Separately expressed T cell receptor α and β chain transgenes exert opposite effects on T cell differentiation and neoplastic transformation

Two aspects of T cell differentiation in T cell receptor (TCR)-transgenic mice, the generation of an unusual population of CD4^?CD8^?TCR⁺ thymocytes and the absence of γδ cells, have been the focus of extensive investigation. To examine the basis for these phenomena, we investigated the effects of separate expression of a transgenic TCR α chain and a transgenic TCR β chain on thymocyte differentiation. Our data indicate that expression of a transgenic TCR α chain causes thymocytes to differentiate into a CD4^?CD8^?TCR⁺ lineage at an early developmental stage, depleting the number of thymocytes that differentiate into the αβ lineage. Surprisingly, expression of the TCR α chain transgene is also associated with the development of T cell lymphosarcoma. In contrast, expression of the transgenic TCR β chain causes immature T cells to accelerate differentiation into the αβ lineage and thus inhibits the generation of γδ cells. Our observations provide a model for understanding T cell differentiation in TCR-transgenic mice.     

Secondary but not primary T cell responses are enhanced in CTLA-4-deficient CD8+ T cells

Negative as well as positive co-stimulation appears to play an important role in controlling T cell activation. CTLA-4 has been proposed to negatively regulate T cell responses. CTLA-4-deficient mice develop a lymphoproliferative disorder, initiated by the activation and expansion of CD4⁺ T cells. To assess the function of CTLA-4 on CD8⁺ T cells, CTLA-4^−/- animals were crossed to an MHC class I-restricted 2C TCR transgenic mouse line. We demonstrate that although the primary T cell responses were similar, the CTLA-4-deficient 2C TCR⁺ CD8⁺ T cells displayed a greater proliferative response upon secondary stimulation than the 2C TCR⁺ CD8⁺ T cells from CTLA-4 wild-type mice. These results suggest that CTLA-4 regulates antigen-specific memory CD8⁺ T cell responses.     

T lymphocyte development in the absence of Fcϵ receptor Iγ subunit: analysis of thymic-dependent and independent αβ and γδ pathways

During fetal development, early thymocyte progenitors transiently express low affinity Fc receptors for IgG (FcγR) of both FcγRII and III isoforms. Only the FcγRIII isoform requires association of an FcγRIII (CD16) α subunit with an FcϵRIγ homodimer for surface expression. To address the role of FcγR in ontogeny, we studied thymic development in FcϵRIγ^−/− mice. We find that day 14.5 CD4⁻CD8⁻ double-negative (DN) fetal thymocytes of FcϵRIγ^−/− mice express mRNA of both FcγRIIb₁ and FcγRIII. Surface expression of FcγRII/III is readily detected on these cells. It appears that FcγRIIb₁, whose surface expression is FcϵRIγ independent, replaces FcγRIII during thymic development in these animals. Moreover, subsequent development into CD4⁺CD8⁺ double-positive and CD4⁺CD8⁻ and CD4⁻CD8⁺ single-positive subsets appears normal even in the absence of FcϵRIγ. However, alterations were noted in adult animals among the DN αβ TCR⁺ thymocytes and peripheral splenic DN T cells as well as CD8αα⁺ intestinal intraepithelial lymphocytes (iIEL). In contrast to conventional T lymphocytes, which do not express either FcγRIII or FcϵRIγ, DN αβ TCR⁺ thymocytes and extrathymically derived αβ TCR⁺ and γδ TCR⁺ CD8αα⁺β⁻ iIEL express TCR which incorporate FcϵRIγ as one of their subunits. Consistent with this, the TCR levels of these cells are lower than the TCR levels on cells from wild-type C57BL/6 mice. Despite the reduction in the level of surface TCR, the development of these cells was unaltered by the absence of FcϵRIγ. Thus, we observed alterations in adult DN αβ TCR⁺ thymocytes, splenic DN αβ TCR⁺ and DN γδ TCR⁺ large granular lymphocytes (LGL), and αβ TCR⁺ and γδ TCR⁺ CD8αα⁺β⁻ iIEL, but no detectable changes in their major fetal thymic developmental pathways. Cultivation of peripheral DN αβ TCR⁺ and DN γδ TCR⁺ cells from FcϵRIγ^−/− mice with interleukin-2 generates LGL which mediate natural killer activity. Unlike LGL from wild-type C57BL/6 mice, LGL from FcϵRIγ^−/− mice lack FcγRIII expression and could not mediate antibody-dependent cellular cytotoxicity through FcγRIII.     

CD5− CD8αβ intestinal intraepithelial lymphocytes (IEL) are induced to express CD5 upon antigen-specific activation: CD5− and CD5+ CD8αβ IEL do not represent separate T cell lineages

We followed αβ T cell receptor (TCR) usage in subsets of gut intraepithelial lymphocytes (IEL) in major histocompatibility complex class I-restricted αβ TCR-transgenic (tg) mice. The proportion of tg αβ TCR⁺ CD8αβ IEL is reduced compared with CD8⁺ splenocytes of the same animal, particularly under conventional conditions of maintenance. Further fractionation of CD8αβ IEL according to the expression level of surface CD5 revealed that in conventionally housed animals tg TCR⁺ CD5^? CD8αβ IEL are as frequent as in specific pathogen-free (SPF) mice, whereas tg TCR⁺ CD5^int or, even more pronounced, tg TCR⁺ CD5^hi CD8αβ IEL are greatly diminished when compared with mice kept under SPF conditions. Upon antigen-specific stimulation of CD5^? CD8αβ IEL in vitro, CD5 surface expression is up-regulated on a large fraction of cells within 48 h. Up-regulation of CD5 surface expression is further enhanced by the presence of the anti-αIEL monoclonal antibody 2E7. This clearly demonstrates that CD5^?, and CD5⁺ CD8αβ IEL cannot be considered as separate T cell lineages.     

Recovery from Mouse Hepatitis Virus Infection Depends on Recruitment of CD8 Cells Rather Than Activation of Intrahepatic CD4αβTCR or NK-T Cells

Mouse hepatitis virus (MHV) provides an excellent animal model for the study of the immunopathological mechanisms involved in hepatic viral diseases. We previously generated an attenuated viral variant, YAC-MHV3, which induces a subclinical disease and recovery within 15 days. In contrast, the L2-MHV3 strain induces the development of a fulminant hepatitis, leading to death within 3 days. In this paper, we document intrahepatic and splenic T cell subpopulations involved in the hepatitis process and viral elimination identified in attenuated or pathogenic MHV3-infected C57BL/6 mice. Percentages of intrahepatic CD4⁺ cells decreased in attenuated YAC-MHV3-infected mice, while they increased in mice infected with pathogenic L2-MHV3, compared with uninfected animals. Moreover, in YAC-MHV3-infected mice, the percentages of intrahepatic CD8⁺ cells slightly decreased at 24 h pi, then increased until 15 days pi. In contrast, the CD4/CD8 ratios of splenic lymphoid subpopulations increased in the first days of infection and returned to normal values at 15 days pi. Intrahepatic NK1.1⁺αβ − TCR^inter cells decreased in both virally infected groups of mice, while CD4⁺αβ − TCR^inter LFA-1^high cells increased in L2-MHV3-infected mice, in contrast with what was seen in YAC-MHV3-infected mice. However, these cells became anergic following Con A or PHA stimulation. Ex vivo studies showed that only the intrahepatic CD8⁺ cells that were increased in YAC-MHV3-infected mice could be stimulated by lectins. In addition, in vitro viral infections revealed that L2-MHV3 viral infection led to an increase of intrahepatic CD4⁺αβ − TCR^inter cells in the absence of CD8⁺ cells only. These results indicate that the attenuated phenotype of the YAC-MHV3 virus is related to two different mechanisms: the first involves no increase of intrahepatic CD4⁺αβ − TCR^inter or NK-T cells, while the second favors the recruitment and activation of CD8⁺ cells in liver. The results are discussed in relation to the integrity of intrahepatic immune tolerance mechanisms and immune-mediated viral elimination.     

Interleukin‐4‐induced loss of CD8 expression and cytolytic function in effector CD8 T cells persists long term in vivo

Activation of naive CD8⁺ T cells in the presence of interleukin‐4 modulates their CD8 co‐receptor expression and functional differentiation, resulting in the generation of CD8^low cells that produce type 2 cytokines and display poor cytolytic and anti‐tumour activity. Although this CD8^low phenotype becomes stable after about a week and can persist with further stimulation in vitro, it is not known whether it can be maintained long term in vivo. Here we report that CD8^low cells derived from oval‐bumin_257–264‐specific T‐cell receptor‐transgenic CD8⁺ T cells activated in the presence of interleukin‐4 could be detected in the spleen for at least 4 months after adoptive transfer into normal mice. A significant proportion of the long‐term surviving cells retained their CD8^low phenotype in vivo and after clonal re‐activation in vitro. Although long‐term surviving CD8^low cells lacked detectable cytolytic activity or perforin expression, they showed some anti‐tumour function in vivo. The persistence of functional cells with a CD8^low phenotype in vivo raises the possibility that such cells can contribute to effector or regulatory responses to tumours or pathogens.     

Existence of a small population of IL-2Rβhi TCRint cells in SCG and MRL-lpr/lpr mice which produce normal Fas mRNA and Fas molecules from the lpr gene

Mice carrying the lpr gene, SCG and MRL-lpr/lpr mice, were used to characterize the phenotype and lpr gene of abnormally proliferating T cells in these mice. A major population which expanded in these mice were T cells expressing intermediate (int) levels of T cell receptor (TCR) (and CD3) and the phenotype of interleukin-2 receptor (IL-2R)β^lo α^? (possibly abnormal TCR^int cells). The levels of TCR^hi cells of thymic origin (generated through the mainstream of T cell differentiation in the thymus) profoundly decreased after the onset of disease. However, a small population of normal TCR^int cells (i.e. IL-2Rβ^hi α^?) were also found to exist in all tested organs. For example, the majority of abnormal IL-2Rβ^lo TCR^int cells were CD4^?8^? CD2^?, while normal IL-2Rβ^hi TCR^int cells were a mixture of single-positive cells (mainly CD8⁺), CD4^?8^? cells and CD2⁺ cells. Moreover, normal TCR^int cells preferentially produced normal Fas mRNA and Fas molecules from the lpr gene. This phenomenon explains the leaky appearance of normal Fas mRNA and Fas molecules in mice carrying the lpr gene. It is suggested that a small population of IL-2Rβ^hiTCR^int cells are resistant to the lpr genetic abnormality.     

Small CD4+8+TCRlow thymocytes contain precursors of mature T cells

To evaluate directly the developmental potential of cortical CD4⁺8⁺ thymocytes, highly purified populations of small, nondividing CD4⁺8⁺TCR^low and large, dividing CD4⁺8⁺TCR^high thymocytes from H-2^d mice expressing a transgenic T cell receptor restricted by H-2D^b (major histocompatibility complex class I) molecules were transferred into the thymus of normal, nonirradiated H-2^b recipient mice. The results show that both populations generate CD4^?8⁺ thymocytes under these conditions, thus providing conclusive evidence that small cortical thymocytes do not represent a “dead end” but an important intermediate stage in T cell development.     

CD4+ Th1 cells promote CD8+ Tc1 cell survival, memory response, tumor localization and therapy by targeted delivery of interleukin 2 via acquired pMHC I complexes

The cooperative role of CD4⁺ helper T (Th) cells has been reported for CD8⁺ cytotoxic T (Tc) cells in tumor eradication. However, its molecular mechanisms have not been well elucidated. We have recently demonstrated that CD4⁺ Th cells can acquire major histocompatibility complex/peptide I (pMHC I) complexes and costimulatory molecules by dendritic cell (DC) activation, and further stimulate naïve CD8⁺ T cell proliferation and activation. In this study, we used CD4⁺ Th1 and CD8⁺ Tc1 cells derived from ovalbumin (OVA)-specific T cell receptor (TCR) transgenic OT II and OT I mice to study CD4⁺ Th1 cell''s help effects on active CD8⁺ Tc1 cells and the molecular mechanisms involved in CD8⁺ Tc1-cell immunotherapy of OVA-expressing EG7 tumors. Our data showed that CD4⁺ Th1 cells with acquired pMHC I by OVA-pulsed DC (DC_OVA) stimulation are capable of prolonging survival and reducing apoptosis formation of active CD8⁺ Tc1 cells in vitro, and promoting CD8⁺ Tc1 cell tumor localization and memory responses in vivo by 3-folds. A combined adoptive T-cell therapy of CD8⁺ Tc1 with CD4⁺ Th1 cells resulted in regression of well-established EG7 tumors (5 mm in diameter) in all 10/10 mice. The CD4⁺ Th1’s help effect is mediated via the helper cytokine IL-2 specifically targeted to CD8⁺ Tc1 cells in vivo by acquired pMHC I complexes. Taken together, these results will have important implications for designing adoptive T-cell immunotherapy protocols in treatment of solid tumors.     

Early Deletion and Late Positive Selection of T-Cells Expressing a Male-Specific Receptor in T-Cell Receptor Transgenic Mice

The ontogeny of T cells in T-cell receptor (TCR) transgenic mice, which express a transgenic αβ heterodimer, specific for the male (H-Y) antigen in association with H-2D^b, was determined. The transgenic α chain was expressed on about 10% of the fetal thymocytes on day 14 of gestation. About 50% of day-15 fetal thymocytes expressed both α and β transchains and virtually all fetal thymocytes expressed the transgenicαβ heterodimer by day 17. The early expression of the transgenic TCR on CD4^-8^- thymocytes prevented the development of γδ cells, and led to accelerated growth of thymocytes and an earlier expression of CD4 and CD8 molecules. Up to day 17, no significant differences in T-cell development could be detected between female and male thymuses. By day 18 of gestation, the male transgenic thymus contained more CD4^-8^- thymocytes than the female transgenic thymus. The preponderance of CD4^-8^- thymocytes in the male transgenic thymus increased until birth and was a consequence of the deletion of the CD4⁺8⁺ thymocytes and their CD4^-8⁺ precursors. By the time of birth, the male transgenic thymus contained half the number of cells as the female transgenic thymus. The deletion of autospecific precursor cells in the male transgenic mouse began only at day 18 of gestation, despite the fact that the ligand could already be detected by day 16.The preferential accumulation of CD4^-8⁺ T cells, which expressed a high density of the transgenic TCR, occurred only after birth and was .obvious in 6-week-old female thymus. These data support the hypothesis that the positive selection of T cells expressing this transgenic heterodimer may involve two steps, i.e., the commitment of CD4⁺8⁺ thymocytes to the CD4^-8⁺ lineage following the interaction of the transgenic TCR with restricting major histocompatibility molecules, followed by a slow conversion of CD4⁺8⁺ thymocytes into CD4^-8⁺ T cells.In normal mice, the precursors of CD⁺4⁺8 and single positive thymocytes have the CD4^-8^- CD3^-J11d⁺ (or M1/69 ⁺) phenotype. Because of the early expression of the transgenic αβ heterodimer, this population was not detected in adult transgenic mice. All CD4^-8^- M1/ 69⁺ cells expressed the transgenic receptor associated with CD3 and could be readily grown in media containing T-cell lectins and interleukin 2.     

Self-Reactivity and the Expression of Memory Markers Vary Independently in MRL-Mp+/+ and MRL-Mp-lpr/lpr Mice

MRL-Mp-lpr/lpr mice contain phenotypically abnormal populations of T cells, and exhibit an SLE-like autoimmune disease in which autoantibodies are a prominent feature. We analyzed the phenotype and T-cell receptor Vß expression pattern in CD4⁺ T cells of this mutant mouse strain to detect abnormalities that could explain the autoimmunity. The CD4⁺ T cells contain two distinct abnormal populations. One of these expresses B220 and HSA, and in these and other respects closely resembles the accumulating CD4^–CD8^– population. The other expresses a high level of CD44 (Pgp-1), and a high level of the 16A epitope of CD45, and so resembles post-activation T cells. Both of these cell types are exclusive to MRL-Mp-lpr/lpr. We also identified V ß5- and V ß11-positive CD4+ T cells, in both MRL-Mp-lpr/lpr and MRL-Mp-+/+ mice. We conclude that autoimmune T cells can be detected in these mice, but that they are not the cause of the accumulation of abnormal CD4⁺ and CD4^–CD8^–cells.     

Phenotypic Characterization of Splenic T Cells from Mice Infected with Plasmodium chabaudi chabaudi

T cells from spleens of mice infected with the erythrocytic stages of Plasmodium chabaudi chabaudi have been analysed with respect to their expression of surface molecules CD3, CD4 and CDS and T-cell receptor (TCR)αβ and γδ. The majority of T cells from infected mice were αβTCR ⁺. However, there was an increase of approximately 8–10-fold in the proportion and total number of γδ T cells. Immunocytochemical analysis of sections of spleens taken from infected C57BL/6 mice during a primary infection showed that this increase took place particularly in the non-lymphoid areas. Within the αβ TCR⁺ T-cell population, both CD4⁺ T cells and CD8⁺ T cells were represented in proportions similar to those observed in normal uninfected mice. Stimulation of splenic T cells from infected mice with P. chabaudi-infected erythrocytes in vitro resulted ina blasted cell population composed predominantly of αβTTCR⁺ T cells with no preferential expansion of γβTCR⁺ T cells. There was no evidence of superantigen-like stimulation of T cells bearing particular Vβ chains of the TCR. The representation of the different Vβ chains within the population was not significantly different from that seen in uninfected mice.     

IL‐15 is critical for the maintenance and innate functions of self‐specific CD8+ T cells

IL‐15 is a pleiotropic cytokine involved in host defense as well as autoimmunity. IL‐15‐deficient mice show a decrease of memory phenotype (MP) CD8⁺ T cells, which develop naturally in naïve mice and whose origin is unclear. It has been shown that self‐specific CD8⁺ T cells developed in male H‐Y antigen‐specific TCR transgenic mice share many similarities with naturally occurring MP CD8⁺ T cells in normal mice. In this study, we found that H‐Y antigen‐specific CD8⁺ T cells in male but not female mice decreased when they were crossed with IL‐15‐deficient mice, mainly due to impaired peripheral maintenance. The self‐specific TCR transgenic CD8⁺ T cells developed in IL‐15‐deficient mice showed altered surface phenotypes and reduced effector functions ex vivo. Bystander activation of the self‐specific CD8⁺ T cells was induced in vivo during infection with Listeria monocytogenes, in which proliferation but not IFN‐γ production was IL‐15‐dependent. These results indicated important roles for IL‐15 in the maintenance and functions of self‐specific CD8⁺ T cells, which may be included in the naturally occurring MP CD8⁺ T‐cell population in naïve normal mice and participate in innate host defense responses.     

T cell development and repertoire of mice expressing a single T cell receptor α chain

We examined T cell development and T cell repertoire in transgenic mice expressing a single T cell receptor (TCR) α chain derived from the H-2D^b -lymphocytic choriomeningitis virus (LCMV)-specific cytolytic T lymphocyte (CTL) clone P14. To generate these α P14 mice, mice transgenic for the P14 TCR α chain were backcrossed to TCR α-deficient mice. Thymi from α P14 mice exhibited a marked decrease of mature CD4⁺8^? and CD8⁺4^? single-positive thymocytes comparable to thymi from TCR α-deficient mice. Correspondingly, the number of peripheral T cells was reduced in the CD4 (tenfold) and in the CD8 (twofold) subsets when compared to normal mice. T cells from α P14 mice generated a primary anti-LCMV CTL response when stimulated in vitro with LCMV in contrast to normal mice which require priming in vivo; elimination of LCMV in vivo was, however, not improved. Flow cytometric analysis of T cells with Vβ-specific antibodies showed a diverse endogenous TCR Vβ repertoire. Functional analysis of the T cell repertoire, however, revealed a strongly reduced (30-fold) allogeneic and the absence of a vesicular stomatitis virus-specific CTL response and an impaired ability to provide T cell help for antibody isotype switching. Thus, T cell selection in the thymus was impaired and the T cell repertoire was limited in mice expressing only one type of TCR α chain.     

Analysis of the inflammatory response in HY-TCR transgenic mice highlights the pathogenic potential of CD4− CD8− T cells

Transgenic mice expressing a rearranged T cell receptor (TCR)-αβ prematurely at the double-negative stage develop an abnormal population of peripheral T cells that lack CD4 and CD8 expression and are hyper-reactive to anti-TCR antibody stimulation. One such example is the HY-TCR transgenic mice. These mice express a TCR transgenic specific for the HY antigen that is expressed in male but not in female mice. As a result, male mice have an abnormal population of HY⁺/CD4^? 8^? or HY⁺/CD4^? CD8^low T cells that are much lower in female mice. In this study, we investigated the potential patho/physiological function of these cells in vivo using a model of delayed-type hypersensitivity (DTH) reaction: the λ-carrageenan-induced paw edema. Interestingly, while both male and female HY-TCR mice develop a classical biphasic inflammatory response to λ-carrageenan, the degree of inflammation in the former was much higher than that in the latter. This was accompanied by a selective expansion of HY⁺/CD4^? 8^? and HY⁺/CD4^? CD8^low T cells in male mice and by a markedly increased production of typical DTH cytokines compared with cells from female mice. These results were specific since analysis of the inflammatory response of HY-TCR transgenic mice subjected to zymosan-induced peritonitis showed no differences between male and female mice. Together, these findings provide novel evidence for the pathological role of self-reactive CD4^? CD8^? T cells, previously described in several autoimmune strains and recently identified in patients suffering from autoimmune diseases such as systemic lupus erythematosus.     

Gender-Related Differences in the Rates of Age Associated Thymic Atrophy

Age associated thymic atrophy has been shown to be linked to problems with rearrangement of the β chain of the T cell receptor (TCR) in male mice during the early phases of the intrathymic T cell developmental pathway. In this study, thymic atrophy in female mice was found to occur at a different rate than in male mice. At 9 months of age there was a significantly greater number of cells in the thymus of female mice compared with male mice, with the major difference found in the CD4⁺CD8⁺ populations. The thymii of female mice at 9 months of age contained double the number of these cells compared with male mice. Analysis of the CD4⁺CD8⁺ cells at 9 months of age demonstrated increased numbers of cells expressing higher levels of CD3 in females compared with males indicating that in females more of these cells were producing successful αβTCR pairings. In F5 transgenic mice comparison of the CD4⁺CD8⁺ population revealed no significant difference in their absolute numbers at 9 months of age. These results indicate that the gender differences at this time point were due to fewer permitted divisions prior to the expression of a selectable TCR α chain within the CD4⁺CD8⁺ populations in male compared with female mice. This gender difference was not due to the action of testosterone and unlikely to be due to differences in the level of oestrogen. The potential mechanisms of this difference may be related to a regulatory feedback of peripheral T cells on the developing thymocyte populations. Such age related changes in the numbers of cells within distinct thymic subpopulations leads to the possibility that the potential repertoire in females is greater than in males later in life.     

Active CD4+ helper T cells directly stimulate CD8+ cytotoxic T lymphocyte responses in wild-type and MHC II gene knockout C57BL/6 mice and transgenic RIP-mOVA mice expressing islet β-cell ovalbumin antigen leading to diabetes

CD4⁺ helper T (Th) cells play crucial role in priming, expansion and survival of CD8⁺ cytotoxic T lymphocytes (CTLs). However, how CD4⁺ Th cell's help is delivered to CD8⁺ T cells in vivo is still unclear. We previously demonstrated that CD4⁺ Th cells can acquire ovalbumin (OVA) peptide/major histocompatibility complex (pMHC I) and costimulatory CD80 by OVA-pulsed DC (DC_OVA) stimulation, and then stimulate OVA-specific CD8⁺ CTL responses in C57BL/6 mice. In this study, we further investigated CD4⁺ Th cell's effect on stimulation of CD8 CTL responses in major histocompatibility complex (MHC II) gene knockout (KO) mice and transgenic rat insulin promoter (RIP)-mOVA mice with moderate expression of self OVA by using CD4⁺ Th cells or Th cells with various gene deficiency. We demonstrated that the in vitro DC_OVA-activated CD4⁺ Th cells (3 × 10⁶ cells/mouse) can directly stimulate OVA-specific CD8⁺ T-cell responses in wild-type C57BL/6 mice and MHC II gene KO mice lacking CD4⁺ T cells. A large amount of CD4⁺ Th cells (12 × 10⁶ cells/mouse) can even overcome OVA-specific immune tolerance in transgenic RIP-mOVA mice, leading to CD8⁺ CTL-mediated mouse pancreatic islet destruction and diabetes. The stimulatory effect of CD4⁺ Th cells is mediated by its IL-2 secretion and CD40L and CD80 costimulations, and is specifically delivered to OVA-specific CD8⁺ T cells in vivo via its acquired pMHC I complexes. Therefore, the above elucidated principles for CD4⁺ Th cells will have substantial implications in autoimmunity and antitumor immunity, and regulatory T-cell-dependent immune suppression.     

CD4-negative cytotoxic T cells with a T cell receptor α/βintermediate expression in CD8-deficient mice

Targeted disruption of the CD8 gene results in a profound block in cytotoxic T cell (CTL) development. Since CTL are major histocompatibility complex (MHC) class I restricted, we addressed the question of whether CD8–/– mice can reject MHC class I-disparate allografts. Studies have previously shown that skin allografts are rejected exclusively by T cells. We therefore used the skin allograft model to answer our question and grafted CD8–/– mice with skins from allogeneic mice deficient in MHC class II or in MHC class I (MHC-I or MHC-II-disparate, respectively). CD8–/– mice rejected MHC-I-disparate skin rapidly even if they were depleted of CD4⁺ cells in vivo (and were thus deficient in CD4⁺ and CD8⁺ T cells). By contrast, CD8+/+ controls depleted of CD4⁺ and CD8⁺ T cells in vivo accepted the MHC-I-disparate skin. Following MHC-I, but not MHC-II stimulation, allograft-specific cytotoxic activity was detected in CD8–/– mice even after CD4 depletion. A population expanded in both the lymph nodes and the thymus of grafted CD8–/– animals which displayed a CD4^?8^?3^intermediateTCRα/β^intermediate phenotype. Indeed its T cell receptor (TCR) density was lower than that of CD4⁺ cells in CD8–/– mice or of CD8⁺ cells in CD8+/+ mice. Our data suggest that this CD4^?8^?T cell population is responsible for the CTL function we have observed. Therefore, MHC class I-restricted CTL can be generated in CD8–/– mice following priming with MHC class I antigens in vivo. The data also suggest that CD8 is needed to up-regulate TCR density during thymic maturation. Thus, although CD8 plays a major role in the generation of CTL, it is not absolutely required.