NK1.1+ T cells in the liver arise in the thymus and are selected by interactions with class I molecules on CD4+CD8+ cells

NK1.1+ T cells represent a specialized T cell subset specific for CD1d, a nonclassical MHC class I-restricting element. They are believed to function as regulatory T cells. NK1.1+ T cell development depends on interactions with CD1d molecules presented by hematopoietic cells rather than thymic epithelial cells. NK1.1+ T cells are found in the thymus as well as in peripheral organs such as the liver, spleen, and bone marrow. The site of development of peripheral NK1.1+ T cells is controversial, as is the nature of the CD1d-expressing cell that selects them. With the use of nude mice, thymectomized mice reconstituted with fetal liver cells, and thymus-grafted mice, we provide direct evidence that NK1.1+ T cells in the liver are thymus dependent and can arise in the thymus from fetal liver precursor cells. We show that the class I+ (CD1d+) cell type necessary to select NK1.1+ T cells can originate from TCRalpha-/- precursors but not from TCRbeta-/- precursors, indicating that the selecting cell is a CD4+CD8+ thymocyte. 5-Bromo-2'-deoxyuridine-labeling experiments suggest that the thymic NK1.1+ T cell population arises from proliferating precursor cells, but is a mostly sessile population that turns over very slowly. Since liver NK1.1+ T cells incorporate 5-bromo-2'-deoxyuridine more rapidly than thymic NK1.1+ T cells, it appears that liver NK1.1+ T cells either represent a subset of thymic NK1.1+ T cells or are induced to proliferate after having left the thymus. The results indicate that NK1.1+ T cells, like conventional T cells, arise in the thymus where they are selected by interactions with restricting molecules.     

CD8+ T cells rapidly acquire NK1.1 and NK cell-associated molecules upon stimulation in vitro and in vivo

NKT cells express both NK cell-associated markers and TCR. Classically, these NK1.1+TCRalphabeta+ cells have been described as being either CD4+CD8- or CD4-CD8-. Most NKT cells interact with the nonclassical MHC class I molecule CD1 through a largely invariant Valpha14-Jalpha281 TCR chain in conjunction with either a Vbeta2, -7, or -8 TCR chain. In the present study, we describe the presence of significant numbers of NK1.1+TCRalphabeta+ cells within lymphokine-activated killer cell cultures from wild-type C57BL/6, CD1d1-/-, and Jalpha281-/- mice that lack classical NKT cells. Unlike classical NKT cells, 50-60% of these NK1.1+TCRalphabeta+ cells express CD8 and have a diverse TCR Vbeta repertoire. Purified NK1.1-CD8alpha+ T cells from the spleens of B6 mice, upon stimulation with IL-2, IL-4, or IL-15 in vitro, rapidly acquire surface expression of NK1.1. Many NK1.1+CD8+ T cells had also acquired expression of Ly-49 receptors and other NK cell-associated molecules. The acquisition of NK1.1 expression on CD8+ T cells was a particular property of the IL-2Rbeta+ subpopulation of the CD8+ T cells. Efficient NK1.1 expression on CD8+ T cells required Lck but not Fyn. The induction of NK1.1 on CD8+ T cells was not just an in vitro phenomenon as we observed a 5-fold increase of NK1.1+CD8+ T cells in the lungs of influenza virus-infected mice. These data suggest that CD8+ T cells can acquire NK1.1 and other NK cell-associated molecules upon appropriate stimulation in vitro and in vivo.     

IL-7 up-regulates IL-4 production by splenic NK1.1+ and NK1.1- MHC class I-like/CD1-dependent CD4+ T cells.

NK T cells are an unusual subset of T lymphocytes. They express NK1. 1 Ag, are CD1 restricted, and highly skewed toward Vbeta8 for their TCR usage. They express the unique potential to produce large amounts of IL-4 and IFN-gamma immediately upon TCR cross-linking. We previously showed in the thymus that the NK T subset requires IL-7 for its functional maturation. In this study, we analyzed whether IL-7 was capable of regulating the production of IL-4 and IFN-gamma by the discrete NK T subset of CD4+ cells in the periphery. Two hours after injection of IL-7 into mice, or after a 4-h exposure to IL-7 in vitro, IL-4 production by CD4+ cells in response to anti-TCR-alphabeta is markedly increased. In contrast, IFN-gamma production remains essentially unchanged. In beta2-microglobulin- and CD1-deficient mice, which lack NK T cells, IL-7 treatment does not reestablish normal levels of IL-4 by CD4+ T cells. Moreover, we observe that in wild-type mice, the memory phenotype (CD62L-CD44+) CD4+ T cells responsible for IL-4 production are not only NK1.1+ cells, but also NK1.1- cells. This NK1.1-IL-4-producing subset shares three important characteristics with NK T cells: 1) Vbeta8 skewing; 2) CD1 restriction as demonstrated by their absence in CD1-deficient mice and relative overexpression in MHC II null mice; 3) sensitivity to IL-7 in terms of IL-4 production. In conclusion, the present study provides evidence that CD4+MHC class I-like-dependent T cell populations include not only NK1.1+ cells, but also NK1.1- cells, and that these two subsets are biased toward IL-4 production by IL-7.     

Emergence of CD8+ T cells expressing NK cell receptors in influenza A virus-infected mice

Both innate and adaptive immune responses play an important role in the recovery of the host from viral infections. In the present report, a subset of cells coexpressing CD8 and NKR-P1C (NK1.1) was found in the lungs of mice infected with influenza A virus. These cells were detected at low numbers in the lungs of uninfected mice, but represented up to 10% of the total CD8(+) T cell population at day 10 postinfection. Almost all of the CD8(+)NK1.1(+) cells were CD8alphabeta(+)CD3(+)TCRalphabeta(+) and a proportion of these cells also expressed the NK cell-associated Ly49 receptors. Interestingly, up to 30% of these cells were virus-specific T cells as determined by MHC class I tetramer staining and by intracellular staining of IFN-gamma after viral peptide stimulation. Moreover, these cells were distinct from conventional NKT cells as they were also found at increased numbers in influenza-infected CD1(-/-) mice. These results demonstrate that a significant proportion of CD8(+) T cells acquire NK1.1 and other NK cell-associated molecules, and suggests that these receptors may possibly regulate CD8(+) T cell effector functions during viral infection.     

NK markers are expressed on a high percentage of virus-specific CD8+ and CD4+ T cells

NK cells have been phenotypically defined by the expression of specific markers such as NK1.1, DX5, and asialo-GM1 (ASGM1). In addition to NK cells, a small population of CD3+ T cells has been shown to express these markers, and a unique subpopulation of NK1. 1+CD3+ T cells that expresses an invariant TCR has been named "NKT cells." Here, we describe NK marker expression on a broad spectrum of MHC class I- and MHC class II-restricted T cells that are induced after acute viral infection. From 5 to >500 days post lymphocytic choriomeningitis virus (LCMV) infection, more than 90% of virus-specific CD8+ and CD4+ T cells coexpress one or more of these three prototypical NK markers. Furthermore, in vivo depletion of NK cells with anti-ASGM1 Ab resulted in the removal of 90% of virus-specific CD8+ T cells and 50-80% of virus-specific CD4+ T cells. This indicates that studies using in vivo depletion to determine the role of NK cells in immune defense could potentially be misinterpreted because of the unintended depletion of Ag-specific T cells. These results demonstrate that NK Ags are widely expressed on the majority of virus-specific T cells and indicate that the NK and T cell lineages may not be as distinct as previously believed. Moreover, the current nomenclature defining NKT cells will require comprehensive modification to include Ag-specific CD8+ and CD4+ T cells that express prototypical NK Ags.     

Splenic NK1.1-negative, TCR alpha beta intermediate CD4+ T cells exist in naive NK1.1 allelic positive and negative mice, with the capacity to rapidly secrete large amounts of IL-4 and IFN-gamma upon primary TCR stimulation

Splenic NK1.1+CD4+ T cells that express intermediate levels of TCR alpha beta molecules (TCRint) and the DX5 Ag (believed to identify an equivalent population in NK1.1 allelic negative mice) possess the ability to rapidly produce high quantities of immunomodulatory cytokines, notably IL-4 and IFN-gamma, upon primary TCR activation in vivo. Indeed, only T cells expressing the NK1.1 Ag appear to be capable of this function. In this study, we demonstrate that splenic NK1.1-negative TCRintCD4+ T cells, identified on the basis of Fc gamma R expression, exist in naive NK1.1 allelic positive (C57BL/6) and negative (C3H/HeN) mice with the capacity to produce large amounts of IL-4 and IFN-gamma after only 8 h of primary CD3 stimulation in vitro. Furthermore, a comparison of the amounts of early cytokines produced by Fc gamma R+CD4+TCRint T cells with NK1. 1+CD4+ or DX5+CD4+TCRint T cells, simultaneously isolated from C57BL/6 or C3H/HeN mice, revealed strain and population differences. Thus, Fc gamma R defines another subpopulation of splenic CD4+TCRint cells that can rapidly produce large concentrations of immunomodulatory cytokines, suggesting that CD4+TCRint T cells themselves may represent a unique family of immunoregulatory CD4+ T cells whose members include Fc gamma R+CD4+ and NK1.1/DX5+CD4+ T cells.     

Development of the CD4 and CD8 lineage of T cells: instruction versus selection

T cells bearing the alpha beta T cell receptor (TCR) can be divided into CD4+8- and CD4-8+ subsets which develop in the thymus from CD4+8+ precursors. The commitment to the CD4 and CD8 lineage depends on the binding of the alpha beta TCR to thymic major histocompatibility complex (MHC) coded class II and class I molecules, respectively. In an instructive model of lineage commitment, the binding of the alpha beta TCR, for instance to class I MHC molecules, would generate a specific signal instructing the CD4+8+ precursors to switch off the expression of the CD4 gene. In a selective model, the initial commitment, i.e. switching off the expression of either the CD4 or the CD8 gene would be a stochastic event which is then followed by a selective step rescuing only CD4+ class II and CD8+ class I specific T cells while CD4+ class I and CD8+ class II specific cells would have a very short lifespan. The selective model predicts that a CD8 transgene which is expressed in all immature and mature T cells should rescue CD4+ class I MHC specific T cells from cell death. We have performed experiments in CD8 transgenic mice which fail to support a selective model and we present data which show that the binding of the alpha beta TCR to thymic class I MHC molecules results in up-regulation of the TCR in the CD4+8+ population. Therefore, these experiments are consistent with an instructive model of lineage commitment.     

IL-15 expands unconventional CD8alphaalphaNK1.1+ T cells but not Valpha14Jalpha18+ NKT cells

Despite recent gains in knowledge regarding CD1d-restricted NKT cells, very little is understood of non-CD1d-restricted NKT cells such as CD8(+)NK1.1(+) T cells, in part because of the very small proportion of these cells in the periphery. In this study we took advantage of the high number of CD8(+)NK1.1(+) T cells in IL-15-transgenic mice to characterize this T cell population. In the IL-15-transgenic mice, the absolute number of CD1d-tetramer(+) NKT cells did not increase, although IL-15 has been shown to play a critical role in the development and expansion of these cells. The CD8(+)NK1.1(+) T cells in the IL-15-transgenic mice did not react with CD1d-tetramer. Approximately 50% of CD8(+)NK1.1(+) T cells were CD8alphaalpha. In contrast to CD4(+)NK1.1(+) T cells, which were mostly CD1d-restricted NKT cells and of which approximately 70% were CD69(+)CD44(+), approximately 70% of CD8(+)NK1.1(+) T cells were CD69(-)CD44(+). We could also expand similar CD8alphaalphaNK1.1(+) T cells but not CD4(+) NKT cells from CD8alpha(+)beta(-) bone marrow cells cultured ex vivo with IL-15. These results indicate that the increased CD8alphaalphaNK1.1(+) T cells are not activated conventional CD8(+) T cells and do not arise from conventional CD8alphabeta precursors. CD8alphaalphaNK1.1(+) T cells produced very large amounts of IFN-gamma and degranulated upon TCR activation. These results suggest that high levels of IL-15 induce expansion or differentiation of a novel NK1.1(+) T cell subset, CD8alphaalphaNK1.1(+) T cells, and that IL-15-transgenic mice may be a useful resource for studying the functional relevance of CD8(+)NK1.1(+) T cells.     

Unusual T cell populations in adult murine bone marrow. Prevalence of CD3+CD4-CD8- and alpha beta TCR+NK1.1+ cells

The T cell populations present in normal murine bone marrow have not been previously analyzed in detail, mainly because of their relative rarity. In order to permit such analyses, bone marrow T cells were enriched by depleting Mac1-positive cells, which constitute 65 to 90% of bone marrow cells (BMC), and then studied by two-color flow cytometry. Analysis of the remaining cells revealed that the T cell profile of adult murine bone marrow is markedly different from that of other lymphoid organs. A very high proportion of bone marrow CD3+ cells (approximately one-third) are CD4-CD8-. CD3+CD4-CD8- cells are much more concentrated among BMC T cells than among thymocytes or splenic T cells, suggesting that bone marrow may be either a site of extrathymic TCR gene rearrangement, or a major site to which such cells home from the thymus. The expression of NK1.1 was also evaluated on Mac1-depleted BMC populations. Surprisingly, up to 39% of alpha beta TCR+ BMC were found to express NK1.1. Most alpha beta TCR+NK1.1+ BMC also expressed CD4 or CD8. NK1.1+ alpha beta TCR+ cells represented a much greater proportion of BMC T cells than of other lymphoid (splenocyte or thymocyte) T cell populations. Mac1-depleted BMC of nude mice contained very few cells with this phenotype. These results are consistent with the hypothesis that NK1.1+ alpha beta TCR+ cells are generated primarily in the thymus of normal animals and migrate preferentially to bone marrow, where they may function as regulatory elements in hematopoiesis.     

Cutting edge: CD4+ T cell control of CD8+ T cell reactivity to a model tumor antigen

Neoantigens resulting from the inherent genomic instability of tumor cells generally do not trigger immune recognition. Similarly, transfection of tumors with model Ags often fails to elicit CD8+ T cell responses or alter a tumor's growth rate or lethality. We report here that the adoptive transfer of activated Th1-type CD4+ T cells specific for a model tumor Ag results in the de novo generation of CD8+ T cells with specificity to that Ag and concomitant tumor destruction. The anti-tumor effects of the CD4+ T cells required the presence of both MHC class I and class II on host cells, as evidenced by experiments in knockout mice, suggesting that CD4+ T cells enhanced the ability of host APC to activate endogenous CD8+ T cells. These results indicate that the apparent inability of tumor cells expressing highly immunogenic epitopes to activate tumor-specific CD8+ T cells can be altered by activated CD4+ T cells.     

Alpha beta TCR+ cells are a minimal fraction of peripheral CD8+ pool in MHC class I-deficient mice

MHC class I molecules play a role in the maintenance of the naive peripheral CD8+ T cell pool. The mechanisms of the peripheral maintenance and the life span of residual CD8+ cells present in the periphery of beta 2-microglobulin-deficient (beta 2m-/-) mice are unknown. We here show that very few CD8+ cells in beta 2m-/- mice coexpress CD8 beta, a marker of the thymus-derived CD8+ T cells. Most of the CD8 alpha+ cells express CD11c and can be found in beta 2m/RAG-2 double-deficient mice, demonstrating that these cells do not require rearranged Ag receptors for differentiation and survival and may be of dendritic cell lineage. Rare CD8 alpha+CD8 beta+ cells can be detected following in vivo alloantigenic stimulation 2 wk after the adult thymectomy. Selective MHC class I expression by bone marrow-derived cells does not lead to an accumulation of CD8 beta+ cells in beta 2m-/- mice. These findings demonstrate that 1) thymic export of CD8+ T cells in beta 2m-/- mice is reduced more severely than previously thought; 2) non-T cells expressing CD8 alpha become prominent when CD8+ T cells are virtually absent; 3) at least some beta 2m-/- CD8+ T cells have a life span in the periphery comparable to wild-type CD8+ cells; and 4) similar ligands induce positive selection in the thymus and survival of CD8+ T cells in the periphery.     

Cross-positive selection of thymocytes expressing a single TCR by multiple major histocompatibility complex molecules of both classes: implications for CD4+ versus CD8+ lineage commitment

This study has investigated the cross-reactivity upon thymic selection of thymocytes expressing transgenic TCR derived from a murine CD8+ CTL clone. The Idhigh+ cells in this transgenic mouse had been previously shown to mature through positive selection by class I MHC, Dq or Lq molecule. By investigating on various strains, we found that the transgenic TCR cross-reacts with three different MHCs, resulting in positive or negative selection. Interestingly, in the TCR-transgenic mice of H-2q background, mature Idhigh+ T cells appeared among both CD4+ and CD8+ subsets in periphery, even in the absence of RAG-2 gene. When examined on beta2-microglobulin-/- background, CD4+, but not CD8+, Idhigh+ T cells developed, suggesting that maturation of CD8+ and CD4+ Idhigh+ cells was MHC class I (Dq/Lq) and class II (I-Aq) dependent, respectively. These results indicated that this TCR-transgenic mouse of H-2q background contains both classes of selecting MHC ligands for the transgenic TCR simultaneously. Further genetic analyses altering the gene dosage and combinations of selecting MHCs suggested novel asymmetric effects of class I and class II MHC on the positive selection of thymocytes. Implications of these observations in CD4+/CD8+ lineage commitment are discussed.     

Thymocyte antigens do not induce tolerance in the CD4+ T cell compartment.

Thymocytes fail to tolerize the developing T cell repertoire to self MHC class I (MHC I) Ags because transgenic (CD2Kb) mice expressing H-2Kb solely in lymphoid cell lineages reject skin grafts mismatched only for H-2Kb. In this study, we examined why thymocytes fail to tolerize the T cell repertoire to self MHC I Ags. The ability of CD2Kb mice to reject H-2Kb skin grafts was age dependent because CD2Kb mice older than 20 wk accepted skin grafts. T cells from younger CD2Kb mice proliferated, but did not develop cytotoxic functions in vitro in response to H-2Kb. Proliferative responses were dominated by H-2Kb-specific, CD4+ T cells rather than CD8+ T cells. Representative CD4+ T cell clones from CD2Kb mice were MHC II restricted and recognized processed H-2Kb. TCR transgenic mice were generated from one CD4+ T cell clone (361) to monitor development of H-2Kb-specific immature thymocytes when all thymic cells or lymphoid cell lineages only expressed H-2Kb. Thymocyte precursors were not eliminated and mice were not tolerant to H-2Kb when Tg361 TCR transgenic mice were intercrossed with CD2Kb mice. In contrast, all thymocyte precursors were eliminated efficiently in thymic microenvironments in which all cells expressed H-2Kb. We conclude that self MHC I Ags expressed exclusively in thymocytes do not induce T cell tolerance because presentation of processed self MHC I Ags on self MHC II molecules fails to induce negative selection of CD4+ T cell precursors. This suggests that some self Ags are effectively compartmentalized and cannot induce self-tolerance in the T cell repertoire.     

Requirement for efficient interactions between CD4 and MHC class II molecules for survival of resting CD4+ T lymphocytes in vivo and for activation-induced cell death

Regulation of homeostasis in the immune system includes mechanisms that promote survival of resting T lymphocytes, and others that control activation-induced cell death (AICD). In this study, we report on the use of a transgenic mouse model to test the role of CD4-MHC class II interactions for the susceptibility of CD4+ T lymphocytes to AICD, and for the survival of resting CD4+ T cells in peripheral lymphoid organs. The only I-Abeta gene expressed in these mice is an Abetak transgene with a mutation that prevents MHC class II molecules from interacting with CD4. We show increased apoptosis in CD4+ T lymphocytes derived from wild-type, but not from mutant Abetak transgenic mice following stimulation with staphylococcal enterotoxin A. Therefore, AICD may be impaired in CD4+ T cells derived from mutant Abetak transgenic mice. Importantly, we observed much higher apoptosis in resting CD4+ T cells from mutant Abetak transgenic mice than from wild-type mice. Furthermore, resting CD4+ T cells from mutant Abetak transgenic mice expressed higher levels of cell surface CD95 (Fas, APO-1). Ab-mediated cross-linking of CD95 further increased apoptosis in CD4+ T cells from mutant Abetak transgenic mice, but not from wild-type mice, suggesting apoptosis involved CD95 signaling. When cocultured with APC-expressing wild-type MHC class II molecules, apoptosis in resting CD4+ T lymphocytes from mutant Abetak transgenic mice was reduced. Our results show for the first time that interactions between CD4 and MHC class II molecules are required for the survival of resting CD4+ T cells in peripheral lymphoid organs.     

Heterogeneity of NK1.1+ T cells in the bone marrow: divergence from the thymus.

NK1.1+ T cells in the mouse thymus and bone marrow were compared because some marrow NK1.1+ T cells have been reported to be extrathymically derived. Almost all NK1.1+ T cells in the thymus were depleted in the CD1-/-, beta2m-/-, and Jalpha281-/- mice as compared with wild-type mice. CD8+NK1.1+ T cells were not clearly detected, even in the wild-type mice. In bone marrow from the wild-type mice, CD8+NK1.1+ T cells were easily detected, about twice as numerous as CD4+NK1.1+ T cells, and were similar in number to CD4-CD8-NK1.1+ T cells. All three marrow NK1.1+ T cell subsets were reduced about 4-fold in CD1-/- mice. No reduction was observed in CD8+NK1.1+ T cells in the bone marrow of Jalpha281-/- mice, but marrow CD8+NK1.1+ T cells were markedly depleted in beta2m-/- mice. All NK1.1+ T cell subsets in the marrow of wild-type mice produced high levels of IFN-gamma, IL-4, and IL-10. Although the numbers of marrow CD4-CD8-NK1.1+ T cells in beta2m-/- and Jalpha281-/- mice were similar to those in wild-type mice, these cells had a Th1-like pattern (high IFN-gamma, and low IL-4 and IL-10). In conclusion, the large majority of NK1.1+ T cells in the bone marrow are CD1 dependent. Marrow NK1.1+ T cells include CD8+, Valpha14-Jalpha281-, and beta2m-independent subsets that are not clearly detected in the thymus.     

Cytolytic mechanisms and expression of activation-regulating receptors on effector-type CD8+CD45RA+CD27- human T cells

Circulating CD8+ T cells with a CD45RA+CD27- phenotype resemble cytolytic effector cells because they express various cytolytic mediators and are able to execute cytotoxicity without prior stimulation in vitro. We here demonstrate that CD8+CD45RA+CD27- T cells can use both granule exocytosis and Fas/Fas ligand pathways to induce apoptosis in target cells. The availability of these cytolytic mechanisms in circulating T cells suggests that the activity of these cells must be carefully controlled to prevent unwanted tissue damage. For this reason, we analyzed the expression of surface receptors that either enhance or inhibit T cell function. Compared with memory-type cells, effector cells were found to express normal levels of CD3epsilon and TCRzeta and relatively high levels of CD8. CTLA-4 was absent from freshly isolated effector cells, whereas a limited number of unstimulated memory cells expressed this molecule. In line with recent findings on CD8+CD28- T cells, CD45RA+CD27- T cells were unique in the abundant expression of NK cell-inhibitory receptors, both of Ig superfamily and C-type lectin classes. Binding of NK cell-inhibitory receptors to classical and nonclassical MHC class I molecules may inhibit the activation of the cytolytic machinery induced by either Ag receptor-specific or nonspecific signals in CD8+CD45RA+CD27- T cells.     

Induction of tumor immunity by removing CD25+CD4+ T cells: a common basis between tumor immunity and autoimmunity.

This study shows that removal of a T cell subpopulation can evoke effective tumor immunity in otherwise nonresponding animals. Elimination of CD25-expressing T cells, which constitute 5-10% of peripheral CD4+ T cells in normal naive mice, elicited potent immune responses to syngeneic tumors in vivo and eradicated them. The responses were mediated by tumor-specific CD8+ CTLs and tumor-nonspecific CD4-8- cytotoxic cells akin to NK cells. Furthermore, in vitro culture of CD25+4+ T cell-depleted splenic cell suspensions prepared from tumor-unsensitized normal mice led to spontaneous generation of similar CD4-8- cytotoxic cells capable of killing a broad spectrum of tumors; reconstitution of CD25+4+ T cells inhibited the generation. In this culture, self-reactive CD25-4+ T cells responding to self peptides/class II MHC complexes on APCs spontaneously proliferated upon removal of CD25+4+ T cells, secreting large amounts of IL-2. The IL-2 thus produced appeared to be responsible for the generation of CD4-8- NK cells as lymphokine-activated killer cells, because direct addition of an equivalent amount of IL-2 to the culture of CD4-8- cells generated similar lymphokine-activated killer/NK cells, whereas coculture of normal CD4-8- cells with CD25-4+ T cells from IL-2-deficient mice did not. Thus, removal of immunoregulatory CD25+4+ T cells can abrogate immunological unresponsiveness to syngeneic tumors in vivo and in vitro, leading to spontaneous development of tumor-specific effector cells as well as tumor-nonspecific ones. This novel way of evoking tumor immunity would help to devise effective immunotherapy for cancer in humans.     

Some cloned murine CD4+ T cells recognize H-2Ld class I MHC determinants directly. Other cloned CD4+ T cells recognize H-2Ld class I MHC determinants in the context of class II MHC molecules.

Murine T lymphocytes recognize nominal Ag presented by class I or class II MHC molecules. Most CD8+ T cells recognize Ag presented in the context of class I molecules, whereas most CD4+ cells recognize Ag associated with class II molecules. However, it has been shown that a proportion of T cells recognizing class I alloantigens express CD4 surface molecules. Furthermore, CD4+ T cells are sufficient for the rejection of H-2Kbm10 and H-2Kbm11 class I disparate skin grafts. It has been suggested that the CD4 component of an anti-class I response can be ascribed to T cells recognizing class I determinants in the context of class II MHC products. To examine the specificity and effector functions of class I-specific HTL, CD4+ T cells were stimulated with APC that differed from them at a class I locus. Specifically, a MLC was prepared involving an allogeneic difference only at the Ld region. CD4+ clones were derived by limiting dilution of bulk MLC cells. Two clones have been studied in detail. The CD4+ clone 46.2 produced IL-2, IL-3, and IFN-gamma when stimulated with anti-CD3 mAb, whereas the CD4+ clone 93.1 secreted IL-4 in addition to IL-2, IL-3, and IFN-gamma. Cloned 46.2 cells recognized H-2Ld directly, whereas recognition of Ld by 93.1 apparently was restricted by class II MHC molecules. Furthermore, cytolysis by both clones 46.2 and 93.1 was inhibited by the anti-CD4 mAb GK1.5. These results demonstrate that CD4+ T cells can respond to a class I difference and that a proportion of CD4+ T cells can recognize class I MHC determinants directly as well as in the context of class II MHC molecules.     

Murine CD160, Ig-like receptor on NK cells and NKT cells, recognizes classical and nonclassical MHC class I and regulates NK cell activation

Human and mouse NK cells use different families of receptors to recognize MHC class I (MHC I) on target cells. Although human NK cells express both Ig-like receptors and lectin-like receptors specific for MHC I, all the MHC I-specific receptors identified on mouse NK cells to date are lectin-like receptors, and no Ig-like receptors recognizing MHC I have been identified on mouse NK cells. In this study we report the first MHC I-specific Ig-like receptor on mouse NK cells, namely, murine CD160 (mCD160). The expression of mCD160 is restricted to a subset of NK cells, NK1.1+ T cells, and activated CD8+ T cells. The mCD160-Ig fusion protein binds to rat cell lines transfected with classical and nonclassical mouse MHC I, including CD1d. Furthermore, the level of mCD160 on NK1.1+ T cells is modulated by MHC I of the host. Overexpression of mCD160 in the mouse NK cell line KY-2 inhibits IFN-gamma production induced by phorbol ester plus ionomycin, whereas it enhances IFN-gamma production induced by NK1.1 cross-linking or incubation with dendritic cells. Cross-linking of mCD160 also inhibits anti-NK1.1-mediated stimulation of KY-2 cells. Anti-mCD160 mAb alone has no effect. Thus, mCD160, the first MHC I-specific Ig-like receptor on mouse NK cells, regulates NK cell activation both positively and negatively, depending on the stimulus.     

CD8+ T cell protective immunity against Chlamydia pneumoniae includes an H2-M3-restricted response that is largely CD4+ T cell-independent

CD8+ T cells are important for immunity to the intracellular bacterial pathogen Chlamydia pneumoniae (Cpn). Recently, we reported that type 1 CD8+ (Tc1) from Cpn-infected B6 mice recognize peptides from multiple Cpn Ags in a classical MHC class Ia-restricted fashion. In this study, we show that Cpn infection also induces nonclassical MHC class Ib-(H2-M3)-restricted CD8+ T cell responses. H2-M3-binding peptides representing the N-terminal formylated sequences from five Cpn Ags sensitized target cells for lysis by cytolytic effectors from the spleens of infected B6 mice. Of these, only peptides fMFFAPL (P1) and fMLYWFL (P4) stimulated IFN-gamma production by infection-primed splenic and pulmonary CD8+ T cells. Studies with Cpn-infected Kb-/-/Db-/- mice confirmed the Tc1 cytokine profile of P1- and P4-specific CD8+ T cells and revealed the capacity of these effectors to exert in vitro H2-M3-restricted lysis of Cpn-infected macrophages and in vivo pulmonary killing of P1- and P4-coated splenocytes. Furthermore, adoptive transfer of P1- and P4-specific CD8+ T cells into naive Kb-/-/Db-/- mice reduced lung Cpn loads following challenge. Finally, we show that in the absence of MHC class Ia-restricted CD8+ T cell responses, CD4+ T cells are largely expendable for the control of Cpn growth, and for the generation, memory maintenance, and secondary expansion of P1- and P4-specific CD8+ T cells. These results suggest that H2-M3-restricted CD8+ T cells contribute to protective immunity against Cpn, and that chlamydial Ags presented by MHC class Ib molecules may represent novel targets for inclusion in anti-Cpn vaccines.