Human invariant V alpha 24-J alpha Q TCR supports the development of CD1d-dependent NK1.1+ and NK1.1- T cells in transgenic mice

A sizable fraction of T cells expressing the NK cell marker NK1.1 (NKT cells) bear a very conserved TCR, characterized by homologous invariant (inv.) TCR V alpha 24-J alpha Q and V alpha 14-J alpha 18 rearrangements in humans and mice, respectively, and are thus defined as inv. NKT cells. Because human inv. NKT cells recognize mouse CD1d in vitro, we wondered whether a human inv. V alpha 24 TCR could be selected in vivo by mouse ligands presented by CD1d, thereby supporting the development of inv. NKT cells in mice. Therefore, we generated transgenic (Tg) mice expressing the human inv. V alpha 24-J alpha Q TCR chain in all T cells. The expression of the human inv. V alpha 24 TCR in TCR C alpha(-/-) mice indeed rescues the development of inv. NKT cells, which home preferentially to the liver and respond to the CD1d-restricted ligand alpha-galactosylceramide (alpha-GalCer). However, unlike inv. NKT cells from non-Tg mice, the majority of NKT cells in V alpha 24 Tg mice display a double-negative phenotype, as well as a significant increase in TCR V beta 7 and a corresponding decrease in TCR V beta 8.2 use. Despite the forced expression of the human CD1d-restricted TCR in C alpha(-/-) mice, staining with mCD1d-alpha-GalCer tetramers reveals that the absolute numbers of peripheral CD1d-dependent T lymphocytes increase at most by 2-fold. This increase is accounted for mainly by an increased fraction of NK1.1(-) T cells that bind CD1d-alpha-GalCer tetramers. These findings indicate that human inv. V alpha 24 TCR supports the development of CD1d-dependent lymphocytes in mice, and argue for a tight homeostatic control on the total number of inv. NKT cells. Thus, human inv. V alpha 24 TCR-expressing mice are a valuable model to study different aspects of the inv. NKT cell subset.     

The CD1d natural killer T-cell antigen presentation pathway is highly conserved between humans and rhesus macaques

Natural killer T (NKT) cells play an important role in controlling cancers, infectious diseases and autoimmune diseases. Although the rhesus macaque is a useful primate model for many human diseases such as infectious and autoimmune diseases, little is known about their NKT cells. We analyzed V alpha 24TCR+ T cells from rhesus macaque peripheral blood mononuclear cells stimulated with alpha-galactosylceramide (alpha-GalCer) and interleukin-2. We found that rhesus macaques possess V alpha 24TCR+ T cells, suggesting that recognition of alpha-GalCer is highly conserved between rhesus macaques and humans. The amino acid sequences of the V-J junction for the V alpha 24TCR of rhesus macaque and human NKT cells are highly conserved (93% similarity), and the CD1d alpha1-alpha2 domains of both species are highly homologous (95.6%). These findings indicate that the rhesus macaque is a useful primate model for understanding the contribution of NKT cells to the control of human diseases.     

Differential responses of invariant V alpha 24J alpha Q T cells and MHC class II-restricted CD4+ T cells to dexamethasone.

NK T cells are a T cell subset in the human that express an invariant alpha-chain (V alpha 24invt T cells). Because of the well-described immunomodulation by glucocorticoids on activation-induced cell death (AICD), the effects of dexamethasone and anti-CD3 stimulation on V alpha 24invt T cell clones and CD4+ T cell clones were investigated. Dexamethasone significantly enhanced anti-CD3-mediated proliferation of V alpha 24invt T cells, whereas CD4+ T cells were inhibited. Addition of neutralizing IL-2 Ab partially abrogated dexamethasone-induced potentiation of V alpha 24invt T cell proliferation, indicating a role for autocrine IL-2 production in corticosteroid-mediated proliferative augmentation. Dexamethasone treatment of anti-CD3-stimulated V alpha 24invt T cells did not synergize with anti-Fas blockade in enhancing proliferation or preventing AICD. The V alpha 24invt T cell response to dexamethasone was dependent on the TCR signal strength. In the presence of dexamethasone, lower doses of anti-CD3 inhibited proliferation of V alpha 24invt T cells and CD4+ T cells; at higher doses of anti-CD3, which caused inhibition of CD4+ T cells, the V alpha 24invt T cell clones proliferated and were rescued from AICD. These results demonstrate significant differences in TCR signal strength required between V alpha 24invt T cells and CD4+ cells, and suggest important immunomodulatory consequences for endogenous and exogenous corticosteroids in immune responses.     

Analysis of human V alpha 24+ CD4+ NKT cells activated by alpha-glycosylceramide-pulsed monocyte-derived dendritic cells

Human V alpha 24+ NKT cells with an invariant TCR (V alpha 24-J alpha Q) have been shown to be specifically activated by synthetic glycolipids such as alpha-galactosylceramide and alpha-glucosylceramide in a CD1d-restricted and V alpha 24 TCR-mediated manner. We recently characterized V alpha 24+ CD4- CD8- double negative (DN) NKT cells using alpha-galactosylceramide-pulsed monocyte-derived dendritic cells. Here, we compare V alpha 24+ CD4+ NKT cells with human V alpha 24+ DN NKT cells from the same donor using alpha-galactosylceramide-pulsed monocyte-derived dendritic cells. Human V alpha 24+ CD4+ NKT cells were phenotypically and functionally similar to the human V alpha 24+ DN NKT cells characterized previously. Both of them use V alpha 24-J alpha Q-V beta 11 TCR and express CD161 (NKR-P1A), but not the other NK receptors tested so far. They also produce cytokines such as IL-4 and IFN-gamma, and, in regard to IL-4 production, V alpha 24+ CD4+ NKT cells produce more IL-4 than V alpha 24+ DN NKT cells. The cells exhibit marked cytotoxic activity against the U937 tumor cell line, but not against the NK target cell line, K562. Although at least some of the factors responsible for the stimulation of V alpha 24+ NKT cells have been clarified, little is known regarding the killing phase of these cells. Here we show that the cytotoxic activity of V alpha 24+ NKT cells against U937 cells is mediated mainly through the perforin pathway and that ICAM-1/LFA-1 as well as CD44/hyaluronic acid interactions are important for the effector phase of V alpha 24+ NKT cell-mediated cytotoxicity against U937 cells.     

The immunopathology of acute experimental allergic encephalomyelitis induced with myelin proteolipid protein. T cell receptors in inflammatory lesions.

To determine whether there is predominance of T cells expressing a particular TCR V beta chain in the inflammatory lesions of an autoimmune disease model, TCR expression was analyzed in central nervous system (CNS) tissues of mice with experimental allergic encephalomyelitis (EAE). Acute EAE was induced in SJL/J mice either by sensitization with a synthetic peptide corresponding to myelin proteolipid protein residues 139-151 or by adoptive transfer of myelin proteolipid protein peptide 139-151-specific encephalitogenic T cell clones. Mice were killed when they showed clinical signs of EAE or by 40 days after sensitization or T cell transfer. Cryostat CNS and lymphoid tissue sections were immunostained with a panel of mAb to T cell markers and proportions of stained cells were counted in inflammatory foci. In mice with both actively induced and adoptively transferred EAE, infiltrates consisted of many CD3+, TCR alpha beta+, and CD4+ cells, fewer CD8+ cells, and small numbers of TCR gamma delta+ cells. Approximately 30% of CD45+ leukocytes in the inflammatory foci were T cells. Cells expressing TCR V beta 2, 3, 4, 6, 7 and 14 were detected in the infiltrates, whereas TCR V beta 8 and 11, which that are deleted in SJL mice, were absent. When EAE was induced by transfer of T cell clones that use either V beta 2, 6, 10, or 17, there was also a heterogeneous accumulation of T cells in the lesions. Similar proportions of TCR V beta+ and gamma delta+ cells were detected in EAE lesions and in the spleens of the mice. Thus, at the time that clinical signs are present in acute EAE, peripherally derived, heterogeneous TCR V beta+ cells are found in CNS lesions, even when the immune response is initiated to a short peptide Ag or by a T cell clone using a single TCR V beta.     

NK T cell-induced protection against diabetes in V alpha 14-J alpha 281 transgenic nonobese diabetic mice is associated with a Th2 shift circumscribed regionally to the islets and functionally to islet autoantigen

The onset of autoimmune diabetes is related to defective immune regulation. Recent studies have shown that NK T cells are deficient in number and function in both diabetic patients and nonobese diabetic (NOD) mice. NK T cells, which are CD1d restricted, express a TCR with an invariant V alpha 14-J alpha 281 chain and rapidly produce large amounts of cytokines. V alpha 14-J alpha 281 transgenic NOD mice have increased numbers of NK T cells and are protected against diabetes onset. In this study we analyzed where and how NK T cells interfere with the development of the anti-islet autoimmune response. NK T cells, which are usually rare in lymph nodes, are abundant in pancreatic lymph nodes and are also present in islets. IL-4 mRNA levels are increased and IFN-gamma mRNA levels decreased in islets from diabetes-free V alpha 14-J alpha 281 transgenic NOD mice; the IgG1/IgG2c ratio of autoantibodies against glutamic acid decarboxylase is also increased in these mice. Treatment with IL-12 (a pro-Th1 cytokine) or anti-IL-4 Ab abolishes the diabetes protection in V alpha 14-J alpha 281 NOD mice. The protection from diabetes conferred by NK T cells is thus associated with a Th2 shift within islets directed against autoantigen such as glutamic acid decarboxylase. Our findings also demonstrate the key role of IL-4.     

The repertoire of killer cell Ig-like receptor and CD94:NKG2A receptors in T cells: clones sharing identical alpha beta TCR rearrangement express highly diverse killer cell Ig-like receptor patterns

Killer cell Ig-like receptor (KIR) and CD94:NKG2A molecules were first defined as human NK cell receptors (NKR), but now are known to be expressed and to function on subpopulations of T cells. Here the repertoires of KIR and CD94:NKG2A expression by T cells from two donors were examined and compared with their previously defined NK cell repertoires. T cell clones generated from peripheral blood of both donors expressed multiple NKR in different combinations and used the range of receptors expressed by NK cells. In both donors alpha beta T cells less frequently expressed the inhibitory receptors CD94:NKG2A and KIR2DL1 than either gamma delta T cells or NK cells. In contrast to NK cells, not all NKR(+) T cells expressed an inhibitory receptor for autologous HLA class I. This lack of specific inhibitory NKR was especially apparent on alpha beta T cells of one donor. Overall, alpha beta T cells exhibited a distinct pattern of NKR expression different from that of gamma delta T and NK cells, which expressed highly similar NKR repertoires. In one donor, analysis of TCR rearrangement revealed a dominant subset of NKR(+) T cells sharing identical TCR alpha- and beta-chains. Remarkably, among 55 T cell clones sharing the same TCR alpha beta rearrangement 18 different KIR phenotypes were seen, suggesting that KIR expression was initiated subsequently to TCR rearrangement.     

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.     

Invariant natural killer T cells from rhesus macaque spleen and peripheral blood are phenotypically and functionally distinct populations

BACKGROUND: Natural killer T cells (NKT) possess dual functions of innate and adaptive immune systems, controlling viral infections and regulating autoimmune diseases. Non-human primates (NHP) are penultimate models for advancing therapeutic immunoregulatory strategies for translational application in humans, though, little is known about NHP NKT cells. Here we characterized rhesus macaque NKT cells ex vivo. METHODS: The frequency, phenotype and intracellular cytokine production of V alpha 24+ 6B11+ invariant NKT (iNKT) cells were analyzed by multi-color flow cytometry. V alpha 24J alpha Q mRNA expression was analyzed by real-time RT-PCR. RESULTS: The frequencies of peripheral blood (PB) and spleen V alpha 24+ 6B11+ iNKT cells were not significantly different. The iNKT cell subset in spleen was significantly increased for CD4+ CD8+ and CD3+ CD56+ co-expression as well as intracellular interleukin-4 production, which was rarely observed in circulating PB. CONCLUSION: Spleen iNKT cells in rhesus macaques are Th2 biased and display phenotypically and functionally distinct profiles from their PB counterpart.     

Role of T cell receptor V beta genes in Theiler's virus-induced demyelination of mice.

Intracerebral infection of certain strains of mice with Theiler's virus results in chronic immune-mediated demyelination in spinal cord. We used mouse mutants with deletion of the V beta class of TCR genes to examine the role of TCR genes in this demyelinating disease which is similar to multiple sclerosis. Quantitative analysis of spinal cord lesions demonstrated a markedly increased number and extent of demyelinated lesions in persistently infected RIII S/J mice which have a massive deletion of the TCR V beta-chain (V beta 5.2, V beta 8.3, V beta 5.1, V beta 8.2, V beta 5.3, V beta 8.1, V beta 13, V beta 12, V beta 11, V beta 9, V beta 6, V beta 15, V beta 17) compared with B10.RIII mice which are of identical MHC haplotype (H-2r) but have normal complement of V beta TCR genes. In contrast, infection of C57L (H-2b) or C57BR (H-2k) mice which have deletion of the V beta TCR genes (V beta 5.2, V beta 8.3, V beta 5.1, V beta 8.2, V beta 5.3, V beta 8.1, V beta 13, V beta 12, V beta 11, and V beta 9) resulted in few demyelinating lesions. Genetic segregation analysis of (B10.RIII x RIII S/J) x RIII S/J backcrossed mice and (B10.RIII x RIII S/J) F2 mice demonstrated correlation of increased susceptibility to demyelination with deletion of TCR V beta genes. The increase in number of demyelinating lesions correlated with increase in number of virus-Ag+ cells in spinal cord. These experiments provide strong evidence that the structural diversity at the TCR beta-complex can influence susceptibility to virus-induced demyelination.