Inducing P-Selectin Ligand Formation in CD8 T Cells: IL-2 and IL-12 Are Active In Vitro but Not Required In Vivo
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免疫学杂志 2005年第7期
Abstract
In vitro studies have demonstrated that IL-2 and IL-12 can support formation of P-selectin ligands (P-SelL) in activated T cells, ligands that are variably required for efficient lymphocyte recruitment to sites of inflammation. To ascertain whether these cytokines were required for P-SelL formation in vivo, TCR transgenic CD8 T cells specific for male Ag (HY) were transferred into male mice under conditions in which either IL-2 and/or IL-15 or IL-12Rp40 were absent. P-SelL formation at day 2 was unperturbed in HY-TCR IL-2null CD8 T cells responding in doubly deficient IL-2nullIL-12null or IL-2nullIL-15null male recipients. HY-specific CD8 T cell proliferative responses detected in both spleen and peritoneum occurred vigorously, but only splenic CD8 T cells up-regulated P-SelL, demonstrating that in vivo induction of P-SelL is an active, nonprogrammed event following T cell activation and that despite the efficacy of IL-2 and IL-12 in supporting P-SelL formation in vitro, these cytokines appear to be dispensable for this purpose in vivo.
Introduction
The choreography of adaptive T cell immunity to foreign Ag generally follows a series of defined steps beginning with T cell activation in the peripheral lymph node by Ag-bearing dendritic cells (DC)3 that had previously emigrated from a peripheral site of innate immune activation. During the process of activation, the T cell acquires a set of instructions to exit the lymph node, to access inflamed sites, and to deliver effector functions appropriately. The extent to which this imprinting process occurs solely during the initial T cell-DC contact or through subsequent influences with cells or environment has not been fully resolved. Nevertheless, the process and form of such instructions represent crucial features required for appropriate spatial delivery of effector functions, the raison d’etre of all adaptive T cell immunity. Regulating selectin ligand formation and integrin expression/activation on T cells are specific examples of instructions received through T cell activation, enabling them to access an inflammatory site and deliver effector functions. On activation, T cells can acquire the ability to form ligands for endothelial expressed E- and P-selectins through de novo expression of a series of glycosyltransferases that branch and extend O-linked carbohydrate on specific glycoprotein substrates. These newly synthesized selectin ligands support rather subtle interactions between T cell and endothelial selectins manifested in transient rolling or tethering behavior along venule walls within an inflammatory site (reviewed in Ref. 1). In many cases, this selectin-mediated rolling and tethering behavior is an essential first step in exiting the vasculature.
Two sets of data have been generated that document distinct methods to differentially induce selectin ligand. The first set of data is based purely on in vitro observations whereby CD4 (2, 3, 4, 5) or CD8 (6, 7, 8, 9, 10) T cells polarized with IL-12 and TGF- or IFN- displayed P-SelL whereas cells polarized by IL-4 and anti-IFN- did not. IL-2 as well has recently been implicated in P SelL formation within activated CD8 T cells (11). These data suggested that cytokines are primary drivers of SelL formation but it has been difficult to conceptualize or demonstrate how and where such extreme polarizing influences might be exerted in vivo. Furthermore, additional observations from in vivo and in vitro polarized T cells, as defined by cytokine gene expression (12, 13, 14) have documented unexpected SelL expression in Th0 or Th2 polarized T cells. These observations suggest that modeling P-SelL induction based on in vitro polarization regimens may not translate reliably to circumstances in vivo. Nevertheless, the in vitro studies demonstrating that IL-2 and IL-12 could direct selectin ligand formation in CD4 and CD8 T cells led to the expectation that these cytokines were similarly influential in vivo and that SelL formation should therefore be compromised in mice lacking these cytokines.
The second set of data was an extension of early in vivo observations that T cells obtained from intestine or skin were inclined to repopulate the same respective site after adoptive transfer (15, 16, 17, 18, 19). More recent investigations have demonstrated differential integrin and SelL expression in vivo depending on the anatomical source of stimulating DCs or responding T cells suggesting that either resident APCs or other local microenvironmental influences can differentially imprint homing properties on responding T cells by selectively directing induction of SelL and integrin expression (14, 20, 21, 22). The respective roles of resident APC (DC) phenotype vs other local environmental influences, including cytokines such as IL-2, IL-12 and cell surface molecules, in determining SelL formation in these in vivo studies has not been resolved (22, 23).
We therefore investigated SelL formation in vivo using mice bearing a transgenic (tg) TCR specific for male Ag (HY) but lacking genes encoding IL-2, IL-15, and/or IL-12. Wild-type or cytokine-deficient TCR tg CD8 T cells from female mice were adoptively transferred into normal or cytokine-deficient male mice, and the formation of P-SelL was monitored as adoptively transferred cells became activated and proliferated. Consistent with recent investigations by others suggesting that the site/source/phenotype of APC can imprint homing properties onto responding T cells, P-SelL formation did not occur in CD8 T cells responding to HY in the peritoneal cavity, whereas P-SelL formation proceeded rapidly and relatively uniformly when TCR tg CD8 T cells were introduced i.v. and reisolated from spleen. Importantly, P-SelL formation on i.v.-transferred CD8 T cells occurred regardless of IL-2, IL-12, or IL-15 cytokine deficiency. Thus, our results indicated that P-SelL formation within activated CD8 T cells in vivo can be differentially induced, independently of T cell activation per se and independently of those cytokines implicated by previous in vitro studies as being involved in P-SelL formation. Because the absence of these cytokines had no detectable impact on P-SelL formation in vivo, our data establish that another distinct signaling event(s) is in fact the primary driver of P-SelL formation in vivo.
Materials and Methods
Mice
Mice 6–12 wk of age were used for analyses. C57BL/6 (B6) mice were bred at the Biomedical Research Centre from founders obtained originally from The Jackson Laboratory. IL-12p40null mice on the B6 background (no. 002693; The Jackson Laboratory) were received and bred as homozygotes. When intercrossed with IL-2null mice, an IL-12 genotype was confirmed by loss of wild-type allele by PCR according to the suppliers’ recommended protocols (sense primer, 5'-AGTGAACCTCACCTGTGACACG-3'; antisense primer, 5'-TCTTTGCACCAGCCATGAGC-3'). IL-2null mice (no. 002252; The Jackson Laboratory) were bred from homozygous studs and heterozygous dams. The offspring genotype was confirmed by PCR according to the suppliers’ recommended procedures using null sense primer 5'-TCGAATTCGCCAATGACAAGACGCT-3', wild-type sense primer 5'-CTA GGCCACAGAATTGAAAGATCT-3', and shared antisense primer 5'-GTAGGTGGAAATTCT AGCATCATCC-3'. IL-15null homozygous mice (no. 004200-M; Taconic, Germantown, NY; Ref. 24) were received and bred as homozygotes. When intercrossed with IL-2null mice, the IL-15 genotype was confirmed by PCR using Neo primer 5'-GAATGGGCTGACCGCTTCCTCG-3', downstream genomic primer 5'-TCATATCCTCTGCACCTTGACTG-3', upstream exon 3 primer 5'-GAGGGCTAAATCTGATGCGTGTG-3', and exon 3 primer 5'-GAGCTGGCTATGGCGATG GGC-3'. PCR was conducted using 1 μl of stock 20 μM primers with 4 μl of extracted DNA per reaction at 94°C for 5 min; 39 cycles of 94°C for 1 min, 62°C for 1 min, and 72°C for 2 min; followed by a 4°C pause. For all genotyping, DNA was isolated with the Sigma REDExtract-N-Amp Tissue PCR kit (XNART). HY-TCR tg genotyping was performed with mAb T3.70-FITC (see Abs) staining of peripheral blood.
Media
Cell suspensions were prepared in RPMI (no. 11875-135l; Life Technologies) supplemented with 8% FCS, 5 x 10–5 M 2-ME, 100 U/ml penicillin, 100 U/ml streptomycin (Stem Cell Technologies), and 2 mM glutamine (Sigma-Aldrich). DMEM (no. 11965-084; Life Technologies) supplemented with 3% FCS was used for Ab staining.
Antibodies
Human IgG1-P-selectin fusion protein (BD Pharmingen; no. 28111A) was detected with PE-conjugated anti-human IgG (Jackson Immunoresearch; no. 109-116-098). CD8-biotin (no. CL8938B; Cedarlane) was detected with CyChrome-conjugated streptavidin (BD Pharmingen; no. 554062). CD8-APC (no. 553035), and CD8-FITC (no. 553031) were obtained from BD Pharmingen, and mAb T3.70 specific for HY TCR tg (25) was conjugated with biotin or Alexafluor 488 in house.
In vivo assessment of P-SelL induction
To circumvent issues of peripheral splenomegaly and lymphadenopathy prevalent in IL-2null mice, thymocytes from HY TCR tg and HY-IL-2null TCR tg mice were used as a source of naive CD8+CD4– HY-specific T cells. Donor mice were 8–12 wk of age as thymi were not grossly perturbed despite peripheral lymphadenopathy, whereas recipient IL-2 deficient mice were 5–7 wk of age as older mice developed increasingly severe splenomegaly that perturbed donor cell responses and made donor cell recovery increasingly difficult. Thymocytes were labeled with CFSE (no. C1157; Molecular Probes) at 107/ml in complete medium for 5 min at room temperature, washed in medium, washed in HBSS (no. H8264; Sigma-Aldrich), and resuspended in HBSS for injection. Twenty million thymocytes were injected i.v. or i.p. Where indicated, synthetic male peptide (prepared in house at the Biomedical Research Centre) H-Lys-Cys-Ser-Arg-Asn-Arg-Gln-Tyr-Leu-OH (26) or male spleen cells were coinjected with CFSE-labeled thymocytes. From 2 to 3 days later (as indicated), recipients were sacrificed. RBC-depleted splenocytes or peritoneal lavage isolates were stained for four-color flow cytometry as follows. CFSE (FL1), human Ig (hIg)-P-Sel + anti-hIg-PE (FL2), T3.70-biotin + SA-CyC (FL3), and CD8-APC (FL4) cytometry was conducted in DMEM supplemented with 3% FCS in the sequence hIg-P-Sel, anti-hIg-PE, NMS, T3.70-biotin+CD8-APC, SA-CyC. Cells were incubated with Abs for 20 min on ice in 96-well round-bottom plates (Nunclon; InterMed). Cells were washed twice and analyzed on a FACSCalibur flow cytometer (BD Biosciences). P-SelL expression was presented either as raw data dot plots or as line graphs of cell division number vs geometric mean P-SelL per division. To exclude spurious geometric mean P-SelL data points in the graphs presented, only data points exhibiting a singular CFSE intensity (i.e., cells that had undergone an equal number of divisions) representing >10% total CFSE+ CD8+T3.70+ gated events were plotted.
Lymphocyte cultures
Con A primary stimulations were conducted with lymph node cells or splenocytes cultured at 106/ml in 4 mg/ml Con A (Sigma-Aldrich) for 48 h at 37°C in 5% CO2. Cultures of 2 or 10 ml were prepared in 24-well Falcon 3047 plates or 6-well Falcon 3046 plates, respectively (BD Biosciences). After 48 h, cells were harvested, washed, counted, and replated in secondary cultures at 0.25 x 106/ml with 2.5% IL-2 supernatants or at 0.05 x 106/ml with 2.5% IL-4 supernatants where indicated. After another 48 h, cells were harvested and stained for flow cytometric analysis with hIg-P-Sel + anti-hIg-PE and CD8-FITC. Primary HY-specific stimulations were conducted with 2000 rad-irradiated splenic DC preparations generated by differential adherence to tissue culture plastic as described previously (27). DC stimulators (4 x 104) were cocultured with 6 x 105 HY TCR tg thymocytes in 96-well flat-bottom tissue culture plates (Nunc; no. 167008) for 3 days and analyzed by flow cytometry with hIg-P-Sel as described above. Cytokines used in these studies included IL-2 and IL-4 obtained as conditioned medium from murine IL-2 or IL-4 cDNA transfectants of myeloma X-653 kindly provided by Fritz Melchers, IL-12 (2 ng/ml; R&D Systems; no. 419-ML), and IL-15 (50 μg/ml; R&D Systems; no. 247–1L).
Results
Role of endogenous IL-2 in vitro
In previous studies using 4-day cultures including a 2-day Con A stimulation (days 0–2), day 2 wash, and subculturing in fresh medium (days 2–4) to activate CD8 T cells, we observed that exogenous IL-2 included during days 2–4 promoted core 2 glycosyltransferase (C2GlcNAcT) activity and formation of P-SelL by day 4 relative to exogenous IL-4 (28). To determine whether endogenous IL-2 produced by responding T cells themselves could drive P-SelL in vitro, the induction of P-SelL was monitored in similar 4-day wild-type B6 vs IL-2null Con A-stimulated cultures. It was established many years ago that IL-2 production in Con A-stimulated cells peaks in the first 18–24 h of culture (29, 30, 31). We therefore focused our analysis of endogenous IL-2 impact by varying conditions during the first 2 days of Con A stimulation and applying constant, IL-2-supplemented conditions from day 2 to day 4. As shown in Fig. 1, induction of P-SelL on CD8+-gated T cells was significantly affected by cytokines present during primary cultures (days 0–2). Several features of these data were noteworthy. 1) P-SelL expression tended to be biphasic. This heterogeneity is a persistent feature of P-SelL induction among in vitro activated CD8 T cells and presumably reflects heterogeneity in either the responding T cells themselves or the microconditions experienced by individual clones present in the culture. 2) When exogenous IL-2 or IL-4 were included in primary cultures (days 0–2), endogenous IL-2 production appeared to have little impact on P-SelL induction insofar as comparable signals were obtained from cultures of B6 or IL-2null tissue. 3) In the absence of exogenous supplements, endogenous IL-2 support of P-SelL induction in B6 vs IL-2null cultures was more evident in cultures of spleen than in cultures of lymph node. This observation was consistent with previous observations that Con A-stimulated spleen is a particularly good source of IL-2 (32) and that P-SelL induction was more evident in Con A-activated cultures of spleen than in those of lymph node (28). In summary, endogenous IL-2 production under specific in vitro conditions of T cell activation could promote P-SelL formation.
FIGURE 1. Endogenous IL-2 can support P-SelL formation by CD8 T cells responding to Con A in vitro. Lymph node (LN) cells or spleen cells (SPL) obtained from IL-2null or B6 (IL-2+) mice were stimulated with Con A for 2 days in complete medium supplemented with no exogenous cytokine (NS) (a–d), exogenous IL-2 (e–h), or exogenous IL-4 (i–l), as indicated. Two days later, cells were subcultured in IL-2-supplemented medium to maintain cell viability for an additional 2 days before analysis for P-SelL expression by flow cytometry gated on CD8+ blasts. This experiment was performed three times with qualitatively similar results.
IL-12 deficiency does not alter P-SelL formation in CD8+ cells responding to Con A in vitro
As described in the introduction, IL-12 had been implicated by previous investigations on P-SelL formation in both CD4 and CD8 T cells. In these studies, exogenous IL-12 was used to supplement medium in Th1/Tc1 polarizing cultures of activated T cells and exhibited significant activity in driving P-SelL formation. When P-SelL formation was evaluated in short term cultures of Con A-activated B6 or IL-12null (p40null) lymphocytes, as shown in Fig. 2, it was evident that IL-12 played no role in this process. Clearly, factors other than IL-12 were responsible for driving P-SelL formation in such short term in vitro responses. The role of IL-12 during short term in vitro activation and P-SelL formation was also assessed in peptide-specific responses by CD8 T cells expressing a tg TCR against the HY. In vitro activation and P-SelL formation in such CD8 T cells was monitored 3–4 days after stimulation with male splenic DCs in the presence of cytokines as indicated in Fig. 3. In this model of short term T cell activation, exogenous IL-12 supplement promoted P-SelL formation when present with either IL-2 or IL-15. However, exogenous IL-12 had no significant impact on P-SelL expression when present alone or with IL-4.
FIGURE 2. P-SelL formation is unperturbed by IL-12p40 deficiency in CD8 T cells responding to Con A in vitro. Lymph node (LN) cells or spleen cells obtained from B6 (IL-12+) (fine line) or IL-12p40null mice (heavy line) were stimulated with Con A for 2 days in complete medium without exogenous cytokine. At day 2, cells were harvested, washed, and subcultured in IL-2 or IL-4 as indicated for an additional 2 days before analysis for P-SelL expression by flow cytometry gated on CD8+ blasts. The experiment was performed twice. In both cases, P-SelL staining profiles for B6 and IL-12null responder populations from both lymph node and spleen overlapped respectively as exemplified in the figure.
FIGURE 3. P-SelL formation induced by exogenous IL-12 is dependent on additional cytokines. DC preparations from male B6 mice were used to stimulate HY-specific CD8 T cells in unsupplemented medium or in medium supplemented with IL-2, IL-4, or IL-15 (a, c, e, and g) as indicated. Parallel cultures were supplemented with additional exogenous IL-12 (b, d, f, and h). P-SelL expression was assessed on day 3 cultures by flow cytometry and gated analysis of CD8+ blasts as shown in A and was further assessed by plotting geometric mean P-SelL per cell division as shown in B. , No cytokine; , IL-2 supplemented; , IL-4 supplemented; , IL-15 supplemented; ?, IL-12 supplemented; , IL-12 + IL-2 supplemented; , IL-12+IL-4 supplemented; , IL-12 + IL-15 supplemented This experiment was performed three times with qualitatively similar results.
In summary, although IL-2, IL-15, and IL-12 could be shown to facilitate P-SelL formation under specific in vitro conditions, some autonomy from these cytokines could also be demonstrated using cytokine-deficient responders in vitro (Figs. 1 and 2). To assess the respective contributions of these cytokines during P-SelL formation in vivo, CD8 T cell responses to HY were therefore examined in mice lacking one, or a combination, of these cytokines.
Uniform and rapid P-SelL formation in male-specific CD8 T cells responding in vivo
HY-specific TCR tg CD8 T cells from female mice become activated and proliferate upon adoptive transfer in male recipient mice. This in vivo response is limited in that clonal expansion peaks within 4–5 days and wanes by day 9 without progressive graft-vs-host disease (33). This clonal exhaustion is presumably due to an inadequate supply of help that supports effector longevity and memory generation (34, 35). Within the first 2 days of in vivo stimulation however, the CD8+ T cell-proliferative response observed in spleen is robust as measured by flow cytometric analysis of clonal expansion (CFSE dye dilution) and blastogenesis among virtually all male-specific (CD8+T3.70high) precursors. As shown in Fig. 4, this response was accompanied by the induction of P-SelL and was distinguished by a number of criteria. 1) On the basis of CFSE dye dilution, P-SelL induction was observed on day 2 and was evident on those cells that had undergone two or more cell divisions. This contrasted with P-SelL formation during in vitro responses that generally required more extended stimulus duration (i.e., day 3 or longer). 2) The staining by P-Sel-hIg was relatively monophasic in contrast to biphasic staining distributions frequently observed after in vitro activation by either Con A or male DCs (Figs. 1 and 2). 3) The tempo and uniformity of P-SelL formation evident on day 2 constituted a pattern that was maintained over a wide range of cell dosages used for adoptive transfer (donor male-specific precursor doses 5 x 106, 5 x 105, 5 x 104; data not shown). Comparable formation of P-SelL was also observed on male-specific CD8 T cells responding in mesenteric and peripheral lymph nodes (data not shown). However, P-SelL induction during in vivo male-specific responses was not invariant given that P-SelL was induced poorly, or not at all, when HY was presented in the peritoneal cavity. As shown in Fig. 4, CD8 T cells responding in the peritoneal cavity to either synthetic male peptide Ag presented by female peritoneal cells or endogenous male peptide presented by male peritoneal cells failed to up-regulate P-SelL. These results reinforce recent observations that CD8 T cells responding in the peritoneum do not form SelL (22).
FIGURE 4. P-SelL formation in vivo is rapid and uniform during anti-HY-specific responses in spleen. Donor CFSE-labeled thymocytes from HY-TCR tg T cells were adoptively transferred iv into male or female B6 recipients for 2 days (a,b,c) whereupon P-SelL expression was evaluated by gated analysis of CD8+T3.70+CFSE+ spleen cells. a, In control female recipients, HY-specific CD8+ cells remained quiescent and P-SelL was not induced. b, In male recipients, HY-specific cells entered the cell cycle, as indicated by CFSE dilution, and effectively generated P-SelL on their surface within several cell divisions. c, Acquisition of P-SelL expression represented as geometric (geo) mean P-SelL for each respective cell division; data shown for one female recipient () and two male recipients (?). d, Donor CD8+T3.70+CFSE+ blasts recovered from spleen after i.v. adoptive transfer into male (?) or female () B6 recipients for 3 days; , P-SelL expressed on donor CD8+T3.70+CFSE+ blasts recovered after i.p. injection into male recipients and assessed on day 3 peritoneal lavage; , P-SelL expressed on donor CD8+T3.70+CFSE+ blasts recovered from peritoneal lavage 3 days after i.p. coinjection of HY thymocytes and 1.4 mg of male peptide into female recipients. The experiments shown in a–c were performed 33 times over a period of 2 years with qualitatively similar results. The results shown in d were performed three times with qualitatively similar results.
P-SelL formation in IL-2-, IL-12-, IL-15-deficient male recipients
IL-12 has been shown to drive P-SelL during in vitro culture of both CD4 and CD8 T cells fostering the widely accepted view that this cytokine is a determinator of P-SelL expression in vivo. Demonstration that IL-2 also promotes P-SelL formation within in vitro activated CD8 T cells led us to question which, if any, of these two cytokines are in fact relevant for P-SelL formation by CD8 T cells activated in vivo. To assess whether P-SelL formation occurred in the absence of IL-2, IL-12, or IL-15, mice deficient in these cytokines were used.
Because IL-12 and IL-15 are thought to be produced by non-T cells, female HY TCR tg thymocytes transferred into male recipients from IL-12null and IL-15null strains were used to investigate the involvement of these cytokines in P-SelL formation. To address the role of IL-2 in P-SelL induction, both donor HY TCR tg T cells and recipient male mice had to be genetically deficient in IL-2 because IL-2 can be produced by both T cells and DCs (36). Thus, recipient IL-2null male mice received donor T cells obtained from IL-2null mice bearing the HY TCR transgene. Donor cells were obtained from thymi of HY or HYIL-2null mice for adoptive transfer experiments for multiple reasons: 1) thymi from these mice contained CD8+CD4– HY-reactive precursors; 2) thymocytes represented a homogeneous source of naive T cells; 3) thymocytes displayed P-SelL formation comparable with that observed by peripheral HY T cells derived from lymph node upon transfer into male recipients (data not shown); and 4) thymic architecture and development in both IL-2null and HY-IL-2null mice were relatively unperturbed by gross lymphadenopathy that typically afflicts peripheral secondary lymphoid tissue of adult mice lacking IL-2.
CFSE was used to track responding CD8 T cells as they advanced through successive cell divisions. In all adoptive transfer combinations including HYIL-2nullIL-2null, HYIL-12null, HYIL-2nullIL-2nullIL-12null, and HYIL-2IL-2nullIL-15null P-SelL formed normally when compared with control wild-type responses in male HYB6 mice. These data, summarized in Fig. 5 and Table I, demonstrated that although IL-2, IL-12, and IL-15 can contribute significantly to P-SelL formation in vitro, these cytokines are not required during P-SelL induction in vivo. P-SelL formation was also unperturbed in IL-12null male mice that received HY TCR tg thymocytes depleted of subpopulations that might express IL-12 (APCs expressing CD11b, CD11c, B220, CD4, and Gr-1; data not shown). That IL-2, IL-12, and IL-15 deficiency had negligible impact on P-SelL formation in the experiments shown in Fig. 5, as measured on a per-cell-division basis, was inconsistent with models based on in vitro studies that these cytokines mediated the signaling events triggering formation of P-SelL in vivo. The possibility exists that these cytokines are indeed irrelevant during this process in vivo.
FIGURE 5. P-SelL formation on responding CD8 T cells in vivo is independent of IL-2, IL-12p40, and IL-15. Three experiments (a–c) are summarized where cytokine-deficient mice were used in assessment of short term P-SelL induction during HY-specific CD8 T cell responses in vivo. Donor CFSE-labeled thymocytes from HY-TCR tg T cells were adoptively transferred i.v. into male or female recipients for 2 days (a and b) or 2.5 days (c). P-SelL expression was evaluated by gated analysis of CD8+T3.70+CFSE+ spleen cells. Values are shown for geometric mean P-SelL expression for HY TCR tg thymocyte donor-derived CD8+T3.70+CFSE+ blasts transiting each cell division; , wild-type HY TCR tg or HY-IL-2nullB6 female; ?, HYB6 male; , HY-IL-2nullB6 male, representative of 14 mice analyzed; , HY-IL-2nullIL-12null male, 6 mice analyzed; , HY-IL-2nullIL-2null male, 8 mice analyzed; , HY-IL-2nullIL-2nullIL-12null male, 3 mice analyzed; , HY-IL-2nullIL-15null male, 5 mice analyzed; , HY-IL-2nullIL-2nullIL-15null male, 4 mice analyzed.
Table I. Geometric mean fluorescence of P-SelL staining on HY TcR-transgenic CD8 T cells from recipient spleensa
Discussion
The view that IL-12 regulates SelL formation originally developed from observations of differential formation of P-SelL on polarized Th1 vs Th2 populations generated in IL-12- vs IL-4-supplemented cultures and the ability of Abs to P-selectin to block recruitment of Th1-inflamed skin (37, 38). Requirements for PSGL1 during skin recruitment of CD4 (39, 40) as well as CD8 T cells (6) established the importance of this SelL, and by association IL-12, in these recruitment processes. Another series of reports established that IL-12 vs IL-4 differentially influenced expression of FucT and C2GlcNAcT1 enzymes in CD4 T cells (3, 4, 5, 41, 42); C2GlcNAcT (43, 44, 45) and FucTs (46, 47) are enzymes required for P-SelL formation respectively. Subsequent reports documented that C2GlcNAcT1 expression was critically dependent on IL-12 signals through STAT4 for C2GlcNAcT expression and SelL formation on in vitro activated CD4 cells (48, 49). IL-2, which had generally been included during these studies on polarized T cell populations, was found to support P-SelL formation in activated CD8 T cells independent of exogenous IL-12 (11). Thus, signals other than IL-12 could effectively support SelL in vitro, at least in CD8 T cells. Furthermore, IL-15 supported CD8 T cell growth and, although less active than IL-2, was also effective at supporting P-SelL expression (11). We have focused on P-SelL formation during the CD8 T cell response, and the aforementioned data prompted us to assess the contributions of these cytokines to P-SelL formation in vivo. The essential observation made in this report is that neither IL-2 nor IL-12 contributed to P-SelL formation in TCR tg CD8 T cells responding to HY in vivo.
A relatively small degree of variability in P-SelL induction was observed in responses between genetically identical mice in individual experiments and more so between experiments. The former variability presumably arose from either general stochastic noise associated with the complex biological process under study (e.g., Fig. 4c) or, in the case of individual mice on the IL-2null background, from sequelae associated with variable severity of lymphadenopathy; the later variability presumably arose from variation in responder cell fitness after CFSE labeling or variation in the precise time point when mice were sacrificed for tissue isolation. Nevertheless, P-SelL formation in all cytokine-deficient combinations tested was surprisingly consistent when compared with CD8 T cell responses in the peritoneum where P-Sel was not induced as shown in Fig. 4d.
With regard to IL-2, we found that endogenously produced IL-2 supported P-SelL formation only under specific conditions of in vitro stimulation: conditions of Con A-activated splenocytes where IL-2 is known to accumulate to relatively high concentrations (50) (D. A. Carlow, personal observations). The rapid 30-min clearance (51) and metabolism (52) of IL-2 in vivo may be responsible for lack of IL-2 impact on P-SelL expression here and in other in vivo CD8 T cell response models in which IL-2-independent and IL-15-independent support for CD8 T cell expansion in secondary lymphoid tissue has been demonstrated (see Fig. 5 and Ref. 53) (35, 54). Consistent with previous observations by others using mice doubly deficient in IL-2 and IL-15, we did note a minor reduction in CD8 T cell in vivo expansion during responses occurring in the absence of both these cytokines (53). Together, these results have led to the conclusion that alternate signals exist in vivo that support the programmed CD8 T cell response (35). The impact of IL-2 on the CD8 T cell response in vitro is interesting in that IL-2 effectively supports both proliferation and P-SelL formation. The fact that in vivo clonal expansion of CD8 T cells occurs effectively when assessed in responding cells isolated from both peritoneum and spleen whereas P-SelL is formed preferentially in cells harvested from spleen suggests that the IL-2-independent factor(s) that support the proliferative response in vivo appear to be distinct from those factors regulating P-SelL formation. Regarding IL-12, although exogenous IL-12 effectively supported P-SelL formation in HY-specific responses to male DC, this was evident only in conjunction with other cytokines IL-2, IL-15, and even IL-4. However, in mice deficient in IL-12p40, shared by both IL-12 and IL-23 (55), P-SelL formation proceeded normally during CD8 T cell responses in vitro (Fig. 2) and in vivo (Fig. 5). In summary, although P-SelL formation during CD8 T cell responses in vivo are independent of both IL-12 and IL-2 it is quite possible that signaling pathways engaged by these cytokines are also efficiently triggered by alternative, as yet unresolved, signaling processes normally operating in vivo.
What are the signals driving P-SelL in CD4 and CD8 T cells in vivo? There are at least three ongoing strategies to address this question that focus on cytokine signaling, APC phenotype/microenvironment, and TCR signaling. First, based on P-SelL induction through IL-12 polarization, in vitro studies are under way to identify relevant downstream signaling events from the IL-2R (e.g., STAT5) or the IL-12R (e.g., STAT4) (1, 49). Signaling pathways unique to IL-2 vs IL-15 (e.g., via FKBP12.6; Ref. 56) may also be relevant given that these cytokines differ in their efficacy of P-SelL induction in vitro (Ref. 11 and this article). Parenthetically, the relevance of IL-2 and IL-12 to P-SelL on CD8 T cells in vivo may be questioned given the results described in this report.
Second, differential SelL/47 formation by APCs located in, or derived from, different anatomical sites (14, 20, 21, 22, 23) may be used to identify relevant properties of APC that can induce P-SelL. However, resolving the relevant phenotypic properties of APC or microenvironmental characteristics responsible may be difficult. For example, it is clear that Ag-bearing DC isolated from different sources can differentially induce SelL on CD8 T cells responding in vitro (21, 22), but it is also clear that DC generated from a single source, cultured bone marrow, can differentially induce SelL on responding CD8 T cells in vivo depending on the route of adoptive transfer (e.g., i.v. vs intradermal) (22). Thus, tissue microenvironmental cues may determine properties expressed by DCs that in turn direct whether or not selectin ligands are formed on responding T cells. Interestingly, Dudda et al. (22) reported that intradermal injection of peptide-pulsed marrow derived dendritic cells induced E-SelL at day 6 on i.v.-injected peptide-Ag-specific CD8 T cells whereas i.v. injection of the same DC preparation did not. Their observations contrast with ours where efficient P-SelL formation occurred on HY-specific CD8 T cells after i.v. adoptive transfer into male mice. Efforts to resolve the Ag presentation requirements for P-SelL induction in response to HY in vivo are ongoing.
Third, signaling events through H-Ras have been implicated in -1,3-fucosyltransferase VII (FucTVII) induction, suggesting that TCR signal strength alone may determine FucTVII expression in Jurkat T cells (57). TCR signal strength has also been implicated in the context of polarization of T cell responses (58) that, as mentioned above, have been correlated with differential SelL formation. Further analysis of the relevant signaling pathways has suggested that concomitant activation of RAF, PI3K, and a third unresolved H-Ras-dependent signaling pathway are required for FucTVII expression (59). The fact that we and others observed that CD8 T cell populations harvested from spleen vs peritoneum both responded robustly to HY (Fig. 4) but exhibited differential P-SelL expression would seem to be less consistent with differential TCR signal strength determining P-SelL expression per se and more consistent with influences of APCs and/or microenvironment (23). All three of these aforementioned approaches are ambitious and carry significant technical challenges for successfully identifying essential molecular interactions and downstream signaling events required for SelL formation in vivo.
In summary, contrary to expectations based on in vitro analyses implicating prominent roles of IL-12 and IL-2 for induction of P-SelL in activated T cells, we find that neither cytokine is required in vivo for this purpose. P-SelL formation was unimpeded in homogenous populations of naive CD8 T cells responding to male Ag after adoptive transfer into male recipients under conditions in which both cytokines were absent. Because P-SelL formation does not occur effectively in CD8 T cells responding to HY in the peritoneum, it appears that P-SelL formation observed in secondary lymphoid tissues is an active process and clearly not an invariant consequence of activation itself. The importance of SelL in T cell recruitment underscores the need to understand what signaling events regulate expression of this structure in vivo.
Disclosures
The authors have no financial conflict of interest.
Acknowledgments
We acknowledge Philip Owen for synthesis of the HY peptide.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by Canadian Institutes for Health Research Grant MOP-53162.
2 Address correspondence and reprint requests to Dr. Douglas A. Carlow or Dr. Hermann J. Ziltener, Biomedical Research Centre, University of British Columbia, 2222 Health Sciences Mall, Vancouver, British Columbia, V6T-1Z3, Canada. E-mail address: doug@brc.ubc.ca or hermann@brc.ubc.ca
3 Abbreviations used in this paper: DC, dendritic cell; C2GlcNAcT, core 2 glycosyltransferase; HY, male antigen; P-SelL, P-ligand; hlg, human Ig; FucTVII, -1,3-fucosyltransferase VII; tg, transgenic.
Received for publication September 13, 2004. Accepted for publication December 17, 2004.
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In vitro studies have demonstrated that IL-2 and IL-12 can support formation of P-selectin ligands (P-SelL) in activated T cells, ligands that are variably required for efficient lymphocyte recruitment to sites of inflammation. To ascertain whether these cytokines were required for P-SelL formation in vivo, TCR transgenic CD8 T cells specific for male Ag (HY) were transferred into male mice under conditions in which either IL-2 and/or IL-15 or IL-12Rp40 were absent. P-SelL formation at day 2 was unperturbed in HY-TCR IL-2null CD8 T cells responding in doubly deficient IL-2nullIL-12null or IL-2nullIL-15null male recipients. HY-specific CD8 T cell proliferative responses detected in both spleen and peritoneum occurred vigorously, but only splenic CD8 T cells up-regulated P-SelL, demonstrating that in vivo induction of P-SelL is an active, nonprogrammed event following T cell activation and that despite the efficacy of IL-2 and IL-12 in supporting P-SelL formation in vitro, these cytokines appear to be dispensable for this purpose in vivo.
Introduction
The choreography of adaptive T cell immunity to foreign Ag generally follows a series of defined steps beginning with T cell activation in the peripheral lymph node by Ag-bearing dendritic cells (DC)3 that had previously emigrated from a peripheral site of innate immune activation. During the process of activation, the T cell acquires a set of instructions to exit the lymph node, to access inflamed sites, and to deliver effector functions appropriately. The extent to which this imprinting process occurs solely during the initial T cell-DC contact or through subsequent influences with cells or environment has not been fully resolved. Nevertheless, the process and form of such instructions represent crucial features required for appropriate spatial delivery of effector functions, the raison d’etre of all adaptive T cell immunity. Regulating selectin ligand formation and integrin expression/activation on T cells are specific examples of instructions received through T cell activation, enabling them to access an inflammatory site and deliver effector functions. On activation, T cells can acquire the ability to form ligands for endothelial expressed E- and P-selectins through de novo expression of a series of glycosyltransferases that branch and extend O-linked carbohydrate on specific glycoprotein substrates. These newly synthesized selectin ligands support rather subtle interactions between T cell and endothelial selectins manifested in transient rolling or tethering behavior along venule walls within an inflammatory site (reviewed in Ref. 1). In many cases, this selectin-mediated rolling and tethering behavior is an essential first step in exiting the vasculature.
Two sets of data have been generated that document distinct methods to differentially induce selectin ligand. The first set of data is based purely on in vitro observations whereby CD4 (2, 3, 4, 5) or CD8 (6, 7, 8, 9, 10) T cells polarized with IL-12 and TGF- or IFN- displayed P-SelL whereas cells polarized by IL-4 and anti-IFN- did not. IL-2 as well has recently been implicated in P SelL formation within activated CD8 T cells (11). These data suggested that cytokines are primary drivers of SelL formation but it has been difficult to conceptualize or demonstrate how and where such extreme polarizing influences might be exerted in vivo. Furthermore, additional observations from in vivo and in vitro polarized T cells, as defined by cytokine gene expression (12, 13, 14) have documented unexpected SelL expression in Th0 or Th2 polarized T cells. These observations suggest that modeling P-SelL induction based on in vitro polarization regimens may not translate reliably to circumstances in vivo. Nevertheless, the in vitro studies demonstrating that IL-2 and IL-12 could direct selectin ligand formation in CD4 and CD8 T cells led to the expectation that these cytokines were similarly influential in vivo and that SelL formation should therefore be compromised in mice lacking these cytokines.
The second set of data was an extension of early in vivo observations that T cells obtained from intestine or skin were inclined to repopulate the same respective site after adoptive transfer (15, 16, 17, 18, 19). More recent investigations have demonstrated differential integrin and SelL expression in vivo depending on the anatomical source of stimulating DCs or responding T cells suggesting that either resident APCs or other local microenvironmental influences can differentially imprint homing properties on responding T cells by selectively directing induction of SelL and integrin expression (14, 20, 21, 22). The respective roles of resident APC (DC) phenotype vs other local environmental influences, including cytokines such as IL-2, IL-12 and cell surface molecules, in determining SelL formation in these in vivo studies has not been resolved (22, 23).
We therefore investigated SelL formation in vivo using mice bearing a transgenic (tg) TCR specific for male Ag (HY) but lacking genes encoding IL-2, IL-15, and/or IL-12. Wild-type or cytokine-deficient TCR tg CD8 T cells from female mice were adoptively transferred into normal or cytokine-deficient male mice, and the formation of P-SelL was monitored as adoptively transferred cells became activated and proliferated. Consistent with recent investigations by others suggesting that the site/source/phenotype of APC can imprint homing properties onto responding T cells, P-SelL formation did not occur in CD8 T cells responding to HY in the peritoneal cavity, whereas P-SelL formation proceeded rapidly and relatively uniformly when TCR tg CD8 T cells were introduced i.v. and reisolated from spleen. Importantly, P-SelL formation on i.v.-transferred CD8 T cells occurred regardless of IL-2, IL-12, or IL-15 cytokine deficiency. Thus, our results indicated that P-SelL formation within activated CD8 T cells in vivo can be differentially induced, independently of T cell activation per se and independently of those cytokines implicated by previous in vitro studies as being involved in P-SelL formation. Because the absence of these cytokines had no detectable impact on P-SelL formation in vivo, our data establish that another distinct signaling event(s) is in fact the primary driver of P-SelL formation in vivo.
Materials and Methods
Mice
Mice 6–12 wk of age were used for analyses. C57BL/6 (B6) mice were bred at the Biomedical Research Centre from founders obtained originally from The Jackson Laboratory. IL-12p40null mice on the B6 background (no. 002693; The Jackson Laboratory) were received and bred as homozygotes. When intercrossed with IL-2null mice, an IL-12 genotype was confirmed by loss of wild-type allele by PCR according to the suppliers’ recommended protocols (sense primer, 5'-AGTGAACCTCACCTGTGACACG-3'; antisense primer, 5'-TCTTTGCACCAGCCATGAGC-3'). IL-2null mice (no. 002252; The Jackson Laboratory) were bred from homozygous studs and heterozygous dams. The offspring genotype was confirmed by PCR according to the suppliers’ recommended procedures using null sense primer 5'-TCGAATTCGCCAATGACAAGACGCT-3', wild-type sense primer 5'-CTA GGCCACAGAATTGAAAGATCT-3', and shared antisense primer 5'-GTAGGTGGAAATTCT AGCATCATCC-3'. IL-15null homozygous mice (no. 004200-M; Taconic, Germantown, NY; Ref. 24) were received and bred as homozygotes. When intercrossed with IL-2null mice, the IL-15 genotype was confirmed by PCR using Neo primer 5'-GAATGGGCTGACCGCTTCCTCG-3', downstream genomic primer 5'-TCATATCCTCTGCACCTTGACTG-3', upstream exon 3 primer 5'-GAGGGCTAAATCTGATGCGTGTG-3', and exon 3 primer 5'-GAGCTGGCTATGGCGATG GGC-3'. PCR was conducted using 1 μl of stock 20 μM primers with 4 μl of extracted DNA per reaction at 94°C for 5 min; 39 cycles of 94°C for 1 min, 62°C for 1 min, and 72°C for 2 min; followed by a 4°C pause. For all genotyping, DNA was isolated with the Sigma REDExtract-N-Amp Tissue PCR kit (XNART). HY-TCR tg genotyping was performed with mAb T3.70-FITC (see Abs) staining of peripheral blood.
Media
Cell suspensions were prepared in RPMI (no. 11875-135l; Life Technologies) supplemented with 8% FCS, 5 x 10–5 M 2-ME, 100 U/ml penicillin, 100 U/ml streptomycin (Stem Cell Technologies), and 2 mM glutamine (Sigma-Aldrich). DMEM (no. 11965-084; Life Technologies) supplemented with 3% FCS was used for Ab staining.
Antibodies
Human IgG1-P-selectin fusion protein (BD Pharmingen; no. 28111A) was detected with PE-conjugated anti-human IgG (Jackson Immunoresearch; no. 109-116-098). CD8-biotin (no. CL8938B; Cedarlane) was detected with CyChrome-conjugated streptavidin (BD Pharmingen; no. 554062). CD8-APC (no. 553035), and CD8-FITC (no. 553031) were obtained from BD Pharmingen, and mAb T3.70 specific for HY TCR tg (25) was conjugated with biotin or Alexafluor 488 in house.
In vivo assessment of P-SelL induction
To circumvent issues of peripheral splenomegaly and lymphadenopathy prevalent in IL-2null mice, thymocytes from HY TCR tg and HY-IL-2null TCR tg mice were used as a source of naive CD8+CD4– HY-specific T cells. Donor mice were 8–12 wk of age as thymi were not grossly perturbed despite peripheral lymphadenopathy, whereas recipient IL-2 deficient mice were 5–7 wk of age as older mice developed increasingly severe splenomegaly that perturbed donor cell responses and made donor cell recovery increasingly difficult. Thymocytes were labeled with CFSE (no. C1157; Molecular Probes) at 107/ml in complete medium for 5 min at room temperature, washed in medium, washed in HBSS (no. H8264; Sigma-Aldrich), and resuspended in HBSS for injection. Twenty million thymocytes were injected i.v. or i.p. Where indicated, synthetic male peptide (prepared in house at the Biomedical Research Centre) H-Lys-Cys-Ser-Arg-Asn-Arg-Gln-Tyr-Leu-OH (26) or male spleen cells were coinjected with CFSE-labeled thymocytes. From 2 to 3 days later (as indicated), recipients were sacrificed. RBC-depleted splenocytes or peritoneal lavage isolates were stained for four-color flow cytometry as follows. CFSE (FL1), human Ig (hIg)-P-Sel + anti-hIg-PE (FL2), T3.70-biotin + SA-CyC (FL3), and CD8-APC (FL4) cytometry was conducted in DMEM supplemented with 3% FCS in the sequence hIg-P-Sel, anti-hIg-PE, NMS, T3.70-biotin+CD8-APC, SA-CyC. Cells were incubated with Abs for 20 min on ice in 96-well round-bottom plates (Nunclon; InterMed). Cells were washed twice and analyzed on a FACSCalibur flow cytometer (BD Biosciences). P-SelL expression was presented either as raw data dot plots or as line graphs of cell division number vs geometric mean P-SelL per division. To exclude spurious geometric mean P-SelL data points in the graphs presented, only data points exhibiting a singular CFSE intensity (i.e., cells that had undergone an equal number of divisions) representing >10% total CFSE+ CD8+T3.70+ gated events were plotted.
Lymphocyte cultures
Con A primary stimulations were conducted with lymph node cells or splenocytes cultured at 106/ml in 4 mg/ml Con A (Sigma-Aldrich) for 48 h at 37°C in 5% CO2. Cultures of 2 or 10 ml were prepared in 24-well Falcon 3047 plates or 6-well Falcon 3046 plates, respectively (BD Biosciences). After 48 h, cells were harvested, washed, counted, and replated in secondary cultures at 0.25 x 106/ml with 2.5% IL-2 supernatants or at 0.05 x 106/ml with 2.5% IL-4 supernatants where indicated. After another 48 h, cells were harvested and stained for flow cytometric analysis with hIg-P-Sel + anti-hIg-PE and CD8-FITC. Primary HY-specific stimulations were conducted with 2000 rad-irradiated splenic DC preparations generated by differential adherence to tissue culture plastic as described previously (27). DC stimulators (4 x 104) were cocultured with 6 x 105 HY TCR tg thymocytes in 96-well flat-bottom tissue culture plates (Nunc; no. 167008) for 3 days and analyzed by flow cytometry with hIg-P-Sel as described above. Cytokines used in these studies included IL-2 and IL-4 obtained as conditioned medium from murine IL-2 or IL-4 cDNA transfectants of myeloma X-653 kindly provided by Fritz Melchers, IL-12 (2 ng/ml; R&D Systems; no. 419-ML), and IL-15 (50 μg/ml; R&D Systems; no. 247–1L).
Results
Role of endogenous IL-2 in vitro
In previous studies using 4-day cultures including a 2-day Con A stimulation (days 0–2), day 2 wash, and subculturing in fresh medium (days 2–4) to activate CD8 T cells, we observed that exogenous IL-2 included during days 2–4 promoted core 2 glycosyltransferase (C2GlcNAcT) activity and formation of P-SelL by day 4 relative to exogenous IL-4 (28). To determine whether endogenous IL-2 produced by responding T cells themselves could drive P-SelL in vitro, the induction of P-SelL was monitored in similar 4-day wild-type B6 vs IL-2null Con A-stimulated cultures. It was established many years ago that IL-2 production in Con A-stimulated cells peaks in the first 18–24 h of culture (29, 30, 31). We therefore focused our analysis of endogenous IL-2 impact by varying conditions during the first 2 days of Con A stimulation and applying constant, IL-2-supplemented conditions from day 2 to day 4. As shown in Fig. 1, induction of P-SelL on CD8+-gated T cells was significantly affected by cytokines present during primary cultures (days 0–2). Several features of these data were noteworthy. 1) P-SelL expression tended to be biphasic. This heterogeneity is a persistent feature of P-SelL induction among in vitro activated CD8 T cells and presumably reflects heterogeneity in either the responding T cells themselves or the microconditions experienced by individual clones present in the culture. 2) When exogenous IL-2 or IL-4 were included in primary cultures (days 0–2), endogenous IL-2 production appeared to have little impact on P-SelL induction insofar as comparable signals were obtained from cultures of B6 or IL-2null tissue. 3) In the absence of exogenous supplements, endogenous IL-2 support of P-SelL induction in B6 vs IL-2null cultures was more evident in cultures of spleen than in cultures of lymph node. This observation was consistent with previous observations that Con A-stimulated spleen is a particularly good source of IL-2 (32) and that P-SelL induction was more evident in Con A-activated cultures of spleen than in those of lymph node (28). In summary, endogenous IL-2 production under specific in vitro conditions of T cell activation could promote P-SelL formation.
FIGURE 1. Endogenous IL-2 can support P-SelL formation by CD8 T cells responding to Con A in vitro. Lymph node (LN) cells or spleen cells (SPL) obtained from IL-2null or B6 (IL-2+) mice were stimulated with Con A for 2 days in complete medium supplemented with no exogenous cytokine (NS) (a–d), exogenous IL-2 (e–h), or exogenous IL-4 (i–l), as indicated. Two days later, cells were subcultured in IL-2-supplemented medium to maintain cell viability for an additional 2 days before analysis for P-SelL expression by flow cytometry gated on CD8+ blasts. This experiment was performed three times with qualitatively similar results.
IL-12 deficiency does not alter P-SelL formation in CD8+ cells responding to Con A in vitro
As described in the introduction, IL-12 had been implicated by previous investigations on P-SelL formation in both CD4 and CD8 T cells. In these studies, exogenous IL-12 was used to supplement medium in Th1/Tc1 polarizing cultures of activated T cells and exhibited significant activity in driving P-SelL formation. When P-SelL formation was evaluated in short term cultures of Con A-activated B6 or IL-12null (p40null) lymphocytes, as shown in Fig. 2, it was evident that IL-12 played no role in this process. Clearly, factors other than IL-12 were responsible for driving P-SelL formation in such short term in vitro responses. The role of IL-12 during short term in vitro activation and P-SelL formation was also assessed in peptide-specific responses by CD8 T cells expressing a tg TCR against the HY. In vitro activation and P-SelL formation in such CD8 T cells was monitored 3–4 days after stimulation with male splenic DCs in the presence of cytokines as indicated in Fig. 3. In this model of short term T cell activation, exogenous IL-12 supplement promoted P-SelL formation when present with either IL-2 or IL-15. However, exogenous IL-12 had no significant impact on P-SelL expression when present alone or with IL-4.
FIGURE 2. P-SelL formation is unperturbed by IL-12p40 deficiency in CD8 T cells responding to Con A in vitro. Lymph node (LN) cells or spleen cells obtained from B6 (IL-12+) (fine line) or IL-12p40null mice (heavy line) were stimulated with Con A for 2 days in complete medium without exogenous cytokine. At day 2, cells were harvested, washed, and subcultured in IL-2 or IL-4 as indicated for an additional 2 days before analysis for P-SelL expression by flow cytometry gated on CD8+ blasts. The experiment was performed twice. In both cases, P-SelL staining profiles for B6 and IL-12null responder populations from both lymph node and spleen overlapped respectively as exemplified in the figure.
FIGURE 3. P-SelL formation induced by exogenous IL-12 is dependent on additional cytokines. DC preparations from male B6 mice were used to stimulate HY-specific CD8 T cells in unsupplemented medium or in medium supplemented with IL-2, IL-4, or IL-15 (a, c, e, and g) as indicated. Parallel cultures were supplemented with additional exogenous IL-12 (b, d, f, and h). P-SelL expression was assessed on day 3 cultures by flow cytometry and gated analysis of CD8+ blasts as shown in A and was further assessed by plotting geometric mean P-SelL per cell division as shown in B. , No cytokine; , IL-2 supplemented; , IL-4 supplemented; , IL-15 supplemented; ?, IL-12 supplemented; , IL-12 + IL-2 supplemented; , IL-12+IL-4 supplemented; , IL-12 + IL-15 supplemented This experiment was performed three times with qualitatively similar results.
In summary, although IL-2, IL-15, and IL-12 could be shown to facilitate P-SelL formation under specific in vitro conditions, some autonomy from these cytokines could also be demonstrated using cytokine-deficient responders in vitro (Figs. 1 and 2). To assess the respective contributions of these cytokines during P-SelL formation in vivo, CD8 T cell responses to HY were therefore examined in mice lacking one, or a combination, of these cytokines.
Uniform and rapid P-SelL formation in male-specific CD8 T cells responding in vivo
HY-specific TCR tg CD8 T cells from female mice become activated and proliferate upon adoptive transfer in male recipient mice. This in vivo response is limited in that clonal expansion peaks within 4–5 days and wanes by day 9 without progressive graft-vs-host disease (33). This clonal exhaustion is presumably due to an inadequate supply of help that supports effector longevity and memory generation (34, 35). Within the first 2 days of in vivo stimulation however, the CD8+ T cell-proliferative response observed in spleen is robust as measured by flow cytometric analysis of clonal expansion (CFSE dye dilution) and blastogenesis among virtually all male-specific (CD8+T3.70high) precursors. As shown in Fig. 4, this response was accompanied by the induction of P-SelL and was distinguished by a number of criteria. 1) On the basis of CFSE dye dilution, P-SelL induction was observed on day 2 and was evident on those cells that had undergone two or more cell divisions. This contrasted with P-SelL formation during in vitro responses that generally required more extended stimulus duration (i.e., day 3 or longer). 2) The staining by P-Sel-hIg was relatively monophasic in contrast to biphasic staining distributions frequently observed after in vitro activation by either Con A or male DCs (Figs. 1 and 2). 3) The tempo and uniformity of P-SelL formation evident on day 2 constituted a pattern that was maintained over a wide range of cell dosages used for adoptive transfer (donor male-specific precursor doses 5 x 106, 5 x 105, 5 x 104; data not shown). Comparable formation of P-SelL was also observed on male-specific CD8 T cells responding in mesenteric and peripheral lymph nodes (data not shown). However, P-SelL induction during in vivo male-specific responses was not invariant given that P-SelL was induced poorly, or not at all, when HY was presented in the peritoneal cavity. As shown in Fig. 4, CD8 T cells responding in the peritoneal cavity to either synthetic male peptide Ag presented by female peritoneal cells or endogenous male peptide presented by male peritoneal cells failed to up-regulate P-SelL. These results reinforce recent observations that CD8 T cells responding in the peritoneum do not form SelL (22).
FIGURE 4. P-SelL formation in vivo is rapid and uniform during anti-HY-specific responses in spleen. Donor CFSE-labeled thymocytes from HY-TCR tg T cells were adoptively transferred iv into male or female B6 recipients for 2 days (a,b,c) whereupon P-SelL expression was evaluated by gated analysis of CD8+T3.70+CFSE+ spleen cells. a, In control female recipients, HY-specific CD8+ cells remained quiescent and P-SelL was not induced. b, In male recipients, HY-specific cells entered the cell cycle, as indicated by CFSE dilution, and effectively generated P-SelL on their surface within several cell divisions. c, Acquisition of P-SelL expression represented as geometric (geo) mean P-SelL for each respective cell division; data shown for one female recipient () and two male recipients (?). d, Donor CD8+T3.70+CFSE+ blasts recovered from spleen after i.v. adoptive transfer into male (?) or female () B6 recipients for 3 days; , P-SelL expressed on donor CD8+T3.70+CFSE+ blasts recovered after i.p. injection into male recipients and assessed on day 3 peritoneal lavage; , P-SelL expressed on donor CD8+T3.70+CFSE+ blasts recovered from peritoneal lavage 3 days after i.p. coinjection of HY thymocytes and 1.4 mg of male peptide into female recipients. The experiments shown in a–c were performed 33 times over a period of 2 years with qualitatively similar results. The results shown in d were performed three times with qualitatively similar results.
P-SelL formation in IL-2-, IL-12-, IL-15-deficient male recipients
IL-12 has been shown to drive P-SelL during in vitro culture of both CD4 and CD8 T cells fostering the widely accepted view that this cytokine is a determinator of P-SelL expression in vivo. Demonstration that IL-2 also promotes P-SelL formation within in vitro activated CD8 T cells led us to question which, if any, of these two cytokines are in fact relevant for P-SelL formation by CD8 T cells activated in vivo. To assess whether P-SelL formation occurred in the absence of IL-2, IL-12, or IL-15, mice deficient in these cytokines were used.
Because IL-12 and IL-15 are thought to be produced by non-T cells, female HY TCR tg thymocytes transferred into male recipients from IL-12null and IL-15null strains were used to investigate the involvement of these cytokines in P-SelL formation. To address the role of IL-2 in P-SelL induction, both donor HY TCR tg T cells and recipient male mice had to be genetically deficient in IL-2 because IL-2 can be produced by both T cells and DCs (36). Thus, recipient IL-2null male mice received donor T cells obtained from IL-2null mice bearing the HY TCR transgene. Donor cells were obtained from thymi of HY or HYIL-2null mice for adoptive transfer experiments for multiple reasons: 1) thymi from these mice contained CD8+CD4– HY-reactive precursors; 2) thymocytes represented a homogeneous source of naive T cells; 3) thymocytes displayed P-SelL formation comparable with that observed by peripheral HY T cells derived from lymph node upon transfer into male recipients (data not shown); and 4) thymic architecture and development in both IL-2null and HY-IL-2null mice were relatively unperturbed by gross lymphadenopathy that typically afflicts peripheral secondary lymphoid tissue of adult mice lacking IL-2.
CFSE was used to track responding CD8 T cells as they advanced through successive cell divisions. In all adoptive transfer combinations including HYIL-2nullIL-2null, HYIL-12null, HYIL-2nullIL-2nullIL-12null, and HYIL-2IL-2nullIL-15null P-SelL formed normally when compared with control wild-type responses in male HYB6 mice. These data, summarized in Fig. 5 and Table I, demonstrated that although IL-2, IL-12, and IL-15 can contribute significantly to P-SelL formation in vitro, these cytokines are not required during P-SelL induction in vivo. P-SelL formation was also unperturbed in IL-12null male mice that received HY TCR tg thymocytes depleted of subpopulations that might express IL-12 (APCs expressing CD11b, CD11c, B220, CD4, and Gr-1; data not shown). That IL-2, IL-12, and IL-15 deficiency had negligible impact on P-SelL formation in the experiments shown in Fig. 5, as measured on a per-cell-division basis, was inconsistent with models based on in vitro studies that these cytokines mediated the signaling events triggering formation of P-SelL in vivo. The possibility exists that these cytokines are indeed irrelevant during this process in vivo.
FIGURE 5. P-SelL formation on responding CD8 T cells in vivo is independent of IL-2, IL-12p40, and IL-15. Three experiments (a–c) are summarized where cytokine-deficient mice were used in assessment of short term P-SelL induction during HY-specific CD8 T cell responses in vivo. Donor CFSE-labeled thymocytes from HY-TCR tg T cells were adoptively transferred i.v. into male or female recipients for 2 days (a and b) or 2.5 days (c). P-SelL expression was evaluated by gated analysis of CD8+T3.70+CFSE+ spleen cells. Values are shown for geometric mean P-SelL expression for HY TCR tg thymocyte donor-derived CD8+T3.70+CFSE+ blasts transiting each cell division; , wild-type HY TCR tg or HY-IL-2nullB6 female; ?, HYB6 male; , HY-IL-2nullB6 male, representative of 14 mice analyzed; , HY-IL-2nullIL-12null male, 6 mice analyzed; , HY-IL-2nullIL-2null male, 8 mice analyzed; , HY-IL-2nullIL-2nullIL-12null male, 3 mice analyzed; , HY-IL-2nullIL-15null male, 5 mice analyzed; , HY-IL-2nullIL-2nullIL-15null male, 4 mice analyzed.
Table I. Geometric mean fluorescence of P-SelL staining on HY TcR-transgenic CD8 T cells from recipient spleensa
Discussion
The view that IL-12 regulates SelL formation originally developed from observations of differential formation of P-SelL on polarized Th1 vs Th2 populations generated in IL-12- vs IL-4-supplemented cultures and the ability of Abs to P-selectin to block recruitment of Th1-inflamed skin (37, 38). Requirements for PSGL1 during skin recruitment of CD4 (39, 40) as well as CD8 T cells (6) established the importance of this SelL, and by association IL-12, in these recruitment processes. Another series of reports established that IL-12 vs IL-4 differentially influenced expression of FucT and C2GlcNAcT1 enzymes in CD4 T cells (3, 4, 5, 41, 42); C2GlcNAcT (43, 44, 45) and FucTs (46, 47) are enzymes required for P-SelL formation respectively. Subsequent reports documented that C2GlcNAcT1 expression was critically dependent on IL-12 signals through STAT4 for C2GlcNAcT expression and SelL formation on in vitro activated CD4 cells (48, 49). IL-2, which had generally been included during these studies on polarized T cell populations, was found to support P-SelL formation in activated CD8 T cells independent of exogenous IL-12 (11). Thus, signals other than IL-12 could effectively support SelL in vitro, at least in CD8 T cells. Furthermore, IL-15 supported CD8 T cell growth and, although less active than IL-2, was also effective at supporting P-SelL expression (11). We have focused on P-SelL formation during the CD8 T cell response, and the aforementioned data prompted us to assess the contributions of these cytokines to P-SelL formation in vivo. The essential observation made in this report is that neither IL-2 nor IL-12 contributed to P-SelL formation in TCR tg CD8 T cells responding to HY in vivo.
A relatively small degree of variability in P-SelL induction was observed in responses between genetically identical mice in individual experiments and more so between experiments. The former variability presumably arose from either general stochastic noise associated with the complex biological process under study (e.g., Fig. 4c) or, in the case of individual mice on the IL-2null background, from sequelae associated with variable severity of lymphadenopathy; the later variability presumably arose from variation in responder cell fitness after CFSE labeling or variation in the precise time point when mice were sacrificed for tissue isolation. Nevertheless, P-SelL formation in all cytokine-deficient combinations tested was surprisingly consistent when compared with CD8 T cell responses in the peritoneum where P-Sel was not induced as shown in Fig. 4d.
With regard to IL-2, we found that endogenously produced IL-2 supported P-SelL formation only under specific conditions of in vitro stimulation: conditions of Con A-activated splenocytes where IL-2 is known to accumulate to relatively high concentrations (50) (D. A. Carlow, personal observations). The rapid 30-min clearance (51) and metabolism (52) of IL-2 in vivo may be responsible for lack of IL-2 impact on P-SelL expression here and in other in vivo CD8 T cell response models in which IL-2-independent and IL-15-independent support for CD8 T cell expansion in secondary lymphoid tissue has been demonstrated (see Fig. 5 and Ref. 53) (35, 54). Consistent with previous observations by others using mice doubly deficient in IL-2 and IL-15, we did note a minor reduction in CD8 T cell in vivo expansion during responses occurring in the absence of both these cytokines (53). Together, these results have led to the conclusion that alternate signals exist in vivo that support the programmed CD8 T cell response (35). The impact of IL-2 on the CD8 T cell response in vitro is interesting in that IL-2 effectively supports both proliferation and P-SelL formation. The fact that in vivo clonal expansion of CD8 T cells occurs effectively when assessed in responding cells isolated from both peritoneum and spleen whereas P-SelL is formed preferentially in cells harvested from spleen suggests that the IL-2-independent factor(s) that support the proliferative response in vivo appear to be distinct from those factors regulating P-SelL formation. Regarding IL-12, although exogenous IL-12 effectively supported P-SelL formation in HY-specific responses to male DC, this was evident only in conjunction with other cytokines IL-2, IL-15, and even IL-4. However, in mice deficient in IL-12p40, shared by both IL-12 and IL-23 (55), P-SelL formation proceeded normally during CD8 T cell responses in vitro (Fig. 2) and in vivo (Fig. 5). In summary, although P-SelL formation during CD8 T cell responses in vivo are independent of both IL-12 and IL-2 it is quite possible that signaling pathways engaged by these cytokines are also efficiently triggered by alternative, as yet unresolved, signaling processes normally operating in vivo.
What are the signals driving P-SelL in CD4 and CD8 T cells in vivo? There are at least three ongoing strategies to address this question that focus on cytokine signaling, APC phenotype/microenvironment, and TCR signaling. First, based on P-SelL induction through IL-12 polarization, in vitro studies are under way to identify relevant downstream signaling events from the IL-2R (e.g., STAT5) or the IL-12R (e.g., STAT4) (1, 49). Signaling pathways unique to IL-2 vs IL-15 (e.g., via FKBP12.6; Ref. 56) may also be relevant given that these cytokines differ in their efficacy of P-SelL induction in vitro (Ref. 11 and this article). Parenthetically, the relevance of IL-2 and IL-12 to P-SelL on CD8 T cells in vivo may be questioned given the results described in this report.
Second, differential SelL/47 formation by APCs located in, or derived from, different anatomical sites (14, 20, 21, 22, 23) may be used to identify relevant properties of APC that can induce P-SelL. However, resolving the relevant phenotypic properties of APC or microenvironmental characteristics responsible may be difficult. For example, it is clear that Ag-bearing DC isolated from different sources can differentially induce SelL on CD8 T cells responding in vitro (21, 22), but it is also clear that DC generated from a single source, cultured bone marrow, can differentially induce SelL on responding CD8 T cells in vivo depending on the route of adoptive transfer (e.g., i.v. vs intradermal) (22). Thus, tissue microenvironmental cues may determine properties expressed by DCs that in turn direct whether or not selectin ligands are formed on responding T cells. Interestingly, Dudda et al. (22) reported that intradermal injection of peptide-pulsed marrow derived dendritic cells induced E-SelL at day 6 on i.v.-injected peptide-Ag-specific CD8 T cells whereas i.v. injection of the same DC preparation did not. Their observations contrast with ours where efficient P-SelL formation occurred on HY-specific CD8 T cells after i.v. adoptive transfer into male mice. Efforts to resolve the Ag presentation requirements for P-SelL induction in response to HY in vivo are ongoing.
Third, signaling events through H-Ras have been implicated in -1,3-fucosyltransferase VII (FucTVII) induction, suggesting that TCR signal strength alone may determine FucTVII expression in Jurkat T cells (57). TCR signal strength has also been implicated in the context of polarization of T cell responses (58) that, as mentioned above, have been correlated with differential SelL formation. Further analysis of the relevant signaling pathways has suggested that concomitant activation of RAF, PI3K, and a third unresolved H-Ras-dependent signaling pathway are required for FucTVII expression (59). The fact that we and others observed that CD8 T cell populations harvested from spleen vs peritoneum both responded robustly to HY (Fig. 4) but exhibited differential P-SelL expression would seem to be less consistent with differential TCR signal strength determining P-SelL expression per se and more consistent with influences of APCs and/or microenvironment (23). All three of these aforementioned approaches are ambitious and carry significant technical challenges for successfully identifying essential molecular interactions and downstream signaling events required for SelL formation in vivo.
In summary, contrary to expectations based on in vitro analyses implicating prominent roles of IL-12 and IL-2 for induction of P-SelL in activated T cells, we find that neither cytokine is required in vivo for this purpose. P-SelL formation was unimpeded in homogenous populations of naive CD8 T cells responding to male Ag after adoptive transfer into male recipients under conditions in which both cytokines were absent. Because P-SelL formation does not occur effectively in CD8 T cells responding to HY in the peritoneum, it appears that P-SelL formation observed in secondary lymphoid tissues is an active process and clearly not an invariant consequence of activation itself. The importance of SelL in T cell recruitment underscores the need to understand what signaling events regulate expression of this structure in vivo.
Disclosures
The authors have no financial conflict of interest.
Acknowledgments
We acknowledge Philip Owen for synthesis of the HY peptide.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by Canadian Institutes for Health Research Grant MOP-53162.
2 Address correspondence and reprint requests to Dr. Douglas A. Carlow or Dr. Hermann J. Ziltener, Biomedical Research Centre, University of British Columbia, 2222 Health Sciences Mall, Vancouver, British Columbia, V6T-1Z3, Canada. E-mail address: doug@brc.ubc.ca or hermann@brc.ubc.ca
3 Abbreviations used in this paper: DC, dendritic cell; C2GlcNAcT, core 2 glycosyltransferase; HY, male antigen; P-SelL, P-ligand; hlg, human Ig; FucTVII, -1,3-fucosyltransferase VII; tg, transgenic.
Received for publication September 13, 2004. Accepted for publication December 17, 2004.
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