B cell survival during anti-CD20 mAb immunotherapy
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免疫学杂志 2005年第7期
Anti-CD20 mAb immunotherapy is used clinically to treat non-Hodgkin’s lymphoma and other B cell diseases. However, the clearance of B cells from locations other than blood and spleen is difficult to monitor. Hamaguchi et al. (p. 4389 ) found that mature B cells, but not pro-B cells, pre-B cells, or immature B cells, were eliminated from the bone marrow, mesenteric lymph nodes, Peyer’s patches, gut-associated intraepithelial cells, and lamina propria cells of wild-type or C3–/– mice by 1 h after i.v. injection of anti-mouse CD20 mAb. No comparable depletion was seen in treated FcR–/– mice. Anti-CD20 mAb treatment led to loss of splenic follicles and almost all of the mature and marginal zone B cells along with transitional T1 and T2 cells; spleen B1a cells were reduced by 70%. Peritoneal B1a, B1b, and B2 cells from mAb-treated mice bound mAb, but their numbers were not reduced until after 28–58 days of treatment. Anti-CD20 mAb treatment did not accelerate depletion of peritoneal CFSE-labeled wild-type or CD20–/– B2 cells introduced into wild-type mice 2–7 days earlier; CFSE-labeled wild-type, but not CD20–/–, B cells that had migrated to the spleen were eliminated almost completely by 7 days after treatment. Intraperitoneal injection of thioglycolate into wild-type mice 1 day before anti-CD20 mAb treatment resulted in a 65% reduction in B2 cell numbers in the peritoneum at day 2 compared with control animals; B1a and B1b cells were deleted by day 7 after treatment. Thioglycolate treatment had no effect in FcR–/– mice. The data suggest that the peritoneal cavity is a protective niche in which B cells evade FcR-dependent immune destruction during anti-CD20 mAb therapy.
Identifying autoreactive CD4+ T cells
The pathology of most autoimmune diseases is mediated by CD4+ T cells that recognize self peptides. Yet details regarding the development of the autoantigen-specific T cells and their role in the autoimmune process are not available. Latham et al. (p. 3978 ) developed a chimeric HLA-DR1 recombinant molecule carrying a collagen II (CII) peptide (DR1-CII) that bound to a CII-specific TCR. DR1-CII tetramers stained CD4+ T cells from lymph nodes of DR1-transgenic mice immunized 10 days earlier with CII to elicit collagen-induced arthritis (CIA). A small population of tetramer-positive cells (1% of CD4+ T cells) was identified and expanded 10-fold by 4 days of stimulation ex vivo with a specific CII peptide. Most of the stimulated tetramer-positive cells expressed the TCR BV13 or BV8 gene segments used by CII peptide-specific DR1-restricted T cell hybridomas. Five days after immunization of DR1 mice with CII, CII peptide-specific cells were detected ex vivo among draining lymph node T cells; cell numbers peaked at 10 days but were still detectable at 130 days. Surface expression of CD62L decreased after day 5, whereas expression of CD44 and CD69 increased. Sorted tetramer-positive cells had higher levels of inflammatory and Th1 cytokine transcripts as measured by real-time PCR. Approximately 74% of CD4+ T cells from joint synovial tissues of mice with CIA expressed BV8 or BV14, and >74% of them bound tetramer. These studies demonstrate that autoimmune T cells that develop in lymph nodes and accumulate in arthritic joints of mice with CIA can be detected by a tetramer-based, ex vivo approach.
TLR9 and bacterial immunomodulation
The intracellular parasite, Propionibacterium acnes, is used clinically to inhibit tumor growth and increase resistance to infectious diseases. Although immunostimulation is mediated by bacterial components, the host receptors have not been identified. Kalis et al. (p. 4295 ) found that mice lacking the intracellular receptor TLR9 did not exhibit the splenomegaly seen in wild-type mice 7 days after treatment with killed P. acnes. Wild-type mice treated with P. acnes had a 100-fold increase in serum TNF- and a 1000-fold increase in serum IFN- at 1 h and 4 h, respectively, after i.v. challenge with LPS, other bacteria, or their components; no elevation in the cytokine levels was seen in similarly treated TLR9–/– animals. Priming with three other pathogenic agents induced strong LPS hypersensitivity in both wild-type and TLR9–/– mice. Whereas wild-type mice had increased splenic expression of IFN- mRNA at day 7 following P. acnes priming compared with treated TLR9-deficient mice, both mouse strains had enhanced TNF- mRNA expression within 1 h of injection with bacteria. All P. acnes-primed wild-type mice survived a challenge with live Salmonella enterica serovar typhimurium, but all challenged P. acnes-treated TLR9–/– mice were terminally ill by day 6. The authors conclude that IFN-, released after P. acnes interacts with TLR9, is responsible for the bacterial-induced immunomodulatory effects.
CD4+ Th cells cross-prime CTL
Memory CD8+ CTL are generated with the participation of CD4+ Th lymphocytes. Although professional APCs are involved in CTL activation, the ability of helper T cells to act as APCs has not been demonstrated. Kennedy et al. (p. 3967 ) pulsed conventional APCs with an immunodominant OVA peptide that bound to MHC class I molecule Kb. Complexes of OVA peptide with Kb, or with a Kb mutant that retained Kb function but could be distinguished from Kb, were detected on H-2b, H-2d, and H-2b/H-2d TCR-transgenic Th lines in the absence of peptide. The predominant complexes on resting or naive Th cells included both acquired and endogenous Kb/OVA peptide, whereas those on Th blasts were predominantly acquired Kb mutant/OVA peptide. Generation of MHC class I/OVA peptide complexes on the Th cells was greater with the OVA peptide covalently linked to a Th epitope (OVA/Th peptide) than with OVA peptide linked to a different I-Ab-restricted Th epitope from OVA. Approximately 35% of draining lymph node cells from TCR-transgenic mice injected with the OVA/Th peptide expressed Kb/OVA peptide complexes. Activated Th cells, incubated with Kb-negative APC loaded with OVA or OVA/Th peptide, were required to induce IFN- production in preactivated TCR-transgenic CTL in the presence of the appropriate peptide. T cell hybridoma cells specific for an MHC class II-restricted Th OVA epitope activated naive TCR-transgenic CTL in the presence of OVA and DC from K–/–D–/– mice. OVA peptide-activated Th cells were as effective as DC in stimulating the CTL and induced high levels of T cell activation markers and functions characteristic of central memory cells. Naive TCR-transgenic cells adoptively transferred into sublethally irradiated wild-type mice proliferated after peptide vaccination and persisted for longer than 10 wk. The authors propose that Th cells act as APC to present MHC class I-restricted epitopes to naive cells to generate central memory CTL.
Kinase regulation of autoimmunity
Signaling by G protein-coupled receptors is attenuated by receptor-specific kinase (GRK)-mediated phosphorylation. Intracellular levels of GRK2 protein determine the response of immune cells to proinflammatory cytokines and chemokines. Vroon et al. (p. 4400 ) found that GRK2 levels in PBMCs from patients with active relapsing-remitting multiple sclerosis (RR-MS) or secondary progressive MS were 40% of levels detected in healthy individuals. GRK2 levels in RR-MS patients in remission were lower than in patients with active RR-MS, in stroke patients with no autoimmunity or in healthy controls. All cell types were affected equally. Heterozygous GRK2 mice had a 50% reduction in GRK2 protein compared with wild-type littermates. Experimental autoimmune encephalomyelitis (EAE) induced in the GRK2+/– mice developed earlier, was not relapsing, and decreased over time. Numbers of infiltrated CD3+ T cells and activated macrophages and microglia were higher in spinal cords of GRK2+/– mice on day 13 after induction of EAE than in wild-type animals. The inflammatory infiltrates disappeared by day 45 after EAE induction in GRK2+/– mice that had become disease-free. Splenocytes from GRK2 heterozygotes and wild-type mice at day 48 postimmunization had similar in vitro proliferative responses and cytokine production when stimulated with the immunizing peptide. The authors conclude that down-regulation of GRK2 expression contributes to disease remission in MS patients and to less severe EAE in mice.
C5 deficiency via exon skipping
Deficiencies in C5 render patients susceptible to recurrent infections, particularly with Neisseriae. To date, only heterozygous nonsense mutations have been detected in C5 disorders. A novel C5 mutation is reported by Pfarr (p. 4172 ). DNA sequencing of all exons, flanking regions, and the promoter of the C5 gene from a patient with trace amounts of C5 in his serum revealed a single nucleotide change in exon 10. The mutation was predicted to result in a conservative substitution of arginine for lysine in codon 372. The patient was homozygous for the mutation; both heterozygous parents and one heterozygous sibling had reduced serum C5 levels. The nucleotide mutation occurred in an exonic splicing enhancer (ESE), a sequence that both encodes the C5 protein and increases mRNA splicing at suboptimal splice sites. Statistical analysis of the mutated sequence located near the 5' splice site suggested that it was less likely to possess ESE activity. PCR amplification of C5 region exon 9 to exon 12 from total RNA of all family members revealed the expected PCR product in all heterozygous family members and in the unaffected sister. The patient and all heterozygous family members had a smaller PCR product that lacked exon 10. The exon skipping created a frameshift with a premature translation STOP codon for the remaining open reading frame, resulting in greatly reduced levels of C5. The data reveal a novel mechanism by which a silent or conservative mutation inactivating an ESE results in a splicing failure.
Treg depletion in HIV infection
T cell activation in chronically infected HIV patients is a predictor of disease progression and death. Although CD4+CD25+CD62L+ regulatory T cells (Tregs) inhibit activation of both CD4+ and CD8+ T cells, their numbers in HIV patients have not been determined. Eggena et al. (p. 4407 ) confirmed that Tregs (CD4+CD25brightCD62Lbright), purified by cell sorting from 81 untreated HIV-positive patients at a clinic in Uganda, suppressed anti-CD3-induced proliferation of CD4+CD25–CD62Lbright T responder cells. The percentage of Tregs in 25 HIV-negative Ugandans was 2.8%. Treg numbers in HIV patients were expressed in several different ways: as an absolute number or as ratios of CD4+, CD8+, or CD3+ cells. Although the numbers varied widely among patients, there was significant correlation between decline in absolute CD4+ T cells and decline in absolute Tregs. Tregs decreased as HIV disease progressed. HIV-positive individuals had increases of 31 and 63% for CD4+ and CD8+ T cell activation, respectively, that were negatively associated with CD4+ T cell count and positively associated with viral load. HIV-negative controls had activation levels of 5% for CD4+ T cells and 13% for CD8+ T cells. The results of the study suggest that Treg depletion results in activation of CD4+ and CD8+ T cells in HIV-infected hosts and directly contributes to AIDS mortality.
Summaries written by Dorothy L. Buchhagen, Ph.D.
Related articles in The JI:
Direct Cross-Priming by Th Lymphocytes Generates Memory Cytotoxic T Cell Responses
Richard Kennedy, Anita H. Undale, William C. Kieper, Matthew S. Block, Larry R. Pease, and Esteban Celis
The JI 2005 174: 3967-3977.
Ex Vivo Characterization of the Autoimmune T Cell Response in the HLA-DR1 Mouse Model of Collagen-Induced Arthritis Reveals Long-Term Activation of Type II Collagen-Specific Cells and Their Presence in Arthritic Joints
Kary A. Latham, Karen B. Whittington, Ruohong Zhou, Zhaohui Qian, and Edward F. Rosloniec
The JI 2005 174: 3978-3985.
Linking C5 Deficiency to an Exonic Splicing Enhancer Mutation
Nicole Pfarr, Dirk Prawitt, Michael Kirschfink, Claudia Schroff, Markus Knuf, Pirmin Habermehl, Wilma Mannhardt, Fred Zepp, William Fairbrother, Michael Loos, Christopher B. Burge, and Joachim Pohlenz
The JI 2005 174: 4172-4177.
Requirement for TLR9 in the Immunomodulatory Activity of Propionibacterium acnes
Christoph Kalis, Marina Gumenscheimer, Nikolaus Freudenberg, Sandrine Tchaptchet, Gy?rgy Fejer, Antje Heit, Shizuo Akira, Chris Galanos, and Marina A. Freudenberg
The JI 2005 174: 4295-4300.
The Peritoneal Cavity Provides a Protective Niche for B1 and Conventional B Lymphocytes during Anti-CD20 Immunotherapy in Mice
Yasuhito Hamaguchi, Junji Uchida, Derek W. Cain, Guglielmo M. Venturi, Jonathan C. Poe, Karen M. Haas, and Thomas F. Tedder
The JI 2005 174: 4389-4399.
G Protein-Coupled Receptor Kinase 2 in Multiple Sclerosis and Experimental Autoimmune Encephalomyelitis
Anne Vroon, Annemieke Kavelaars, Volker Limmroth, Maria Stella Lombardi, Marion U. Goebel, Anne-Marie Van Dam, Marc G. Caron, Manfred Schedlowski, and Cobi J. Heijnen
The JI 2005 174: 4400-4406.
Depletion of Regulatory T Cells in HIV Infection Is Associated with Immune Activation
Mark P. Eggena, Banson Barugahare, Norman Jones, Martin Okello, Steven Mutalya, Cissy Kityo, Peter Mugyenyi, and Huyen Cao
The JI 2005 174: 4407-4414.
Identifying autoreactive CD4+ T cells
The pathology of most autoimmune diseases is mediated by CD4+ T cells that recognize self peptides. Yet details regarding the development of the autoantigen-specific T cells and their role in the autoimmune process are not available. Latham et al. (p. 3978 ) developed a chimeric HLA-DR1 recombinant molecule carrying a collagen II (CII) peptide (DR1-CII) that bound to a CII-specific TCR. DR1-CII tetramers stained CD4+ T cells from lymph nodes of DR1-transgenic mice immunized 10 days earlier with CII to elicit collagen-induced arthritis (CIA). A small population of tetramer-positive cells (1% of CD4+ T cells) was identified and expanded 10-fold by 4 days of stimulation ex vivo with a specific CII peptide. Most of the stimulated tetramer-positive cells expressed the TCR BV13 or BV8 gene segments used by CII peptide-specific DR1-restricted T cell hybridomas. Five days after immunization of DR1 mice with CII, CII peptide-specific cells were detected ex vivo among draining lymph node T cells; cell numbers peaked at 10 days but were still detectable at 130 days. Surface expression of CD62L decreased after day 5, whereas expression of CD44 and CD69 increased. Sorted tetramer-positive cells had higher levels of inflammatory and Th1 cytokine transcripts as measured by real-time PCR. Approximately 74% of CD4+ T cells from joint synovial tissues of mice with CIA expressed BV8 or BV14, and >74% of them bound tetramer. These studies demonstrate that autoimmune T cells that develop in lymph nodes and accumulate in arthritic joints of mice with CIA can be detected by a tetramer-based, ex vivo approach.
TLR9 and bacterial immunomodulation
The intracellular parasite, Propionibacterium acnes, is used clinically to inhibit tumor growth and increase resistance to infectious diseases. Although immunostimulation is mediated by bacterial components, the host receptors have not been identified. Kalis et al. (p. 4295 ) found that mice lacking the intracellular receptor TLR9 did not exhibit the splenomegaly seen in wild-type mice 7 days after treatment with killed P. acnes. Wild-type mice treated with P. acnes had a 100-fold increase in serum TNF- and a 1000-fold increase in serum IFN- at 1 h and 4 h, respectively, after i.v. challenge with LPS, other bacteria, or their components; no elevation in the cytokine levels was seen in similarly treated TLR9–/– animals. Priming with three other pathogenic agents induced strong LPS hypersensitivity in both wild-type and TLR9–/– mice. Whereas wild-type mice had increased splenic expression of IFN- mRNA at day 7 following P. acnes priming compared with treated TLR9-deficient mice, both mouse strains had enhanced TNF- mRNA expression within 1 h of injection with bacteria. All P. acnes-primed wild-type mice survived a challenge with live Salmonella enterica serovar typhimurium, but all challenged P. acnes-treated TLR9–/– mice were terminally ill by day 6. The authors conclude that IFN-, released after P. acnes interacts with TLR9, is responsible for the bacterial-induced immunomodulatory effects.
CD4+ Th cells cross-prime CTL
Memory CD8+ CTL are generated with the participation of CD4+ Th lymphocytes. Although professional APCs are involved in CTL activation, the ability of helper T cells to act as APCs has not been demonstrated. Kennedy et al. (p. 3967 ) pulsed conventional APCs with an immunodominant OVA peptide that bound to MHC class I molecule Kb. Complexes of OVA peptide with Kb, or with a Kb mutant that retained Kb function but could be distinguished from Kb, were detected on H-2b, H-2d, and H-2b/H-2d TCR-transgenic Th lines in the absence of peptide. The predominant complexes on resting or naive Th cells included both acquired and endogenous Kb/OVA peptide, whereas those on Th blasts were predominantly acquired Kb mutant/OVA peptide. Generation of MHC class I/OVA peptide complexes on the Th cells was greater with the OVA peptide covalently linked to a Th epitope (OVA/Th peptide) than with OVA peptide linked to a different I-Ab-restricted Th epitope from OVA. Approximately 35% of draining lymph node cells from TCR-transgenic mice injected with the OVA/Th peptide expressed Kb/OVA peptide complexes. Activated Th cells, incubated with Kb-negative APC loaded with OVA or OVA/Th peptide, were required to induce IFN- production in preactivated TCR-transgenic CTL in the presence of the appropriate peptide. T cell hybridoma cells specific for an MHC class II-restricted Th OVA epitope activated naive TCR-transgenic CTL in the presence of OVA and DC from K–/–D–/– mice. OVA peptide-activated Th cells were as effective as DC in stimulating the CTL and induced high levels of T cell activation markers and functions characteristic of central memory cells. Naive TCR-transgenic cells adoptively transferred into sublethally irradiated wild-type mice proliferated after peptide vaccination and persisted for longer than 10 wk. The authors propose that Th cells act as APC to present MHC class I-restricted epitopes to naive cells to generate central memory CTL.
Kinase regulation of autoimmunity
Signaling by G protein-coupled receptors is attenuated by receptor-specific kinase (GRK)-mediated phosphorylation. Intracellular levels of GRK2 protein determine the response of immune cells to proinflammatory cytokines and chemokines. Vroon et al. (p. 4400 ) found that GRK2 levels in PBMCs from patients with active relapsing-remitting multiple sclerosis (RR-MS) or secondary progressive MS were 40% of levels detected in healthy individuals. GRK2 levels in RR-MS patients in remission were lower than in patients with active RR-MS, in stroke patients with no autoimmunity or in healthy controls. All cell types were affected equally. Heterozygous GRK2 mice had a 50% reduction in GRK2 protein compared with wild-type littermates. Experimental autoimmune encephalomyelitis (EAE) induced in the GRK2+/– mice developed earlier, was not relapsing, and decreased over time. Numbers of infiltrated CD3+ T cells and activated macrophages and microglia were higher in spinal cords of GRK2+/– mice on day 13 after induction of EAE than in wild-type animals. The inflammatory infiltrates disappeared by day 45 after EAE induction in GRK2+/– mice that had become disease-free. Splenocytes from GRK2 heterozygotes and wild-type mice at day 48 postimmunization had similar in vitro proliferative responses and cytokine production when stimulated with the immunizing peptide. The authors conclude that down-regulation of GRK2 expression contributes to disease remission in MS patients and to less severe EAE in mice.
C5 deficiency via exon skipping
Deficiencies in C5 render patients susceptible to recurrent infections, particularly with Neisseriae. To date, only heterozygous nonsense mutations have been detected in C5 disorders. A novel C5 mutation is reported by Pfarr (p. 4172 ). DNA sequencing of all exons, flanking regions, and the promoter of the C5 gene from a patient with trace amounts of C5 in his serum revealed a single nucleotide change in exon 10. The mutation was predicted to result in a conservative substitution of arginine for lysine in codon 372. The patient was homozygous for the mutation; both heterozygous parents and one heterozygous sibling had reduced serum C5 levels. The nucleotide mutation occurred in an exonic splicing enhancer (ESE), a sequence that both encodes the C5 protein and increases mRNA splicing at suboptimal splice sites. Statistical analysis of the mutated sequence located near the 5' splice site suggested that it was less likely to possess ESE activity. PCR amplification of C5 region exon 9 to exon 12 from total RNA of all family members revealed the expected PCR product in all heterozygous family members and in the unaffected sister. The patient and all heterozygous family members had a smaller PCR product that lacked exon 10. The exon skipping created a frameshift with a premature translation STOP codon for the remaining open reading frame, resulting in greatly reduced levels of C5. The data reveal a novel mechanism by which a silent or conservative mutation inactivating an ESE results in a splicing failure.
Treg depletion in HIV infection
T cell activation in chronically infected HIV patients is a predictor of disease progression and death. Although CD4+CD25+CD62L+ regulatory T cells (Tregs) inhibit activation of both CD4+ and CD8+ T cells, their numbers in HIV patients have not been determined. Eggena et al. (p. 4407 ) confirmed that Tregs (CD4+CD25brightCD62Lbright), purified by cell sorting from 81 untreated HIV-positive patients at a clinic in Uganda, suppressed anti-CD3-induced proliferation of CD4+CD25–CD62Lbright T responder cells. The percentage of Tregs in 25 HIV-negative Ugandans was 2.8%. Treg numbers in HIV patients were expressed in several different ways: as an absolute number or as ratios of CD4+, CD8+, or CD3+ cells. Although the numbers varied widely among patients, there was significant correlation between decline in absolute CD4+ T cells and decline in absolute Tregs. Tregs decreased as HIV disease progressed. HIV-positive individuals had increases of 31 and 63% for CD4+ and CD8+ T cell activation, respectively, that were negatively associated with CD4+ T cell count and positively associated with viral load. HIV-negative controls had activation levels of 5% for CD4+ T cells and 13% for CD8+ T cells. The results of the study suggest that Treg depletion results in activation of CD4+ and CD8+ T cells in HIV-infected hosts and directly contributes to AIDS mortality.
Summaries written by Dorothy L. Buchhagen, Ph.D.
Related articles in The JI:
Direct Cross-Priming by Th Lymphocytes Generates Memory Cytotoxic T Cell Responses
Richard Kennedy, Anita H. Undale, William C. Kieper, Matthew S. Block, Larry R. Pease, and Esteban Celis
The JI 2005 174: 3967-3977.
Ex Vivo Characterization of the Autoimmune T Cell Response in the HLA-DR1 Mouse Model of Collagen-Induced Arthritis Reveals Long-Term Activation of Type II Collagen-Specific Cells and Their Presence in Arthritic Joints
Kary A. Latham, Karen B. Whittington, Ruohong Zhou, Zhaohui Qian, and Edward F. Rosloniec
The JI 2005 174: 3978-3985.
Linking C5 Deficiency to an Exonic Splicing Enhancer Mutation
Nicole Pfarr, Dirk Prawitt, Michael Kirschfink, Claudia Schroff, Markus Knuf, Pirmin Habermehl, Wilma Mannhardt, Fred Zepp, William Fairbrother, Michael Loos, Christopher B. Burge, and Joachim Pohlenz
The JI 2005 174: 4172-4177.
Requirement for TLR9 in the Immunomodulatory Activity of Propionibacterium acnes
Christoph Kalis, Marina Gumenscheimer, Nikolaus Freudenberg, Sandrine Tchaptchet, Gy?rgy Fejer, Antje Heit, Shizuo Akira, Chris Galanos, and Marina A. Freudenberg
The JI 2005 174: 4295-4300.
The Peritoneal Cavity Provides a Protective Niche for B1 and Conventional B Lymphocytes during Anti-CD20 Immunotherapy in Mice
Yasuhito Hamaguchi, Junji Uchida, Derek W. Cain, Guglielmo M. Venturi, Jonathan C. Poe, Karen M. Haas, and Thomas F. Tedder
The JI 2005 174: 4389-4399.
G Protein-Coupled Receptor Kinase 2 in Multiple Sclerosis and Experimental Autoimmune Encephalomyelitis
Anne Vroon, Annemieke Kavelaars, Volker Limmroth, Maria Stella Lombardi, Marion U. Goebel, Anne-Marie Van Dam, Marc G. Caron, Manfred Schedlowski, and Cobi J. Heijnen
The JI 2005 174: 4400-4406.
Depletion of Regulatory T Cells in HIV Infection Is Associated with Immune Activation
Mark P. Eggena, Banson Barugahare, Norman Jones, Martin Okello, Steven Mutalya, Cissy Kityo, Peter Mugyenyi, and Huyen Cao
The JI 2005 174: 4407-4414.