Cellular basis of skin allograft rejection across a class I major histocompatibility barrier in mice depleted of CD8+ T cells in vivo.

The present study was undertaken to define the cellular mechanisms involved in the rejection of major histocompatibility complex (MHC) class I disparate skin grafts by mice depleted of CD8+ T cells in vivo. Mice were effectively depleted of CD8+ T cells by adult thymectomy followed by in vivo administration of anti-CD8 monoclonal antibody (mAb) and then engrafted with allogeneic skin. We found that CD8 depleted mice did reject MHC class I disparate skin grafts, but only when the grafts also expressed additional alloantigens. Despite the marked depletion of CD8+ T cells in these mice, we found that their rejection of MHC class I disparate grafts was mediated by CD8+ cytolytic T lymphocyte (CTL) effectors that had escaped depletion. These CD8+ CTL effectors were unique in that: (a) their generation was dependent upon the injected anti-CD8 mAb and upon exposure to class I MHC alloantigens expressed on the engrafted skin, and (b) their effector function was resistant to blockade by anti-CD8 mAb. We observed that the additional alloantigens coexpressed on MHC class I disparate grafts that triggered graft rejection in CD8-depleted mice could be MHC-linked or not and that they functioned in these rejection responses to activate third party specific CD4+ T helper (Th) cells to provide helper signals for the generation of CD8+ anti-CD8 resistant CTL effector cells. Thus, mice depleted of CD8+ T cells by thymectomy and in vivo administration of anti-CD8 mAb harbor a unique population of anti-CD8 resistant, CD8+ effector cells that mediate anti-MHC class I responses in vivo and in vitro, but require help from third party specific Th cells to do so.     

Skin graft rejection by beta 2-microglobulin-deficient mice.

Mice homozygous for a beta 2-microglobulin (beta 2-m) gene disruption lack beta 2-m protein and are deficient for functional major histocompatibility complex class I (MHC-I) molecules. The mutant mice have normal numbers of CD4+8- T helper cells, but lack MHC-I-directed CD4-8+ cytotoxic T lymphocytes (CTLs). In this study we used the beta 2-m mutant mice to study the importance of MHC-I-directed immunity in skin graft rejection. Our results indicate that MHC-I-directed CD8+ CTLs are not essential in the rejection of allografts with whole MHC or multiple minor H differences. However, the absence of MHC-I-guided immunity profoundly reduces the ability of mutant mice to reject H-Y disparate grafts. In addition, we show that natural killer cells which vigorously reject MHC-I-deficient bone marrow grafts, are not effective in the destruction of MHC-I-deficient skin grafts.     

Mechanism of the rejection of major histocompatibility complex class I-disparate murine skin grafts: rejection can be mediated by CD4+ cells activated by allo-class I + II antigen in CD8+ cell-depleted hosts.

In the preceding article, we analyzed the immunohistochemical rejection mechanism of major histocompatibility complex (MHC) class I (H-2K)-disparate murine skin grafts, and showed that only CD8+ cells infiltrated at the site of the epithelial tissue of MHC class I-disparate graft. We also showed that perfect survival of MHC class I-disparate grafts were attained in thymectomized recipients treated with anti-Lyt-2 monoclonal antibody. In this report, we showed that these long-surviving allo-class I grafts were rejected in the absence of CD8+ cells by stimulation with allo-MHC class I + II-disparate graft as the second stimulation. Furthermore, it was immunohistochemically revealed that under that condition, a large number of CD4+ cells infiltrated into the epithelial tissue of these long-surviving class I grafts, which were going to be rejected 2-5 d after the transplantation of a second graft with MHC class I + II difference. This result directly shows that CD4+ cells are able to became effectors for the rejection of allo-MHC class I (H-2K) skin graft.     

Differential involvement of CD4+ cells in mediating skin graft rejection against different amounts of transgenic H-2K(b) antigen.

Differential involvement of CD4+ cells in mediating class I-disparate skin graft rejection was investigated using quantitatively different Kb transgenic mice as donors under conditions in which CD8+ cells were blocked in vivo by administration of anti-CD8 monoclonal antibody (mAb). Tg.H-2Kb-1 and -2 are C3H transgenic mice with 14 and 4 copies, respectively, of the H-2Kb gene. Cell surface expression of Kb antigen and the Kb antigenicity of skin for eliciting graft rejection with homozygous and heterozygous transgenic mice were correlated with the copy number. In vivo administration of anti-Lyt-2.1 (CD8) mAb markedly prolonged survival of heterozygous and homozygous C3H Tg.H-2Kb-2 skin grafted onto C3H mice, but prolonged survival of heterozygous Tg.H-2Kb-1 skin grafts much less and did not prolong survival of homozygous Tg.H-2Kb-1 grafts. Administration of anti-L3T4 (CD4) mAb alone did not have any effect on skin graft rejection. Administration of anti-L3T4 (CD4) mAb with anti-Lyt-2.1 (CD8) mAb blocked rejection in all combinations. These findings indicate that a quantitative difference of class I antigen caused differential activation of CD4+ cells under conditions in which CD8+ cells were blocked.     

Specific unresponsiveness in rats with prolonged cardiac allograft survival after treatment with cyclosporine. III. Further characterization of the CD4+ suppressor cell and its mechanisms of action

The cellular basis of the specific unresponsiveness that develops in DA rats treated with cyclosporine (CSA) for 10 d after grafting a PVG heart was examined using an adoptive transfer assay. CD4+ cells from rats with long survival grafts specifically lack the capacity to restore PVG heart graft rejection, and can also inhibit the capacity of naive T cells to restore rejection, while in the first few weeks post-transplant, both CD4+ and CD8+ T cells from CSA-treated hosts have the capacity to effect PVG graft rejection. In this study, we demonstrated the CD4+ suppressor cells also had the capacity to inhibit restoration of rejection by CD4+ cells from CSA-treated DA rats recently transplanted with PVG hearts, and from rats sensitized to third party, but not from those specifically sensitized to PVG. They also inhibited the capacity of both naive CD8+ and sensitized CD8+ cells to effect rejection. These results showed that the CD4+ suppressor cell was capable of overriding the capacity to effect rejection of the CD4+ cell and activated CD8+ cells that were present in the CSA-treated host shortly after transplantation. The failure of naive CD8+ cells to augment suppression and the capacity of CD4+ suppressor cells to transfer unresponsiveness to irradiated hosts in which regeneration of CD8+ cells was abolished by thymectomy suggested that it was the CD4+ cell alone that mediated suppression. However, the failure of CD4+ suppressor cells to reinduce unresponsiveness in irradiated hosts whose CD8+ cells had been depleted by therapy with the mAb MRC Ox8 showed that a radioresistant CD8+ cell was required to reestablish the state of specific unresponsiveness. The induction of CD4+ suppressor cells in thymectomized hosts suggested that these cells were derived from long-lived CD4+ lymphocytes. However, their sensitivity to cyclophosphamide and their loss of suppressor function both after removal of the graft and after 3 d in culture demonstrated that the suppressor cell itself had a short lifespan. The CD4+ suppressor was shown to be MRC Ox22+ (CD45R+), MRC Ox17+ (MHC class II), and MRC Ox39+ (CD25, IL-2-R). These studies demonstrated the CD4+ suppressive cell identified in rats with specific unresponsiveness induced by CSA therapy had many features of the suppressor inducer cell identified in in vitro studies of the alloimmune response.(ABSTRACT TRUNCATED AT 400 WORDS)     

Long-term survival of skin allografts induced by donor splenocytes and anti-CD154 antibody in thymectomized mice requires CD4(+) T cells, interferon-gamma, and CTLA4.

Treatment of C57BL/6 mice with one transfusion of BALB/c spleen cells and anti-CD154 (anti-CD40-ligand) antibody permits BALB/c islet grafts to survive indefinitely and BALB/c skin grafts to survive for approximately 50 d without further intervention. The protocol induces long-term allograft survival, but the mechanism is unknown. We now report: (a) addition of thymectomy to the protocol permitted skin allografts to survive for > 100 d, suggesting that graft rejection in euthymic mice results from thymic export of alloreactive T cells. (b) Clonal deletion is not the mechanism of underlying long-term graft survival, as recipient thymectomized mice were immunocompetent and harbor alloreactive T cells. (c) Induction of skin allograft acceptance initially depended on the presence of IFN-gamma, CTLA4, and CD4(+) T cells. Addition of anti-CTLA4 or anti-IFN-gamma mAb to the protocol was associated with prompt graft rejection, whereas anti-IL-4 mAb had no effect. The role of IFN-gamma was confirmed using knockout mice. (d) Graft survival was associated with the absence of IFN-gamma in the graft. (e) Long-term graft maintenance required the continued presence of CD4(+) T cells. The results suggest that, with modification, our short-term protocol may yield a procedure for the induction of long-term graft survival without prolonged immunosuppression.     

Interleukin 7 induces CD4+ T cell-dependent tumor rejection

The potential of interleukin 7 (IL-7) to induce an antitumor response in vivo was analyzed. Therefore, the IL-7 gene was expressed in the plasmacytoma cell line J558L. Although the growth of IL-7-producing cells was not retarded in vitro, the IL-7-producing cells were completely rejected upon injection into mice. Tumor rejection was observed only in syngeneic but not in nude mice. The tumor-suppressive effect could be abolished by the parallel injection of an anti-IL-7 monoclonal antibody. Immunohistochemical analysis revealed IL-7-dependent infiltration of the tumor tissue by CD4+ and CD8+ T lymphocytes, and also type 3 complement receptor-positive (CR3+) cells, predominantly macrophages. Depletion of T cell subsets in tumor-bearing mice showed the absolute dependence of the antitumor response on CD4+ cells, whereas tumor rejection was unaffected by depletion of CD8+ cells. In addition to CD4+ cells, CR3+ cells were also needed for tumor rejection. The antitumor effect of IL-7 was confirmed by expression of the IL-7 gene in a second tumor cell line of different cellular origin. Together, our results demonstrate that a high local IL-7 concentration at the tumor site obtained by tumor cell-targeted gene transfer leads to tumor rejection involving a cellular mechanism that seems to be different from the ones observed in analogous experiments with other cytokines.     

Immunity to a pulmonary Cryptococcus neoformans infection requires both CD4+ and CD8+ T cells

The role of CD4+ and CD8+ T cells in mediating pulmonary clearance of a cryptococcal infection was investigated. Intratracheal inoculation of BALB/c and C.B-17 mice with a moderately virulent strain of Cryptococcus neoformans (52D) resulted in a pulmonary infection, which was cleared by a T cell-dependent mechanism. During this clearance, there was a significant influx of both CD4+ and CD8+ T cells into the lungs. Depletion of CD4+ T cells by injections of CD4-specific monoclonal antibody (mAb) prevented pulmonary clearance and also resulted in significant colonization of the brain and spleen of infected mice. CD4 depletion did not prevent the influx of CD8+ T cells into the lungs. Surprisingly, depletion of CD8+ T cells by mAb also ablated pulmonary clearance. CD8-depleted mice also had a small but significant increase in brain and spleen colony-forming unit compared to control mice by the end of the study. CD4+ T cell pulmonary influx was independent of the presence of CD8+ T cells. The lungs of T cell-depleted mice were examined histologically. CD4+ and CD8+ T cells each mediated a degree of inflammatory influx seen in the lungs of infected mice and raised the possibility that CD4+ and CD8+ T cells may synergize to generate the inflammatory response in the lungs. Numerous phagocytized but intact cryptococci were seen in the inflammatory foci of CD8-depleted mice but not in control or CD4-depleted mice. We propose that CD4+ T cells may recruit and activate effector phagocytes while CD8+ T cells predominantly function to lyse cryptococcus-laden unactivated phagocytes similar to the function of CD8+ T cells during listeria and mycobacteria infections.     

The CD8 population in CD4-deficient mice is heavily contaminated with MHC class II-restricted T cells

In experiments to study the impact of deficiency in CD4+ T cell help on the magnitude of CD8+ cytotoxic T cell response to pathogens, it was noted that in CD4 gene knockout mice, the CD8 population made significant responses to several nominally major histocompatibility complex (MHC) class II-restricted epitopes in addition to the expected responses to MHC class I-restricted epitopes. A similar response by CD8+ T cells to class II-restricted epitopes was not observed in wild-type mice, or in mice that had been acutely depleted of CD4+ T cells just before the immunization. Coincident with this unexpected response to class II-restricted epitopes, it was also observed that the CD8+ response to the class I-restricted epitopes was consistently lower in CD4-/- mice than in wild-type mice. Further experiments suggested that these two observations are linked and that the CD8 population in CD4-/- mice may contain a majority of T cells that were actually selected by recognition of MHC class II molecules in the thymus. These results have implications for understanding CD4 versus CD8 lineage commitment in the thymus, and for the practical use of CD4-/- mice as models of helper deficiency.     

CD103 expression is required for destruction of pancreatic islet allografts by CD8(+) T cells

The mechanisms by which CD8 effector populations interact with epithelial layers is a poorly defined aspect of adaptive immunity. Recognition that CD8 effectors have the capacity to express CD103, an integrin directed to the epithelial cell-specific ligand E-cadherin, potentially provides insight into such interactions. To assess the role of CD103 in promoting CD8-mediated destruction of epithelial layers, we herein examined the capacity of mice with targeted disruption of CD103 to reject pancreatic islet allografts. Wild-type hosts uniformly rejected islet allografts, concomitant with the appearance of CD8(+)CD103(+) effectors at the graft site. In contrast, the majority of islet allografts transplanted into CD103(-/-) hosts survived indefinitely. Transfer of wild-type CD8 cells into CD103(-/-) hosts elicited prompt rejection of long-surviving islet allografts, whereas CD103(-/-) CD8 cells were completely ineffectual, demonstrating that the defect resides at the level of the CD8 cell. CD8 cells in CD103(-/-) hosts exhibited normal effector responses to donor alloantigens in vitro and trafficked normally to the graft site, but strikingly failed to infiltrate the islet allograft itself. These data establish a causal relationship between CD8(+)CD103(+) effectors and destruction of graft epithelial elements and suggest that CD103 critically functions to promote intragraft migration of CD8 effectors into epithelial compartments.     

Phenotype, specificity, and function of T cell subsets and T cell interactions involved in skin allograft rejection

In the present study we used an adoptive transfer model with athymic nude mice to characterize the T cells involved in initiating and mediating skin allograft rejection. It was found that skin allograft rejection in nude mice required the transfer of immunocompetent T cells and that such reconstitution did not itself stimulate the appearance of T cells derived from the nude host. Reconstitution with isolated populations of Lyt-2+/L3T4- T cells resulted in the rapid rejection of MHC class I-disparate skin allografts, whereas reconstitution with isolated populations of L3T4+/Lyt-2- T cells resulted in the rapid rejection of MHC class II-disparate and minor H-disparate skin allografts. By correlating these rejection responses with the functional capabilities of antigen-specific T cells contained within the reconstituting Lyt-2+ and L3T4+ T cell populations, it was noted that skin allografts were only rejected by mice that, as shown by in vitro assessment, contained both lymphokine-secreting Th cells and lymphokine-responsive Tk cells specific for the alloantigens of the graft. The ability of two such functionally distinct T cell subsets to interact in vivo to reject skin allografts was directly demonstrated in H-Y-specific rejection responses by taking advantage of the fact that H-Y-specific Th cells are L3T4+ while H-Y specific Tk cells are Lyt-2+. Finally, the importance of in vivo interactions between functionally distinct Th/T-inducer cells and T killer (Tk)/T-effector cells in skin allograft rejection was demonstrated by the observation that normal B6 mice retain Qala and Kbm6 skin allografts because of a selective deficiency in antigen-specific Th cells, even though they contain T-effector cells that, when activated, are able to reject such allografts. Thus, the ability to reject skin allografts is neither unique to a specialized subset of T cells with a given Lyt phenotype, nor unique to a specialized subset of helper-independent effector T cells with so-called dual function capability. Rather, skin allograft rejection can be mediated by in vivo collaborations between T-inducer cells and T-effector cells, and the two interacting T cell subsets can express different Lyt phenotypes as well as different antigen specificities.     

Both intrathymic and peripheral selection modulate the differential expression of V beta 5 among CD4+ and CD8+ T cells.

Murine T cells expressing V beta 5 are characterized by (a) intrathymic deletion in the presence of I-E and products of endogenous mouse mammary tumor viruses, and (b) a greater representation in CD8+ relative to CD4+ peripheral T cells, thought to be due to more efficient intrathymic positive selection on class I rather than class II major histocompatibility complex antigens. We have engineered mice that are transgenic for a rearranged gene encoding a V beta 5+ beta chain of the T cell receptor for antigen. Deletion is not predicted in I-E- V beta 5+ transgenic mice, and until the age of 2 wk, the CD4/CD8 ratio of peripheral T cells is > 3:1 and indistinguishable between transgenic and nontransgenic mice. Transgenic mice then show a rapid, age-dependent decline in the ratio of CD4+ to CD8+ T cells in the lymphoid periphery, reaching a low of 1:10 by 7 mo of age. Furthermore, the percent of peripheral CD4+ cells that express the transgene drops with age, reaching a low of about 60% at 7 mo, while the percent of CD8+ cells that express V beta 5 remains greater than 95% at all ages. The lymphoid periphery is implicated in this selection against CD4+ V beta 5+ T cells as it occurs more rapidly in thymectomized transgenic mice, and can be delayed in mice whose peripheral T cells are replaced by recent thymic emigrants after depletion by in vivo treatment with anti-Thy-1 antibodies. These results indicate that the relative expression of V beta 5 in T cell subsets can be influenced not only intrathymically in I-E+ V beta 5+ transgenic mice, but also by events in the periphery, in the absence of I-E expression.     

FBL-reactive CD8+ cytotoxic and CD4+ helper T lymphocytes recognize distinct Friend murine leukemia virus-encoded antigens

Immunization of C57BL/6 (B6) mice with FBL, a Friend murine leukemia virus (F-MuLV), induces both tumor-specific cytolytic CD8+ (CTL) and lymphokine-producing CD4+ Th that are effective in adoptive therapy of B6 mice bearing disseminated FBL leukemia. The current study evaluated the F-MuLV antigenic determinants expressed on FBL that are recognized by FBL-reactive CD8+ and CD4+ T cells. To identify the specificity of the FBL-reactive CD8+ CTL, Fisher rat embryo fibroblast (FRE) cells transfected with plasmids encoding F-MuLV gag or envelope (env) gene products plus the class I-restricting element Db were utilized. FBL-reactive CTL recognized FRE target cells transfected with the F-MuLV gag-encoded gene products, but failed to recognize targets expressing F-MuLV env. Attempts to generate env-specific CD8+ CTL by immunization with a recombinant vaccinia virus containing an inserted F-MuLV env gene were unsuccessful, despite the generation of a cytolytic response to vaccinia epitopes, implying that B6 mice fail to generate CD8+ CTL to env determinants. By contrast, CD4+ Th clones recognized FRE target cells transfected with env and not gag genes, and immunization with the recombinant vaccinia virus induced an env-specific CD4+ T cell response. These data show that in a Friend retrovirus-induced tumor model in which tumor rejection can be mediated by either CTL or Th, antigens derived from discrete retroviral proteins are predominantly responsible for activation of each T cell subset.     

CD4+8- thymocytes bearing major histocompatibility complex class I-restricted T cell receptors: evidence for homeostatic control of early stages of CD4/CD8 lineage development.

During thymus development CD4+ CD8+ precursor cells differentiate into mature CD4+ and CD8+ T cells expressing T cell receptors (TCR) that recognize foreign antigens in association with major histocompatibility complex (MHC) class II or I molecules, respectively. Studies with TCR transgenic mice have shown that the accumulation of mature CD4+ and CD8+ thymocytes is strongly skewed by the MHC restriction specificity of the TCR, thus suggesting that commitment of CD4+ CD8+ precursors to the CD4 or CD8 lineage is a direct consequence of TCR/MHC interactions. However, we show here that CD4+ cells expressing an inappropriate (MHC class I-specific) TCR appear transiently in the neonatal thymus of TCR transgenic mice and can also be found in the periphery of adult TCR transgenic recombination-deficient SCID mice. These data argue that the early stages of CD4 and CD8 lineage development in the thymus are (at least in part) controlled by homeostatic mechanisms independent of appropriate TCR/MHC interactions.     

Antitumor efficacy of CD137 ligation is maximized by the use of a CD137 single-chain Fv-expressing whole-cell tumor vaccine compared with CD137-specific monoclonal antibody infusion

Tumor-destructive immune responses can be generated by engaging CD137 (4-1BB) via infusing a monoclonal antibody specific for CD137 or vaccinating with a single-chain Fv (scFv) CD137-expressing whole-cell tumor vaccine. We assessed whether such a vaccine can induce tumor rejection in the neu-transgenic (neu-Tg) mouse breast cancer model and compared the antitumor efficacy of vaccination with the infusion of a CD137-specific antibody. Mammary carcinoma cells (MMC) from a neu-Tg mouse were transfected to stably express surface scFv derived from the anti-CD137 rat hybridoma 1D8 or 3H3. The anti-CD137 scFv-expressing cells were rejected when transplanted into neu-Tg mice by a mechanism that involved both CD4(+) and CD8(+) T cells, and vaccination with such cells delayed the outgrowth of MMC cells transplanted 3 days previously. T cells from neu-Tg mice that had been vaccinated proliferated and produced IFN-gamma when stimulated by MMC but not by antigen-negative variant breast cancer cells that did not express the neu tumor antigen. In addition, antibodies binding to the MMC but not to antigen-negative variant cells were detected in sera from some but not all of the immunized mice. Complete regression of s.c. transplanted MMC tumors was observed in mice repeatedly immunized against MMC-1D8 starting on the day the MMC cells were transplanted. In contrast, repeated administration of either of two different anti-CD137 monoclonal antibodies did not induce complete tumor regression, although tumor growth was delayed.     

An early CD4+ T cell-dependent immunoglobulin A response to influenza infection in the absence of key cognate T-B interactions

Contact-mediated interactions between CD4+ T cells and B cells are considered crucial for T cell-dependent B cell responses. To investigate the ability of activated CD4+ T cells to drive in vivo B cell responses in the absence of key cognate T-B interactions, we constructed radiation bone marrow chimeras in which CD4+ T cells would be activated by wild-type (WT) dendritic cells, but would interact with B cells that lacked expression of either major histocompatibility complex class II (MHC II) or CD40. B cell responses were assessed after influenza virus infection of the respiratory tract, which elicits a vigorous, CD4+ T cell-dependent antibody response in WT mice. The influenza-specific antibody response was strongly reduced in MHC II knockout and CD40 knockout mice. MHC II-deficient and CD40-deficient B cells in the chimera environment also produced little virus-specific immunoglobulin (Ig)M and IgG, but generated a strong virus-specific IgA response with virus-neutralizing activity. The IgA response was entirely influenza specific, in contrast to the IgG2a response, which had a substantial nonvirus-specific component. Our study demonstrates a CD4+ T cell-dependent, antiviral IgA response that is generated in the absence of B cell signaling via MHC II or CD40, and is restricted exclusively to virus-specific B cells.     

Genetic engineering of murine CD8+ and CD4+ T cells for preclinical adoptive immunotherapy studies

T-cell receptor (TCR) gene therapy enables for the rapid creation of antigen-specific T cells from mice of any strain and represents a valuable tool for preclinical immunotherapy studies. Here, we describe the superiority of γ-retroviral vectors compared with lentiviral vectors for transduction of murine T cells and surprisingly illustrate robust gene-transfer into phenotypically naive/memory-stem cell like (TN/TSCM; CD62L(hi)/CD44(low)) and central memory (TCM; CD62L(hi)/CD44(hi)) CD8+ T cells using murine stem cell-based γ-retroviral vectors (MSGV1). We created MSGV1 vectors for a major histocompatibility complex-class I-restricted TCR specific for the melanocyte-differentiation antigen, glycoprotein 100 (MSGV1-pmel-1), and a major histocompatibility complex-class II-restricted TCR specific for tyrosinase-related protein-1 (MSGV1-TRP-1), and found that robust gene expression required codon optimization of TCR sequences for the pmel-1 TCR. To test for functionality, we adoptively transferred TCR-engineered T cells into mice bearing B16 melanomas and observed delayed growth of established tumors with pmel-1 TCR engineered CD8+ T cells and significant tumor regression with TRP-1 TCR transduced CD4 T cells. We simultaneously created lentiviral vectors encoding the pmel-1 TCR, but found that these vectors mediated low TCR expression in murine T cells, but robust gene expression in other murine and human cell lines. These results indicate that preclinical murine models of adoptive immunotherapies are more practical using γ-retroviral rather than lentiviral vectors.     

Identification of pre-T cells in human peripheral blood. Extrathymic differentiation of CD7+CD3- cells into CD3+ gamma/delta+ or alpha/beta+ T cells

CD7+CD3- cells purified (greater than 99.99%) by FACS from the peripheral blood of healthy adults include precursors for mature T cells that have the capacity to differentiate into TCR-1+ or TCR-2+ CD3+ cells. Extrathymic differentiation was demonstrable from all eight healthy donors in the presence of a high concentration of IL-2, mitogenic levels of PHA, and irradiated blood mononuclear feeder cells, after a lag of approximately 40 d in vitro. The extrathymic T (ET) cells were predominantly TCR-1+, although TCR-2+ cells were also derived. ET TCR-1+ cells were CD4-CD8-, CD4-CD8DIM+, and CD4+CD8-, and were distinguished from natural T TCR-2+ cells by a variety of cell surface markers. The ET cells had phenotypes generally displayed by normal mature T cells, although the CD5DIM+ on ET cells was more typical of thymocytes. Acquisition of CD3 on purified CD7+CD3- cells was not due to antigenic modulation or growth of contaminants, and ET cells could be demonstrated at the clonal level. Studies in athymic mice and bone marrow recipients support the view that extrathymic maturation does occur in vivo. Whether the CD7+CD3- cell population was unexposed to the thymus, or exposed but not processed, is unknown. In any case, unusual or "forbidden" autoreactive specificities are predicted since ET cells differentiate without thymic selection of the TCR.     

Regulatory functions of CD8+CD28- T cells in an autoimmune disease model

CD8+ T cell depletion renders CD28-deficient mice susceptible to experimental autoimmune encephalomyelitis (EAE). In addition, CD8-/-CD28-/- double-knockout mice are susceptible to EAE. These findings suggest a role for CD8+ T cells in the resistance of CD28-deficient mice to disease. Adoptive transfer of CD8+CD28- T cells into CD8-/- mice results in significant suppression of disease, while CD8+CD28+ T cells demonstrate no similar effect on the clinical course of EAE in the same recipients. In vitro, CD8+CD28- but not CD8+CD28+ T cells suppress IFN-gamma production of myelin oligodendrocyte glycoprotein-specific CD4+ T cells. This suppression requires cell-to-cell contact and is dependent on the presence of APCs. APCs cocultured with CD8+CD28- T cells become less efficient in inducing a T cell-dependent immune response. Such interaction prevents upregulation of costimulatory molecules by APCs, hence decreasing the delivery of these signals to CD4+ T cells. These are the first data establishing that regulatory CD8+CD28- T cells occur in normal mice and play a critical role in disease resistance in CD28-/- animals.