Regulation of mucosal responses by CD4+ T lymphocytes: effects of anti-L3T4 treatment on the gastrointestinal immune system.

The role of CD4+ T cells in the gastrointestinal (GI) immune system in vivo was studied in mice selectively depleted of this subset by treatment with monoclonal anti-L3T4 (GK1.5) mAb. Treatment of young BALB/c mice with weekly injections of anti-L3T4 mAb resulted in a selective depletion of CD4+ T cells in both IgA effector (lamina propria regions of the intestine; LP) and inductive (Peyer's patch; PP) sites. However, levels of CD3+CD4-CD8+ and CD4-CD8- (double negative) T cells remained constant or increased. When sections of small intestine were assessed for the isotype of Ig-containing cells, normal mice contained predominantly IgA plasma cells with small numbers of IgM and IgG plasma cells while anti-L3T4 treatment dramatically reduced the numbers of IgA plasma cells. When numbers of IgA-producing cells were assessed by the isotype-specific ELISPOT assay, the LPL of anti-L3T4 mAb-treated mice showed an 80% reduction in the number of IgA spot-forming cells. The effect of anti-L3T4 mAb treatment on IgA inductive sites was also studied and this treatment reduced the overall size of PP as well as the germinal centers in this tissue. Although anti-L3T4 treatment depleted CD3+CD4+ T cells in PP, the relative frequency of surface IgA-positive (slgA+) B cells in this tissue did not change. These results show that repeated injection of anti-L3T4 mAb results in a CD4+ T cell deficiency in both IgA inductive (PP) and effector (LP) sites. The depletion of CD4+ T cells resulted in reductions in the numbers of mature IgA plasma cells present in the LP of gut-associated tissues, and reduced the overall size of PP including germinal centers, but did not affect the frequency of sIgA+ B cells in this IgA inductive site.     

Opposing roles of interferon-gamma on CD4+ T cell-mediated graft-versus-host disease: effects of conditioning.

Although alloreactive T cells are required for the induction of graft-versus-host disease (GVHD), other factors can influence outcome in murine models of the disease. Lethal total body irradiation (TBI) conditioning regimens followed by reconstitution with allogeneic lymphohematopoietic cells results in the generation of donor anti-host cytotoxic T lymphocyte (CTL)-mediated solid organ (gut, liver, skin) destruction. In contrast, donor anti-host CTL-mediated hematopoietic failure is the primary cause of morbidity following sublethal TBI. To determine the role of interferon (IFN)-gamma in graft-versus-host reactions against hematopoietic and solid organ targets, we used IFN-gamma knockout mice as donors in both lethal TBI and bone marrow transplantation (BMT) rescue and sublethal TBI models. In this report, we show that CD4+ T cells from IFN-gamma knockout (KO) mice resulted in accelerated GVHD after lethal TBI/BMT using a single major histocompatibility class II mismatch model. In marked contrast, the use of these same IFN-gamma KO CD4+ donor cells in combination with sublethal TBI significantly ameliorated GVHD-associated mortality. In these recipients, severe anemia, bone marrow aplasia, and intestinal lesions were observed in the presence but not the absence of donor-derived IFN-gamma. Administration of anti-IFN-gamma antibodies to sublethally irradiated recipients of wild-type donor cells confirmed the role of IFN-gamma depletion in CD4+ T cell-mediated GVHD. In conclusion, the extent of conditioning markedly affects the role of IFN-gamma in GVHD lesions mediated by CD4+ T cells. In models using sublethal TBI, the absence of IFN-gamma is protective from GVHD, whereas in lethal TBI situations, the loss is deleterious.     

CD4+CD25+ T lymphocytes in human tonsils suppress the proliferation of CD4+CD25- tonsil cells

Animal studies define CD4+CD25+ T cells as a subset that protect against autoimmune inflammation. We wanted to investigate whether CD4+CD25+ T cells from patients with recurrent tonsillitis could suppress the proliferation of other tonsil cells, in vitro, as this immunological tissue also may serve as a model for chronic inflammation. Tonsil CD4+CD25+ cells markedly suppressed the proliferation of CD4+CD25- T cells in Concanavalin A-stimulated cocultures compared with cultures containing CD4+CD25- T cells only. The suppression exerted by the CD4+CD25+ cells was abrogated if these cells were irradiated before coculture or if interleukin (IL)-2 was added to the culture medium. CD4+CD25+ T cells proliferated poorly in response to mitogen, when cultured alone. Substitution with CD4+CD25+ T cells isolated from peripheral blood, enriched by similar methods, did not downregulate the proliferation of CD4+CD25- responder cells from tonsils. The augmented suppressive ability of tonsil CD4+CD25+ T cells compared with cells of this phenotype from blood, on CD4+CD25- responder cells from tonsils, suggests that there may be a functional difference between CD25+ cells from the two locations. In conclusion, CD4+CD25+ T cells from inflamed tonsils distinctly suppressed T-cell responses to mitogen in vitro, pointing to a regulatory role for CD4+CD25+ cells retrieved from inflammatory reactions in humans.     

CD25+CD4+ regulatory T cells suppress CD4+ T cell-mediated pulmonary hyperinflammation driven by Pneumocystis carinii in immunodeficient mice

The CD4(+) T cell-mediated inflammatory response to Pneumocystis carinii (PC) critically contributes to the clinical severity of PC pneumonia. It has been suggested that lymphopenic conditions predispose individuals to this immunopathology, although the mechanisms remain poorly understood. Another set of evidence indicates that a subpopulation of CD4(+) T cells constitutively expressing the CD25 molecule prevent lymphopenia-induced autoimmunity and inflammatory bowel disease. We tested the ability of this CD25(+)CD4(+) population to regulate CD4(+) T cell-mediated inflammatory response to PC. Adoptive transfer of CD25(-)CD4(+) cells into PC-infected recombination-activating gene-2-deficient mice led to lethal pneumonia within 13 days post-transfer. PC infection appeared to trigger CD25(-)CD4(+) cells, since recipients with reduced PC load survived up to 5 weeks after transfer. In contrast, transfer of CD25(+)CD4(+) cells did not induce lethal pneumonia and prevented the development of the disease induced by CD25(-)CD4(+) cells. Furthermore, CD25(-)CD4(+) cells reduced the PC load in the lung, while CD25(+)CD4(+) cells suppressed this immune response. Our results indicate an essential role for CD25(+)CD4(+) T cells in the control of PC-driven immunopathology, and suggest that in immunocompromised hosts PC pneumonia may result from a deficiency in regulatory T cells.     

Antigen presenting cells integrate opposing signals from CD4+ and CD8+ regulatory T lymphocytes to arbitrate the outcomes of immune responses.

An individual's set of polymorphic HLA class II and I molecules is known to select the T cell repertoire in the thymus and to present processed antigenic peptides (pAg) to mature peripheral CD4+ T helper (Th) and CD8+ T cytotoxic (Th) cells in the periphery. This review highlights new studies which address how antigen presenting cells (APC) integrate the responses of cognate Th and T suppressor (Ts) cells to determine the outcome of immune responses. Together with other findings, these studies emphasize that understanding the mechanism of immune processes requires consideration of HLA molecules in the context of the peptides they bind, the antigen presenting cells (APC) that express them and the T lymphocytes that recognize them. The activities of lymphocyte and APC surface structures are becoming integrated into a physiological understanding of the cellular interactions between regulatory and effector T cells with APC.     

Immunization with soluble simian virus 40 large T antigen induces a specific response of CD3+ CD4- CD8+ cytotoxic T lymphocytes in mice.

C57BL/6 (B6) mice (H-2b) were immunized with the large tumor antigen (T Ag) of simian virus 40 (SV40). Intraperitoneal or subcutaneous sensitization with soluble T Ag specifically primed cytotoxic lymphocyte precursors (CTLp). T Ag-specific cytotoxic T lymphocytes (CTL) were detected in a cytotoxicity assay after specific in vitro restimulation of effector cell populations from mice immunized with 2-10 micrograms purified, soluble T Ag and boosted with an injection of 2 micrograms T Ag 2-4 weeks after priming. Cells used for in vitro restimulation and as targets in cytotoxicity assays were syngeneic (B6-derived) RBL5 lymphoma cells expressing SV40 T Ag after transfection with a T Ag-encoding expression vector. Effector cells of this response were H-2 class I-restricted CD3+ CD4-CD8+ CTL. The magnitude of the anti-T Ag CTL response of B6 mice stimulated by soluble virus protein was comparable to the anti-T Ag CTL response of SV40-infected B6 mice. Injections of denatured or native T Ag protein primed CTLp equally well, but immunization with an equal dose of antigen emulsified in incomplete Freund's adjuvants inefficiently stimulated CTLp.     

Assessment of alloreactive T cell subpopulations of aged mice in vivo. CD4+ but not CD8+ T cell-mediated rejection response declines with advanced age

The present investigation explored age-related alterations in T cell populations mediating allospecific responses in vivo. Healthy aged and young H-2^b and H-2^bxH-2^k mice were engrafted with major histocompatibility complex (MHC) class II-disparate bm12 skin, rejection of which requires CD4⁺ T cells, and MHC class I-disparate bm1 skin, rejection of which requires CD8⁺ T cells. Aged mice of both genders exhibited prolonged survival of bm12 skin grafts relative to their young counterparts but rejected bm1 skin grafts at a rate equivalent to that of young mice. Consistent with prolonged survival of bm12 skin grafts, markedly diminished levels of Ia^bm12 CTL activity were elicited from T cells of aged mice in vitro. However, no such decline was observed in the level of K^bm1 CTL from T cells of aged mice. The alterations in Ia^bm12 allospecific responses were not attributable to quantitative changes in CD4⁺ T cells of aged mice, and addition of soluble T cell helper factors to response cultures of aged mice did not augment Ia^bm12 cytotoxic T lymphocytes activity. These data demonstrate that aging fundamentally affects CD4⁺ T cell-mediated allospecific responses particularly in vivo, and that deficient generation of soluble T cell helper factors alone cannot explain this deficit.     

Sequential interaction of macrophages, initiator T lymphocytes and recruited T lymphocytes in a cell-mediated immune response to soluble antigen.

We have investigated the sequential interaction of macrophages, initiator T lymphocytes (ITL) and recruited T lymphocytes (RTL) in the development of a T cell-mediated response to soluble antigens. Macrophages were pulsed with soluble antigens and used to sensitize ITL in vitro. The ITL were irradiated to prevent their proliferation and then injected into the footpads of syngeneic mice. Sensitized ITL were found to recruit immunospecific RTL in the draining lymph nodes, as determined by a thymidine uptake assay of the lymph node cells in vitro. The richest source of lymphocytes with ITL activity was the thymus, and progressively less activity was detectable among spleen or lymph node lymphocytes. The magnitude of the subsequent RTL response could be modified by genetic differences between the ITL and the antigen-pulsed macrophages that were used to sensitize them. Thus, ITL conveyed an immunogenic signal to RTL whose magnitude reflected the genotype of the macrophages, but whose specificity was directed by determinants of the soluble antigen.     

Analysis of the roles of CD4+ and CD8+ T cells in autoimmune diabetes of NOD mice using transfer to NOD athymic nude mice.

The NOD mouse, which spontaneously develops insulitis and overt diabetes, is a model of autoimmune type I diabetes mellitus. For the precise analysis of the roles of CD4+ and CD8+ T cells in the pathogenesis of this mouse, these subsets must be transferred into recipients that are completely free of T cells and pathological changes. We used athymic NOD nude mice, which congenitally lack mature T cells and are free of insulitis and hyperglycemia up to the age of 60 weeks, as recipients for this purpose. To the nude recipients we transferred either one of a highly purified CD4+ or CD8+ T cell subset derived from non-diabetic female NOD mice; any in vivo increase in the contaminating T cell subsets was prevented by injecting the antibody homologous to it. Most of the T cell-reconstituted recipients were treated with cyclophosphamide to promote the onset of overt diabetes. Transfer of the CD8+ T cell subset alone did not induce insulitis or hyperglycemia. In contrast, transfer of the CD4+ T cell subset alone produced insulitis, but not hyperglycemia, in all the recipients. However, the subsequent transfer of CD8+ T cells into CD4+ T cell-reconstituted recipients induced severe insulitis and hyperglycemia in almost all the recipients. In these diabetic recipients, we observed severe damage of the pancreatic islets and the infiltration of a large number of CD8+ T cells into the remaining islets; insulin-secreting beta cells were no longer detected. These results suggest that CD4+ T cells play a predominant role in the development of insulitis and that CD8+ T cells migrate into the islets and are subsequently, with the aid of CD4+ T cells, differentiated into killer cells which act against beta cells.     

T cell-mediated immune responses of lupus-prone BXSB mice and other murine strains.

Cellular-mediated immunity in the newly described BXSB strain of mice, which is prone to autoimmune disease, has been compared with that of two other strains, C57Bl/6 and 129/J. Quantificaiton of cytotoxic T cell responses to alloantigens and viruses (lymphocytic choriomeningitis and vaccinia virus) showed no difference in the kinetics of appearance and relative activity of cytotoxic T cells per spleen between the young and old BXSB and the control mice. The T cell-dependent primary footpad swelling after local injection with lymphocytic choriomeningitis virus was within the same range for all strains tested with respect to kinetics, but the size was greater by two-fold in C57Bl/6 mice. The susceptibility to systemic infection and subsequent induction of lymphocytes immune to Listeria monocytogenes were about equivalent in all strains. However, clearance of Listeria by the reticuloendothelial system and early non-immune bactericidal activity of the young and old BXSB were significantly lower than in the control strains. The results indicate that the cellular-mediated immunity (CMI) of BXSB mice compared favourably with that of other strains and that there is no apparent differences between CMI of BXSB mice before the onset of disease and during the course of disease. The role of the reduced reticuloendothelial function of BXSB mice in their autoimmune disease or in their high susceptibility to infection remains to be determined.     

CD4+CD25+免疫调节性T细胞对CD4+T细胞介导的移植物抗宿主病预防作用的研究

目的研究CD4+CD25+免疫调节性T(Treg)细胞在小鼠骨髓移植后,对移植物抗宿主病的预防作用及其作用机制.方法用C3H(H-2k)小鼠骨髓作为供体,提取C3H(H-2k)小鼠CD4+T及CD4+CD25+T细胞,C3H×B6(H-2k/b)F1小鼠为骨髓移植的受者.在受者接受致死量全身放射后,输注供者去除T细胞的骨髓(ATBM),使其造血功能重建(ATBM组).于不同的实验组给予CD4+(CD4组)T细胞,CD4+CD25+T(CD25组)或二者同时输注(CD4/CD25组).观察各组小鼠移植物抗宿主病(GVHD)的发生率.结果所有10只ATBM组小鼠至骨髓移植后60天仍全部存活,无GVHD发生;所有10只CD4组小鼠在骨髓移植10天内全部死于GVHD(P＜0.01);所有5只CD25组小鼠于骨髓移植后60天仍全部存活,无明显GVHD发生(P＞0.05);同样,所有6只CD4/CD25组小鼠至骨髓移植后仍全部存活,无明显GVHD发生(P＞0.05).结论在同种异基因小鼠的骨髓移植模型中,CD4+CD25+T不诱导GVHD的发生,并有预防CD4+T细胞介导的GVHD发生的作用.     

Immunological properties of Fc receptor on lymphocytes. VIII. The behaviour of FcR+ and FcR- T cells in cell-mediated immune responses.

The roles of splenic FcR+ and FcR- T cells from mice immunized either with allogeneic cells or with LCMV or stimulated either with MMC-treated allogeneic cells or TNP-modified syngeneic cells in vitro were examined for cell-mediated cytolytic responses. Effective lysis was observed in the FcR- cell fraction enriched with nylon wool eluted T cells in all experiments. Killer cells were generated from the FcR- T-cell fraction after exposure to either allo-, virus- or chemically modified antigen. On the other hand, the lytic activity of the FcR+ T-cell fraction, was low but nevertheless still significant. However, this weak cytotoxicity was increased by 24 h of incubation, although the recovery of living cells was found to be significantly lower in the FcR+ than in the FcR- T-cell fraction. This suggested that non-killer cells, which could interfere with the activity of pre-killer cells, were perhaps preferentially eliminated after binding with the immune complexes. Moreover, almost complete inhibition of lytic activity was achieved in allo-activated FcR+ T, but not FcR- T-cell fraction, after they had been treated with immune complexes, implying a functional significance of FcR in the manifestation of lytic activity.     

Multiple roles for CD4+ T cells in anti-tumor immune responses

Summary: Our understanding of the importance of CD4⁺ T cells in orchestrating immune responses has grown dramatically over the past decade. This lymphocyte family consists of diverse subsets ranging from interferon-γ (IFN-γ)-producing T-helper 1 (Th1) cells to transforming growth factor-β (TGF-β)-secreting T-regulatory cells, which have opposite roles in modulating immune responses to pathogens, tumor cells, and self-antigens. This review briefly addresses the various T-cell subsets within the CD4⁺ T-cell family and discusses recent research efforts aimed at elucidating the nature of the 'T-cell help' that has been shown to be essential for optimal immune function. Particular attention is paid to the role of Th cells in tumor immunotherapy. We review some of our own work in the field describing how CD4⁺ Th cells can enhance anti-tumor cytotoxic T-lymphocyte (CTL) responses by enhancing clonal expansion at the tumor site, preventing activation-induced cell death and functioning as antigen-presenting cells for CTLs to preferentially generate immune memory cells. These unconventional roles for Th lymphocytes, which require direct cell-to-cell communication with CTLs, are clear examples of how versatile these immunoregulatory cells are.     

Masking of veto function in vivo by activated CD4+ T lymphocytes

Donor CD8+ T lymphocytes injected into recipient mice incompatible at major histocompatibility complex (MHC) class I genes induce donor-specific CTL nonresponsiveness, attributed to the veto function of donor cells. Here we show that conditions leading to strong activation of CD4+ T cells, namely the presence in the recipient of foreign MHC class II determinants, lead to the apparent loss of veto function of donor cells. This "masking" of veto function is dependent on the dose of foreign MHC class II present. Veto function can be partially restored by treatment of recipients in vivo with CD4-specific antibody, a measure which has been shown to eliminate the function of CD4+ T cells in vivo. We conclude that CD4+ T cells activated by contact with antigen can interfere with the veto function of CD8+ T cells. Consequences of this finding are: (a) veto function of a sample cell population can be overlooked when activation of CD4+ T cells occurs simultaneously. (b) The balance between veto function of recipient cells and its abrogation might be responsible for the kind of graft-vs.-host reaction generated (CD8+ T cell-mediated and frequently lethal or CD4+ T cell-mediated and not lethal) when parental T cells are injected into recipients incompatible at MHC class I and class II genes. (c) A possible contribution of veto cells should be considered in several protocols in which donor hemopoetic cells were used in conjunction with CD4-specific antibodies to induce transplantation tolerance. (d) Veto function in vivo does not require a contribution of CD4+ T cells.     

Antigen presentation by B lymphocytes to CD4+ T lymphocytes in vivo: importance for B lymphocyte and T lymphocyte activation.

B lymphocytes, like macrophages and dendritic cells, can present antigen to CD4+ T cells. Antigen presentation by B cells is essential for the generation of an in vivo T cell dependent antibody response, and repeated antigen presentation by B cells to T cells is necessary to induce B cell clonal expansion. Presentation of antigen by resting B cells to unprimed T cells tolerizes T cells, while anti-IgD antibody activates B cells and allows B cell antigen presentation that productively activates T cells. However, activation is not all that is required for B cells to productively present antigen to T cells.     

The selective antigen-presenting cell capacity of activated B lymphocytes in HLA-II-restricted responses of CD4+ T lymphocytes

We examined the role of antigen-presenting B lymphocytes using panels of antigen-specific CD4+8-T-lymphocyte clones (TLC). All 19 TLC showed a class II major histocompatibility complex-encoded (HLA-II) restricted proliferation to antigen presented by antigen-presenting cells (APC) from the monocyte fraction of peripheral blood. Only six TLC were effectively activated by antigen presented by autologous B lymphocytes activated by EBV transformation. This failure of B lymphocytes was not due to: (i) a high degree of cell surface sialic acid; (ii) a low expression of the cell surface proteins HLA-II, ICAM-1 or LFA-3 that restrict antigen presentation; (iii) lack of secretion of the cytokine IL-1 or other soluble factors that may be required as secondary signals; or (iv) induction of incomplete T-cell activation resulting in the production of growth factor interleukin-2 (IL-2) or the expression of receptors for IL-2 only. These data suggest the involvement of another cell surface interaction in antigen presentation acting besides the interactions known so far.