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PubMed1995

Carrier-reactive hapten-specific cytotoxic T lymphocyte clones originate from a highly preselected T cell repertoire: implications for chemical-induced self-reactivity.

We have recently described trinitrophenyl (TNP)-specific cytotoxic T lymphocyte (CTL) clones from C57BL/6 mice specific for hapten-modified peptides bearing a TNP-lysine in a peripheral position, i.e. in position 7 of H-2Kb-bound octapeptides. CTL recognition of such determinants is always sequence-dependent due to co-recognition of TNP as well as amino acid side chains of the carrier peptide. By the use of glycine-based designer peptides for primary induction of CTL in vitro, we have identified two sub-epitopes on individual position 7-haptenated peptides that form two TcR contact points and which can be independently recognized by cloned CTL. One of these sub-epitopes is represented by the hapten itself, the other by the amino acids tyrosine and lysine in positions 3 and 4 of the carrier peptide, respectively. Immunization with such TNP-modified peptides frequently results in the specific induction of CTL also reacting with the unmodified carrier peptides. DNA sequence analyses of the TcR revealed an extraordinary similarity of several independent TcR of CTL from individual mice and induced with different TNP-peptides. These receptor similarities clearly correlate with structural elements common to the immunizing peptides and suggest their origin from positive thymic selection of TcR on Kb-associated associated self-peptides bearing Tyr in position 3. Our data provide additional information concerning the topology of TcR binding to peptide/MHC complexes with, but also without, TNP. They also indicate a mechanism which might explain the potential of chemicals or drugs to induce autoimmune phenomena.

Amino Acid Sequence↗
PubMed1997

Molecular cloning of the mouse proteasome subunits MC14 and MECL-1: reciprocally regulated tissue expression of interferon-gamma-modulated proteasome subunits.

The primary structures of the interferon-gamma-inducible mouse 20S proteasome subunit MECL-1 and its alternate homolog MC14 were determined. Northern analysis of mouse tissues revealed that MECL-1 mRNA predominantly occurred in thymus, lymph nodes, and spleen, whereas small amounts were detected in non-lymphoid tissues such as kidney, muscle, and testis. Unexpectedly, probing RNA blots with MC14 showed that tissues with high MECL-1 expression contained little MC14 and vice versa. A very similar reciprocal tissue expression was subsequently found for the homologous subunit pairs LMP2 and delta as well as LMP7 and MB1. The subunit protein composition of 20S proteasomes purified from liver, thymus, and lung reflected RNA expression. The impact of a regulated reciprocal tissue expression is discussed with respect to thymic selection and the induction of tolerance in potentially autoreactive T cells.

Amino Acid Sequence↗
PubMed2003

Death of peripheral CD8+ T cells in the absence of MHC class I is Fas-dependent and not blocked by Bcl-xL.

Productive immune responses require an appropriate environment to support peripheral CD8(+) T cell survival. Although host MHC class I molecules appear to be required for this process, the cellular and molecular requirements have not been comprehensively studied. Using adoptive transfer of 2C/recombinase-activating gene-2 (RAG-2)(-/-) TCR-transgenic T cells, we found that the survival of both naive and effector CD8(+) T cells was dependent upon host expression of the same MHC class I alleles that supported thymic selection. Expression of appropriate MHC class Iby either bone marrow- or non-bone-marrow-derived cells was sufficient, suggesting that professional antigen-presenting cells were not mandatory. In contrast to MHC class I, neither T cell expression of CD28 nor host expression of ICAM-1 was required for peripheral T cell survival. Finally, T cell death in the absence of appropriate host MHC class I was overcome by elimination of Fas signaling but not by overexpression of Bcl-x(L) by CD8(+) T cells. These results suggest that, in the absence of a survival signal provided by engagement of host MHC/self peptide complexes, CD8(+) T cells die via a Fas-dependent, mitochondria-independent pathway.

Adoptive Transfer↗
PubMed2004

Characterization of MHC- and TCR-binding residues of the myelin oligodendrocyte glycoprotein 38-51 peptide.

Myelin oligodendrocyte glycoprotein (MOG) is a major experimental autoimmune encephalomyelitis (EAE) antigen in H-2b mice and a potential autoantigen in multiple sclerosis. How well MOG peptides bind to MHC and how TCR recognize the peptide/MHC complex have important implications for thymic selection as well as T cell activation in the periphery. In this study, we have characterized amino acids in the MOG(38-51) peptide important for peptide binding to I-Ab, and for TCR recognition of the peptide/MHC complex. We found that the amino acids R41, F44, R46 and V47 constituted the major TCR contact residues, as alanine substitution at these positions abrogated T cell responses without decreasing their binding affinity to I-Ab. In addition, G38 and W39 were found to be minor TCR contact residues. Finally, substituting tyrosine for alanine at position 40 decreased binding to I-Ab by approximately 50% and prevented induction of T cell responses in C57BL/6J mice upon immunization. Thus, Y40 is the dominant MHC-binding residue of the MOG(38-51) peptide and most likely occupies the p1 pocket of I-Ab. Our results could be useful to design peptides with altered agretopes and epitopes of the MOG(38-51) peptide to study their therapeutic potential in the EAE model.

Amino Acids↗
PubMed2003

Characterization of HLA DR3/DQ2 transgenic mice: a potential humanized animal model for autoimmune disease studies.

Linkage studies indicate close associations of certain HLA alleles with autoimmune diseases. To better understand how specific HLA alleles are related to disease pathogenesis, we have generated an HLA DR3/DQ2 transgenic mouse utilizing a 550-kb yeast artificial chromosome (YAC) construct containing the complete DRalpha, DRbeta1, DRbeta3, DQalpha, and DQbeta regions. The transgenic mouse (4D1/C2D) in an I-Abeta(o) background appears healthy with no signs of autoimmune diseases. Lymphoid tissues as well as CD4(+) T cells develop normally. Characterization of the transgene expression demonstrates that approximately 90% of B cells express high levels of DR3 and 50-70% of B cells express DQ2. CD11c(+) dendritic cells express high levels of DR and DQ. Approximately 12-18% of resting T cells are positive for DR expression, and further up-regulation to 40-50% expression is seen upon activation with anti-CD3/anti-CD28 mAb. These results suggest that the transgenic construct confers a high fidelity to the normal human temporal and spatial expression profile. Analysis of T cell receptor repertoire in transgenic mice confirms that DR3/DQ2 are able to mediate thymic selection. Furthermore, transgenic mice respond to a DR3-restricted antigen, demonstrating antigen processing and presentation by antigen-presenting cells (APC). Purified T cells from ovalbumin (OVA)-immunized 4D1 mice respond to human APC co-cultured with OVA, suggesting appropriate antigen/DR3 or DQ2 recognition by murine T cells. Immunoglobulin isotype switching is also observed, indicating functional T-B cognate interactions. Thus, the DR3/DQ2 transgenic mouse has normal lymphoid development and functionality that are mediated by HLA transgenes and can be used to investigate HLA-associated immunological questions.

Animals↗
PubMed1993

Abnormal T cell development in CD3-zeta-/- mutant mice and identification of a novel T cell population in the intestine.

The T cell antigen receptor (TCR)-associated invariable membrane proteins (CD3-gamma, -delta, -epsilon and -zeta) are critical to the assembly and cell surface expression of the TCR/CD3 complex and to signal transduction upon engagement of TCR with antigen. Disruption of the CD3-zeta gene by homologous recombination resulted in a structurally abnormal thymus which primarily contained CD4- CD8- and TCR/CD3very lowCD4+CD8+ cells. Spleen and lymph nodes of CD3-zeta-/- mutant mice contained a normal number and ratio of CD4+ and CD8+ single positive cells that were TCR/CD3very low. These splenocytes did not respond to antibody cross-linking or mitogenic triggering. The V beta genes of CD4-CD8- and CD4+CD8+ thymocytes and splenic T cells were productively rearranged. These data demonstrated that (i) in the absence of the CD3-zeta chain, the CD4- CD8- thymocytes could differentiate to CD4+CD8+ TCR/CD3very low thymocytes, (ii) that thymic selection might have occurred, (iii) but that the transition to CD4+CD8- and CD4-CD8+ cells took place at a very low rate. Most strikingly, intraepithelial lymphocytes (IELs) isolated from the small intestine or the colon expressed normal levels of TCR/CD3 complexes on their surface which contained Fc epsilon RI gamma homodimers. In contrast to CD3-zeta containing IELs, these cells failed to proliferate after triggering with antibody cross-linking or mitogen. In comparison to thymus-derived peripheral T cells in the spleen and lymph nodes, the preferential expression of normal levels of TCR/CD3 in intestinal IELs suggested they mature via an independent extrathymic pathway.

Animals↗
PubMed1996

Arrest of in vitro T cell differentiation of normal bone marrow-derived CD34+ stem cells with thymic epithelial fragments from children with AIDS.

A novel approach is presented to assess the ability of thymic tissues obtained from children with end stage AIDS to attract normal bone marrow (BM)-derived CD34+ (lineage negative) stem cells (SCs) and support lymphopoiesis in vitro. Chemokinesis of BM-derived CD34+ SCs was analyzed by time-lapse videomicroscopy to ascertain whether an alteration in SC motility could contribute to abnormal thymopoiesis under conditions of HIV infection. The migration of SCs derived from an HIV+ donor into thymic tissue was not significantly altered compared to normal controls, as were normal SCs migrating toward thymic epithelial cell monolayers derived from an HIV+ patient. Thymic tissue obtained from children with AIDS contained nests of CD34+ SCs identified by immunofluorescence, indicating SC homing to the thymus is apparently supported in HIV infection. The ability of HIV-affected thymic epithelial fragments to support lymphopoiesis was determined by examining the initial thymocyte populations present, compared to thymocytes produced de novo in T cell-depleted thymic fragments, following a single pulse of lineage negative CD34+ CD38- SCs. In comparison to normal controls, thymocytes derived from the HIV-affected thymic epithelial fragment coculture had an increased percentage of triple negative thymocytes (28% of lymphocytes from HIV-affected tissue versus 1.5% in controls, p < 0.01) and a decreased percentage of double and single positive CD4+ thymocytes. However, CD3+CD8+ TCR alpha beta + expression was comparable to control cultured thymic epithelial fragments indicating that HIV-affected thymic epithelia were capable of supporting the development of the CD8+ lineage. In an effort to extend the information obtained to date from the histological examination of HIV-affected thymic tissue, select patient thymic tissues were maintained in culture to evaluate the capacity of undifferentiated thymic epithelial cell guirlandes to differentiate in vitro. A partial regeneration of certain subpopulations of the thymic epithelium defined by TE-4 monoclonal antibody (mAb) and CDR2 mAbs occurred during the in vitro culture. The epithelial and mesenchymal components of thymic tissues were distinguished by immunostaining for keratins (indicative of epithelium) and vimentin (a mesenchymal marker). Further evaluation of the modulation of HIV thymus, with respect to the testing of new therapeutic strategies on SCs, will be possible with this in vitro model.

ADP-ribosyl Cyclase↗
PubMed2002

Quantifying the strength of ligand antagonism in TCR triggering.

Triggering of the T cell receptor (TCR) may be antagonized by ligands that are slight variants of the immunogenic peptide. This paper proposes a mathematical model to quantify the strength of the antagonistic effect. The model is based on the kinetics of association and dissociation of TCR and peptide/major histocompatibility (pMHC) molecules, and incorporates TCR triggering according to a kinetic proofreading mechanism. Model analysis indicates that while the average lifetime of the TCR/pMHC complex is the basic determinant of the contribution to TCR triggering made by the ligand, the affinity of the ligand and its MHC presentation level are also important. However, these contributions depend on the kinetic limitation regime. There is a continuum of limitation regimes, at the extremes of which are found TCR limitation and MHC limitation. Both ligand affinity and TCR and pMHC densities determine whether TCR triggering is TCR limited or MHC limited. The changing importance of affinity and antigen presentation level under various kinetic limitation regimes may explain the respective roles of antagonistic and agonistic self peptides in thymic selection. Moreover, TCR down-regulation under TCR-limited conditions may allow the T cell to differentiate between the average lifetime of the TCR/pMHC complex and the presentation level of the ligand. A method for experimental differentiation between passive and active antagonistic effects is proposed which exploits the differences between TCR and MHC limitation.

Antigen-Presenting Cells↗
PubMed1996

Multiple levels of regulation for self-tolerance in beef insulin transgenic mice.

To characterize the mechanism(s) of tolerance toward soluble self-antigens (Ags), beef insulin (BI) transgenic (Tg) mice were generated in which the transgene was expressed in pancreatic beta-cells. Our previous data showed that: (i) Ag-specific tolerance can be induced and/or maintained in peripheral T cells in thymectomized BI-Tg mice and (ii) CD4+ Th2 regulatory T cells are involved in maintaining peripheral tolerance (by anti-BI antibody response). In this paper, we have further characterized the relationship of low levels of BI expression (10(-10)-10(-11) M) in Th1/Th2 activation. In addition, we have explored intrathymic events associated with tolerance to self-Ags not expressed in the thymus and/or to circulating self-Ags. Limiting dilution analysis showed that there was a significantly higher frequency of BI-specific Th2 cells in Tg mice with a corresponding higher frequency of Th1 cells in non-Tg mice. While there was no transgene expression in the thymus (by RT-PCR), independent studies showed that BI can be processed and presented in the Tg thymus, which correlated with the Ag-specific hyporesponsiveness of mature thymocyes detected in vitro. High-dose rIL-2 (150 U/ml) was able to restore in vitro peripheral T cell response of Tg mice to levels comparable to those of the non-Tg control. Collectively, our data suggest that: (i) there is a differential activation of BI-specific Th1/Th2 cells in vivo in the presence of low Ag concentration; (ii) the thymus may play a role in self-tolerance to Ags whose expression in adults is restricted to the periphery; and (iii) multiple levels of regulation such as thymic selection, peripheral anergy, and active suppression may be involved in tolerance to BI in BI-Tg mice.

Animals↗
PubMed1998

The association of MHC with autoimmune diseases: understanding the pathogenesis of autoimmune diabetes.

The current paradigm of MHC and disease association is efficient binding of autoantigens by disease-associated MHC molecules leading to a T cell-mediated immune response and resultant autoimmune sequelae. Data presented here offer a different model for this association of MHC with autoimmune diabetes. This new explanation suggests that the association of MHC with autoimmunity results from "altered" thymic selection in which high-affinity self-reactive (potentially autoreactive) T cells escape negative selection. This model offers an explanation for the requirement of homozygous MHC class II expression in NOD mice (and in man) in susceptibility to IDDM.

Animals↗
PubMed2001

A reliable and safe T cell repertoire based on low-affinity T cell receptors.

Antigens are presented to T cells as short peptides bound to MHC molecules on the surface of body cells. The binding between MHC/peptides and T cell receptors (TCRs) has a low affinity and is highly degenerate. Nevertheless, TCR-MHC/peptide recognition results in T cell activation of high specificity. Moreover, the immune system is able to mount a cellular response when only a small fraction of the MHC molecules on an antigen-presenting cell is occupied by foreign peptides, while autoimmunity remains relatively rare. We consider how to reconcile these seemingly contradictory facts using a quantitative model of TCR signalling and T cell activation. Taking into account the statistics of TCR recognition and antigen presentation, we show that thymic selection can produce a working T cell repertoire which will produce safe and effective responses, that is, recognizes foreign antigen presented at physiological levels while tolerating self. We introduce "activation curves" as a useful tool to study the repertoire's statistical activation properties.

Humans↗
PubMed1992

A Tcra congenic mouse: V alpha epitope expression is influenced by both Tcra haplotypes and background genes.

A T-cell receptor alpha chain locus (Tcra) congenic mouse is described. The Tcraa haplotype of BALB/c (donor strain) was bred on to B10.D2 (background strain, Tcrab haplotype) by using a Bgl I Tcra-C restriction fragment length polymorphism. Tcraa/b heterozygous offspring from the eleventh backcross generation were brother-sister mated to obtain Tcra-Ca homozygous animals. The resulting congenic line, B10.D2.C-Tcraa/Bo carries a recombination between the Tcra and the hr loci; thus, the transferred differential segment is the centromeric 18-27 cM of the BALB/c chromosome 14. Analysis with a multitude of Tcra-V and Tcrd-V probes demonstrates that the complete Tcraa haplotype is contained within this differential segment. Lymph node T cells of BALB/c (Tcraa) B10.D2 (Tcrab) and B10.D2.C-Tcraa were stained with anti-V alpha 8 (KT50, KT65), anti-V alpha 3.2 (RR3-16) and anti-V alpha 11.1 and 2 (RR8-1) monoclonal antibodies. We find that the frequencies of V alpha epitope expression are highly Tcra haplotype-dependent even though an influence of background genes is also observed. Thus, Tcra-V germline differences may possibly influence the T cell repertoire, in addition to the already well known positive and negative thymic selections. Tcra haplotype does not influence the frequencies of V beta utilization. However, BALB/c mice have fewer V beta 11+ T cells than B10.D2 and B10.D2-Tcraa, therefore, the BALB/c genome must harbor a V beta 11 deleting gene(s) in addition to those described so far.

Animals↗
PubMed1998

The role of caspases in T cell development and the control of immune responses.

Apoptosis is responsible for the removal of potentially autoreactive or useless T cells during thymic selection and activated T cells in the periphery. Specific families of receptors, kinases, transcription factors, and cysteine proteases, termed caspases, are involved in the apoptotic cascade leading to proteolysis of specific substrates and to morphological changes associated with programmed cell death. Although common members of the apoptotic cascade are shared between different cell types, it appears that cell-specific factors can influence the response to a given apoptotic stimuli. Characterization and understanding of the basic mechanisms involved in the different pathways protecting or leading to cell death may provide novel ways to control inappropriate apoptosis involved in several diseases.

Animals↗
PubMed1999

Spontaneous T-cell proliferation in the non-obese diabetic mouse to a peptide from the unique class II MHC molecule, I-Ag7, which is also protective against the development of autoimmune diabetes.

AIMS/HYPOTHESIS: Major histocompatibility complex class II molecules present antigenic peptides to T-cells and have an important role in T-cell thymic education. The mechanism by which major histocompatibility complex alleles confer a high genetic risk for autoimmune diabetes is not known. One hypothesis is that during positive thymic selection, the peripheral T-cell repertoire is modelled by major histocompatibility complex-restricted presentation of self major histocompatibility complex molecule-derived peptides, some of which mimic tissue autoantigens. The sequence similarity between a known T-cell epitope of glutamic acid decarboxylase-65, 509:VPPSLRTLED and the non-obese diabetic mouse class II major histocompatibility complex molecule I-Ag7 86:VPTSLRRLEQ is consistent with this. METHODS: We measured spontaneous proliferation of peripheral T-cells from non-obese diabetic mice and other, non-diabetes-prone strains, to the I-Ag7 86-101 and glutamic acid decarboxylase-65(509-524) peptides, binding of these peptides to intact I-Ag7 and assessed the effect of tolerance induction on diabetes development, by injecting young non-obese diabetic mice with high doses of peptide. RESULTS: T-cells from non-obese diabetic, but not other mice strains, spontaneously proliferate to the I-Ag7 86-101 and glutamic acid decarboxylase-65(509-524) peptides, but not control peptides. Both test peptides bind I-Ag7. Tolerance induction prolongs diabetes-free survival in non-obese diabetic mice when either the I-Ag7 86-101 or glutamic acid decarboxylase-65(509-524) peptide, but not control peptide, is used. CONCLUSION/INTERPRETATION: A peptide from the unique class II major histocompatibility complex, diabetes-susceptibility molecule, I-Ag7, presented by I-Ag7 is a target of T-cell responses in diabetes-prone nonobese diabetic mice and tolerance induction against the peptide offers appreciable protection against the development of diabetes.

Amino Acid Sequence↗
PubMed2000

Soluble donor MHC class I gene transfer to thymus promotes allograft survival in a high-responder heart transplant model.

Thymic selection of self and non-self-reactive lymphocytes is a process that may be targeted to induce donor-specific immunologic unresponsiveness in organ transplantation. In the present study, gene transfer was used to preexpose the recipient thymus to soluble donor-specific MHC class I molecules prior to heart transplantation in the high-responder ACI (RT1a) to Lewis (RT1l) rat strain combination. Specifically, cultured Lewis hepatocytes were transfected with DNA encoding a secreted form of the donor allo-MHC class I antigen, RT1.Aa. Seven days prior to ACI heart transplantation, genetically altered recipient-strain hepatocytes were injected into the thymus of Lewis recipients which also received a dose of antilymphocyte serum (ALS). Results showed that treatment with both ALS and soluble donor MHC-expressing hepatocytes prolonged transplant survival time by twofold, compared to injection of control hepatocytes and ALS. Therefore, intrathymic gene therapy delivery of soluble donor MHC molecules may be useful for promoting allograft survival in heart transplantation.

Animals↗
PubMed2001

Existence of MHC class I-restricted alloreactive CD4+ T cells reacting with peptide transporter-deficient cells.

It is generally accepted that as the result of positive thymic selection, CD8-expressing T cells recognize peptide antigens presented in the context of MHC class I molecules and CD4-expressing T cells interact with peptide antigens presented by MHC class II molecules. Here we report the generation of TCRalpha/beta(+), CD3(+), CD4(+), CD8(-), MHC class I-restricted alloreactive T-cell clones which were induced using peripheral blood mononuclear cells from healthy individuals following in vitro stimulation with transporter associated with antigen processing (TAP)-deficient cell lines T2. The CD4(+) T-cell clones showed an HLA-A2.1-specific proliferative response against T2 cells which was inhibited by anti-CD3 and anti-CD4 monoclonal antibodies. These results suggest that interaction of the TCR with peptide-bound HLA class I molecules contributes to antigen-specific activation of these co-receptor-mismatched T-cell clones. Antigen recognition by alloreactive MHC class I-restricted CD4(+) T cells was inhibited by removing peptides bound to HLA molecules on T2 cells suggesting that the alloreactive CD4(+) T cells recognize peptides that bind in a TAP-independent manner to HLA-A2 molecules. The existence of such MHC class I-restricted CD4(+) T cells which can recognize HLA-A2 molecules in the absence of TAP function may provide a basis for the development of immunotherapy against TAP-deficient tumor variants which would be tolerant to immunosurveillance by conventional MHC class I-restricted cytotoxic lymphocytes.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

In vitro construction of graded thymus chimeras.

A simple in vitro approach is described for constructing chimeric thymi in which the degree of chimeric contribution can be tightly controlled. This is achieved by protease-assisted dissociation of fresh fetal thymic tissue, mixing the resulting cell suspensions at the desired ratios, and subsequent reaggregation. Analysis of these graded chimeras shows that stromal elements become evenly interspersed and form a microenvironment able to support the maturation of endogenous T cell precursors. Chimeras between normal and MHC-deficient thymi have been used to demonstrate the rescue of mutant, allotype-marked thymocytes by wild-type stromal cells. Other potential applications of the method include quantitative studies on positive and negative thymic selection, the functional role of defined stromal cell types in these processes, and the analysis of mutants (either natural or engineered) by complementation.

Animals↗
PubMed1985

Expression of antigen-specific, major histocompatibility complex-restricted receptors by cortical and medullary thymocytes in situ.

We have examined the distribution of the antigen-specific, major histocompatibility complex-restricted receptor on mouse thymocytes in situ, using immunohistochemical techniques and the monoclonal antibody KJ16-133. This antibody reacts with the beta chain of the receptors on about 20% of peripheral murine T cells. Of the cortical thymocytes reacting with KJ16-133, cells with only cytoplasmic staining were most frequently observed. Such cytoplasmic staining was not observed in the medulla. Occasional cortical cells had low levels of surface expression, which was almost invariably patched in the region of contact with epithelial cell processes. KJ16-133+ medullary thymocytes had high levels of uniform surface labeling. These results suggest that thymic selection of MHC restriction and/or tolerance may occur in the cortex, where the receptors on maturing thymocytes interact with MHC proteins on epithelial cells.

Animals↗