Paradoxical intrathymic positive selection in mice with only a covalently presented agonist peptide.

The Y-Ae mAb and the 1H3.1 alphabeta T cell antigen receptor (TCR) are both specific for the I-Ealpha52-68 peptide bound to the I-A(b) major histocompatibility complex (MHC) class II molecule. Antigen-presenting cells (APCs) from I-A(b+) mice with a natural or transgenic (Tg) I-Ealpha chain activate mature 1H3.1 T cells and cause the deletion of 1H3.1 TCR Tg thymocytes. However, 1H3.1 T cells were neither activated nor inactivated by confrontation with APCs from I-Ab-Ep mice in which I-A(b) molecules are occupied only by the covalently associated Ealpha52-68 peptide. Instead, immature 1H3.1 TCR Tg thymocytes were efficiently positively selected into the CD4 lineage in the I-Ab-Ep thymus. This selection relied on specific recognition of the Ealpha52-68/I-A(b) complex because it was blocked by Y-Ae. 1H3.1 TCR Tg T cells maturing in the I-Ab-Ep thymus efficiently populated the periphery, displayed a naive phenotype, and were specifically reactive to the Ealpha52-68 peptide or to I-A(b+)I-Ealpha(+) APCs, indicating that 1H3.1 T cells were not antagonized in I-Ab-Ep mice. The data identify major histocompatibility complex class II molecules with only a covalently attached self-peptide as a ligand for in vivo positive selection of T cells specific for the same peptide.

CD62L is required on effector cells for local interactions in the CNS to cause myelin damage in experimental allergic encephalomyelitis.

Adhesion molecules are believed to facilitate infiltration of leukocytes into the CNS of mice with experimental allergic encephalomyelitis (EAE). The role of the adhesion molecule CD62L (L-selectin) in the immunopathology of EAE is not known. To study this, we crossed CD62L-deficient mice with myelin basic protein-specific TCR (MBP-TCR) transgenic mice. CD62L-deficient MBP-TCR transgenic mice failed to develop antigen-induced EAE, and, despite the presence of leukocyte infiltration, damage to myelin in the CNS was not seen. EAE could, however, be induced in CD62L-deficient mice upon adoptive transfer of wild-type macrophages. Our results suggest that CD62L is not required for activation of autoimmune CD4 T cells but is important for the final destructive function of effector cells in the CNS and support a novel mechanism whereby CD62L expressed on effector cells is important in mediating myelin damage.

A role for accessibility to self-peptide-self-MHC complexes in intrathymic negative selection.

Whether intrathymic-positive and -negative selection of conventional alpha beta T cells occur in anatomically distinct sites is a matter of debate. By using a system composed of two distinct immune receptors, the Y-Ae mAb and the 1H3.1 (V alpha 1/V beta 6) TCR, both directed against the 52--68 fragment of the I-E alpha-chain (E alpha 52--68) bound to I-A(b), we examined the occurrence of negative selection imposed in vivo by a self-peptide-self-MHC class II complex with differential tissue expression. 1H3.1 TCR-transgenic (Tg) mice were bred to mice having an I-E alpha transgene with expression directed to all MHC class II-positive cells, restricted to thymic epithelial cells, or restricted to B cells, dendritic cells, and medullary thymic epithelial cells. All 1H3.1 TCR/I-E alpha double-Tg mice revealed a severely diminished thymic cellularity. Their lymph node cells were depleted of V beta 6(+)CD4(+) cells and were unresponsive to E alpha 52--68 in vitro. The absolute number of CD4(+)CD8(+) thymocytes was drastically reduced in all combinations, indicating that negative selection caused by an endogenously expressed self-determinant can effectively occur in the thymic cortex in vivo. Moreover, both cortical epithelial cells and, interestingly, the few cortical dendritic cells were able to support negative selection of CD4(+)CD8(+) thymocytes, albeit with a distinct efficiency. Collectively, these observations support a model where, in addition to the avidity of the thymocyte/stromal cell interaction, in vivo negative selection of autoreactive TCR-Tg T cells is determined by accessibility to self-peptide-self-MHC complexes regardless of the anatomical site.

Alteration at a single amino acid residue in the T cell receptor alpha chain complementarity determining region 2 changes the differentiation of naive CD4 T cells in response to antigen from T helper cell type 1 (Th1) to Th2.

To study whether changes in the structure of a T cell receptor (TCR) at a single peptide-contacting residue could affect T cell priming with antigenic peptide, we made transgenic mice with a point mutation in the TCR alpha chain of the D10.G4.1 (D10) TCR and bred them to D10 beta chain transgenic mice. The mutation consisted of a leucine to serine substitution at position 51 (L51S), which we had already established contacted the second amino acid of the peptide such that the response to the reference peptide was reduced by approximately 100-fold. A mutation in the reference peptide CA134-146 (CA-WT) from the arginine at peptide position 2 to glycine (R2G) restored full response to this altered TCR. When we examined in vitro priming of naive CD4 T cells, we observed that the response to doses of CA-WT that induced T helper cell type 1 (Th1) responses in naive CD4 T cells from mice transgenic for the D10 TCR gave only Th2 responses in naive CD4 T cells derived from the L51S. However, when we primed the same T cells with the R2G peptide, we observed Th1 priming in both D10 and L51S naive CD4 T cells. We conclude from these data that a mutation in the TCR at a key position that contacts major histocompatibility complex-bound peptide is associated with a shift in T cell differentiation from Th1 to Th2.

Variability of invariant mouse CD3epsilon chains detected by anti-CD3 antibodies.

Current models of the TCR / CD3 complex assume that, in mature peripheral T lymphocytes, variability is restricted to the alpha beta (or gamma delta) chains of the TCR heterodimer responsible for antigen recognition, whereas the CD3 polypeptides involved in signal transmission are invariant. Here we show that mouse CD4(+) T lymphocytes and T cell lines are bound with different avidity by anti-CD3 monoclonal antibodies. These findings cannot be accounted for by allelic differences between CD3 chains, by the nature of the TCR chains, or by the ratio of CD3epsilon delta to CD3epsilon gamma chain pairing. Rather, they are linked to heterogeneity of the N-terminal region of CD3epsilon chains, as detected by peptide-specific antibodies. In turn, these differences among CD3epsilon chains correlate with variations in the strength of TCR / CD3 interaction. N-terminal CD3epsilon heterogeneity is not due to alternative splicing mechanisms, but rather involves digestion by metalloproteases, as suggested by reverse transcription-PCR amplification and by the effect of protease inhibitors, respectively. Based on these data, we propose a model linking CD3epsilon N-terminal variability with altered CD3 recognition by monoclonal antibodies and TCR / CD3 interaction. This model suggests the possibility of distinct spatial arrangements of the TCR / CD3 complex.

Functional and phenotypic evidence for presentation of E alpha 52-68 structurally related self-peptide(s) in I-E alpha-deficient mice.

The Y-Ae mAb and the 1H3.1 TCR-alpha beta (V alpha 1/V beta 6) are two immune receptors specific for I-Ab MHC class II molecules complexed to the 52-68 fragment of the alpha-chain of I-E class II molecules (the E alpha 52-68 peptide). A profound intrathymic negative selection occurs in 1H3.1 TCR transgenic mice in the presence of an I-E alpha transgene. The administration of mAbs to 1H3.1/I-E alpha double-transgenic newborn mice reveals that Y-Ae, but not the isotype-matched anti-I-E Y17 mAb, rescues a significant number of mature (V beta 6highCD4+CD8-) thymocytes and allows the detection of E alpha 52-68-reactive T cells in the periphery. These observations indicate that deletion of autoreactive T cells can be specifically inhibited in vivo by an mAb specific for the deleting self-peptide:self-MHC class II complex. Similar inhibition experiments indicate that C57BL/6 (I-Ab+/I-E alpha-) mice constitutively express an E alpha-independent, Y-Ae-recognizable epitope(s). This finding is confirmed by the phenotypic analysis of mature (MHC class II high) C57BL/6 bone marrow-derived dendritic cells. Collectively, these observations further illustrate the peptide specificity of negative selection and demonstrate that MHC class II-positive cells from unmanipulated C57BL/6 mice that lack a functional I-E alpha gene can assemble one or more self-peptide:I-Ab complexes recognizable by the E alpha 52-68:I-Ab complex-specific Y-Ae mAb.

Expression of the novel T cell activation molecule hpH4 in HIV-infected patients: correlation with disease status.

We have described hpH4, a surface glycoprotein selectively expressed by activated T cells and mature thymocytes and displaying weak lateral association with CD4. The hpH4 expression pattern and biochemical features, together with analysis of its tryptic digest by peptide mass searching using MALDI-MS, suggested that it is a novel molecule. The aim of this work was to evaluate the peripheral blood T cell expression of hpH4 in HIV-infected patients and the interplay between HIV gp120 and hpH4, since both molecules interact with CD4. hpH4 expression during HIV-1 infection was evaluated by assessing 55 patients at various disease stages and following up 3 patients with primary infection and 3 patients with AIDS. hpH4 expression displayed a peak in the early phase of primary infection, dropped to control levels in the asymptomatic phase, and was newly expressed, at low levels, as AIDS developed. The expression kinetics were different than those shown by HLA-DR, CD25, and CD38. The most striking findings were the transient hpH4 expression peak displayed in the earliest stage, which was unique for hpH4. Incubation of T cells from normal donors with HIV gp120 induced transient hpH4 expression in resting CD4+ T cells and potentiated the hpH4 lateral association with CD4 in activated T cells. Moreover, hpH4 triggering inhibited gp120-induced death of CD4+ cells. Therefore, H4 expression may be a response to avoid apoptosis induced by HIV products.

An islet-specific CD8+ T cell hybridoma generated from non-obese diabetic mice recognizes insulin as an autoantigen.

Although CD8+ T cells play a major role in beta cell destruction in insulin-dependent diabetes in the non-obese diabetic mouse, the T cell autoantigen(s) recognized by such cells remains to be identified. Therefore, an islet-reactive, CD8+ T cell line was generated from islet-infiltrating cells and hybridized by fusion with a CD8+ alphabeta TCR- BW5147 thymoma. In the presence of islets, none of the 12 CD3+ CD8+ T cell hybridomas isolated secreted IL-2/IL-4 or IFNgamma but three were islet specific, as shown by activation induced cell death. Subclone 4A7.7.15 recognized only islets expressing H-2Kd, demonstrated islet-specific inhibition of proliferation and concomitant partial arrest in the G2/M phase of the cell cycle. Further analysis using a panel of cell lines, expressing H-2Kd, and transfected with the cDNA for various putative autoantigens in type 1 diabetes showed that 4A7.7.15 recognizes insulin as an antigen.

Characterization of a novel human surface molecule selectively expressed by mature thymocytes, activated T cells and subsets of T cell lymphomas.

We have previously characterized mouse H4 (mH4), a surface glycoprotein recognized by the C398.4A monoclonal antibody. We now show that C398.4A also binds its human putative homolog (hpH4). Both hpH4 and mH4 (1) are selectively expressed by activated T cells and mature thymocytes, (2) are disulfide-linked dimers of two chains (29/37 kDa in humans, 25/29 kDa in mice), whose N-deglycosylation produces a single band at 20 - 21 kDa, and (3) display a low association with CD4 and the TCR. The expression pattern of hpH4 and its biochemical features showed that it is different from other known activation molecules, and this was confirmed when analysis of the tryptic digest of the hpH4 29-kDa band by peptide mass searching using matrix-assisted laser desorption ionization mass spectrometry did not reveal any significant homology with other molecules. In normal lymphoid tissue, hpH4 is expressed by T cells located at the periphery of lymph node germinal centers and paracortical areas. In T cell neoplasia, expression of hpH4 clusters with a subset of peripheral T cell lymphomas with a large-cell component, and with cases of angioimmunoblastic T cell lymphomas. Overall, these data provide evidence for a novel T cell activation molecule that could help in the phenotypic categorization of T cell malignancies.

Cross-antagonism of a T cell clone expressing two distinct T cell receptors.

Inhibition of T cell activation can be mediated by analogs of the original antigenic peptide (TCR antagonists). Here, a T cell clone expressing two distinct TCR was used to investigate whether such inhibition involves an active mechanism by examining whether an antagonist for one TCR could influence responses stimulated by the other TCR engaging its agonist. Our results demonstrate functional cross-inhibition under these conditions involving the ability of antagonist: TCR interactions to diminish Lck enzymatic activity associated with the agonist-recognizing second TCR, apparently through enhancement of SHP-1 association with these receptors. Our findings reveal that inhibition of cellular responses by antagonists arises at least in part from active negative regulation of proximal TCR signaling and identify elements of the biochemical process.

Alpha(1,3)-fucosyltransferase VII and alpha(2,3)-sialyltransferase IV are up-regulated in activated CD4 T cells and maintained after their differentiation into Th1 and migration into inflammatory sites.

Activated Th1 CD4 T cells bind to P-selectin and migrate into inflamed tissue, whereas Th2 cells do not. We show that alpha(1, 3)-fucosyltransferase VII (FucT-VII) and alpha(2, 3)-sialyltransferase IV (ST3GalIV), which are crucial for the biosynthesis of functional P-selectin ligands, are absent in naive CD4 T cells, but are rapidly up-regulated upon activation. Th1 or Th2 differentiation in the presence of polarizing cytokines leads to down-regulation of FucT-VII mRNA selectively in Th2 but not in Th1 cells. Influencing the differentiation by varying the priming dose of antigenic peptide results in similar FucT-VII down-regulation only in Ag-specific Th2 cells. ST3GalIV levels remain elevated. FucT-VII and ST3GalIV mRNAs are also up-regulated by Th1 cells primed in vivo and recruited into the lymph nodes draining delayed-type hypersensitivity sites. We identify FucT-VII gene expression as a principal difference between Th1 and Th2 cells, and underscore the importance of FucT-VII and ST3GalIV expression for the biosynthesis of functional selectin ligands.

Identification of an MHC class I-restricted autoantigen in type 1 diabetes by screening an organ-specific cDNA library.

Type 1 diabetes is an autoimmune disease in which the insulin-producing pancreatic beta cells are destroyed at an early age by an immune process that involves both CD4 and CD8 T lymphocytes. The identification of autoantigens in diabetes is very important for the design of antigen-specific immunotherapy. By screening a pancreatic islet cDNA library, we have identified the autoantigen recognized by highly pathogenic CD8 T cells in the non-obese diabetic mouse, one of the best animal models for human diabetes. This is the first identification, to our knowledge, of a CD8 T-cell epitope in an autoimmune disease. The peptide recognized by the cells is in the same region of the insulin B chain as the epitope recognized by previously isolated pathogenic CD4 T cells. This has very important implications for the potential use of insulin in preventative therapy.

Presentation of the self antigen myelin basic protein by dendritic cells leads to experimental autoimmune encephalomyelitis.

Bone marrow (BM)-derived dendritic cells (DC) are potent stimulators of naive CD4+ T cell activation. Because DC are efficient at Ag processing and could potentially present self Ags, we investigated the role of DC in the presentation of an encephalitogenic peptide from myelin basic protein (Ac1-11) in the induction of experimental autoimmune encephalomyelitis (EAE). To determine if DC could prime for EAE, we transferred DC pulsed with Ac1-11 or with medium alone into irradiated mice in combination with CD4+ T cells isolated from a mouse transgenic for a TCR specific for Ac1-11 + I-Au. Mice transferred with Ac1-11-pulsed DC developed EAE 7-10 days later, whereas mice receiving medium-pulsed DC did not. By day 15, all mice given peptide-loaded DC had signs of tail and hind limb paralysis, and by day 20 infiltration of Ac1-11-specific CD4+ T cells was detected in the brain parenchyma. We also demonstrated interactions between Ac1-11-pulsed DC and Ac1-11-specific T cells in the lymph nodes 24 h following adoptive transfer of both cell populations. These data show that DC can efficiently present the self Ag myelin basic protein Ac1-11 to Ag-specific T cells in the periphery of mice to induce EAE.

Evidence for Fas-dependent and Fas-independent mechanisms in the pathogenesis of experimental autoimmune encephalomyelitis.

To determine whether Fas or Fas ligand (FasL) plays a role in susceptibility to experimental autoimmune encephalomyelitis (EAE), we bred a TCR transgenic mouse specific for the Ac1-11 peptide of myelin basic protein to mice with inactivating mutations in Fas (lpr) or FasL (gld). Disease induction by peptide immunization in such mice produced similar disease scores, demonstrating that Fas/FasL interactions were not necessary to generate EAE. However, adoptive transfer experiments showed evidence that these interactions can play a role in the pathogenesis of EAE, shown most dramatically by the absence of disease following transfer of cells from a normal myelin basic protein TCR transgenic mouse into a Fas-deficient lpr recipient. Furthermore, transfer of cells lacking FasL (gld) into normal or gld recipients gave a diminished disease score. Thus, Fas/FasL interactions can play a role in the pathogenesis of EAE, but they are not required for disease to occur.

T-cell development: a role for self-peptides in positive selection.

An important issue for immunologists is the difference between the two main processes that determine the mature repertoire of T-cell receptors, termed positive and negative selection. Recent papers have addressed the role of self-peptides in the process of positive selection.

STAT5 interaction with the T cell receptor complex and stimulation of T cell proliferation.

The role of STAT (signal transducer and activator of transcription) proteins in T cell receptor (TCR) signaling was analyzed. STAT5 became immediately and transiently phosphorylated on tyrosine 694 in response to TCR stimulation. Expression of the protein tyrosine kinase Lck, a key signaling protein in the TCR complex, activated DNA binding of transfected STAT5A and STAT5B to specific STAT inducible elements. The role of Lck in STAT5 activation was confirmed in a Lck-deficient T cell line in which the activation of STAT5 by TCR stimulation was abolished. Expression of Lck induced specific interaction of STAT5 with the subunits of the TCR, indicating that STAT5 may be directly involved in TCR signaling. Stimulation of T cell clones and primary T cell lines also induced the association of STAT5 with the TCR complex. Inhibition of STAT5 function by expression of a dominant negative mutant STAT5 reduced antigen-stimulated proliferation of T cells. Thus, TCR stimulation appears to directly activate STAT5, which may participate in the regulation of gene transcription and T cell proliferation during immunological responses.

Innate immune recognition and control of adaptive immune responses.

The immune system of higher vertebrates consists of two components: innate and adaptive. The innate immune system relies on a set of germ-line encoded receptors that recognize conserved molecular patterns found only in microorganisms. The adaptive immune system uses somatically generated antigen receptors which are clonally distributed on the two types of lymphocytes: T cells and B cells. These antigen receptors are generated by random processes and, as a consequence, the general design of the adaptive immune system is based on clonal selection of lymphocytes expressing receptors with particular specificities. Here we discuss the essential role of the innate immune system in the clonal selection of lymphocytes and activation of the adaptive immune responses.