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PubMed1995

Severe colitis in mice with aberrant thymic selection.

Tg epsilon 26 mice display an arrest very early in T cell development that has a profound effect on the architecture of thymic stromal cells. We have recently demonstrated that transplantation of wild-type bone marrow cells restores the thymic microenvironment of fetal but not adult Tg epsilon 26 mice. Here, we report that T cell-reconstituted adult Tg epsilon 26 mice develop a spontaneous wasting syndrome characterized by extensive inflammation of the colon, resembling human ulcerative colitis. Colitis in these animals was marked by substantial infiltration of the colon by activated thymus-derived CD4+ T cells. Importantly, bone marrow-transplanted Tg epsilon 26 mice previously engrafted with a fetal Tg epsilon 26 thymus did not develop colitis. These results suggest that T cells selected in an aberrant thymic microenvironment contain a population of cells able to induce severe colitis that can be prevented by T cells that have undergone normal thymic development.

Animals↗
PubMed2005

Molecular signature of recent thymic selection events on effector and regulatory CD4+ T lymphocytes.

Natural CD4+CD25+ regulatory T lymphocytes (Treg) are key protagonists in the induction and maintenance of peripheral T cell tolerance. Their thymic origin and biased repertoire continue to raise important questions about the signals that mediate their development. We validated analysis of MHC class II capture by developing thymocytes from thymic stroma as a tool to study quantitative and qualitative aspects of the cellular interactions involved in thymic T cell development and used it to analyze Treg differentiation in wild-type mice. Our data indicate that APCs of bone marrow origin, but, surprisingly and importantly, not thymic epithelial cells, induce significant negative selection among the very autoreactive Treg precursors. This fundamental difference between thymic development of regulatory and effector T lymphocytes leads to the development of a Treg repertoire enriched in cells specific for a selected subpopulation of self-Ags, i.e., those specifically expressed by thymic epithelial cells.

Animals↗
PubMed2003

Disparate peptide-dependent thymic selection outcomes in beta2M-deficient mice versus TAP-1-deficient mice: implications for repertoire formation.

Fetal thymic organ cultures of N15-transgenic RAG-2-/- H-2b mice on normal, beta-2 microglobulin (beta2M)-/- or transporter associated with antigen processing (rAP-1)-/- MHCl-deficient backgrounds were used to examine differentiation of thymocytes bearing a TCR specific for a viral peptide bound to H-2Kb. Strong agonists mediate negative selection in all mice whereas weak agonists are positively selecting in beta2MW-/- mice but negatively selecting on TAP-1-/- or normal backgrounds. Very weak agonists and very weak antagonists are generally without effect in beta2M-/- mice yet foster differentiation in TAP-1-/- animals. The 20-40-fold reduction in beta2M4-/- thymic H-2Kb surface expression suggests that the avidity of the TCR for peptide-MHCI accounts for these differences, consistent with effects of TCR density and individual thymic-peptide abundance in peptide-MHC complexes. TCR-self-MHC interaction dominates Kb-based selection, subtly modulated by peptides as revealed by X-ray crystallography.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Impaired immune responses toward alloantigens and tumor cells but normal thymic selection in mice deficient in the beta2 integrin leukocyte function-associated antigen-1.

We have generated mice deficient in the beta2 integrin LFA-1 by targeted disruption of the CD11a gene in embryonic stem cells. In vitro LFA-1 -/- cells exhibit a delayed proliferative response toward alloantigens in the MLR. In vivo the host-vs-graft reaction toward injected allogeneic cells is also reduced. Alloantigen-specific CTLs generated from LFA-1 -/- mice are impaired in their cytotoxic activity toward allogeneic spleen cells as well as cell line targets. The proliferative response of LFA-1 -/- splenocytes following stimulation by LPS, PMA plus ionomycin, or immobilized anti-CD3epsilon mAb is normal, but Con A-stimulated proliferation is greatly diminished. We observe typical edema formation in a delayed type hypersensitivity reaction to SRBC with normal extravasation of leukocytes and demonstrate recruitment of neutrophils to an LPS-induced inflammatory site in these mice, suggesting that LFA-1 does not play an essential role in lymphocyte homing and leukocyte extravasation. We further show that LFA-1 -/- mice are susceptible to metastasis of B16 melanoma tumors, although their in vitro NK cell activity appears normal. A study of LFA-1 -/- mice expressing transgenic TCRs indicates that thymic maturation and selection of T cells are unaffected by the loss of LFA-1. Our results indicate that LFA-1 is important for alloantigen-triggered T cell proliferation and cytotoxicity, for Con A stimulation of T cells, and in tumor rejection. It does not appear to play an essential role in lymphocyte homing and leukocyte extravasation or in T cell maturation and selection in the thymus.

Animals↗
PubMed1993

Thymic microenvironmental abnormalities and thymic selection in NZB.H-2bm12 mice.

We have taken advantage of an extensive panel of mAb directed to thymic epithelial and nonepithelial stromal cells to examine the expression of these Ag from day 16 of gestation through 6 mo of age in NZB(H-2d), NZB.H-2b, NZB.H-2bm12, C57BL/6(H-2b), and C57BL/6.H-2bm12 mice. In addition, by triple color flow cytometry we have examined the expression of cell surface markers defining distinct stages of intrathymic T cell maturation. New Zealand mice demonstrated three abnormalities. MTS 10 normally stains thymic medullary and subcapsular epithelium. However, New Zealand mice demonstrated striking irregular medullary epithelial cell shape whereas their subcapsular epithelium remained normal. Moreover, New Zealand mice, unlike controls, were found to have MTS 10+ epithelial cells within the cortex. Additionally, MTS 39 and MTS 44, which normally stain reticular cortical epithelium, produced a striking different staining pattern in New Zealand mouse thymus, including the presence of large cortical epithelial cellfree regions, so-called "cortical holes." MTS 33 normally stains cortical thymocytes but in New Zealand mice, there was a severe decrease of MTS 33+ cells. There was also an increase of CD3lowCD4+CD8+ cells in NZB mice, which may include many predeletion thymocytes. Finally, there was a significant increase of CD3highCD4+CD8- cells in NZB.H-2bm12 and C57BL/6.H-2bm12 mice compared with NZB.H-2b and C57BL/6(H-2b) mice. We postulate that these microenvironmental alterations in NZB mice contribute to and reflect altered T cell differentiation, thereby predisposing them to autoimmune disease. Moreover, the increased proportion of CD3highCD4+CD8- cells associated with the H-2bm12 mutation may be involved in the remarkably different profiles of disease between NZB.H-2b and NZB.H-2bm12 mice.

Animals↗
PubMed1996

Regulation of AP-1 and NFAT transcription factors during thymic selection of T cells.

The ability of thymocytes to express cytokine genes changes during the different stages of thymic development. Although CD4- CD8- thymocytes are able to produce a wide spectrum of cytokines in response to a T-cell receptor (TcR)-independent stimulus, as they approach the double-positive (DP) CD4+ CD8+ stage, they lose the ability to produce cytokine. After the DP stage, thymocytes become single-positive CD4+ or CD8+ thymocytes which reacquire the ability to secrete cytokines. In an attempt to understand the molecular basis of this specific regulatin, we use AP-1-luciferase and newly generated NFAT-luciferase transgenic mice to analyze the transcriptional and DNA-binding activities of these two transcription factors that are involved in the regulation of cytokine gene expression. Here, we show that both AP-1 and NFAT transcriptional activities are not inducible in the majority of DP cells but that during the differentiation of DP cells to the mature single-positive stage, thymocytes regain this inducibility. Subpopulation analysis demonstrates that this inducibility is reacquired at the DP stage before the down-modulation of one of the coreceptors. Indeed AP-1 inducibility, just like the ability to express the interleukin-2 gene, is reacquired during the differentiation of DP TcRlow CD69low heat-stable antigen (HSA)high thymocytes to DP TcRhigh CD69high HSAhigh cells, which is considered to be the consequence of the first signal that initiates positive selection. We therefore propose that the inability of DP thymocytes to induce AP-1 and NFAT activities is one of the causes for the lack of cytokine gene expression at this stage and that this inducibility is reacquired at the latest stage of DP differentiation as a consequence of positive selection. This could be a mechanism to prevent the activation of DP thymocytes before selection has taken place.

Animals↗
PubMed2000

Effects of a constitutively active form of calcineurin on T cell activation and thymic selection.

Calcineurin is a calcium/calmodulin-dependent phosphatase whose activity is required for the induction of T cell lymphokine production and proliferation. Although its specific role in T cell development is less well defined, studies with the immunosuppressive drugs cyclosporin A and FK-506 suggest that it is involved in both positive and negative selection of immature thymocytes. To more completely characterize a role for calcineurin in T cell development in vivo, we have generated transgenic mice that express an activated form of this enzyme in thymocytes and peripheral T cells. We find that the transgene causes a block in early thymic development, resulting in a reduction in the steady-state number of CD4 and CD8 double positives, but not on the number of mature T cells. We also find that thymocytes and mature T cells expressing this transgene are more sensitive to signals through their TCR. In thymocytes this sensitivity difference is manifested as an increase in positive selection, although negative selection seems to remain unaffected. Therefore, these studies confirm and extend past reports that suggested a role for calcineurin in thymic development and selection.

Animals↗
PubMed1999

Cutting edge: thymic selection and autoreactivity are regulated by multiple coreceptors involved in T cell activation.

Immune responses are shaped by several processes that promote responses to pathogens and hinder responses to self. One mechanism that contributes to this polarization in response is negative selection, in which thymocytes that can respond to self-peptide/MHC complexes are deleted from the T cell repertoire. I found here that several coreceptors known to contribute to mature T cell activation also participate in negative selection. Interestingly, these molecules appeared to act in a cooperative fashion. Blocking the contribution of these molecules in fetal thymus organ culture not only prevented negative selection in the CD4+ lineage, but also induced the appearance of autoreactive thymocytes. This is the first demonstration that blocking coreceptor interactions during thymic development can produce autoreactive T cells. The contribution of negative selection to the mature T cell repertoire and to autoimmunity is discussed in light of these results.

Animals↗
PubMed1994

Thymic selection of immunoglobulin idiotype specific T-cells.

The thymus is the major site for differentiation and maturation of developing T-cells. The diversity in the T-cell repertoire is generated during ontogeny by rearrangements of T-cell receptor gene segments that encode clonally distributed receptors. Most of the newly produced thymocytes that bear alpha beta TCR die in the thymus as a consequence of at least two selective processes. Those that can react to self MHC antigens survive (positive selection), but only if the binding affinity for the same or other self MHC (probably complexed with self peptides) molecules is not too high (negative selection). As a result, each T-cell that has acquired functional competence in the thymus should have a certain degree of self-MHC specificity. Our comprehension of thymic development has been aided by the study of transgenic mice that bear functionally rearranged TCR or Ig lambda light chain genes. To study positive selection we analyzed transgenic mice with alpha beta TCR genes isolated from a T-cell clone specific for an idiotypic peptide of lambda 2(315) (a mutant of the lambda 2 Ig chain) bound to the E(d) MHC class II molecule. The results suggest that positive selection of some thymocytes might be weak, reminiscent of a 'struggle for survival', since the selected population was small and had high levels of transgenic receptor and coreceptor (CD4) molecules. The thymic tolerance mechanism was also investigated using a combination of both TCR and lambda 2(315) transgenic mice. The clonal deletion of Id specific thymocytes was influenced by the production site as well as the concentration of the lambda 2(315) antigen. In conclusion, the interaction avidity of a single thymocyte, which is important for selective processes, might be affected by the expression levels of each interacting component: TCR, CD4 or CD8 and MHC molecule and self antigen.

Animals↗
PubMed1996

Molecular defects in TCRBV genes preclude thymic selection and limit the expressed TCR repertoire.

A prerequisite for the assembly of a functional TCR is the rearrangement of gene segments to result in in-frame transcripts that can vary in length across the CDR3 region. Selection for in-frame 3-bp spaced rearrangements is observed for functional TCRB genes in thymocyte DNA and mRNA transcripts from PBMC. Previous analyses of the expressed human TCRBV gene repertoire have suggested that BV10S1 and BV19S1 gene segments may be expressed at very low levels or not at all in some individuals. CDR3 size analysis for BV10 and BV19 transcripts and thymic DNA rearrangements revealed no such selection of in-frame 3-bp spaced rearrangements. Comparison of the BV19 leader intron sequence with consensus 5'-splice signal sequences suggested that the mature mRNA for this gene would contain the unspliced leader intron. Sequencing of BV19 transcripts from PBMC confirmed that the intron was not spliced, resulting in a predicted translation product that terminates prematurely. Both genomic DNA and mRNA were analyzed for the BV10 gene. The leader sequence contained a single extra base, which would result in a shift in the V region reading frame upon conventional mRNA splicing. This gene is predicted to be nonfunctional due to the presence of a stop codon in the V gene segment just after the splice signal. A splice variant that uses an alternative 3'-splice site further downstream in the V region was also detected. This variant is predicted to be nonfunctional due to the presence of an in-frame stop codon in the V region. These processing defects are sufficient to abrogate positive selection. Therefore, the conclusions drawn from previous studies of the expressed T cell repertoire in normal and disease states based on the presumed functional status of these two genes need to be reassessed.

Base Sequence↗
PubMed1988

Thymic selection process induced by hybrid antibodies.

Thymus-derived (T) lymphocytes using the alpha beta T-cell antigen receptor (TCR) recognize fragmented antigen in conjunction with surface molecules encoded by genes of the major histocompatibility complex (MHC). Peripheral T lymphocytes preferentially see antigen presented by self rather than by foreign MHC molecules, and autoreactive T lymphocytes are deleted. Thus, the peripheral T-lymphocyte repertoire is skewed towards recognition of antigen in the context of self-MHC and towards tolerance to self-antigens. During T-lymphocyte development in the thymus, this repertoire is formed by the interaction of TCR with MHC molecules resulting in positive and negative selection phenomena. Hybrid antibodies (HAbs) that carry binding sites to the TCR and to a surface marker on another cell can engage all T lymphocytes regardless of their specificity. It should be possible to mimic selection processes in normal animals with HAb that specifically link members of a TCR family to MHC molecules on the thymic stroma. We have probed T-lymphocyte development with HAbs linking V beta 8-positive TCR to either class I or class II MHC products in thymic organ culture. Thymocytes exposed to either HAb in an early stage of maturation respond with a significant increase in the frequency of V beta 8-carrying cells. At a later stage of development V beta 8-positive thymocytes are depleted. These results illustrate the succession of positive and negative selection in the developing thymus of normal mice.

Animals↗
PubMed1994

Thymic selection and adaptability of cytotoxic T lymphocyte responses in transgenic mice expressing a viral protein in the thymus.

Upon primary challenge with lymphocytic choriomeningitis virus (LCMV), H-2d (BALB/cByJ) mice mount a cytotoxic T lymphocyte (CTL) response to a single immunodominant domain of the viral nucleoprotein (NP) but no detectable response to the viral glycoprotein (GP). To manipulate this CTL response, the viral NP gene was expressed in the thymus and peripheral T lymphocytes using the murine Thy1.2 promoter. As a result, such Thy1.2-NP (H-2d) transgenic (tg) mice deleted their high-affinity anti-LCMV-NP CTL, but generated equal numbers of lower-affinity NP CTL. Further, they made an alternative anti-LCMV-GP CTL response that is not normally found in non-tg mice indicating a hierarchial control of the CTL response. Unlike the H-2d mice, H-2b (C57Bl/6J) mice normally mount a CTL response to both LCMV-GP and -NP. When the LCMV-NP was expressed using the Thy1.2 promoter in these H-2b mice, the LCMV-NP-specific CTL response was completely aborted and no CTL to new, alternative viral epitopes were generated. Dilutions of H-2b or H-2d NP peptides indicated that 3-4 logs less H-2b NP peptide was required to sensitize syngeneic target cells for CTL-specific lysis, suggesting that the differing affinities of H-2b and H-2d major histocompatibility complex molecules for their peptides likely account for the total removal of NP CTL in the H-2b mice but only partial removal in H-2d mice made to express thymic NP. Thymic grafting experiments done with thymi from newborn Thy1.2-NP tg mice show that selection processes studied in this model are of central (thymic) origin and are not caused by Thy1.2-positive LCMV-NP-expressing T lymphocytes in the periphery.

Adaptation, Physiological↗
PubMed1995

An essential role for gp39, the ligand for CD40, in thymic selection.

The interactions between CD40 on B cells and its ligand gp39 on activated T helper cells are known to be essential for the development of thymus-dependent humoral immunity. However, CD40 is also functionally expressed on thymic epithelial cells and dendritic cells, suggesting that gp39-CD40 interactions may also play a role in thymic education, the process by which self-reactive cells are deleted from the T cell repertoire. Six systems of negative selection were studied for their reliance on gp39-CD40 interactions to mediate negative selection. In all cases, when the antigen/superantigen was endogenously expressed (in contrast to exogenously administered), negative selection was blocked by loss of gp39 function. Specifically, blockade of gp39-CD40 interactions prevented the deletion of thymocytes expressing V beta 3, V beta 11, and V beta 12, specificities normally deleted in BALB/c mice because of the endogenous expression of minor lymphocyte-stimulating determinants. Independent verification of a role of gp39 in negative selection was provided by studies in gp39-deficient mice where alterations in T cell receptor (TCR) V beta expression were also observed. Studies were also performed in the AND TCR transgenic (Tg) mice, which bear the V alpha 11, V beta 3 TCR and recognize both pigeon cytochrome c (PCC)/IEk and H-2As. Neonatal administration of anti-gp39 to AND TCR Tg mice that endogenously express H-2As or endogenously produce PCC prevented the deletion of TCR Tg T cells. In contrast, deletion mediated by high-dose PCC peptide antigen (administered exogenously) in AND TCR mice was unaltered by administration of anti-gp39. In addition, deletion by Staphylococcus enterotoxin B in conventional mice was also unaffected by anti-gp39 administration. gp39 expression was induced on thymocytes by mitogens or by antigen on TCR Tg thymocytes. Immunohistochemical analysis of B7-2 expression in the thymus indicated that, in the absence of gp39, B7-2 expression was substantially reduced. Taken together, these data suggest that gp39 may influence negative selection through the regulation of costimulatory molecule expression. Moreover, the data support the hypothesis that, for negative selection to some endogenously produced antigens, negative selection may be dependent on TCR engagement and costimulation.

Amino Acid Sequence↗
PubMed2003

Thymic selection revisited: how essential is it?

Intrathymic T cell development represents one of the best studied paradigms of mammalian development. Lymphoid committed precursors enter the thymus and the Notch1 receptor plays an essential role in committing them to the T cell lineages. The pre-T cell receptor (TCR), as an autonomous cell signaling receptor, commits cells to the alphabeta lineage while its rival, the gammadeltaTCR, is involved in generating the gammadelta lineage of T cells. Positive and negative selection of immature alphabetaTCR-expressing cells are essential mechanisms for generating mature T cells, committing them to the CD4 and CD8 lineages and avoiding autoimmunity. Additional lineages of alphabetaT cells, such as the natural killer T cell lineage and the CD25+ regulatory T cell lineage, are formed when the alphabetaTCR encounters specific ligands in suitable microenvironments. Thus, positive selection and receptor-instructed lineage commitment represent a hallmark of the thymus. Ectopically expressed organ-specific antigens contribute to thymic self-nonself discrimination, which represents an essential feature for the evolutionary fitness of mammalian species.

Animals↗
PubMed1998

Normal thymic selection of TCR transgenic CD4 T cells, but impaired survival in the periphery despite the presence of selecting MHC molecules.

In this paper, we investigate selection in the thymus and survival in the periphery of CD4 T cells, which carry a major histocompatibility class II-restricted transgenic TCR (A18 TCRtg) specific for a natural self Ag, the fifth component of complement (C5). A18 TCRtg thymocytes develop normal numbers of CD4 single-positive (SP) thymocytes, but do not show pronounced overselection as do some other TCR transgenic strains. CD4 SP cells are mature as judged by termination of CD8 synthesis, resistance to cortisone, and functional competence. The kinetics of positive selection, determined by BrdU labeling, are very fast. CD4 SP thymocytes are demonstrable within 2 days of labeling, and within 8 days after labeling a large proportion (20%) of lymph node T cells are recent thymic emigrants. The high number of recent thymic emigrants suggests rapid turnover of CD4 T cells in the periphery, which was confirmed by thymectomy and determination of CD4 T cell life spans. A18 TCRtg T cells have a t(1/2) of approximately 6 wk, despite the presence of selecting MHC molecules. This explains the failure to accumulate high numbers of peripheral T cells and suggests that the MHC-bound ligand(s) responsible for initiating survival signals is limiting for the selection and maintenance of A18 transgenic CD4 T cells.

Animals↗
PubMed2001

A critical role for CD2 in both thymic selection events and mature T cell function.

To examine the function of CD2 in vivo, N15 TCR transgenic (tg) RAG-2(-/-) H-2(b) mice bearing a single TCR specific for the vesicular stomatitis virus octapeptide bound to the H-2K(b) molecule were compared on a wild-type or CD2(-/-) background. In N15tg RAG-2(-/-) CD2(-/-) mice, thymic dysfunction is evident by 6 wk with a pre-TCR block in the CD4(-)CD8(-) double-negative thymocytes at the CD25(+)CD44(-) stage. Moreover, mature N15tg RAG-2(-/-) CD2(-/-) T cells are approximately 100-fold less responsive to vesicular stomatitis virus octapeptide and unresponsive to weak peptide agonists, as judged by IFN-gamma production. Repertoire analysis shows substantial differences in Valpha usage between non-tg C57BL/6 (B6) and B6 CD2(-/-) mice. Collectively, these findings show that CD2 plays a role in pre-TCR function in double-negative thymocytes, TCR selection events during thymocyte development, and TCR-stimulated cytokine production in mature T cells.

Animals↗
PubMed1991

The V beta 17+ T cell repertoire: skewed J beta usage after thymic selection; dissimilar CDR3s in CD4+ versus CD8+ cells.

To ascertain how the actual repertoire of T cell receptors (TCRs) deviates from the theoretical, we have generated a large number of junctional region sequences from TCRs carrying the V beta 17 variable region. The greater than 600 sequences analyzed represent transcripts from nine different cell populations, permitting several comparisons: transcripts from an expressed vs. a non-expressed V beta 17 allele, those from E+ vs. E- mice, transcripts from immature vs. mature thymocytes, those from thymic vs. peripheral T cells, and those from CD4+ vs. CD8+ cells. These comparisons have allowed us to distinguish between the influence of molecular events involved in TCR gene rearrangement and that of various selection events that shape the T cell repertoire. Our most striking findings are: (a) that J beta usage is markedly skewed, partly due to recombination mechanics and partly due to selection forces: in particular, those mediated by the class II E molecule in the thymus; and (b) that TCRs on CD4+ and CD8+ cells show intriguing dissimilarities. In addition, we present evidence that N nucleotide additions occur with clear biases, probably due to idiosyncrasies of the recombination enzymes, and provide arguments that TCR and immunoglobulin CDR3s have distinct structures.

Alleles↗
PubMed1997

Induction of the early growth response (Egr) family of transcription factors during thymic selection.

There is little known about the regulation of gene expression during TCR-mediated differentiation of immature CD4+8+ (double positive) thymocytes into mature T cells. Using the DPK CD4+8+ thymocyte precursor cell line, we demonstrate that the early growth response-1 gene (Erg-1), encoding a zinc finger transcription factor, is rapidly upregulated after TCR stimulation. We also report that Egr-1 is expressed by a subset of normal double positive thymocytes in the thymic cortex, as well by a majority of medullary single positive thymocytes. Expression of Egr-1 is dramatically reduced in the thymus of major histocompatibility complex knockout mice, but can be induced by anti-CD3 antibody stimulation of isolated thymocytes from these animals. These and other data suggest that high level expression of Egr-1 in the thymus is a consequence of selection. A similar pattern of expression is found for family members Egr-2 and Egr-3. Using the DPK cell line, we also demonstrate that expression of Egr-1, 2, and 3 is dependent upon ras activation, as is the initiation of differentiation to a single positive cell. In contrast, the calcineurin inhibitor cyclosporin A, which inhibits DPK cell differentiation as well as positive selection, inhibits expression of Egr-2 and Egr-3, but not Egr-1. The identification of the Egr family in this context represents the first report of a link between the two known signaling pathways involved in positive selection and downstream transcriptional regulators.

Animals↗