Regulation of apoptosis in mature alphabeta+CD4-CD8- antigen-specific suppressor T cell clones

The regulation of apoptosis in mature CD4+ or CD8+ alphabeta+ T cells has been well studied. How the survival and death is regulated in peripheral CD4-CD8- (double negative, DN) alphabeta+ T cells remains unknown. Recent studies suggest that peripheral DN T cells may play an important role in the regulation of the immune responses mediated by CD4+ or CD8+ T cells. Here, we used immunosuppressive DN T cell clones to elucidate the mechanisms involved in the regulation of death and survival of alphabeta+ DN T cells. The DN T cell clones were generated from the spleen cells of 2C transgenic mice, which express the transgenic TCR specific for Ld and permanently accepted Ld+ skin allografts after pretransplant infusion of Ld+ lymphocytes. We report that 1) the mature DN T cells are highly resistant to TCR cross-linking-induced apoptosis in the presence of exogenous IL-4; 2) Fas/Fas-ligand and TNF-alpha/TNFR pathways do not play an apparent role in regulating apoptosis in DN T cells; 3) the DN T cells constitutively express a high level of Bcl-xL, but not Bcl-2; 4) both Bcl-xL and Bcl-2 are up-regulated following TCR-cross-linking; and 5) IL-4 stimulation significantly up-regulates Bcl-xL and c-Jun expression and leads to mitogen-activated protein kinase phosphorylation in DN T cells, which may contribute to the resistance to apoptosis in these T cells. Taken together, these results provide us with an insight into how mature DN T cells resist activation-induced apoptosis to provide a long-term suppressor function in vivo.     

Self-specific MHC class II-restricted CD4-CD8- T cells that escape deletion and lack regulatory activity

In the presence of the I-Ealpha protein, transgenic (Tg) mice expressing the 1H3.1 alphabeta TCR that is specific for the Ealpha52-68:I-A(b) complex display drastic intrathymic deletion. Although peripheral T cells from these mice remained unresponsive to the Ealpha52-68:I-A(b) complex, they contained a subpopulation able to specifically react to this complex in the presence of exogenous IL-2, indicating that some 1H3.1 alphabeta TCR Tg T cells have escaped clonal deletion and efficiently populated the periphery. IL-2-dependent, Ealpha52-68:I-A(b) complex-responsive T cells were CD4-CD8- and expressed the 1H3.1 alphabeta TCR. Such T cells could develop intrathymically, did not show sign of regulatory/suppressor activity, displayed a typical naive phenotype, and seemed to persist in vivo over time. CD4-CD8- TCR Tg T cells were also detected when the surface density of the deleting ligand was increased on MHC class II+ cells. In addition, the development of CD4-CD8- 1H3.1 alphabeta TCR Tg T cells could be supported by I-A(b) molecules. These observations indicate that CD4 surface expression neither specifies, nor is required for, the thymic export of mature thymocytes expressing a MHC class II-restricted alphabeta TCR. The data also show that, although the avidity of the interaction involved in intrathymic deletion is significantly lower than that involved in mature T cell activation, its range can be large enough to be influenced by the presence or absence of coreceptors. Finally, the margin created by the absence of CD4 coreceptor was substantial because it could accommodate various amounts of the deleting ligand on thymic stromal cells.     

A model for the origin of TCR-alphabeta+ CD4-CD8- B220+ cells based on high affinity TCR signals

The origin of TCR-alphabeta+ CD4-CD8- cells is unclear, yet accumulating evidence suggests that they do not represent merely a default pathway of unselected thymocytes. Rather, they arise by active selection as evidenced by their absence in mice lacking expression of class I MHC. TCR-alphabeta+ CD4-CD8- cells also preferentially accumulate in mice lacking expression of Fas/APO-1/CD95 (lpr) or Fas-ligand (gld), suggesting that this subset might represent a subpopulation destined for apoptosis in normal mice. Findings from mice bearing a self-reactive TCR transgene support this view. In the current study we observe that in normal mice, TCR-alphabeta+ CD4-CD8- thymocytes contain a high proportion of cells undergoing apoptosis. The apoptotic subpopulation is further identified by its expression of B220 and IL2Rbeta and the absence of surface CD2. The CD4-CD8- B220+ phenotype is also enriched in T cells that recognize endogenous retroviral superantigens, and can be induced in TCR transgenic mice using peptide/MHC complexes that bear high affinity, but not low affinity, for TCR. A model is presented whereby the TCR-alphabeta+ CD2- CD4-CD8- B220+ phenotype arises from high intensity TCR signals. This model is broadly applicable to developing thymocytes as well as mature peripheral T cells and may represent the phenotype of self-reactive T cells that are increased in certain autoimmune conditions.     

TCR/self-antigen interactions drive double-negative T cell peripheral expansion and differentiation into suppressor cells.

Mature CD4-CD8- alphabeta+ T cells (DNTC) in the periphery of TCR transgenic mice are resistant to clonal deletion in cognate Ag-expressing (Ag+) mice. Previously, we have characterized DNTC populations bearing the alloreactive 2C TCR in Ag-free (Ag-) and Ag+ mice. Despite appearing functionally anergic when challenged with cognate Ag in vitro, Ag-experienced DNTC exhibit markers of activation/memory, a lowered threshold of activation, ex vivo cytolytic activity, and the ability to rapidly secrete IFN-gamma. Remarkably, these memory-like DNTC also possess potent immunoregulatory properties, competing effectively for bystander-produced IL-2 and suppressing autoreactive CD8+ T cell proliferation via a Fas/FasL-dependent cytolytic mechanism. The fact that DNTC recovered from Ag+ mice possess markers and attributes characteristic of naive CD8+ T cells that have undergone homeostasis-induced proliferation suggested that they may be derived from a similar peripheral expansion process. Naive DNTC adoptively transferred into Ag-bearing hosts rapidly acquire markers and functional attributes of DNTC that have continually developed in the presence of Ag. Thus, the peripheral selection and maintenance of such autoreactive cells may serve to negatively regulate potential autoimmune T cell responses.     

Cytokine production by mature and immature CD4-CD8- T cells. Alpha beta-T cell receptor+ CD4-CD8- T cells produce IL-4.

This study follows our previous investigation describing the production of four cytokines (IL-2, IL-4, IFN-gamma, and TNF-alpha) by subsets of thymocytes defined by the expression of CD3, 4, 8, and 25. Here we investigate in greater detail subpopulations of CD4-CD8- double negative (DN) thymocytes. First we divided immature CD25-CD4-CD8-CD3- (CD25- triple negative) (TN) thymocytes into CD44+ and CD44- subsets. The CD44+ population includes very immature precursor T cells and produced high titers of IL-2, TNF-alpha, and IFN-gamma upon activation with calcium ionophore and phorbol ester. In contrast, the CD44- subset of CD25- TN thymocytes did not produce any of the cytokines studied under similar activation conditions. This observation indicates that the latter subset, which differentiates spontaneously in vitro into CD4+CD8+, already resembles CD4+CD8+ thymocytes (which do not produce any of the tested cytokines). We also subdivided the more mature CD3+ DN thymocytes into TCR-alpha beta- and TCR-gamma delta-bearing subsets. These cells produced cytokines upon activation with solid phase anti-CD3 mAb. gamma delta TCR+ DN thymocytes produced IL-2, IFN-gamma and TNF-alpha, whereas alpha beta TCR+ DN thymocytes produced IL-4, IFN-gamma, and TNF-alpha but not IL-2. We then studied alpha beta TCR+ DN T cells isolated from the spleen and found a similar cytokine production profile. Furthermore, splenic alpha beta TCR+ DN cells showed a TCR V beta gene expression profile reminiscent of alpha beta TCR+ DN thymocytes (predominant use of V beta 8.2). These observations suggest that at least some alpha beta TCR+ DN splenocytes are derived from alpha beta TCR+ DN thymocytes and also raises the possibility that these cells may play a role in the development of Th2 responses through their production of IL-4.     

p59fyn (Fyn) promotes the survival of anergic CD4-CD8- alpha beta TCR+ cells but negatively regulates their proliferative response to antigen stimulation

T cell anergy is characterized by alterations in TCR signaling that may play a role in controlling the unresponsiveness of the anergic cell. We have addressed questions regarding the importance of the Src kinase p59(fyn) (Fyn) in this process by using Fyn null mice. We demonstrate that a mature population of CD4(-)CD8(-) alphabeta TCR(+) anergic T cells lacking Fyn have a substantial recovery of their proliferation defect in response to Ag stimulation. This recovery cannot be explained by ameliorated production of IL-2, and the improved proliferation correlates with an enhanced ability of the Fyn(-/-) anergic T cells to up-regulate the high affinity IL-2 receptor. We also observe that anergic CD4(-)CD8(-) alphabeta TCR(+) T cells have a heightened survival ability that is partially dependent on the elevated levels of Fyn and IL-2 receptor beta-chain expressed by these cells. The enhanced survival correlates with an increased capacity of the anergic cells to respond to IL-15. We conclude that Fyn plays an important role in aspects of T cell anergy pertaining to TCR signaling and to cell survival.     

Characterization of defective CD4-CD8- T cells in murine tumors generated independent of antigen specificity

Immune-based therapy confers limited benefits to hosts bearing late-stage tumors. Mounting evidence points to local suppression of T cell function as the most substantial barrier to effective antitumor immunity in hosts with large tumor burdens. Despite this, events responsible for locally defective T cells and immune suppression in tumors remain unclear. We describe in this study a predominant T cell population localized within two murine tumors that is characterized by expression of apoptotic markers and TCRalphabeta/CD3, but not CD4, CD8, or NK-associated markers. These defective cells resembled double negative (DN) T cells in lpr mice, harbored defects in the expression of T cell signaling molecules, and produced the anti-inflammatory cytokine, IL-10. Conditions known to increase or decrease the accumulation of lpr DN T cells had corresponding effects on local DN tumor infiltrating lymphocyte (TIL) levels and inversely impacted host survival. Adoptive transfer into s.c. tumors demonstrated that naive CD8(+) T cells were highly susceptible to becoming DN TIL, and local supplementation of tumors with nontumor Ag-bearing MHC class I-expressing fibroblasts decreased both this susceptibility and endogenous DN TIL levels. These findings identify a major defective T cell population with suppressive potential within tumors. The data also suggest that local T cell defectiveness is controlled by the tumor environment independent of cognate Ag specificity per se. Decreasing defective DN TIL levels by increasing noncognate peptide MHC class I availability, or modulating TCR or cytokine signaling may facilitate host survival by bolstering endogenous immunity to late-stage tumors, and may help improve therapeutic tumor vaccines.     

Thymic selection and peripheral activation of CD8 T cells by the same class I MHC/peptide complex

Thymic selection is controlled by the interaction between TCR and MHC/peptide. Strength and quality of the signal determine whether thymocytes are selected or deleted. The factors that contribute to this signal remain poorly defined. Here we show that fetal thymic organ cultures (FTOCs) derived from OT-I transgenic mice (the OT-I TCR is restricted by K(b)-SIINFEKL) on a K(b)D(b-/-) background support positive selection, but only when provided with soluble H-2K(b)-SIINFEKL complexes. Selection of CD8 T cells is independent of the valency of the ligand or its capability to coengage CD8 molecules. Both CD8alphaalpha and CD8alphabeta T cells are selected by H-2K(b)-SIINFEKL, but only CD8alphabeta cells are capable of releasing IFN-gamma in response to the same ligand. The alpha(4)beta(7) integrin is up-regulated on postselection thymocytes from FTOCs. After adoptive transfer, FTOC-derived OT-I CD8 T cells divide in response to the agonist peptide SIINFEKL. These results establish that CD8 T cells responsive to their nominal peptide-Ag can be generated in FTOC supplemented with soluble MHC class I molecules equipped with the same peptide.     

Naive CD8+ T cells do not require costimulation for proliferation and differentiation into cytotoxic effector cells

Most current models of T cell activation postulate a requirement for two distinct signals. One signal is delivered through the TCR by engagement with peptide/MHC complexes, and the second is delivered by interaction between costimulatory molecules such as CD28 and its ligands CD80 and CD86. Soluble peptide/MHC tetramers provide an opportunity to test whether naive CD8+ T cells can be activated via the signal generated through the TCR-alphabeta in the absence of any potential costimulatory molecules. Using T cells from two different TCR transgenic mice in vitro, we find that TCR engagement by peptide/MHC tetramers is sufficient for the activation of naive CD8+ T cells. Furthermore, these T cells proliferate, produce cytokines, and differentiate into cytolytic effectors. Under the conditions where anti-CD28 is able to enhance proliferation of normal B6 CD4+, CD8+, and TCR transgenic CD8+ T cells with anti-CD3, we see no effect of anti-CD28 on proliferation induced by tetramers. The results of this experiment argue that given a strong signal delivered through the TCR by an authentic ligand, no costimulation is required.     

Suppressor effector function of CD4+CD25+ immunoregulatory T cells is antigen nonspecific

CD4+CD25+ T cells represent a unique population of "professional" suppressor T cells that prevent induction of organ-specific autoimmune disease. In vitro, CD4+CD25+ cells were anergic to simulation via the TCR and when cultured with CD4+CD25- cells, markedly suppressed polyclonal T cell proliferation by specifically inhibiting the production of IL-2. Suppression was cytokine independent, cell contact dependent, and required activation of the suppressors via their TCR. Further characterization of the CD4+CD25+ population demonstrated that they do not contain memory or activated T cells and that they act through an APC-independent mechanism. CD4+CD25+ T cells isolated from TCR transgenic (Tg) mice inhibited responses of CD4+CD25- Tg T cells to the same Ag, but also inhibited the Ag-specific responses of Tg cells specific for a distinct Ag. Suppression required that both peptide/MHC complexes be present in the same culture, but the Ags could be presented by two distinct populations of APC. When CD4+CD25+ T cells were cultured with anti-CD3 and IL-2, they expanded, remained anergic, and in the absence of restimulation via their TCR, suppressed Ag-specific responses of CD4+CD25- T cells from multiple TCR transgenics. Collectively, these data demonstrate that CD4+CD25+ T cells require activation via their TCR to become suppressive, but once activated, their suppressor effector function is completely nonspecific. The cell surface molecules involved in this T-T interaction remain to be characterized.     

Induction of tolerance in CD8+ T cells to a transgenic autoantigen expressed in the liver does not require cross-presentation

Cross-presentation of normal self and candidate tumor Ags by bone marrow (BM)-derived APCs that have not been activated has been demonstrated as a major mechanism contributing to acquisition of tolerance by mature T cells that first encounter an Ag in the periphery (cross-tolerance). Following adoptive transfer of naive TCR-transgenic CD8(+) T cells into a host expressing a transgenic Ag that is a potentially targetable tumor Ag in normal hepatocytes as a self-Ag, we found that the majority of Ag-specific CD8(+) T cells were deleted, with the remaining cells rendered anergic. Studies in BM chimeric mice and with purified cell populations demonstrated that these events were not dependent on cross-presentation by BM-derived APCs including Kupffer cells or liver sinusoidal endothelial cells, and apparently can occur entirely as a consequence of direct recognition of Ag endogenously processed and presented by hepatocytes. Direct recognition of Ag-expressing hepatocytes in vivo induced a proliferative response and up-regulation of activation markers in responding CD8(+) T cells, but proliferating cells did not accumulate, with most cells rapidly eliminated, and the persisting T cells lost the capacity to proliferate in response to repeated Ag stimulation. The results suggest that parenchymal tissues may retain the capacity to directly regulate in vivo responses to self-Ags processed and presented in the context of class I MHC molecules.     

Double-negative T regulatory cells can develop outside the thymus and do not mature from CD8+ T cell precursors

Recent studies have demonstrated that activated peripheral alphabeta TCR+ CD3+ CD4- CD8- NK1.1- (double-negative, DN) regulatory T cells (Tregs) from both mice and humans are able to down-regulate immune responses in vitro and in vivo. However, the origin and developmental requirements of functional DN Tregs remain unclear. In this study, we investigated the requirement for CD8 expression as well as the presence of a thymus for the development of functional DN Tregs. We demonstrate that DN Tregs exist in CD8-deficient mice and that stimulation of CD8+ T cells in vivo with TCR-specific Ag does not convert CD8+ T cells into DN Tregs. In addition, we found that DN T cells are present in the spleens and lymph nodes of thymectomized mice that are irradiated and reconstituted with T cell-depleted bone marrow cells. Interestingly, DN Tregs that develop in thymectomized mice can suppress syngeneic CD8+ T cells more effectively than those that develop in sham-thymectomized mice. Taken together, our data suggest that DN Tregs are not derived from CD8+ T cell precursors and that functional DN Tregs may preferentially develop outside of the thymus. These data suggest that DN Tregs may represent a developmentally and functionally unique cell population.     

Impaired clonal expansion in athymic nude CD8+CD4- T cells

A comparative study of the phenotype and immune functions of highly purified CD8+CD4- T cells obtained from the spleen and thymus of normal mice and from the spleen of athymic nude mice was conducted. Of seven individual normal and nude mice examined, the range of V beta 8+ cells among CD8+ T cells was a heterogeneous 4.3 to 30.5% for athymic nude mice and a much more uniform spread from 14.7 to 18.5% for normal mice. In six of the seven nude mice examined, the fraction of V beta 8+ cells was below the lower limit of the V beta 8 distribution in normal mice. However, one of the seven nude mice contained nearly twice the percentage of normal V beta 8+ cells. A reduction in the density of V beta 8 as well as CD3 Ag expression was also observed in athymic CD8+CD4- cells although an Ly-6-linked Ag, B4B2 displayed a highly increased expression. Considering the battery of Ag analyzed in entirety, athymic CD8+CD4- T cells were clearly distinct from their "counterpart" CD8+CD4- T cells isolated from either thymus or spleen of normal (euthymic) mice. Anti-CD3-mediated triggering of the TCR:CD3 complex caused extensive clonal proliferation in cultures to which single responding CD8+ T cells had been deposited. Under identical conditions, however, anti-CD3 caused little, if any clonal expansion in CD8+ cells from athymic nude mice. Highly purified athymic CD8+CD4- cells produced readily detectable IL-2R expression and IL-2 synthesis and secretion upon stimulation by anti-CD3 and by Con A. Production of IL-2 by purified athymic CD8+CD4- cells was due to CD8+CD4- cells and not due to a minor population of contaminating CD8- cells as anti-CD8 + C treatment completely abrogated the ability of athymic CD8+CD4- cells to produce IL-2. Despite IL-2 production and IL-2R expression by athymic nude CD8+CD4- T cells in response to anti-CD3 and to Con A, an impaired proliferative response followed.     

Developmental and functional analyses of CD8(+) NK1.1(+) T cells in class-I-restricted TCR transgenic mice.

Using a class-I-restricted T cell receptor (TCR) transgenic mice (Tgm), 2C (Valpha3.1/Vbeta 8.2, specific for L(d) + LSPFPFDL), the development and cytokine production of tg-TCR(+) NKT cells were analyzed. We found that CD8(+) or double negative (DN) NKT cells constituted a major population of NKT cells in the H-2(b/b) 2C Tgm (positive selecting background) or the H-2(b/d) 2C Tgm (negative selecting background), respectively. Virtually no NKT cells were generated in the H-2(k/k) 2C Tgm (neutral selecting background). CD8(+) NKT cells in the H-2(b/b) 2C Tgm expressed CD8alphabeta heterodimers, whereas those in the H-2(b/d) 2C Tgm expressed CD8alphaalpha homodimers. These findings suggest that development of a subpopulation of NKT cells is influenced by the H-2 molecules. Upon stimulation with anti-CD3 mAb, tg-TCR(+) NKT cells generated in the H-2(b/b) and H-2(b/d) backgrounds produced IFN-gamma, but not IL-4.     

Recognition of a shared human prostate cancer-associated antigen by nonclassical MHC-restricted CD8+ T cells

To identify prostate cancer-associated Ags, tumor-reactive T lymphocytes were generated using iterative stimulations of PBMC from a prostate cancer patient with an autologous IFN-gamma-treated carcinoma cell line in the presence of IL-2. A CD8+ T cell line and TCR alphabeta+ T cell clone were isolated that secreted IFN-gamma and TNF-alpha in response to autologous prostate cancer cells but not to autologous fibroblasts or lymphoblastoid cells. However, these T cells recognized several normal and malignant prostate epithelial cell lines without evidence of shared classical HLA molecules. The T cell line and clone also recognized colon cancers, but not melanomas, sarcomas, or lymphomas, suggesting recognition of a shared epithelium-associated Ag presented by nonclassical MHC or MHC-like molecules. Although Ag recognition by T cells was inhibited by mAb against CD8 and the TCR complex (anti-TCR alphabeta, CD3, Vbeta12), it was not inhibited by mAb directed against MHC class Ia or MHC class II molecules. Neither target expression of CD1 molecules nor HLA-G correlated with T cell recognition, but beta2-microglobulin expression was essential. Ag expression was diminished by brefeldin A, lactacystin, and cycloheximide, but not by chloroquine, consistent with an endogenous/cytosolic Ag processed through the classical class I pathway. These results suggest that prostate cancer and colon cancer cells can process and present a shared peptidic Ag to TCR alphabeta+ T cells via a nonclassical MHC I-like molecule yet to be defined.     

Functional characterization of MHC class II-restricted CD8+CD4- and CD8-CD4- T cell responses to infection in CD4-/- mice

Classical CD4(+) and CD8(+) T cells recognize Ag presented by MHC class II (MHCII) and MHC class I (MHCI), respectively. However, our results show that CD4(-/-) mice mount a strong, readily detectable CD8(+) T cell response to MHCII-restricted epitopes after a primary bacterial or viral infection. These MHCII-restricted CD8(+)CD4(-) T cells are more similar to classical CD8(+) T cells than to CD4(+) T cells in their expression of effector functions during a primary infection, yet they also differ from MHCI-restricted CD8(+) T cells by their inability to produce high levels of the cytolytic molecule granzyme B. After resolution of a primary infection, epitope-specific MHCII-restricted T cells in CD4(-/-) mice persist for a long period of time as memory T cells. Surprisingly, upon reinfection the secondary MHCII-restricted response in CD4(-/-) mice consists mainly of CD8(-)CD4(-) T cells. In contrast to CD8(+) T cells, MHCII-restricted CD8(-)CD4(-) T cells are capable of producing IL-2 in addition to IFN-gamma and thus appear to have attributes characteristic of CD4(+) T cells rather than CD8(+) T cells. Therefore, MHCII-restricted T cells in CD4(-/-) mice do not share all phenotypic and functional characteristics with MHCI-restricted CD8(+) T cells or with MHCII-restricted CD4(+) T cells, but, rather, adopt attributes from each of these subsets. These results have implications for understanding thymic T cell selection and for elucidating the mechanisms regulating the peripheral immune response and memory differentiation.     

Thymus and autoimmunity: production of CD25+CD4+ naturally anergic and suppressive T cells as a key function of the thymus in maintaining immunologic self-tolerance

This study shows that the normal thymus produces immunoregulatory CD25+4+8- thymocytes capable of controlling self-reactive T cells. Transfer of thymocyte suspensions depleted of CD25+4+8- thymocytes, which constitute approximately 5% of steroid-resistant mature CD4+8- thymocytes in normal naive mice, produces various autoimmune diseases in syngeneic athymic nude mice. These CD25+4+8- thymocytes are nonproliferative (anergic) to TCR stimulation in vitro, but potently suppress the proliferation of other CD4+8- or CD4-8+ thymocytes; breakage of their anergic state in vitro by high doses of IL-2 or anti-CD28 Ab simultaneously abrogates their suppressive activity; and transfer of such suppression-abrogated thymocyte suspensions produces autoimmune disease in nude mice. These immunoregulatory CD25+4+8- thymocytes/T cells are functionally distinct from activated CD25+4+ T cells derived from CD25-4+ thymocytes/T cells in that the latter scarcely exhibits suppressive activity in vitro, although both CD25+4+ populations express a similar profile of cell surface markers. Furthermore, the CD25+4+8- thymocytes appear to acquire their anergic and suppressive property through the thymic selection process, since TCR transgenic mice develop similar anergic/suppressive CD25+4+8- thymocytes and CD25+4+ T cells that predominantly express TCRs utilizing endogenous alpha-chains, but RAG-2-deficient TCR transgenic mice do not. These results taken together indicate that anergic/suppressive CD25+4+8- thymocytes and peripheral T cells in normal naive mice may constitute a common T cell lineage functionally and developmentally distinct from other T cells, and that production of this unique immunoregulatory T cell population can be another key function of the thymus in maintaining immunologic self-tolerance.     

Antigen feeding increases frequency and antigen-specific proliferation ability of intraepithelial CD4+ T cells in alphabeta T cell receptor transgenic mice

To study how intestinal intraepithelial lymphocytes (IEL) are affected by orally ingested antigen, the phenotypes and responses of the IEL in mice expressing a transgenic T cell receptor alphabeta (TCR alphabeta) specific for ovalbumin (OVA) were analyzed after feeding OVA. In the OVA-fed mice, the abundance of alphabeta-IEL as a proportion of the total IEL population increased and the frequency of CD4+ cells increased within the TCR alphabeta+ IEL population. CD4(+) IEL from OVA-fed transgenic mice proliferated in vitro more markedly in response to antigen stimulation than IEL from mice fed the control diet. These results indicate that antigen-specific proliferation of CD4+ IEL was amplified as a result of oral administration of antigen.     

4-1BB ligand induces cell division, sustains survival, and enhances effector function of CD4 and CD8 T cells with similar efficacy

A costimulatory member of the TNFR family, 4-1BB, is expressed on activated T cells. Although some reports have suggested that 4-1BB is primarily involved in CD8 T cell activation, in this report we demonstrate that both CD4 and CD8 T cells respond to 4-1BB ligand (4-1BBL) with similar efficacy. CD4 and CD8 TCR transgenic T cells up-regulate 4-1BB, OX40, and CD27 and respond to 4-1BBL-mediated costimulation during a primary response to peptide Ag. 4-1BBL enhanced proliferation, cytokine production, and CTL effector function of TCR transgenic T cells. To compare CD4 vs CD8 responses to 4-1BBL under similar conditions of antigenic stimulation, we performed MLRs with purified CD4 or CD8 responders from CD28(+/+) and CD28(-/-) mice. We found that CD8 T cells produced IL-2 and IFN-gamma in a 4-1BBL-dependent manner, whereas under the same conditions the CD4 T cells produced IL-2 and IL-4. 4-1BBL promoted survival of CD4 and CD8 T cells, particularly at late stages of the MLR. CD4 and CD8 T cells both responded to anti-CD3 plus s4-1BBL with a similar cytokine profile as observed in the MLR. CD4 and CD8 T cells exhibited enhanced proliferation and earlier cell division when stimulated with anti-CD3 plus anti-CD28 compared with anti-CD3 plus 4-1BBL, and both subsets responded comparably to anti-CD3 plus 4-1BBL. These data support the idea that CD28 plays a primary role in initial T cell expansion, whereas 4-1BB/4-1BBL sustains both CD4 and CD8 T cell responses, as well as enhances cell division and T cell effector function.