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.     

The repertoire of CD4+ CD28- T cells in rheumatoid arthritis.

BACKGROUND: While oligoclonality of circulating CD4- CD8 and of CD8+ T cells is not uncommon, clonal dominance within the CD4 compartment is not frequently found in healthy individuals. In contrast, the majority of patients with rheumatoid arthritis (RA) have clonally expanded CD4+ T cell populations. Previous studies have demonstrated that these clonogenic CD4+ T cells do not express the CD28 molecule. To examine the correlation between CD28 expression and clonal proliferation, we have analyzed the T cell receptor (TCR) diversity of CD4+ CD28- T cells in normal individuals and in RA patients. MATERIAL AND METHODS: The size of the peripheral blood CD4+ CD28- compartment was determined in 30 healthy individuals and 30 RA patients by two-color FACS analysis. In 10 RA patients and five controls with more than 2.5% CD4+ CD28- T cells, TCR BV gene segment usage was analyzed with 19 BV-specific antibodies. Oligoclonality was assessed in sorted CD4+ CD28+ and CD28- T cells using TCR BV-BC-specific polymerase chain reaction and size fractionation. Clonal dominance was confirmed by direct sequencing. RESULTS: The CD4+ CD28- T cell compartment was expanded to more than 2.5% in 70% of the RA patients and 30% of the normal individuals. Compared with the CD4+ CD28+ T cells, the TCR BV gene segment usage among CD4+ CD28- cells was grossly skewed with the dominance of single BV elements. Molecular TCR analysis provided evidence for oligoclonality in 17 of 21 expanded BV elements. In two unrelated RA patients who shared both HLA-DRB1 alleles, the TCR beta-chain sequences of dominant clonotypes were highly conserved. CONCLUSIONS: Oligoclonality is a characteristic feature of CD4+ CD28- T cells which are expanded in some healthy individuals and in the majority of RA patients. The lack of CD28 expression is a common denominator of CD4+, CD8+, and CD4- CD8- T cells prone to develop clonal dominance. The limited TCR diversity of clonal CD4+ CD28- populations in RA patients suggests that these T cells recognize a limited spectrum of antigens. The fact that the majority of individuals with marked expansions and oligoclonality of CD4+ CD28- T cells are RA patients suggests a role for these unusual lymphocytes in the pathogenetic events leading to RA.     

CD4-CD8- human T cells: phenotypic heterogeneity and activation requirements of freshly isolated "double-negative" T cells

In the present study we have analyzed the in vitro activation requirements of freshly isolated CD4-CD8- "double-negative" (DN) human peripheral blood T cells. DN cells were isolated from E+ cells by removal of CD4+, CD8+, and CD16+ cells through consecutive steps of C'-mediated lysis and panning. While the majority (79.0 +/- 12.0%) of DN cells were TCR gamma delta+ as shown by staining with mAb TCR delta-1, a minor fraction (6.7 +/- 4.7%) expressed TCR alpha beta as revealed by staining with mAb BMA031. Within the gamma delta+ DN fraction, most cells reacted with mAb Ti gamma A which delineates a V gamma 9JPC gamma 1 epitope, whereas a minor fraction stained with mAb delta TCS-1 which identifies a V delta 1J delta 1 epitope. Functional studies performed at low cell number (1000) per microculture indicated that DN cells can be activated by anti-CD3 mAb, PHA and allogeneic stimulator cells, provided that exogenous growth factors are supplied. Both rIl-2 and rIl-4 acted as efficient growth factors for DN cells, and a synergistic stimulatory effect of rIl-2 and rIl-4 was observed when DN cells were cocultured with allogeneic LCL stimulator cells. As compared to unseparated E+ cells, isolated DN responder cells had a reduced capacity to secrete Il-2 upon PHA stimulation in the presence of LCL feeder cells. The majority of DN cells maintained their CD3+ CD4-CD8- phenotype upon coculture with allogeneic LCL stimulator cells. These data demonstrate that CD3+ DN cells in human peripheral blood are heterogeneous with respect to TCR expression. In addition, they show that freshly isolated DN cells are deficient in Il-2 production but may be normally stimulated by anti-CD3, PHA, or alloantigen if exogenous growth factors (rIL-2 and/or rIl-4) are provided.     

CD1d-independent developmental acquisition of prompt IL-4 gene inducibility in thymus CD161(NK1)-CD44lowCD4+CD8- T cells is associated with complementarity determining region 3-diverse and biased Vbeta2/Vbeta7/Vbeta8/Valpha3.2 T cell receptor usage

Among Ag-inexperienced naive T cells, the CD1d-restricted NKT cell that uses invariant TCR-alpha-chain is the most widely studied cell capable of prompt IL-4 inducibility. We show in this study that thymus CD161-CD44lowCD4+CD8- T cells promptly produce IL-4 upon TCR stimulation, a response that displays biased Vbeta(2/7/8) and Valpha3.2 TCR usage. The association of Vbeta family bias and IL-4 inducibility in thymus CD161-CD44lowCD4+CD8- T cells is found for B6, B10, BALB/c, CBA, B10.A(4R), and ICR mouse strains. Despite reduced IL-4 inducibility, there is a similarly biased Vbeta(2/7/8) TCR usage by IL-4 inducibility+ spleen CD161-CD44lowCD4+CD8- T cells. Removal of alpha-galacotosylceramide/CD1d-binding cells from CD161-CD44lowCD4+CD8- thymocytes does not significantly affect their IL-4 inducibility. The development of thymus CD161-CD44lowCD4+CD8- T cells endowed with IL-4 inducibility and their associated use of Vbeta(2/7/8) are beta2-microglobulin-, CD1d-, and p59fyn-independent. Thymus CD161-CD44lowCD4+CD8- T cells produce low and no IFN-gamma inducibility in response to TCR stimulation and to IL-12 + IL-18, respectively, and they express diverse complementarity determining region 3 sequences for both TCR-alpha- and -beta-chains. Taken together, these results demonstrate the existence of a NKT cell distinct, TCR-repertoire diverse naive CD4+ T cell subset capable of prompt IL-4 inducibility. This subset has the potential to participate in immune response to a relatively large number of Ags. The more prevalent nature of this unique T cell subset in the thymus than the periphery implies roles it might play in intrathymic T cell development and may provide a framework upon which mechanisms of developmentally regulated IL-4 gene inducibility can be studied.     

A single cell analysis of TCR AV24AJ18+ DN T cells.

The T-cell receptor (TCR) BV gene of human TCR AV24+ double-negative (DN) T cells, a novel subset of natural killer (NK) T cells, was investigated by single-cell sorting and single-cell polymerase chain reaction (PCR) methods. Seven of eleven TCR AV24+ DN T-cell clones utilized TCR BV8, three BV9, and one BV6. Six of seven TCR AV24/BV8+ DN T-cell clones had identical TCR beta and alpha chains, indicating that they were the same clone. All three TCR AV24/BV9+ DN T-cell clones also demonstrated the same amino acids in the CDR3 region. These findings strongly suggest that the usage of TCR beta and alpha chains on TCR AV24+ DN T cells is extremely restricted, supporting the notion that these cells recognize highly limited T-cell epitopes on antigens. All TCR AV24+ clones expressed the NKR-P1A mRNA, and so were true NK T cells. IL-2 and IL-4 mRNAs were detected in all clones, suggesting that the majority of these cells were Th0-type T cells. Six clones overexpressed Fas-ligand (Fas-L) mRNA and Fas antigen was detected on all clones at the mRNA level. In conclusion, TCR AV24+ DN T cells might recognize restricted T-cell epitopes on antigens and function as Th0-type T cells, inducer cells to Th1- or Th2-type T cells (regulatory T cells), and as Fas-L-positive cytolytic T cells.     

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.     

Phenotype, ontogeny, and repertoire of CD4-CD8- T cell receptor alpha beta + thymocytes. Variable influence of self-antigens on T cell receptor V beta usage

We have characterized CD4-CD8- double negative (DN) thymocytes that express TCR-alpha beta and represent a minor thymocyte subpopulation expressing a markedly skewed TCR repertoire. We found that DN TCR-alpha beta + thymocytes resemble mature T cells in that they (a) are phenotypically CD2hiCD5hiQa2+HSA-, (b) appear late in ontogeny, and (c) are susceptible to cyclosporin A-induced maturation arrest. In addition, we found that DNA sequences 5' to the CD8 alpha gene were demethylated relative to their germline state, suggesting that DN TCR-alpha beta + thymocytes are derived from cells that had at one time expressed their CD8 alpha gene locus. Because DN TCR-alpha beta + thymocytes are known to express an unusual TCR repertoire with significant overexpression of V beta 8, we were interested in examining the possible role played by self-Ag in shaping their TCR repertoire. It has been suggested that DN TCR-alpha beta + thymocytes are derived from potentially self-reactive thymocytes that have escaped clonal deletion by down-regulating their surface expression of CD4 and/or CD8 determinants. However, apparently inconsistent with such an hypothesis, we found that the frequency of DN thymocytes expressing various anti-self TCR (V beta 6, V beta 8.1, V beta 11, V beta 17a) were not increased in strains expressing their putative self-Ag, but instead were either unaffected or significantly reduced in those strains. With regard to V beta 8 expression among DN TCR-alpha beta + thymocytes, V beta 8 overexpression in DN TCR-alpha beta + thymocytes appeared to be independent of, and superimposed on, the developmental appearance of the basic DN thymocyte repertoire. Even though V beta 8 overexpression appeared to be generated by a mechanism distinct from that generating the rest of the DN TCR-alpha beta + thymocyte repertoire, we found that super-Ag against which V beta 8 TCR react introduced into the neonatal differentiation environment also significantly reduced, rather than increased, the frequency of DN TCR-alpha beta + V beta 8+ thymocytes. Thus, the present study is consistent with DN TCR-alpha beta + thymocytes being mature cells derived from CD8+ precursors, and documents that their TCR repertoire can be influenced, at least negatively, by either self-Ag or Ag introduced into the neonatal differentiation environment. However, we found no evidence to support the hypothesis that DN TCR-alpha beta + thymocytes are enriched in cells expressing TCR reactive against self-Ag.     

Thymic selection in CD8 transgenic mice supports an instructive model for commitment to a CD4 or CD8 lineage

Immature thymocytes, which coexpress CD4 and CD8, give rise to mature CD4+CD8- and CD4-CD8+ T cells. Only those T cells that recognize self-MHC are selected to mature, a process known as positive selection. The specificity of the T cell antigen receptor (TCR) for class I or class II MHC influences the commitment to a CD4 or CD8 lineage. This may occur by a directed mechanism or by stochastic commitment followed by a selection step that allows only CD8+, class I-specific and CD4+, class II-specific cells to survive. We have generated a mouse line expressing a CD8 transgene under the control of the T cell-specific CD2 regulatory sequences. Although constitutive CD8 expression does not affect thymic selection of CD4+ cells, selection of a class I-specific TCR in the CD8 subset is substantially improved. This outcome is consistent with a model for positive selection in which selection occurs at a developmental stage in which both CD4 and CD8 are expressed, and positive selection by class I MHC generates an instructive signal that directs differentiation to a CD8 lineage.     

Immunoregulatory function of CD3+, CD4-, and CD8- T cells. Gamma delta T cell receptor-positive T cells from nude mice abrogate oral tolerance

Previous work has shown that abrogation of oral tolerance is mediated by T cells which are found in the CD3+, L3T4- (CD4-), and Lyt-2- (CD8-) subset (termed double-negative; DN) in mice. Inasmuch as it is known that athymic, nude (nu/nu) mice possess Thy 1+, CD4-, and CD8- T cells which also exhibit a functionally rearranged TCR gamma-chain, we investigated whether this subset of nude T cells contained functional immunoregulatory cells. In this report, we examined the phenotype and distribution of CD3+ T cells in the spleen and in the mesenteric and peripheral lymph nodes of BALB/c nu/nu mice in comparison with normal mice (+/+). In the spleens of nude mice, the predominant CD3+ T cell subpopulation was DN. Further, in mesenteric and peripheral lymph nodes, approximately one-third and one-half of the CD3+ T cells were double negative, respectively. In contrast, CD3+, DN T cells represent a small subpopulation in normal (+/+) mice. We next showed that functional regulatory T cells which possess the ability to abrogate oral tolerance were induced in nu/nu mice by Ag priming. BALB/c nude mice were immunized with SRBC, and the splenic CD3+, Vicia villosa-adherent cells were obtained by panning. Adoptive transfer of CD3+, V. villosa-adherent T cells to orally tolerant BALB/c mice restored responsiveness to SRBC, whereas V. villosa nonadherent cells were without effect. In other experiments, CD3+ T cells from the spleens of SRBC-primed mice were further enriched for the CD5+, DN phenotype and adoptive transfer of this subset completely abrogated oral tolerance to SRBC. To characterize the nature of the TCR expressed on these CD3+, DN T cells, we developed a rabbit antibody to a synthetic peptide (residues 209-218: Tyr-Ala-Asn-Ser-Phe-Asn-Asn-Glu-Lys-Leu) which was synthesized from a deduced sequence of the murine delta-gene. Immunoprecipitation of a cell membrane fraction from CD3+, DN T cells with anti-delta TCR antibody isolated a 45-kDa band. Furthermore, immunoprecipitation of these cells with anti-CD3 (145-2C11) revealed bands at 45 and 35 kDa (corresponding to delta- and gamma-chains, respectively). Taken together, these results are the first to show that gamma delta-TCR bearing CD3+, CD4-, and CD8- T cells are functional and reverse oral tolerance when adoptively transferred.     

Nonencephalitogenic CD4-CD8- V alpha 2V beta 8.2+ anti-myelin basic protein rat T lymphocytes inhibit disease induction.

Recently there has been a number of reports suggesting that CD4-CD8- T cells participate in the processes of inflammatory reaction. In an attempt to delineate the distinctive functions of double negative (DN) T lymphocytes in an autoimmune-induced disease, we isolated and cloned such T cells, along with control CD4+ cells, from Lewis rats immunized with guinea-pig myelin basic protein in CFA. Both clones proliferated in response to the guinea-pig myelin basic protein and its synthetic encephalitogenic peptide, and expressed the same TCR V genes homologous to the mouse V alpha 2 and V beta 8.2 families that appear to be the defining entity of experimental autoimmune encephalomyeltis (EAE). Moreover, the TCR D and J region gene products of the DN cell were found to be similar to another encephalitogenic rat T cell clone. The two T clones did not differ markedly in their ability to produce TNF and IL-2 and to adhere to vascular wall-derived extracellular matrix- and laminin-coated plates. Surprising, therefore, was the finding that, although the CD4+ T lymphocytes were capable of inducing EAE, the DN cells did not elicit disease but rather inhibited subsequent EAE induction. Thus, TCR V alpha 2V beta 8.2 and its junctional region gene products are not the only prerequisite segment for a T cell to become encephalitogenic. We suggest that the important determinants of the T cell ability to induce disease are features of the T cell, other than or in addition to, the T cell receptor.     

Human CD4- CD8- alpha beta+ T cells express a functional T cell receptor and can be activated by superantigens.

The CD4 and CD8 molecules play an important role in the stimulation of T cells and in the process of thymic education. Most mature T cells express the alpha beta TCR and either CD4 or CD8; however, there is a small population of alpha beta+ TCR T cells that lack both CD4 and CD8. Little is known of the biology of the CD4- CD8- (double-negative) alpha beta+ TCR T cells or the nature of the Ag to which they may respond. These cells not only represent a novel population of T cells but also provide useful biologic tools to study the roles that CD4 and CD8 play in T cell activation. In this study we have addressed two questions. Firstly, whether CD4- CD8- alpha beta+ TCR T cells have functionally active TCR and, secondly, whether CD4 or CD8 is required for the activation of T cells by bacterial enterotoxins. Six double-negative alpha beta+ TCR T cell clones, propagated from two healthy donors, were challenged with a panel of nine bacterial enterotoxins. The V alpha and V beta usage of their TCR was determined by polymerase chain reaction. All of the CD4-CD8- clones proliferated in response to at least one of the enterotoxins, in a V beta-specific manner. The proliferative response of the CD4-CD8- alpha beta+ TCR T cell clones was similar in magnitude to that exhibited by CD4+ T cell clones of known V beta expression. These data clearly show that the CD4 and CD8 molecules are not required for the activation of untransformed human T cells by bacterial enterotoxins. Furthermore, these results indicate that CD4-CD8- alpha beta+ TCR T cells, normally present in all individuals, are not functionally silent, because they can be stimulated via their TCR. Their physiologic role, like that of gamma delta T cells, remains to be elucidated.     

Analysis of the functional capabilities of CD3+CD4-CD8- and CD3+CD4+CD8+ human T cell clones

The functional capabilities of human peripheral blood CD3+CD4-CD8- and CD3+CD4+CD8+ T cell clones were examined. The clones were generated by culturing purified populations of CD3+CD4-CD8- and CD3+CD4+CD8+ T cells at limiting dilution (0.3 cell/well) in the presence of PHA, rIL-2, and irradiated PBMC as feeders. Twelve CD3+CD4-CD8- and 5 CD3+CD4+CD8+ clones were generated. Clonality was documented by analyzing TCR gamma- and beta-chain rearrangement patterns. All CD3+CD4-CD8- clones were stained by the TCR-delta 1 mAb that identifies a framework epitope of the TCR delta-chain, but not by mAb WT31 that identifies the TCR-alpha beta on mature T cells. In contrast, the CD3+CD4+CD8+ clones were all stained by WT31 and not by TCR-delta 1. All 17 clones were screened for various functional activities. Each secreted IL-2, IFN-gamma, and lymphotoxin/TNF-like factors when stimulated with immobilized mAb to CD3 (64.1), albeit in varying quantities. These clones secreted far less IL-2 and IFN-gamma than CD3+CD4+CD8- or CD3+CD4-CD8+ alpha beta expressing clones, but comparable amounts of lymphotoxin/TNF. All clones also functioned as MHC-unrestricted cytotoxic cells. This activity was comparable to that mediated by the CD3+CD4+CD8- or CD3+CD4-CD8+ alpha beta clones. Nine of 12 CD3+CD4-CD8- and 4 of 5 CD3+CD4+CD8+ clones were able to support B cell differentiation when activated by immobilized anti-CD3, but usually not as effectively as the CD3+CD4+CD8- or CD3+CD4-CD8+ alpha beta clones. The differences in the functional capabilities of the various clones could not be accounted for by alterations in the signaling capacity of the CD3 molecular complex as mAb to CD3 induced comparable increases in intracellular free calcium in each clone examined. When clones were stimulated with PWM, each suppressed B cell differentiation supported by mitomycin C-treated fresh CD4+ T lymphocytes. Suppression was dependent on the number of clone cells added to culture, but could be observed with as few as 12,500 cells per microtiter well. Phenotypic analysis of the clones revealed that all expressed CD29, CD11b, and the NKH1 surface Ag. These results demonstrate that the CD3+CD4-CD8- and CD3+CD4+CD8+ T cell clones exhibit many of the functional characteristics of mature T cells, although they produce IL-2 and IFN-gamma and provide help for B cell differentiation less effectively than CD3+CD4+CD8- and CD3+CD4-CD8+ alpha beta T cell clones.     

Evidence for two subgroups of CD4-CD8- NKT cells with distinct TCR alpha beta repertoires and differential distribution in lymphoid tissues

NKT cells are a subset of T lymphocytes that is mainly restricted by the nonclassical MHC class I molecule, CD1d, and that includes several subpopulations, in particular CD4+ and CD4-CD8- (DN) cells. In the mouse, differential distribution of these subpopulations as well as heterogeneity in the expression of various markers as a function of tissue localization have been reported. We have thus undertaken a detailed study of the DN NKT cell subpopulation. With a highly sensitive semiquantitative RT-PCR technique, its TCR repertoire was characterized in various tissues. We found that mouse DN NKT cells are a variable mixture of two subgroups, one bearing the invariant Valpha14 chain paired to rearranged Vbeta2, Vbeta7, Vbeta8.1, Vbeta8.2, or Vbeta8.3 beta-chains and the other exhibiting unskewed alpha- and beta-chains. The proportion of these subgroups varies from about 100:0 in thymus, 80:20 in liver, and 50:50 in spleen to 20:80% in bone marrow, respectively. Finally, further heterogeneity in the tissue-derived DN NKT cells was discovered by sequencing extensively Vbeta8.2-Jbeta2.5 rearrangements in individual mice. Despite a few recurrences in TCR sequences, we found that each population exhibits its own and broad TCRbeta diversity.     

B7-CD28 interaction promotes proliferation and survival but suppresses differentiation of CD4-CD8- T cells in the thymus

Costimulatory molecules play critical roles in the induction and effector function of T cells. More recent studies reveal that costimulatory molecules enhance clonal deletion of autoreactive T cells as well as generation and homeostasis of the CD25(+)CD4(+) regulatory T cells. However, it is unclear whether the costimulatory molecules play any role in the proliferation and differentiation of T cells before they acquire MHC-restricted TCR. In this study, we report that targeted mutations of B7-1 and B7-2 substantially reduce the proliferation and survival of CD4(-)CD8(-) (double-negative (DN)) T cells in the thymus. Perhaps as a result of reduced proliferation, the accumulation of RAG-2 protein in the DN thymocytes is increased in B7-deficient mice, which may explain the increased expression of TCR gene and accelerated transition of CD25(+)CD44(-) (DN3) to CD25(-)CD44(-) (DN4) stage. Qualitatively similar, but quantitatively less striking effects were observed in mice with a targeted mutation of CD28, but not CTLA4. Taken together, our results demonstrate that the development of DN in the thymus is subject to modulation by the B7-CD28 costimulatory pathway.     

In vitro differentiation from a pluripotent human CD4+CD8+ thymic cloned cell into four phenotypically distinct subsets

Human thymic cell differentiation is almost totally unknown. In the present study we developed an in vitro system using human thymic cloned cells to analyze precursor-progeny relationship. We obtained several CD4+CD8+ double positive thymic clones that could give rise after several weeks in culture only to either CD4 or CD8 single positive clones. By contrast we isolated a unique pluripotent thymic double positive clone, termed B12, which differentiated into four phenotypically distinct T cell clones, namely double-positive CD4+CD8+, double-negative CD4-CD8- or either single-positive phenotype. We derived stable subclones representative of each phenotype and we showed by molecular analysis that they expressed the same TCR. Utilization of either CD3 or anticlonotypic mAb revealed that this TCR expressed by the four subclones was functional.     

Liver CD4-CD8- NK1.1+ TCR alpha beta intermediate cells increase during experimental malaria infection and are able to exhibit inhibitory activity against the parasite liver stage in vitro

Experimental infection of C57BL/6 mice by Plasmodium yoelii sporozoites induced an increase of CD4-CD8- NK1.1+ TCR alpha beta int cells and a down-regulation of CD4+ NK1.1+ TCR alpha beta int cells in the liver during the acute phase of the infection. These cells showed an activated CD69+, CD122+, CD44high, and CD62Lhigh surface phenotype. Analysis of the expressed TCRV beta segment repertoire revealed that most of the expanded CD4-CD8- (double-negative) T cells presented a skewed TCRV beta repertoire and preferentially used V beta 2 and V beta 7 rather than V beta 8. To get an insight into the function of expanded NK1.1+ T cells, experiments were designed in vitro to study their activity against P. yoelii liver stage development. P. yoelii-primed CD3+ NK1.1+ intrahepatic lymphocytes inhibited parasite growth within the hepatocyte. The antiplasmodial effector function of the parasite-induced NK1.1+ liver T cells was almost totally reversed with an anti-CD3 Ab. Moreover, IFN-gamma was in part involved in this antiparasite activity. These results suggest that up-regulation of CD4-CD8- NK1.1+ alpha beta T cells and down-regulation of CD4+ NK1.1+ TCR alpha beta int cells may contribute to the early immune response induced by the Plasmodium during the prime infection.     

Valpha24-JalphaQ-independent,CD1d-restricted recognition of alpha-galactosylceramide by human CD4(+) and CD8alphabeta(+) T lymphocytes

Human CD1d molecules present an unknown ligand, mimicked by the synthetic glycosphingolipid alpha-galactosylceramide (alphaGC), to a highly conserved NKT cell subset expressing an invariant TCR Valpha24-JalphaQ paired with Vbeta11 chain (Valpha24(+)Vbeta11(+) invariant NK T cell (NKT(inv))). The developmental pathway of Valpha24(+)Vbeta11(+)NKT(inv) is still unclear, but recent studies in mice were consistent with a TCR instructive, rather than a stochastic, model of differentiation. Using CD1d-alphaGC-tetramers, we demonstrate that in humans, TCR variable domains other than Valpha24 and Vbeta11 can mediate specific recognition of CD1d-alphaGC. In contrast to Valpha24(+)Vbeta11(+)NKT(inv) cells, Valpha24(-)/CD1d-alphaGC-specific T cells express either CD8alphabeta or CD4 molecules, but they are never CD4 CD8 double negative. We show that CD8alphabeta(+)Valpha24(-)/CD1d-alphaGC-specific T cells exhibit CD8-dependent specific cytotoxicity and have lower affinity TCRs than Valpha24(+)/CD1d-alphaGC-specific T cells. In conclusion, our results demonstrate that, contrary to the currently held view, recognition of CD1d-alphaGC complex in humans is not uniformly restricted to the Valpha24-JalphaQ/Vbeta11 NKT cell subset, but can be mediated by a diverse range of Valpha and Vbeta domains. The existence of a diverse repertoire of CD1d-alphaGC-specific T cells in humans strongly supports their Ag-driven selection.