant bias in TCR usage (41), a previous study in Shigella (16) showed that the in vitro TCR V repertoire was similar to that observed in vivo. By comparing the in vivo and in vitro changes in the TCR V repertoire over time, the authors concluded that the in vitro response induced by Shigella antigens showed a TCR V distribution similar to that observed in vivo. Interestingly, the changes in TCR V usage observed in that study peaked 8 to 11 days after the onset of disease, returning to baseline levels after 30 days. Islam et al. hypothesized that this kinetics was either due to homing of circulating cells to the gut-associated lymphoid tissues or, alternatively, due to apoptotic cell death (16). Interestingly, in the present study, we also observed that the expansion of some V specificities after one cycle of in vitro restimulation (about day 8) was followed by contraction and/or stabilization of these same V specificities during the next rounds of in vitro restimulation. Of note is that after one cycle of in vitro restimulation, we observed stabilization in the cytotoxicity of and IFN- production by serovar Typhi-specific CD8+ T cells, despite an increase in the percentage of CD8+ T cells up to the third week of restimulation. These results may reflect the loss of some clones (e.g., by apoptosis death or loss of specificity) and their replacement by others following successive in vitro restimulations, a phenomenon frequently observed with T-cell clones. Finally, it should be emphasized that it is presently not possible to conclusively assess to what extent the specificities that we observed following in vitro expansion are representative of those present in vivo following immunization. This assertion is based on the lack of a complete understanding of the full complement of immune responses to serovar Typhi following immunization that depend, at least in part, on the set of antigens presented during the various stages of in vivo serovar Typhi infection (50), the presence of variables during in vitro expansion that are difficult to control, and technological limitations. These limitations include the following: (i) in most cases, TCR V antigen-specific T-cell precursors exist at frequencies below the level of detection by standard ex vivo methodologies, e.g., tetrameric complexes; (ii) even in the case of high in vivo frequency of certain TCR V antigen-specific T-cell precursors, T-cell stimulation may require a particular TCR conformation or conformational change that is found only on the surface of certain presenting cells (6); and (iii) while Bouneaud et al. (2) showed that nearly half of their tetramer-binding T cells were unable to expand in response to physiological levels of antigen, Bullock et al. (4) showed that the reverse may also occur, i.e., T cells that show strong functional reactivity towards target cells may not necessarily be detected ex vivo by major histocompatibility complex-tetramer staining.
It is well established that lymphocytes continuously recirculate from peripheral blood to secondary lymphoid tissues (3). This recirculation is essential for homing of effector memory cells, which do not express CCR7 or CD27 and are mostly CD45RO+ CD62Llow, to effector sites (9, 13, 37). Serovar Typhi cells are highly invasive bacteria that rapidly and efficiently colonize and invade the mucosa of the small intestine following ingestion (19). Thus, in Salmonella infections, it is likely that specific memory effector T cells in circulation will be homing to effector sites in the gut where they might encounter Salmonella antigens. Selective homing of effector memory cells to the lamina propria of the small intestine is driven, to a large extent, by the expression of the integrin 47, which binds MAdCAM-1, a molecule selectively expressed in the venules of normal and inflamed intestinal endothelium (5). In this regard, a study by Lundin et al. has shown that immunization with the Ty21a typhoid vaccine induces antigen-specific T cells that preferentially express integrin 47 and are found in circulation up to 21 days following immunization (23). However, other molecules have also recently been shown to participate in this homing process. For example, expression of CCR9 in subsets of circulating integrin 47hi or E7 (CD103) lymphocytes plays an important role in homing of these cells to the small intestine (13, 18, 43). Thus, it was important to evaluate the patterns of expression of these homing molecules in V serovar Typhi-specific CD8+ T cells. We observed that T-cell clones, regardless of their TCR V usage, exhibited an effector memory phenotype (i.e., CCR7– CD27– CD45RO+ CD62Llow) and coexpressed high levels of integrin 47, intermediate levels of CCR9, and low levels of CD103 gut homing molecules, suggesting that these TCR V-specific clones have the potential to home to the gut-associated lymphoid tissues.
It has been reported that only a few V families predominate in the human gut mucosa (1), with the most frequent being V 1, V 2, V 3, and V 6 (49). Because we studied peripheral blood, we have to consider the possibility that our observations might not reflect the full spectrum of TCR V usage by serovar Typhi-specific CD8+ T cells in the gut microenvironment in vivo. However, our observations that (i) serovar Typhi-specific CD8+ T cells from Ty21a vaccinees exhibited gut homing potential and (ii) except for V 6, which was not present in our MAb panel, we observed all other V specificities (V 1, V 2, and V 3, with the most prominent changes in V 2 and V 3) support the contention that the TCR V usage that we observed in circulation might reflect to a large extent that found in the gut mucosa microenvironment.
Taken together, our observations suggest that the CD8+ effector memory T-cell subpopulations of the restricted TCR V specificities described in this paper are likely to be the effector cells that mediate anti-serovar Typhi long-term cell-mediated immune responses in the local microenvironment of the gut mucosa following oral immunization with attenuated strains of serovar Typhi.
ACKNOWLEDGMENTS
We are indebted to the volunteers who allowed us to perform this study. We also thank Bernadette McConnell and the staff from the Blood Bank of the University of Maryland hospital for their help in collecting leukapheresis specimens and Regina Harley for excellent technical assistance in flow cytometric determinations and sorting.
This work was supported by a grant from the National Institute of Allergy and Infectious Diseases, NIH (R01 AI36525 to M.B.S.).
REFERENCES
1. Blumberg, R. S., C. E. Yockey, G. G. Gross, E. C. Ebert, and S. P. Balk. 1993. Human intestinal intraepithelial lymphocytes are derived from a limited number of T cell clones that utilize multiple V beta T cell receptor genes. J. Immunol. 150:5144-5153.
2. Bouneaud, C., P. Kourilsky, and P. Bousso. 2000. Impact of negative selection on the T cell repertoire reactive to a self-peptide: a large fraction of T cell clones escapes clonal deletion. Immunity 13:829-840.
3. Brandtzaeg, P., I. N. Farstad, and G. Haraldsen. 1999. Regional specialization in the mucosal immune system: primed cells do not always home along the same track. Immunol. Today 20:267-277.
4. Bullock, T. N., D. W. Mullins, T. A. Colella, and V. H. Engelhard. 2001. Manipulation of avidity to improve effectiveness of adoptively transferred CD8(+) T cells for melanoma immunotherapy in human MHC class I-transgenic mice. J. Immunol. 167:5824-5831.
5. Butcher, E. C., M. Williams, K. Youngman, L. Rott, and M. Briskin. 1999. Lymphocyte trafficking and regional immunity. Adv. Immunol. 72:209-253.
6. Coles, R. M., C. M. Jones, A. G. Brooks, P. U. Cameron, W. R. Heath, and F. R. Carbone. 2003. Virus infection expands a biased subset of T cells that bind tetrameric class I peptide complexes. Eur. J. Immunol. 33:1557-1567.
7. Davis, M. M., and P. J. Bjorkman. 1988. T-cell antigen receptor genes and T-cell recognition. Nature 334:395-402.
8. Ericson, M. L., N. A. Mooney, and D. J. Charron. 1996. Prokaryotic production of the human T cell receptor Vbeta2 chain and binding to toxic-shock syndrome toxin-1. Gene 168:257-260.
9. Esser, M. T., R. D. Marchese, L. S. Kierstead, L. G. Tussey, F. Wang, N. Chirmule, and M. W. Washabaugh. 2003. Memory T cells and vaccines. Vaccine 21:419-430.
10. Gagnon, S. J., A. Leporati, S. Green, S. Kalayanarooj, D. W. Vaughn, H. A. Stephens, S. Suntayakorn, I. Kurane, F. A. Ennis, and A. L. Rothman. 2001. T cell receptor Vbeta gene usage in Thai children with dengue virus infection. Am. J. Trop. Med. Hyg. 64:41-48.
11. Gambon-Deza, F., M. Pacheco Carracedo, T. Cerd á Mota, and J. Montes Santiago. 1995. Lymphocyte populations during tuberculosis infection: V repertoires. Infect. Immun. 63:1235-1240.
12. Gomez-Duarte, O. G., M. F. Pasetti, A. Santiago, M. B. Sztein, S. L. Hoffman, and M. M. Levine. 2001. Expression, extracellular secretion, and immunogenicity of the Plasmodium falciparum sporozoite surface protein 2 in Salmonella vaccine strains. Infect. Immun. 69:1192-1198.
13. Hamann, A., D. P. Andrew, D. Jablonski-Westrich, B. Holzmann, and E. C. Butcher. 1994. Role of alpha 4-integrins in lymphocyte homing to mucosal tissues in vivo. J. Immunol. 152:3282-3293.
14. Hingorani, R., I. H. Choi, P. Akolkar, B. Gulwani-Akolkar, R. Pergolizzi, J. Silver, and P. K. Gregersen. 1993. Clonal predominance of T cell receptors within the CD8+ CD45RO+ subset in normal human subjects. J. Immunol. 151:5762-5769.
15. Huleatt, J. W., I. Pilip, K. Kerksiek, and E. G. Pamer. 2001. Intestinal and splenic T cell responses to enteric Listeria monocytogenes infection: distinct repertoires of responding CD8 T lymphocytes. J. Immunol. 166:4065-4073.
16. Islam, D., B. Wretlind, A. A. Lindberg, and B. Christensson. 1996. Changes in the peripheral blood T-cell receptor V repertoire in vivo and in vitro during shigellosis. Infect. Immun. 64:1391-1399.
17. Ivanoff, B., M. M. Levine, and P. H. Lambert. 1994. Vaccination against typhoid fever: present status. Bull. W. H. O. 72:957-971.
18. Kantele, A., J. Zivny, M. Hakkinen, C. O. Elson, and J. Mestecky. 1999. Differential homing commitments of antigen-specific T cells after oral or parenteral immunization in humans. J. Immunol. 162:5173-5177.
19. Levine, M., and M. Sztein. 2000. Shigella, Salmonella typhi, and Escherichia coli, p. 171-194. In M. Cunningham and R. Fujinami (ed.), Effects of microbes on the immune system. Lippincott Williams & Wilkins, Philadelphia, Pa.
20. Levine, M. M., C. Ferreccio, P. Abrego, O. S. Martin, E. Ortiz, and S. Cryz. 1999. Duration of efficacy of Ty21a, attenuated Salmonella typhi live oral vaccine. Vaccine 17:S22-S27.
21. Levine, M. M., and O. S. Levine. 1997. Influence of disease burden, public perception, and other factors on new vaccine development, implementation, and continued use. Lancet 350:1386-1392.
22. Lim, A., L. Trautmann, M. A. Peyrat, C. Couedel, F. Davodeau, F. Romagne, P. Kourilsky, and M. Bonneville. 2000. Frequent contribution of T cell clonotypes with public TCR features to the chronic response against a dominant EBV-derived epitope: application to direct detection of their molecular imprint on the human peripheral T cell repertoire. J. Immunol. 165:2001-2011.
23. Lundin, B. S., C. Johansson, and A.-M. Svennerholm. 2002. Oral immunization with a Salmonella enterica serovar Typhi vaccine induces specific circulating mucosa-homing CD4+ and CD8+ T cells in humans. Infect. Immun. 70:5622-5627.
24. Maus, M. V., A. K. Thomas, D. G. Leonard, D. Allman, K. Addya, K. Schlienger, J. L. Riley, and C. H. June. 2002. Ex vivo expansion of polyclonal and antigen-specific cytotoxic T lymphocytes by artificial APCs expressing ligands for the T-cell receptor, CD28 and 4-1BB. Nat. Biotechnol. 20:143-148.
25. McCloskey, T. W., V. Haridas, R. Pahwa, and S. Pahwa. 2002. T cell receptor V beta repertoire of the antigen specific CD8 T lymphocyte subset of HIV infected children. AIDS 16:1459-1465.
26. Meuer, S. C., K. A. Fitzgerald, R. E. Hussey, J. C. Hodgdon, S. F. Schlossman, and E. L. Reinherz. 1983. Clonotypic structures involved in antigen-specific human T cell function. Relationship to the T3 molecular complex. J. Exp. Med. 157:705-719.
27. Ohara-Nemoto, Y., and M. Kaneko. 1996. Expression of T-cell receptor V beta 2 and type 1 helper T-cell-related cytokine mRNA in streptococcal pyrogenic exotoxin-C-activated human peripheral blood mononuclear cells. Can. J. Microbiol. 42:1104-1111.
28. Ohmen, J. D., P. F. Barnes, C. L. Grisso, B. R. Bloom, and R. L. Modlin. 1994. Evidence for a superantigen in human tuberculosis. Immunity 1:35-43.
29. Paliard, X., S. G. West, J. A. Lafferty, J. R. Clements, J. W. Kappler, P. Marrack, and B. L. Kotzin. 1991. Evidence for the effects of a superantigen in rheumatoid arthritis. Science 253:325-329.
30. Pannetier, C., J. Even, and P. Kourilsky. 1995. T-cell repertoire diversity and clonal expansions in normal and clinical samples. Immunol. Today 16:176-181.
31. Pasetti, M. F., R. J. Anderson, F. R. Noriega, M. M. Levine, and M. B. Sztein. 1999. Attenuated guaBA Salmonella typhi vaccine strain CVD 915 as a live vector utilizing prokaryotic or eukaryotic expression systems to deliver foreign antigens and elicit immune responses. Clin. Immunol. 92:76-89.
32. Pasetti, M. F., M. M. Levine, and M. B. Sztein. 2003. Animal models paving the way for clinical trials of attenuated Salmonella enterica serovar Typhi live oral vaccines and live vectors. Vaccine 21:401-418.
33. Pasetti, M. F., R. Salerno-Gonalves, and M. B. Sztein. 2005. Mechanisms of adaptive immunity that prevent colonization at mucosal surfaces, p. 35-47. In J. P. Nataro, P. S. Cohen, H. L. T. Mobley, and J. N. Weiser (ed.), Colonization of mucosal surfaces. American Society for Microbiology, Washington, D.C.
34. Salerno-Goncalves, R., M. Fernandez-Vina, D. M. Lewinsohn, and M. B. Sztein. 2004. Identification of a human HLA-E-restricted CD8+ T cell subset in volunteers immunized with Salmonella enterica serovar Typhi strain Ty21a typhoid vaccine. J. Immunol. 173:5852-5862.
35. Salerno-Goncalves, R., M. F. Pasetti, and M. B. Sztein. 2002. Characterization of CD8(+) effector T cell responses in volunteers immunized with Salmonella enterica serovar Typhi strain Ty21a typhoid vaccine. J. Immunol. 169:2196-2203.
36. Salerno-Goncalves, R., T. L. Wyant, M. F. Pasetti, M. Fernandez-Vina, C. O. Tacket, M. M. Levine, and M. B. Sztein. 2003. Concomitant induction of CD4(+) and CD8(+) T cell responses in volunteers immunized with Salmonella enterica serovar Typhi strain CVD 908-htrA. J. Immunol. 170:2734-2741.
37. Sallusto, F., D. Lenig, R. Forster, M. Lipp, and A. Lanzavecchia. 1999. Two subsets of memory T lymphocytes with distinct homing potentials and effector functions. Nature 401:708-712.
38. Seth, A., L. J. Stern, T. H. Ottenhoff, I. Engel, M. J. Owen, J. R. Lamb, R. D. Klausner, and D. C. Wiley. 1994. Binary and ternary complexes between T-cell receptor, class II MHC and superantigen in vitro. Nature 369:324-327.
39. Shaw, S. K., and M. B. Brenner. 1995. The beta 7 integrins in mucosal homing and retention. Semin. Immunol. 7:335-342.
40. Soroosh, P., F. Shokri, M. Azizi, and M. Jeddi-Tehrani. 2003. Analysis of T-cell receptor beta chain variable gene segment usage in healthy adult responders and nonresponders to recombinant hepatitis B vaccine. Scand. J. Immunol. 57:423-431.
41. Sourdive, D. J., K. Murali-Krishna, J. D. Altman, A. J. Zajac, J. K. Whitmire, C. Pannetier, P. Kourilsky, B. Evavold, A. Sette, and R. Ahmed. 1998. Conserved T cell receptor repertoire in primary and memory CD8 T cell responses to an acute viral infection. J. Exp. Med. 188:71-82.
42. Stewart-Jones, G. B., A. J. McMichael, J. I. Bell, D. I. Stuart, and E. Y. Jones. 2003. A structural basis for immunodominant human T cell receptor recognition. Nat. Immunol. 4:657-663.
43. Svensson, M., J. Marsal, A. Ericsson, L. Carramolino, T. Broden, G. Marquez, and W. W. Agace. 2002. CCL25 mediates the localization of recently activated CD8alphabeta(+) lymphocytes to the small-intestinal mucosa. J. Clin. Investig. 110:1113-1121.
44. Sztein, M. B., M. K. Tanner, Y. Polotsky, J. M. Orenstein, and M. M. Levine. 1995. Cytotoxic T lymphocytes after oral immunization with attenuated vaccine strains of Salmonella typhi in humans. J. Immunol. 155:3987-3993.
45. Sztein, M. B., S. S. Wasserman, C. O. Tacket, R. Edelman, D. Hone, A. A. Lindberg, and M. M. Levine. 1994. Cytokine production patterns and lymphoproliferative responses in volunteers orally immunized with attenuated vaccine strains of Salmonella typhi. J. Infect. Dis. 170:1508-1517.
46. Tomai, M. A., J. A. Aelion, M. E. Dockter, G. Majumdar, D. G. Spinella, and M. Kotb. 1991. T cell receptor V gene usage by human T cells stimulated with the superantigen streptococcal M protein. J. Exp. Med. 174:285-288.
47. van den Beemd, R., P. P. Boor, E. G. van Lochem, W. C. Hop, A. W. Langerak, I. L. Wolvers-Tettero, H. Hooijkaas, and J. J. van Dongen. 2000. Flow cytometric analysis of the Vbeta repertoire in healthy controls. Cytometry 40:336-345.
48. van den Elsen, P. J., and A. Rudensky. 2004. Antigen processing and recognition. Recent developments. Curr. Opin. Immunol. 16:63-66.
49. Van Kerckhove, C., G. J. Russell, K. Deusch, K. Reich, A. K. Bhan, H. DerSimonian, and M. B. Brenner. 1992. Oligoclonality of human intestinal intraepithelial T cells. J. Exp. Med. 175:57-63.
50. Waterman, S. R., and D. W. Holden. 2003. Functions and effectors of the Salmonella pathogenicity island 2 type III secretion system. Cell. Microbiol. 5:501-511.
51. Wick, M. J. 2002. The role of dendritic cells during Salmonella infection. Curr. Opin. Immunol. 14:437-443.
52. Wick, M. J. 2003. The role of dendritic cells in the immune response to Salmonella. Immunol. Lett. 85:99-102.
53. Zissel, G., I. Baumer, B. Fleischer, M. Schlaak, and J. Muller-Quernheim. 1997. TCR V beta families in T cell clones from sarcoid lung parenchyma, BAL, and blood. Am. J. Respir. Crit. Care Med. 156:1593-1600.(Rosangela Salerno-Gonalve)
ABSTRACT
CD8+ T cells are likely to play an important role in host defense against Salmonella enterica serovar Typhi by several effector mechanisms, including lysis of infected cells (cytotoxicity) and gamma interferon (IFN-) secretion. In an effort to better understand these responses, we studied the T-cell receptor (TCR) repertoire of serovar Typhi-specific CD8+ T cells in humans. To this end, we determined the TCR beta chain (V) usage of CD8+ T cells from three volunteers orally immunized with Ty21a typhoid vaccine by flow cytometry using a panel of monoclonal antibodies. Although TCR V usage varied among volunteers, we identified oligoclonal V subset expansions in individual volunteers (V 2, 5.1, 8, 17, and 22 in volunteer 1; V 1, 2, 5.1, 14, 17, and 22 in volunteer 2; and V 3, 8, 14, and 16 in volunteer 3). These subsets were antigen specific, as shown by cytotoxicity and IFN- secretion assays on V sorted cells and on T-cell clones derived from these volunteers. Moreover, eight-color flow cytometric analysis showed that these clones exhibited a T effector memory phenotype (i.e., CCR7– CD27– CD45RO+ CD62L–) and coexpressed gut homing molecules (e.g., high levels of integrin 47, intermediate levels of CCR9, and low levels of CD103). In conclusion, our results show that long-term T-cell responses to serovar Typhi in Ty21a vaccinees are oligoclonal, involving multiple TCR V families. Moreover, these serovar Typhi-specific CD8+ T cells bearing defined V specificities are phenotypically and functionally consistent with T effector memory cells with preferential gut homing potential.
INTRODUCTION
Typhoid fever remains an important public health priority, particularly in developing countries, with an estimated 16 million new cases annually and 600,000 deaths (17, 21). The appearance of antibiotic-resistant strains of Salmonella enterica serovar Typhi, the causative agent of typhoid fever, has added new urgency for the development of improved typhoid vaccines. New-generation attenuated serovar Typhi vaccine strains have the potential to become the preferred public health tool to immunize against typhoid fever because of their ability to elicit long-lasting protective systemic and mucosal immune responses. In addition, significant efforts have been focused in recent years on the use of attenuated serovar Typhi vaccines as carriers of foreign antigens to deliver foreign genes because of their ability to gain access to dendritic cells (51, 52) and deliver antigens or DNA coding for the antigens (12, 31, 32). These characteristics make the use of Salmonella live vectors particularly attractive for mass immunization programs. Moreover, live attenuated serovar Typhi vaccines are expected to have relatively low manufacturing costs compared to many other types of vaccines.
CD8+ T cells might play an important role in host defense against serovar Typhi by several effector mechanisms, including lysis of infected cells and gamma interferon (IFN-) secretion (35, 36, 44, 45). These CD8+ T cells recognize antigenic peptides bound to self-major histocompatibility complex via T-cell antigen receptor (TCR) (26, 48) on the surface of infected cells. The TCR on most peripheral T cells is a heterodimeric molecule, composed of (V) and (V) chains (7). Functionally, V has been associated with the antigen recognition process, and specific recognition of peptide results in the clonal expansion of a subset of V antigen-specific T cells (7, 26). Thus, direct examination of TCR V specificities has been used to identify and track oligoclonal antigen-specific T-cell responses during infection and after immunization (1, 14, 30). Approximately 60 TCR V gene segments are known in humans (30). TCR V usage has been studied in various systems, and either broad or restricted patterns have been identified in a number of areas, including autoimmunity (29, 53), response to viral (10, 25, 40, 42) or bacterial (8, 11, 16, 27) antigens, and tumor immunity (22).
The induction of strong, long-lived immunologic T- and B-cell memory responses is of paramount importance in the development of effective vaccines. Thus, in the present study, we analyzed the TCR V gene usage of specific CD8+ T cells from healthy adults 5 to 40 months after oral immunization with serovar Typhi strain Ty21a, the only licensed attenuated typhoid vaccine, to gain further insights into the clonality and characteristics of T-cell responses to serovar Typhi. Although the responses were oligoclonal, we observed that CD8+ T cells from several V families participated in the anti-serovar Typhi response. Moreover, we studied the induction of memory T-cell subsets and the homing potential of these cells. The specificity of the expanded cell subsets was demonstrated by functional assays on V sorted cells and on clones. Moreover, we observed that V serovar Typhi-specific CD8+ T cells express surface molecules associated with a T memory effector phenotype with gut homing potential.
MATERIALS AND METHODS
Subjects. Three healthy volunteers, 32 to 41 years old, recruited from the Baltimore-Washington area and University of Maryland at Baltimore campus, participated in this study. Volunteers were vaccinated following the routine immunization schedule for the Ty21a typhoid vaccine, i.e., four spaced doses of 2 x 109 to 6 x 109 CFU of Ty21a at an interval of 48 h between doses (20, 35). Volunteers were screened for good health by medical history, physical examination, normal laboratory tests, including blood counts, and the absence of antibiotic treatment at the times of leukapheresis. The purpose of this study was explained to volunteers, and they gave informed, signed consent. Cells from volunteers 1, 2, and 3 were collected 5, 25, and 40 months after immunization, respectively, to perform a time course analysis of the immune responses to serovar Typhi. Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation (35).
Target and stimulator cells. Blasts were established from PBMC isolated from Ty21a vaccinees following standard procedures (35, 36, 44) and maintained in RPMI 1640 (Gibco, Grand Island, New York) supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, 50 μg/ml gentamicin, 2 mM L-glutamine, 2.5 mM sodium pyruvate, 10 mM HEPES buffer, 1% nonessential amino acids, and 10% heat-inactivated fetal bovine serum (R10) plus 20 IU/ml recombinant human interleukin-2 (rhIL-2) (Boehringer GmbH, Mannheim, Germany). Blasts were infected by incubating in RPMI (without antibiotics) for 3 h at 37°C with wild-type serovar Typhi strain ISP1820 (35, 36). In some experiments, the following day, blasts were -irradiated (4,000 rads) and used as stimulators. For cytotoxic T lymphocyte (CTL) assays, infected and noninfected cells were labeled with 200 μCi of sodium chromate (51Cr) (Amersham Pharmacia Biotech, Piscataway, New Jersey) for 1 h at 37°C, washed, and used as targets.
To confirm that targets were infected with serovar Typhi, cells were stained with anti-CSA-1-fluorescein isothiocyanate (FITC) (KPL, Gaithersburg, MD) and analyzed by flow cytometry (35, 36).
Effector cells. For cytotoxic assays, both ex vivo and in vitro-expanded PBMC from immunized volunteers were used as effectors. In vitro-expanded effectors were obtained using a modification of a previously described technique (35, 36). Briefly, PBMC were restimulated weekly with irradiated autologous serovar Typhi-infected blasts at an effector-to-stimulator cell ratio of 7:1 for 3 weeks in R10 containing 20 IU/ml of rhIL-2. Aliquots of these effector cells were frozen weekly before each new restimulation. The expression of surface molecules on these cell lines was determined by flow cytometry. To this end, effectors were stained with a panel of anti-TCR V monoclonal antibodies (MAbs) (TCR V Repertoire kit, IO Test Beta Mark; Beckman-Coulter, Miami, FL) and combinations of MAbs to CD3 (clone UCHT1), CD4 (clone IM0704), CD25 (clone B1.49.9), CD28 (clone IM1236), HLA-DR (clone H279) (Beckman-Coulter), CD8 (clone SK1), CD56 (clone B159), CD38 (clone HB7), CD45RO (clone UCHL-1), and CD62L (clone Dreg-56) (BD Pharmingen, San Diego, CA). MAbs or the corresponding isotype controls conjugated to the following fluorochromes were used in these studies: FITC, phycoerythrin (PE), energy-coupled dye (ECD) (PE-Texas Red conjugate), peridinin chlorophyll protein (PerCP), PE-Cy5 conjugate, PE-Cy7 conjugate, allophycocyanin (APC), and APC-Cy7 conjugate or biotin (followed by streptavidin-Pacific blue [Molecular Probes, Eugene, Oregon]). The TCR V panel used in these studies included MAbs that recognize 24 different V specificities (i.e., V 5.3, V 7.1, V 3, V 9, V 17, V 16, V 13.1, V 13.6, V 8, V 18, V 5.1, V 20, V 5.2, V 2, V 12, V 23, V 1, V 21.3, V 11, V 22, V 14, V 13.2, V 4, and V 7.2) (see Table 1 for more details), which cover 60% of the known specificities (47) (Table 1). Samples were run in a MoFlo flow cytometer/cell sorter system (Dako-Cytomation, Fort Collins, CO) and analyzed using the WinList flow cytometry software analysis package (Verity Software House, Topsham, ME).
For IFN- enzyme-linked immunospot (ELISPOT) assays, both ex vivo and in vitro-expanded PBMC (as described above) were also used as effectors.
T-cell cloning. T cells from volunteers 1 and 3 were expanded for one cycle in the presence of serovar Typhi-infected autologous blasts and cloned by limiting dilution and polyclonal stimulation with 2.5 x 105 irradiated allogeneic blasts and 2.5 x 105 K562/CD32/4-1 BBL cells (kindly provided by M. Mauer and C. June, University of Pennsylvania, Philadelphia, PA) prepulsed with both anti-CD3 (clone OKT3; ATCC, Rockville, MD) and anti-CD28 (clone 9.3; BD Pharmingen) MAbs (0.5 μg/ml each) (24). These cell lines and clones were cultured in R10 supplemented with 60 IU/ml of rhIL-2 for 10 days and expanded by multiple cycles of in vitro restimulation every 10 days with allogeneic blasts and K562/CD32/4-1 BBL cells at a 2:1 ratio of T cells:stimulator cells. The expression of surface homing and/or effector memory molecules was assessed in these T-cell clones by eight-color flow cytometry (MoFlo). The following MAbs (coupled to the indicated fluorochromes) were used: integrin 47 (clone ACT-1) was kindly provided by W. Newman, Leukosite, Cambridge, MA, and conjugated to Alexa 488 using an Alexa 488 labeling kit (Molecular Probes); CD27-PE (clone M-T271); CD62L-PE-Cy5 (clone Dreg-56); CCR7-PE-Cy7 (clone 3D12) and CD8-APC-Cy7 (clone Sk1) (BD Pharmingen); CD45RO-ECD (clone UCHL-1) and CD103-biotin (clone 2G5) (Beckman-Coulter); and CCR9-APC (clone 112509; R&D Systems, Minneapolis, MN). Alexa 405-streptavidin was purchased from Molecular Probes.
Cytotoxicity (chromium release test). As previously described in detail, cytotoxicity against uninfected or serovar Typhi-infected targets was determined by using a 4-h 51Cr release assay (34-36, 44). Specific cytotoxicity mediated by effector cells was calculated by substracting the lysis of uninfected targets from the lysis of serovar Typhi-infected targets. Lysis of noninfected targets was generally less than 10%. The cutoff for positive responses in CTL assays was defined as >10% specific cytotoxicity (34-36, 44).
IFN- ELISPOT assays. The human IFN- ELISPOT assay was carried out as previously described (34-36). Briefly, PBMC from immunized volunteers were incubated with uninfected or serovar Typhi-infected stimulator cells. Effector cells cultured without stimulator cells or with CD3/CD28 beads (0.6 μl/ml; Dynal) were used as negative and positive controls, respectively. After 16 h, cultures were assayed for IFN- by ELISPOT. Anti-IFN- (5 μg/ml, clone 2G1; Endogen, Woburn, MA) and biotinylated anti-IFN- (2 μg/ml, clone B133.5; Endogen) MAbs were used as capture and detection reagents, respectively. Complexes were detected by incubation with avidin-peroxidase conjugate (Sigma), and TrueBlue reagent (KPL) was used as a substrate. Each sample was tested in triplicate. Spots were enumerated using a stereomicroscope. Net frequencies were calculated as previously described (34-36). The cutoff for ELISPOT assays was established as the frequency of IFN- spot-forming cells/106 PBMC in cocultures of effectors with noninfected targets plus 3 standard errors (SE).
Statistical analysis. All tests were performed using SigmaStat software (version 3.0; SPSS Science software products, Chicago, IL). P values that were <0.05 were considered significant.
RESULTS
Generation of serovar Typhi-specific polyclonal cell lines for V analysis. Our initial approach to evaluating the V repertoire of specific T cells elicited in humans following immunization with the attenuated Ty21a typhoid vaccine involved the enrichment of serovar Typhi-specific T cells by repeated cycles of in vitro restimulation with serovar Typhi-infected polyclonal autologous blasts. We have previously reported the use of serovar Typhi-specific T-cell lines following one cycle of in vitro stimulation with serovar Typhi-infected autologous polyclonal blasts to demonstrate specific cytotoxic activity and IFN- production in clinical trials of attenuated serovar Typhi oral live vector vaccines (34-36). In the present studies, serovar Typhi-specific polyclonal cell lines from three Ty21a typhoid vaccinees generated following one cycle of in vitro stimulation were further expanded by weekly restimulation with irradiated autologous serovar Typhi-infected blasts for up to 3 weeks before specificity and TCR V repertoire analysis. Aliquots of these effector cells were frozen weekly before each new restimulation, and the phenotype of these cell lines was determined. The specificity of these cell lines was evaluated by measuring their ability to secrete IFN- and to kill serovar Typhi-infected targets.
Secretion of IFN- by effector cells in response to stimulation with serovar Typhi-infected targets was measured by ELISPOT in ex vivo PBMC and in polyclonal cell lines after one, two, and three cycles of in vitro restimulation. Marked increases in the frequency of IFN--secreting cells in response to stimulation with serovar Typhi-infected targets were observed. The largest increase of IFN- production occurred after one cycle of in vitro restimulation. Similarities in the levels of IFN- production following two and three cycles of in vitro restimulation suggest a stabilization of these responses with continued in vitro stimulation. Compared with preimmunization levels without in vitro restimulation, postimmunization PBMC without in vitro restimulation showed a 3-fold increase, while polyclonal cell lines showed an average 35-fold increase in serovar Typhi-responsive cells (63-, 24-, and 19-fold after one, two, and three cycles of in vitro restimulation, respectively) (Fig. 1). This corresponds to 0.3 to 1% of the total cell population (2,853 to 9,640 spots/106 cells in these polyclonal cell lines).
51Cr release assays and correlation with IFN--secreting effector T-cell populations. Cytolytic function of the polyclonally expanded cell lines was measured by standard 51Cr release assays. After 3 weeks of in vitro restimulation with autologous serovar Typhi-infected targets, we observed that these cell lines maintain high levels of CTL activity against serovar Typhi-infected targets. As shown in Fig. 2, similar results were obtained in volunteers 1 and 2. Consistent with our previous results, ex vivo PBMC from both volunteers were unable to lyse autologous blasts infected with serovar Typhi (35, 36). However, both volunteers showed increased levels of cytotoxic activity after in vitro restimulation. Although CTL activity is at best semiquantitative, it is important to note that, as observed with IFN- production, the highest increase of the cytotoxicity levels occurred after one in vitro restimulation followed by stabilization after two and three cycles of in vitro restimulation (e.g., volunteer 2 exhibited 33, 17, and 17% of specific lysis at an effector-to-target cell [E:T] ratio of 3:1 after one, two, and three in vitro restimulation cycles, respectively). Moreover, as we have previously described (35), when we compared the results obtained by ELISPOT with those obtained by 51Cr release assays, a positive correlation (R2 = 0.910, P < 0.046) was observed between the frequencies of serovar Typhi antigen-responsive cells, as evidenced by IFN- production and the cytotoxic activity of these cell lines at E:T ratios of 3:1 (data not shown).
Taken together, the results from IFN- ELISPOT and CTL assays clearly show that the polyclonal cell lines remain functional after repeated cycles of specific antigenic stimulation.
Characterization of the effector population. We next investigated which cell populations in PBMC from immunized volunteers were preferentially expanded after several cycles of in vitro restimulation with serovar Typhi-infected autologous blasts. In agreement with previous results using a similar system (34-36), a major expansion in CD8+ T-cell populations, as determined by flow cytometry, occurred during in vitro restimulations. The percentage of CD8+ T-cell populations increased progressively after each additional in vitro restimulation (e.g., 26, 44, and 63% after one, two, and three cycles of in vitro restimulation, respectively). These increases were accompanied by concomitant decreases in the percentage of CD4+ cells at the same time points (28, 13, and 9% after one, two, and three cycles of in vitro restimulation, respectively) (Fig. 3). No changes were observed in the percentage of NK (CD56+) or T cells bearing / T-cell receptors (Fig. 3).
To further characterize the composition of polyclonal cell lines after in vitro restimulation, the proportion of cells expressing molecules involved in T-cell activation and those associated with memory effector T cells in populations following three cycles of in vitro expansion were compared to those present in PBMC without in vitro restimulation. To this end, PBMC from Ty21a vaccinees ex vivo or cocultured with stimulators for up to 3 weeks were stained with MAbs to CD3, CD4, CD8, CD56, CD25, CD28, CD38, CD45RO, CD62L, and HLA-DR and analyzed by multicolor flow cytometry. The results are shown in Table 2. Of the various CD3+ CD8+ cell subpopulations expressing CD45RO+, a T-cell memory marker, only the percentages of CD38+ and HLA-DR+ differed significantly between ex vivo and in vitro-restimulated PBMC. Five-color flow cytometric analysis showed that in vitro-expanded CD3+ CD8+ CD45RO+ T cells contained a greater proportion of cells bearing activation molecules (HLA-DR, 97%; CD38+, 39%) than did the initial ex vivo population (HLA-DR, 24%; CD38+, 9%). Smaller increases were also observed in the percentages of CD3+ CD4+ CD45RO+ populations coexpressing HLA-DR and CD38 after each cycle of restimulation (Table 2). Of note is that no major differences were observed in the expression of CD62L and CD28 in either cell population (Table 2).
Taken together, the results from the functional assays and the analysis of surface expression of activation/memory molecules suggest that activated CD8+ T cells coexpressing memory markers remain functional after several rounds of in vitro stimulation and are therefore appropriate for studying the TCR V repertoire induced by immunization of volunteers with the Ty21a typhoid vaccine.
Flow cytometric V analysis in polyclonal cell lines. Following functional validation and characterization of the polyclonal cell lines, we next evaluated the TCR V repertoire of these populations. First, we examined whether CD8+ T cells with a broad TCR V repertoire or a limited selected repertoire are involved in the host's serovar Typhi response after administration of the Ty21a typhoid vaccine. To this end, we studied the TCR V usage of these cell lines by flow cytometry using a panel of 24 MAbs which cover 60% of the known specificities (47).
Five-color cytofluorometric analysis was performed to determine whether CD4+ or CD8+ T cells of defined V were preferentially expanded by in vitro restimulation. Changes above the mean plus 3 SE of the ex vivo effector cells (controls) were defined as being increased. Although TCR V diversity varied greatly from volunteer to volunteer, we observed the oligoclonal expansion of particular V subsets in each individual (V 2, 5.1, 8, 17, and 22 in volunteer 1; V 1, 2, 5.1, 14, 17, and 22 in volunteer 2; and V 3, 8, 14, and 16 in volunteer 3). As shown in Fig. 4, volunteer 1 exhibited a marked increase in the percentage of CD8+ T cells expressing TCR V 17 after three in vitro restimulations (26.4%) compared to postimmunization levels without in vitro restimulation (3.0%), whereas CD4+ V 17+ cells did not change significantly over time (5.8, 5.4, 5.4, and 5.8% ex vivo and after one, two, and three restimulations, respectively) (data not shown). In volunteer 2, a major increase in the percentage of CD8+ T cells expressing TCR V 2 was found even after a single cycle of in vitro restimulation (14.3%), and it continued to rise after three successive cycles of in vitro restimulation (20.5%), compared to postimmunization levels without in vitro restimulation (4.1%) (Fig. 4). CD4+ V 2+ cells did not change significantly over time (4.2, 6.3, 5.3, and 6.5% ex vivo and after one, two, and three restimulations, respectively) (data not shown). Taken together, the most prominent changes were observed in the V 2, 3, 5.1, 14, 16, and 17 families. Increases in V 2, V 5.1, and V 17 were observed in both volunteer 1 and volunteer 2. Of note is that although in volunteer 3 the V 2 and V 17 changes did not reach the mean percentage plus 3 SE of ex vivo effector cells used as a cutoff level of positivity, increases of 5 to 18 times over the values observed ex vivo without restimulation were observed after the first and second restimulations (i.e., for V 2, 0.1, 1.43, and 1.85% and for V 17, 0.19, 1.02, and 2.43% of positive cells were observed ex vivo, after one cycle, and after two cycles, respectively).
It is important to note that except for the overrepresentation of V 13.2 in volunteer 2 and V 12 in volunteer 3, the reported distributions of V usage observed in ex vivo effectors (controls) are comparable to the V usage previously reported for healthy controls (Table 1) (47). Moreover, the sum average of the percentages of all V specificities in ex vivo CD8+ PBMC after immunization was 69% (volunteer 1, 72%; volunteer 2, 72%; and volunteer 3, 61%), further supporting the contention that the V usage in these volunteers is similar to that previously reported for healthy individuals (60% of CD8+ PBMC) (Table 1) (47).
We next evaluated whether, following the remarkable expansion of CD8+ TCR V 2 T cells, they remain functional in mediating CTL activity against serovar Typhi-infected cells. To this end, all three in vitro-restimulated postimmunization PBMC aliquots from volunteer 2 were pooled and surface-stained for CD8, CD56, CD3, V 2, V 5.2, and V 12 and the TCR V 2+ CD8+ CD56– CD3+ and TCR V 2– CD8+ CD56– CD3+ populations sorted (Fig. 5A). The CTL activity of sorted as well as unsorted populations was measured by 51Cr release assays against serovar Typhi-infected autologous blasts. More than 10% specific lysis was observed in both sorted and unsorted PBMC at an E:T ratio of 3:1 (Fig. 5B). These results demonstrate that both TCR V 2+ CD3+ CD8+ and TCR V 2– CD3+ CD8+ populations are functional and specific for serovar Typhi-infected targets. Moreover, these results also indicate that TCR V specificities other than V 2 are also able to mediate CTL activity. Taken together, these observations support the notion that more than one TCR V family is involved in the host's response to serovar Typhi after vaccination with the Ty21a typhoid vaccine.
TCR V repertoire and expression of molecules involved in homing and effector and memory function in T-cell clones specific for serovar Typhi antigens. To confirm the V usage of T cells elicited by immunization, as well as to identify additional TCR V specificities that could be involved in specific responses to serovar Typhi, T-cell clones were derived from PBMC from volunteers 2 and 3. T-cell clones were derived by limiting dilution, as described in Materials and Methods, and established cell lines were examined for TCR V usage, a procedure that also helped to confirm the clonality of the populations. The functional activity of these T-cell clones was examined by specific cytotoxicity and frequency of IFN--secreting cells against serovar Typhi-infected targets by 51Cr release and IFN- ELISPOT assays, respectively. Figure 6 summarizes the IFN- production, cytotoxicity, and TCR V usage of eight CD8+ T-cell clones. In ELISPOT assays, two CD4+ T-cell clones (TP1-24 and TS1-24) were used as negative controls. The clones that either killed infected targets or produced IFN- were considered responders. Among the six responder CD8+ T-cell clones (TP1-6, TP1-16, TP1-38, TP1-39, TP1-40, and TS1-27), one clone expressed V 7.2, one expressed V 3, and two expressed V 16. For two clones (TP1-16 and TP1-38), no reactivity was observed with any of the anti-TCR V MAbs in our panel. This is likely the result of the lack of a MAb for the appropriate V specificity in the panel, which in the case of volunteer 3 identified 61% of the TCR V repertoire. Of note is that while some T-cell clones (e.g., TP1-38 and TS1-27) exhibited both CTL and IFN- production capabilities, other clones (e.g., TP1-16) were only either cytotoxic or able to produce IFN-, suggesting heterogeneity in the effector function of different T-cell subpopulations following immunization with serovar Typhi vaccines.
It is widely recognized that oral immunization induces effector memory cells carrying distinct homing receptors (e.g., integrin 47, CCR9, and CD103) that might endow the effector cells with the capability to migrate preferentially to the gut (13, 18, 33, 39). Thus, it was important to assess whether these molecules are present on the T-cell clones from volunteers immunized orally with the Ty21a typhoid vaccine described above. Moreover, it was also important to determine whether these T-cell clones exhibit an effector memory phenotype. To this end, clones were stained with MAbs to CD8, CD27, CCR7, CD45RO, CD62L, 47, CCR9, and CD103 and analyzed by eight-color flow cytometry. Despite the observed functional and TCR V usage differences among these clones, there were remarkable similarities in their phenotype and expression of homing molecules. All serovar Typhi-specific T-cell clones were found to be CCR7– CD27– CD45RO+ CD62Llow, i.e., exhibit an effector memory phenotype (9, 37). Moreover, clones expressed high levels of integrin 47 (>92%), moderate levels of CCR9 (8 to 35%), and low levels of CD103 (1 to 8%) (Fig. 7). Taken together, these results confirm an oligoclonal expansion of V serovar Typhi-specific CD8+ T cells, which have a phenotype consistent with T effector memory cells with preferential gut homing potential.
DISCUSSION
In the present study, we evaluated the TCR V specificities of CD8+ T cells from three healthy adults orally immunized with the attenuated serovar Typhi Ty21a strain typhoid vaccine. PBMC from Ty21a typhoid vaccinees were expanded by weekly restimulation with irradiated autologous serovar Typhi-infected blasts for up to 3 weeks and used as effector cells. We found that CD8+ T cells from several V families participated in the anti-serovar Typhi response. Although TCR V usage varied from one volunteer to another, our results are consistent with an oligoclonal V subset expansion in each volunteer (V 2, 5.1, 8, 17, and 22 in volunteer 1; V 1, 2, 5.1, 14, 17, and 22 in volunteer 2; and V 3, 8, 14, and 16 in volunteer 3). Moreover, we observed that these subsets were antigen specific, as shown by functional assays on cells sorted based on their V expression and on T-cell clones. Furthermore, we show that V serovar Typhi-specific CD8+ T cells exhibit a phenotype consistent with effector memory cells with gut homing potential.
Restricted T-cell V repertoires have been shown in infections with bacteria, including Streptococcus (27, 46), Staphylococcus (8, 38), Mycobacterium tuberculosis (11, 28), Listeria (15), and Shigella (16). Although the TCR V repertoire observed with these pathogens was somewhat diverse, TCR V 2 and V 3 usage appears to be favored. The observation in our studies with volunteers immunized with serovar Typhi that V 2 and V 3 specificities were preferentially used appears to support this notion. Interestingly, a study with Shigella (16), a pathogen that shares 80% homology with serovar Typhi, showed a pattern of TCR V usage surprisingly similar to that observed in our expanded T-cell lines. Despite the fact that in this study a limited panel composed of just nine MAbs to different TCR V specificities was used (compared to the 24 V specificities evaluated in our study), the increases in TCR V 2, V 3, V 5.1, V 13.6, and V 17 reported were remarkably similar to the ones observed in our study (TCR V 1, V 2, V 3, V 5.1, V 8, V 14, V 16, V 17, and V 22). Taken together, these observations support the notion that it is likely that T-cell responses against some, if not most, bacteria might be oligoclonal in nature and use a relatively restricted set of TCR V specificities.
The aim of the studies presented in this paper was to analyze the TCR V usage in healthy adults 5 to 40 months after oral immunization with serovar Typhi strain Ty21a to evaluate the persistence of serovar Typhi-specific responses over time. Because the postimmunization specimens were collected long after the T-cell memory contraction phase took place, it is not surprising that we did not observe differences when comparing the ex vivo TCR V usage before and after immunization in two of the volunteers (volunteers 1 and 2) (data not shown). To overcome this difficulty, we evaluated the V repertoire of specific T cells after repeated cycles of in vitro restimulation with serovar Typhi-infected polyclonal autologous blasts. Although this procedure does not allow us to estimate their frequency in vivo, available evidence with other gram-negative bacteria suggests that it should not have negatively affected the identification of at least some of the different specificities elicited by immunization with serovar Typhi. In this context, although some reports studying virus-specific CTL populations showed that in vitro expansion of lymphocytes can introduce a significant bias in TCR usage (41), a previous study in Shigella (16) showed that the in vitro TCR V repertoire was similar to that observed in vivo. By comparing the in vivo and in vitro changes in the TCR V repertoire over time, the authors concluded that the in vitro response induced by Shigella antigens showed a TCR V distribution similar to that observed in vivo. Interestingly, the changes in TCR V usage observed in that study peaked 8 to 11 days after the onset of disease, returning to baseline levels after 30 days. Islam et al. hypothesized that this kinetics was either due to homing of circulating cells to the gut-associated lymphoid tissues or, alternatively, due to apoptotic cell death (16). Interestingly, in the present study, we also observed that the expansion of some V specificities after one cycle of in vitro restimulation (about day 8) was followed by contraction and/or stabilization of these same V specificities during the next rounds of in vitro restimulation. Of note is that after one cycle of in vitro restimulation, we observed stabilization in the cytotoxicity of and IFN- production by serovar Typhi-specific CD8+ T cells, despite an increase in the percentage of CD8+ T cells up to the third week of restimulation. These results may reflect the loss of some clones (e.g., by apoptosis death or loss of specificity) and their replacement by others following successive in vitro restimulations, a phenomenon frequently observed with T-cell clones. Finally, it should be emphasized that it is presently not possible to conclusively assess to what extent the specificities that we observed following in vitro expansion are representative of those present in vivo following immunization. This assertion is based on the lack of a complete understanding of the full complement of immune responses to serovar Typhi following immunization that depend, at least in part, on the set of antigens presented during the various stages of in vivo serovar Typhi infection (50), the presence of variables during in vitro expansion that are difficult to control, and technological limitations. These limitations include the following: (i) in most cases, TCR V antigen-specific T-cell precursors exist at frequencies below the level of detection by standard ex vivo methodologies, e.g., tetrameric complexes; (ii) even in the case of high in vivo frequency of certain TCR V antigen-specific T-cell precursors, T-cell stimulation may require a particular TCR conformation or conformational change that is found only on the surface of certain presenting cells (6); and (iii) while Bouneaud et al. (2) showed that nearly half of their tetramer-binding T cells were unable to expand in response to physiological levels of antigen, Bullock et al. (4) showed that the reverse may also occur, i.e., T cells that show strong functional reactivity towards target cells may not necessarily be detected ex vivo by major histocompatibility complex-tetramer staining.
It is well established that lymphocytes continuously recirculate from peripheral blood to secondary lymphoid tissues (3). This recirculation is essential for homing of effector memory cells, which do not express CCR7 or CD27 and are mostly CD45RO+ CD62Llow, to effector sites (9, 13, 37). Serovar Typhi cells are highly invasive bacteria that rapidly and efficiently colonize and invade the mucosa of the small intestine following ingestion (19). Thus, in Salmonella infections, it is likely that specific memory effector T cells in circulation will be homing to effector sites in the gut where they might encounter Salmonella antigens. Selective homing of effector memory cells to the lamina propria of the small intestine is driven, to a large extent, by the expression of the integrin 47, which binds MAdCAM-1, a molecule selectively expressed in the venules of normal and inflamed intestinal endothelium (5). In this regard, a study by Lundin et al. has shown that immunization with the Ty21a typhoid vaccine induces antigen-specific T cells that preferentially express integrin 47 and are found in circulation up to 21 days following immunization (23). However, other molecules have also recently been shown to participate in this homing process. For example, expression of CCR9 in subsets of circulating integrin 47hi or E7 (CD103) lymphocytes plays an important role in homing of these cells to the small intestine (13, 18, 43). Thus, it was important to evaluate the patterns of expression of these homing molecules in V serovar Typhi-specific CD8+ T cells. We observed that T-cell clones, regardless of their TCR V usage, exhibited an effector memory phenotype (i.e., CCR7– CD27– CD45RO+ CD62Llow) and coexpressed high levels of integrin 47, intermediate levels of CCR9, and low levels of CD103 gut homing molecules, suggesting that these TCR V-specific clones have the potential to home to the gut-associated lymphoid tissues.
It has been reported that only a few V families predominate in the human gut mucosa (1), with the most frequent being V 1, V 2, V 3, and V 6 (49). Because we studied peripheral blood, we have to consider the possibility that our observations might not reflect the full spectrum of TCR V usage by serovar Typhi-specific CD8+ T cells in the gut microenvironment in vivo. However, our observations that (i) serovar Typhi-specific CD8+ T cells from Ty21a vaccinees exhibited gut homing potential and (ii) except for V 6, which was not present in our MAb panel, we observed all other V specificities (V 1, V 2, and V 3, with the most prominent changes in V 2 and V 3) support the contention that the TCR V usage that we observed in circulation might reflect to a large extent that found in the gut mucosa microenvironment.
Taken together, our observations suggest that the CD8+ effector memory T-cell subpopulations of the restricted TCR V specificities described in this paper are likely to be the effector cells that mediate anti-serovar Typhi long-term cell-mediated immune responses in the local microenvironment of the gut mucosa following oral immunization with attenuated strains of serovar Typhi.
ACKNOWLEDGMENTS
We are indebted to the volunteers who allowed us to perform this study. We also thank Bernadette McConnell and the staff from the Blood Bank of the University of Maryland hospital for their help in collecting leukapheresis specimens and Regina Harley for excellent technical assistance in flow cytometric determinations and sorting.
This work was supported by a grant from the National Institute of Allergy and Infectious Diseases, NIH (R01 AI36525 to M.B.S.).
REFERENCES
1. Blumberg, R. S., C. E. Yockey, G. G. Gross, E. C. Ebert, and S. P. Balk. 1993. Human intestinal intraepithelial lymphocytes are derived from a limited number of T cell clones that utilize multiple V beta T cell receptor genes. J. Immunol. 150:5144-5153.
2. Bouneaud, C., P. Kourilsky, and P. Bousso. 2000. Impact of negative selection on the T cell repertoire reactive to a self-peptide: a large fraction of T cell clones escapes clonal deletion. Immunity 13:829-840.
3. Brandtzaeg, P., I. N. Farstad, and G. Haraldsen. 1999. Regional specialization in the mucosal immune system: primed cells do not always home along the same track. Immunol. Today 20:267-277.
4. Bullock, T. N., D. W. Mullins, T. A. Colella, and V. H. Engelhard. 2001. Manipulation of avidity to improve effectiveness of adoptively transferred CD8(+) T cells for melanoma immunotherapy in human MHC class I-transgenic mice. J. Immunol. 167:5824-5831.
5. Butcher, E. C., M. Williams, K. Youngman, L. Rott, and M. Briskin. 1999. Lymphocyte trafficking and regional immunity. Adv. Immunol. 72:209-253.
6. Coles, R. M., C. M. Jones, A. G. Brooks, P. U. Cameron, W. R. Heath, and F. R. Carbone. 2003. Virus infection expands a biased subset of T cells that bind tetrameric class I peptide complexes. Eur. J. Immunol. 33:1557-1567.
7. Davis, M. M., and P. J. Bjorkman. 1988. T-cell antigen receptor genes and T-cell recognition. Nature 334:395-402.
8. Ericson, M. L., N. A. Mooney, and D. J. Charron. 1996. Prokaryotic production of the human T cell receptor Vbeta2 chain and binding to toxic-shock syndrome toxin-1. Gene 168:257-260.
9. Esser, M. T., R. D. Marchese, L. S. Kierstead, L. G. Tussey, F. Wang, N. Chirmule, and M. W. Washabaugh. 2003. Memory T cells and vaccines. Vaccine 21:419-430.
10. Gagnon, S. J., A. Leporati, S. Green, S. Kalayanarooj, D. W. Vaughn, H. A. Stephens, S. Suntayakorn, I. Kurane, F. A. Ennis, and A. L. Rothman. 2001. T cell receptor Vbeta gene usage in Thai children with dengue virus infection. Am. J. Trop. Med. Hyg. 64:41-48.
11. Gambon-Deza, F., M. Pacheco Carracedo, T. Cerd á Mota, and J. Montes Santiago. 1995. Lymphocyte populations during tuberculosis infection: V repertoires. Infect. Immun. 63:1235-1240.
12. Gomez-Duarte, O. G., M. F. Pasetti, A. Santiago, M. B. Sztein, S. L. Hoffman, and M. M. Levine. 2001. Expression, extracellular secretion, and immunogenicity of the Plasmodium falciparum sporozoite surface protein 2 in Salmonella vaccine strains. Infect. Immun. 69:1192-1198.
13. Hamann, A., D. P. Andrew, D. Jablonski-Westrich, B. Holzmann, and E. C. Butcher. 1994. Role of alpha 4-integrins in lymphocyte homing to mucosal tissues in vivo. J. Immunol. 152:3282-3293.
14. Hingorani, R., I. H. Choi, P. Akolkar, B. Gulwani-Akolkar, R. Pergolizzi, J. Silver, and P. K. Gregersen. 1993. Clonal predominance of T cell receptors within the CD8+ CD45RO+ subset in normal human subjects. J. Immunol. 151:5762-5769.
15. Huleatt, J. W., I. Pilip, K. Kerksiek, and E. G. Pamer. 2001. Intestinal and splenic T cell responses to enteric Listeria monocytogenes infection: distinct repertoires of responding CD8 T lymphocytes. J. Immunol. 166:4065-4073.
16. Islam, D., B. Wretlind, A. A. Lindberg, and B. Christensson. 1996. Changes in the peripheral blood T-cell receptor V repertoire in vivo and in vitro during shigellosis. Infect. Immun. 64:1391-1399.
17. Ivanoff, B., M. M. Levine, and P. H. Lambert. 1994. Vaccination against typhoid fever: present status. Bull. W. H. O. 72:957-971.
18. Kantele, A., J. Zivny, M. Hakkinen, C. O. Elson, and J. Mestecky. 1999. Differential homing commitments of antigen-specific T cells after oral or parenteral immunization in humans. J. Immunol. 162:5173-5177.
19. Levine, M., and M. Sztein. 2000. Shigella, Salmonella typhi, and Escherichia coli, p. 171-194. In M. Cunningham and R. Fujinami (ed.), Effects of microbes on the immune system. Lippincott Williams & Wilkins, Philadelphia, Pa.
20. Levine, M. M., C. Ferreccio, P. Abrego, O. S. Martin, E. Ortiz, and S. Cryz. 1999. Duration of efficacy of Ty21a, attenuated Salmonella typhi live oral vaccine. Vaccine 17:S22-S27.
21. Levine, M. M., and O. S. Levine. 1997. Influence of disease burden, public perception, and other factors on new vaccine development, implementation, and continued use. Lancet 350:1386-1392.
22. Lim, A., L. Trautmann, M. A. Peyrat, C. Couedel, F. Davodeau, F. Romagne, P. Kourilsky, and M. Bonneville. 2000. Frequent contribution of T cell clonotypes with public TCR features to the chronic response against a dominant EBV-derived epitope: application to direct detection of their molecular imprint on the human peripheral T cell repertoire. J. Immunol. 165:2001-2011.
23. Lundin, B. S., C. Johansson, and A.-M. Svennerholm. 2002. Oral immunization with a Salmonella enterica serovar Typhi vaccine induces specific circulating mucosa-homing CD4+ and CD8+ T cells in humans. Infect. Immun. 70:5622-5627.
24. Maus, M. V., A. K. Thomas, D. G. Leonard, D. Allman, K. Addya, K. Schlienger, J. L. Riley, and C. H. June. 2002. Ex vivo expansion of polyclonal and antigen-specific cytotoxic T lymphocytes by artificial APCs expressing ligands for the T-cell receptor, CD28 and 4-1BB. Nat. Biotechnol. 20:143-148.
25. McCloskey, T. W., V. Haridas, R. Pahwa, and S. Pahwa. 2002. T cell receptor V beta repertoire of the antigen specific CD8 T lymphocyte subset of HIV infected children. AIDS 16:1459-1465.
26. Meuer, S. C., K. A. Fitzgerald, R. E. Hussey, J. C. Hodgdon, S. F. Schlossman, and E. L. Reinherz. 1983. Clonotypic structures involved in antigen-specific human T cell function. Relationship to the T3 molecular complex. J. Exp. Med. 157:705-719.
27. Ohara-Nemoto, Y., and M. Kaneko. 1996. Expression of T-cell receptor V beta 2 and type 1 helper T-cell-related cytokine mRNA in streptococcal pyrogenic exotoxin-C-activated human peripheral blood mononuclear cells. Can. J. Microbiol. 42:1104-1111.
28. Ohmen, J. D., P. F. Barnes, C. L. Grisso, B. R. Bloom, and R. L. Modlin. 1994. Evidence for a superantigen in human tuberculosis. Immunity 1:35-43.
29. Paliard, X., S. G. West, J. A. Lafferty, J. R. Clements, J. W. Kappler, P. Marrack, and B. L. Kotzin. 1991. Evidence for the effects of a superantigen in rheumatoid arthritis. Science 253:325-329.
30. Pannetier, C., J. Even, and P. Kourilsky. 1995. T-cell repertoire diversity and clonal expansions in normal and clinical samples. Immunol. Today 16:176-181.
31. Pasetti, M. F., R. J. Anderson, F. R. Noriega, M. M. Levine, and M. B. Sztein. 1999. Attenuated guaBA Salmonella typhi vaccine strain CVD 915 as a live vector utilizing prokaryotic or eukaryotic expression systems to deliver foreign antigens and elicit immune responses. Clin. Immunol. 92:76-89.
32. Pasetti, M. F., M. M. Levine, and M. B. Sztein. 2003. Animal models paving the way for clinical trials of attenuated Salmonella enterica serovar Typhi live oral vaccines and live vectors. Vaccine 21:401-418.
33. Pasetti, M. F., R. Salerno-Gonalves, and M. B. Sztein. 2005. Mechanisms of adaptive immunity that prevent colonization at mucosal surfaces, p. 35-47. In J. P. Nataro, P. S. Cohen, H. L. T. Mobley, and J. N. Weiser (ed.), Colonization of mucosal surfaces. American Society for Microbiology, Washington, D.C.
34. Salerno-Goncalves, R., M. Fernandez-Vina, D. M. Lewinsohn, and M. B. Sztein. 2004. Identification of a human HLA-E-restricted CD8+ T cell subset in volunteers immunized with Salmonella enterica serovar Typhi strain Ty21a typhoid vaccine. J. Immunol. 173:5852-5862.
35. Salerno-Goncalves, R., M. F. Pasetti, and M. B. Sztein. 2002. Characterization of CD8(+) effector T cell responses in volunteers immunized with Salmonella enterica serovar Typhi strain Ty21a typhoid vaccine. J. Immunol. 169:2196-2203.
36. Salerno-Goncalves, R., T. L. Wyant, M. F. Pasetti, M. Fernandez-Vina, C. O. Tacket, M. M. Levine, and M. B. Sztein. 2003. Concomitant induction of CD4(+) and CD8(+) T cell responses in volunteers immunized with Salmonella enterica serovar Typhi strain CVD 908-htrA. J. Immunol. 170:2734-2741.
37. Sallusto, F., D. Lenig, R. Forster, M. Lipp, and A. Lanzavecchia. 1999. Two subsets of memory T lymphocytes with distinct homing potentials and effector functions. Nature 401:708-712.
38. Seth, A., L. J. Stern, T. H. Ottenhoff, I. Engel, M. J. Owen, J. R. Lamb, R. D. Klausner, and D. C. Wiley. 1994. Binary and ternary complexes between T-cell receptor, class II MHC and superantigen in vitro. Nature 369:324-327.
39. Shaw, S. K., and M. B. Brenner. 1995. The beta 7 integrins in mucosal homing and retention. Semin. Immunol. 7:335-342.
40. Soroosh, P., F. Shokri, M. Azizi, and M. Jeddi-Tehrani. 2003. Analysis of T-cell receptor beta chain variable gene segment usage in healthy adult responders and nonresponders to recombinant hepatitis B vaccine. Scand. J. Immunol. 57:423-431.
41. Sourdive, D. J., K. Murali-Krishna, J. D. Altman, A. J. Zajac, J. K. Whitmire, C. Pannetier, P. Kourilsky, B. Evavold, A. Sette, and R. Ahmed. 1998. Conserved T cell receptor repertoire in primary and memory CD8 T cell responses to an acute viral infection. J. Exp. Med. 188:71-82.
42. Stewart-Jones, G. B., A. J. McMichael, J. I. Bell, D. I. Stuart, and E. Y. Jones. 2003. A structural basis for immunodominant human T cell receptor recognition. Nat. Immunol. 4:657-663.
43. Svensson, M., J. Marsal, A. Ericsson, L. Carramolino, T. Broden, G. Marquez, and W. W. Agace. 2002. CCL25 mediates the localization of recently activated CD8alphabeta(+) lymphocytes to the small-intestinal mucosa. J. Clin. Investig. 110:1113-1121.
44. Sztein, M. B., M. K. Tanner, Y. Polotsky, J. M. Orenstein, and M. M. Levine. 1995. Cytotoxic T lymphocytes after oral immunization with attenuated vaccine strains of Salmonella typhi in humans. J. Immunol. 155:3987-3993.
45. Sztein, M. B., S. S. Wasserman, C. O. Tacket, R. Edelman, D. Hone, A. A. Lindberg, and M. M. Levine. 1994. Cytokine production patterns and lymphoproliferative responses in volunteers orally immunized with attenuated vaccine strains of Salmonella typhi. J. Infect. Dis. 170:1508-1517.
46. Tomai, M. A., J. A. Aelion, M. E. Dockter, G. Majumdar, D. G. Spinella, and M. Kotb. 1991. T cell receptor V gene usage by human T cells stimulated with the superantigen streptococcal M protein. J. Exp. Med. 174:285-288.
47. van den Beemd, R., P. P. Boor, E. G. van Lochem, W. C. Hop, A. W. Langerak, I. L. Wolvers-Tettero, H. Hooijkaas, and J. J. van Dongen. 2000. Flow cytometric analysis of the Vbeta repertoire in healthy controls. Cytometry 40:336-345.
48. van den Elsen, P. J., and A. Rudensky. 2004. Antigen processing and recognition. Recent developments. Curr. Opin. Immunol. 16:63-66.
49. Van Kerckhove, C., G. J. Russell, K. Deusch, K. Reich, A. K. Bhan, H. DerSimonian, and M. B. Brenner. 1992. Oligoclonality of human intestinal intraepithelial T cells. J. Exp. Med. 175:57-63.
50. Waterman, S. R., and D. W. Holden. 2003. Functions and effectors of the Salmonella pathogenicity island 2 type III secretion system. Cell. Microbiol. 5:501-511.
51. Wick, M. J. 2002. The role of dendritic cells during Salmonella infection. Curr. Opin. Immunol. 14:437-443.
52. Wick, M. J. 2003. The role of dendritic cells in the immune response to Salmonella. Immunol. Lett. 85:99-102.
53. Zissel, G., I. Baumer, B. Fleischer, M. Schlaak, and J. Muller-Quernheim. 1997. TCR V beta families in T cell clones from sarcoid lung parenchyma, BAL, and blood. Am. J. Respir. Crit. Care Med. 156:1593-1600.(Rosangela Salerno-Gonalve)