Peripheral antigen display by lymph node stroma promotes T cell tolerance to intestinal self

The intestinal epithelium functions to absorb nutrients and to protect the organism against microbes. To prevent autoimmune attack on this vital tissue, T cell tolerance to intestinal self-antigens must be established. Central tolerance mechanisms involve medullary thymic epithelial cells (mTECs), which use endogenously expressed peripheral-tissue antigens (PTAs) to delete self-reactive thymocytes. The prevailing model for the induction of peripheral tolerance involves cross-presentation of tissue antigens by quiescent dendritic cells. Here we show that lymph node stromal cells present endogenously expressed PTAs to T cells. Moreover, antigen presentation by lymph node stroma is sufficient to induce primary activation and subsequent tolerance among CD8(+) T cells. Thus, lymph node stromal cells are functionally akin to mTECs and provide a direct strategy for purging the peripheral repertoire of self-reactive T cells.     

CD8+ T Cell Exhaustion During Persistent Viral Infection is Regulated Independently of the Virus-Specific T Cell Receptor

During chronic viral infections, responses by virus-specific CD8⁺ T cells become marginalized by the acquisition of functional defects and reduced cell numbers in a process defined as T cell exhaustion. Similarly, T cell tolerance to self-antigen is also characterized by impaired effector function and eventual deletion of self-reactive T cells. Induction of both tolerance and exhaustion involve many shared inhibitory mechanisms, thus similar therapeutic approaches have proven effective in these distinct environments. We previously demonstrated that tolerant self-reactive CD8⁺ T cells expressing dual-T cell receptors (i.e., dual-TCR) could be rescued by immunization through a second TCR specific for a foreign antigen. These data revealed that T cell tolerance was regulated at the level of the self-reactive TCR. Here, dual-TCR CD8⁺ T cells were used to examine if exhaustion during persistent viral infection could be rescued by an analogous strategy of immunization through a second TCR not involved in recognition of virus. In direct contrast to the rescue achievable in tolerant CD8⁺ T cells, exhausted T cells were equally impaired through both TCR. These findings suggest that exhaustion is maintained by defects downstream of the virus-specific TCR, and establish that exhaustion and tolerance are distinctly regulated states of T cell dysfunction.     

Lymph node stroma broaden the peripheral tolerance paradigm

Research into how self-reactive T cells are tolerized in lymph nodes has focused largely on dendritic cells (DCs). We now know that lymph node stromal cells (LNSC) are important mediators of deletional tolerance to peripheral tissue-restricted antigens (PTAs), which are constitutively expressed and presented by LNSCs. Of the major LNSC subsets, fibroblastic reticular cells and lymphatic endothelial cells are known to directly induce tolerance of responding na?ve CD8 T cells. The biological outcome of this interaction fills a void otherwise not covered by DCs or thymic stromal cells. These findings, we suggest, necessitate a broadening of peripheral tolerance theory to include steady-state presentation of clinically relevant PTA to na?ve CD8 T cells by lymph node-resident stroma.     

Peripheral tolerance of CD4 T cells following local activation in adolescent mice

In addition to thymic T cell selection, post-thymic mechanisms of tolerance induction are required to eliminate autoreactive T cells with specificities for peripheral self antigens. While CD8⁺ T cells can recognize their target antigen on a wide variety of cell types, CD4⁺ T cells generally depend on the presence of specialized antigen-presenting cells. Because of this fundamental difference in antigen recognition peripheral tolerance of CD4⁺ T cells appears more difficult to achieve than of CD8⁺ T cells. Utilizing T cell receptor (TCR)-transgenic mice in which CD4⁺ T cells specific for a pancreatic β cell neoantigen (the simian virus 40 T antigen) are constantly generated at low frequency, we have now established a mouse model of peripheral, tissue-specific CD4⁺ T cell tolerance. In these animals, tolerance is preceded by a phase of activation of the autoreactive T cells as characterized by up-regulation of CD69 and CD44, and down-regulation of the L-selectin lymph node homing receptor. T antigen-specific T cells bearing this phenotype can be detected in the local lymphoid environment of the pancreas but not in more remote locations like axillary or inguinal lymph nodes. The proportion of activated, autoreactive T cells is maximal at 2–3 weeks of age, after which these cells are gradually deleted from the peripheral lymphocyte pool. We further demonstrate that deletion of the autoreactive T cells does not occur in TCR-tansgenic mice bred to the RAG-1-deficient background in which the transgenic T cells represent the only functional lymphocyte population.     

Induction of peripheral CD4+ T‐cell tolerance and CD8+ T‐cell cross‐tolerance by dendritic cells

DC can present and cross‐present self‐antigens to autoreactive CD4⁺ and CD8⁺ T cells, respectively, and incapacitate them by inducing anergy, deletion or converting them into Treg. In this review, we summarize the recent progress in immune tolerance research, which has been achieved by employing antigen‐ and TCR‐transgenic mice. We cover the numerous discoveries that have furthered our knowledge of the DC subsets and maturation pathways involved in tolerance; the signals, such as CD70, TGF‐β, B7‐H1/PD‐L1, which dictate the decision between immunity and tolerance; and the in vivo role of DC in the maintenance of CD4⁺ T‐cell tolerance and CD8⁺ T‐cell cross‐tolerance.     

Thymic and peripheral apoptosis of antigen-specific T cells might cooperate in establishing self tolerance

Aside from CD4⁺CD8⁺ double-positive (DP) thymocytes, the subpopulations of T lineage cells affected by negative selection are unknown. To address whether this process occurs in more mature cell types, we have compared the responses of purified single-positive (SP) murine thymocytes and peripheral T cells to the superantigen staphylococcal enterotoxin B (SEB) utilizing as antigen-presenting cells (APC) a fibroblast cell line expressing transfected I-E^k class II molecules. Whereas ∽ 70% of SEB-reactive SP thymocytes, either CD4⁺ or CD8⁺, undergo programmed cell death (apoptosis) and, therefore, negative selection, CD4⁺ and CD8⁺ antigen-specific peripheral T cells are predominantly activated and proliferate to APC+SEB. Thus, mature thymocytes and peripheral T cells, with identical patterns and levels of expression of CD4, CD8 and T cell receptor (TCR), are programmed to elicit different responses followingTCR stimulation. Unexpectedly, however activation of peripheral T cells was preceded by deletion of a large fraction of Vβ8⁺ T lymphocytes (SEB specific). This surprising phenomenon was also observed in in vivo studies: in fact, administration of SEB to adult mice resulted in depletion of the majority of antigen-specific T cells from the peripheral lymphoid tissues analyzed (lymph nodes and spleen). This depletion is the consequence of deletion as indicated by program cell death of Vβ8⁺ T cells and is followed by proliferation of the remaining SEB-reactive T cells. Clonal elimination of peripheral T cells may represent a mechanism by which tolerance to self antigens never expressed in and/or exported to the thymus is achieved.     

Central tolerance: Clonal deletion or clonal arrest?

Studies in various experimental animals have shown that developing T cells with specificity for self antigens can be prevented from maturation at an early stage of development. While several in vitro and in vivo experiments have shown that the mechanism of silencing autospecific T cells is the deletion of immature CD4⁺8⁺ thymocytes other experiments were interpreted to indicate that tolerance could also result from developmental arrest of more immature CD4^?8⁺ thymocytes not involving cell death. Here we show that immature CD4^?8⁺ cells when confronted with T cell receptor ligands in vitro neither survive nor differentiate into cells which cannot be deleted, indicating that clonal elimination rather than developmental arrest is the mechanism of central tolerance of all immature T cells.     

Selective antigen‐specific CD4+ T‐cell,but not CD8+ T‐ or B‐cell,tolerance corrupts cancer immunotherapy

Self‐tolerance, presumably through lineage‐unbiased elimination of self‐antigen‐specific lymphocytes (CD4⁺ T, CD8⁺ T, and B cells), creates a formidable barrier to cancer immunotherapy. In contrast to this prevailing paradigm, we demonstrate that for some antigens, self‐tolerance reflects selective elimination of antigen‐specific CD4⁺ T cells, but preservation of CD8⁺ T‐ and B‐cell populations. In mice, antigen‐specific CD4⁺ T‐cell tolerance restricted CD8⁺ T‐ and B‐cell responses targeting the endogenous self‐antigen guanylyl cyclase c (GUCY2C) in colorectal cancer. Although selective CD4⁺ T‐cell tolerance blocked GUCY2C‐specific antitumor immunity and memory responses, it offered a unique solution to the inefficacy of GUCY2C vaccines through recruitment of self‐antigen‐independent CD4⁺ T‐cell help. Incorporating CD4⁺ T‐cell epitopes from foreign antigens into vaccines against GUCY2C reconstituted CD4⁺ T‐cell help, revealing the latent functional capacity of GUCY2C‐specific CD8⁺ T‐ and B‐cell pools, producing durable antitumor immunity without autoimmunity. Incorporating CD4⁺ T‐cell epitopes from foreign antigens into vaccines targeting self‐antigens in melanoma (Trp2) and breast cancer (Her2) produced similar results, suggesting selective CD4⁺ T‐cell tolerance underlies ineffective vaccination against many cancer antigens. Thus, identification of self‐antigens characterized by selective CD4⁺ T‐cell tolerance and abrogation of such tolerance through self‐antigen‐independent T‐cell help is essential for future immunotherapeutics.     

Antigens expressed by myelinating glia cells induce peripheral cross‐tolerance of endogenous CD8+ T cells

Auto‐reactivity of T cells is largely prevented by central and peripheral tolerance. Nevertheless, immunization with certain self‐antigens emulsified in CFA induces autoimmunity in rodents, suggesting that tolerance to some self‐antigens is not robust. To investigate the fate of nervous system‐specific CD8⁺ T cells, which only recently came up as being important contributors for MS pathogenesis, we developed a mouse model that allows inducible expression of lymphocytic choriomeningitis virus‐derived CD8⁺ T‐cell epitopes specifically in oligodendrocytes and Schwann cells, the myelinating glia of the nervous system. These transgenic CD8⁺ T‐cell epitopes induced robust tolerance of endogenous auto‐reactive T cells, which proved thymus‐independent and was mediated by cross‐presenting bone‐marrow‐derived cells. Immunohistological staining of secondary lymphoid organs demonstrated the presence of glia‐derived antigens in DC, suggesting that peripheral tolerance of CD8⁺ T cells results from uptake and presentation by steady state DC.     

CD8+ T cells specific for β cells encounter their cognate antigens in the islets of NOD mice

CD8⁺ T cells play a key role in the initiation of insulitis. However, the site(s) where naive CD8⁺ T cells encounter β‐cell antigens and the mechanism(s) by which β‐cell autoimmunity is initiated remain to be determined. In the current study, an adoptive transfer model was employed assessing the initial site of priming and the nature of antigen recognition by naive β‐cell‐specific CD8⁺ T cells. Temporal analysis demonstrated that unlike CD4⁺ T cells that are primed in the draining pancreatic lymph nodes, initial proliferation of transferred CD8⁺ T cells was detected in the islets. These results indicate that in our model, naive β‐cell‐specific CD8⁺ T cells encounter β‐cell antigens in the islets. Furthermore, ectopic expression of CD80 by β cells accelerated the onset of insulitis mediated by β‐cell‐specific CD8⁺ T cells, but had no effect on CD4⁺ T‐cell‐mediated diabetes, suggesting an antigenic interaction between β cells and naive CD8⁺ T cells. However, it remains to be determined whether the initiation of insulitis in spontaneous diabetes is the result of a cognate interaction between naive CD8⁺ T cells and islet β cells.     

Lymph-borne CD8α+ dendritic cells are uniquely able to cross-prime CD8+ T cells with antigen acquired from intestinal epithelial cells

Cross-presentation of cellular antigens is crucial for priming CD8⁺ T cells, and generating immunity to intracellular pathogens—particularly viruses. It is unclear which intestinal phagocytes perform this function in vivo. To address this, we examined dendritic cells (DCs) from the intestinal lymph of IFABP-tOVA 232-4 mice, which express ovalbumin in small intestinal epithelial cells (IECs). Among lymph DCs (LDCs) only CD103⁺ CD11b⁻ CD8α⁺ DCs cross-present IEC-derived ovalbumin to CD8⁺ OT-I T cells. Similarly, in the mesenteric lymph nodes (MLNs), cross-presentation of IEC–ovalbumin was limited to the CD11c⁺ MHCII^hi CD8α⁺ migratory DCs, but absent from all other subsets, including the resident CD8α^hi DCs. Crucially, delivery of purified CD8α⁺ LDCs, but not other LDC subsets, into the MLN subcapsular lymphatic sinus induced proliferation of ovalbumin-specific, gut-tropic CD8⁺ T cells in vivo. Finally, in 232-4 mice treated with R848, CD8α⁺ LDCs were uniquely able to cross-prime interferon γ-producing CD8⁺ T cells and drive their migration to the intestine. Our results clearly demonstrate that migrating CD8α⁺ intestinal DCs are indispensable for cross-presentation of cellular antigens and, in conditions of inflammation, for the initial differentiation of effector CD8⁺ T cells. They may therefore represent an important target for the development of antiviral vaccinations.     

PTPN2 restrains CD8+ T cell responses after antigen cross-presentation for the maintenance of peripheral tolerance in mice

Antigen cross-presentation by dendritic cells is crucial for priming cytotoxic CD8⁺ T cells to invading pathogens and tumour antigens, as well as mediating peripheral tolerance to self-antigens. The protein tyrosine phosphatase N2 (PTPN2) attenuates T cell receptor (TCR) signalling and tunes CD8⁺ T cell responses in vivo. In this study we have examined the role of PTPN2 in the maintenance of peripheral tolerance after the cross-presentation of pancreatic β-cell antigens. The transfer of OVA-specific OT-I CD8⁺ T cells (C57BL/6) into RIP-mOVA recipients expressing OVA in pancreatic β-cells only results in islet destruction when OVA-specific CD4⁺ T cells are co-transferred. Herein we report that PTPN2-deficient OT-I CD8⁺ T cells transferred into RIP-mOVA recipients acquire CTL activity and result in β cell destruction and the development of diabetes in the absence of CD4⁺ help. These studies identify PTPN2 as a critical mediator of peripheral T cell tolerance limiting CD8⁺ T cell responses after the cross-presentation of self-antigens. Our findings reveal a mechanism by which PTPN2 SNPs might convert a tolerogenic CD8⁺ T cell response into one capable of causing the destruction of pancreatic β-cells. Moreover, our results provide insight into potential approaches for enhancing T cell-mediated immunity and/or T cell adoptive tumour immunotherapy.     

Human primary adenotonsillar naïve phenotype CD45RA CD4 T lymphocytes undergo apoptosis upon stimulation with a high concentration of CD3 antibody

Young children need to develop immune tolerance to harmless foreign antigens such as digested nutrients and various inhaled airborne antigens. Because of its anatomical location, pharyngeal adenotonsillar tissue is a potential site for the establishment of this immune tolerance. To characterize possible mechanisms of peripheral immune tolerance, we studied human primary adenotonsillar naïve phenotype CD45RA⁺ CD4⁺ T cells, which represent cells that have not previously encountered foreign antigens. It was found that these CD45RA⁺ CD4⁺ T cells expressed higher levels of the activation marker CD69 as compared with peripheral blood CD45RA⁺ CD4⁺ T cells. Upon stimulation with a high concentration of CD3 antibody, which mimics the encounter of a high antigen dose, adenotonsillar CD45RA⁺ CD4⁺ T lymphocytes, but not peripheral blood CD45RA⁺ CD4⁺ T cells, underwent apoptosis. After 6 h stimulation with a high concentration of CD3 antibody, over 25% of the cells were apoptotic. Interfering with the Fas–FasL interaction with recombinant Fas or an antibody against Fas-ligand partially inhibited apoptosis. Our study results suggest that high concentrations of antigens, such as various nutrients and airborne antigens, may induce peripheral immune tolerance by selectively deleting naïve phenotype CD45RA⁺ CD4⁺ T cells via T-cell receptor-triggered apoptosis in human adenotonsillar tissue.     

Alloreactive CD8 T cells rescued from apoptosis during co‐stimulation blockade by Toll‐like receptor stimulation remain susceptible to Fas‐induced cell death

Blockade of co‐stimulatory signals to T cells is extremely effective for the induction of transplantation tolerance in immunologically naive rodents. However, infections and inflammation compromise the efficacy of co‐stimulation blockade regimens for the induction of tolerance, thereby stimulating the rejection of allografts. Previous studies have shown that stimulation of innate immunity abrogates tolerance induction by preventing the deletion of alloreactive CD8⁺ T cells that normally occurs during co‐stimulation blockade. Although inflammation prevents the deletion of alloreactive T cells during co‐stimulation blockade, it is not known if this resistance to cell death is the result of a mechanism intrinsic to the T cell. Here, we used syngeneic bone marrow chimeric mice that contain a trace population of T‐cell receptor transgenic alloreactive CD8⁺ T cells to investigate the early apoptotic signature and activation status of alloreactive T cells following exposure to inflammatory signals during co‐stimulation blockade with an antibody specific for CD154. Our findings revealed that the presence of bacterial lipopolysaccharide during co‐stimulation blockade enhanced the early activation of alloreactive CD8⁺ T cells, as indicated by the up‐regulation of CD25 and CD69, suppressed Fas ligand expression, and prevented apoptotic cell death. However, alloreactive CD8⁺ T cells from lipopolysaccharide‐treated mice remained sensitive to Fas‐mediated apoptosis in vitro. These findings suggest that alloreactive T cells rescued from deletion during co‐stimulation blockade by inflammation are still sensitive to pro‐apoptotic signals and that stimulating these apoptotic pathways during co‐stimulation blockade may augment the induction of tolerance.     

Plasmacytoid dendritic cells induce tolerance predominantly by cargoing antigen to lymph nodes

Plasmacytoid dendritic cells (pDCs) have been shown to induce tolerance to innocuous antigens. Their migratory properties allow them to take up antigens from the periphery and transport them to the draining lymph nodes or to the thymus. However, pDC‐T‐cell interaction in the primary and secondary lymphoid organs still remains poorly defined. In this study, we show that resting pDCs loaded with exogenous antigen could induce tolerance when transferred intralymphatically into a single lymph node of wild‐type C57BL/6 mice. However, this was a result of antigen transfer from pDCs to endogenous antigen presenting cells and subsequent abortive proliferation of cognate CD4⁺ T cells. pDCs could not directly induce the proliferation of CD4⁺ T cells, as observed in mice lacking MHC class II gene. Moreover, pDCs failed to make physical contacts with OT‐II cells as revealed by two‐photon imaging. Thus, the role of resting pDCs in tolerance induction seems to be independent of its direct interaction with cognate CD4⁺ T cells.     

A Vβ.2-specific superantigen from exogenous mouse mammary tumor virus carried by FM mice

A number of endogenous mouse mammary tumor virus (MMTV) proviruses encode superantigen that have the ability to stimulate T cells with a certain T cell receptor (TCR) β-chain variable region (Vβ) and to mediate the Vβ-specific clonal deletion. The tumorigenic milk-borne MMTV carried by C3H and GR mice also have superantigenic properties in vivo. In the present study we identified and characterized a novel Vβ8.2-specific superantigen of exogenous MMTV carried by FM mice. The open reading frame (ORF) in the 3′ long terminal repeat of the MMTV was cloned by polymerase chain reaction with primers corresponding to conserved regions spanning the ORF coding region. Sequence analysis of the ORF revealed that there is no sequence identical to those in other known MMTV in the carboxy terminus implicated in TCR Vβ recognition. Subcutaneous injection of the virus into adult BALB/c mice induced an approximately three- to fourfold enlargement of draining lymph nodes and a substantial increase of Vβ8.2⁺ CD4⁺ T cells in the lymph nodes within 6 days. The exposure of newborn BALB/c mice to the virus by foster nursing resulted in a marked deletion of Vβ8.2⁺ cells both in CD4⁺ and CD8⁺ T cells. Thus, a novel milk-borne MMTV in FM mice expresses strong superantigenic properties capable of stimulating Vβ8.2⁺ T cells. Vβ8.2⁺ T cells have been demonstrated to be frequently involved in recognition of conventional antigens and responsible for autoimmune diseases such as experimental allergic encephalomyelitis. Therefore, the MMTV (FM) may provide a new mouse model system for inducing immunodeficiency or autoimmune disease by retroviral infection.     

MHC I tetramer staining tends to overestimate the number of functionally relevant self-reactive CD8 T cells in the preimmune repertoire

Previous studies that used peptide-MHC (pMHC) tetramers (tet) to identify self-specific T cells have questioned the effectiveness of thymic-negative selection. Here, we used pMHCI tet to enumerate CD8 T cells specific for the immunodominant gp33 epitope of lymphocytic choriomeningitis virus glycoprotein (GP) in mice transgenically engineered to express high levels of GP as a self-antigen in the thymus. In GP-transgenic mice (GP⁺), monoclonal P14 TCR⁺ CD8 T cells that express a GP-specific TCR could not be detected by gp33/D^b-tet staining, indicative of their complete intrathymic deletion. By contrast, in the same GP⁺ mice, substantial numbers of polyclonal CD8 T cells identifiable by gp33/D^b-tet were present. The gp33-tet staining profiles of polyclonal T cells from GP⁺ and GP-negative (GP⁻) mice were overlapping, but mean fluorescence intensities were ∼15% lower in cells from GP⁺ mice. Remarkably, the gp33-tet⁺ T cells in GP⁺ mice failed to clonally expand after lymphocytic choriomeningitis virus infection, whereas those of GP⁻ mice did so. In Nur77^GFP-reporter mice, dose-dependent responses to gp33 peptide-induced TCR stimulation revealed that gp33-tet⁺ T cells with high ligand sensitivity are lacking in GP⁺ mice. Hence, pMHCI tet staining identifies self-specific CD8 T cells but tends to overestimate the number of truly self-reactive cells.     

A role for Th2 cytokines in the suppression of CD8+ T cell-mediated graft rejection

A major histocompatibility complex (MHC) class I-specific T cell receptor (TCR)-transgenic mouse was used to study classical-type transplantation tolerance in the adult. Engraftment of MHC class I-incompatible bone marrow and tolerance to donor-type skin grafts were obtained using dimethylmyeleran (DMM) as a myeloablative agent and a non-depleting anti-CD8 monoclonal antibody (mAb) as the sole immunosuppressant. Surprisingly, bone marrow engraftment was facilitated by host CD4⁺ T cells, a subset normally considered unable to reject class I MHC-incompatible grafts. A combination of mAb to interleukins (IL)?4 and ?10 antagonized the “permissive” effects of host CD4⁺ T cells, indicating a possible role for Th2-type immunoregulation that can act on CD8⁺ T cells in this form of transplantation tolerance. The fate of graft-reactive T cells was monitored using anti-clonotypic antibodies. It was observed that bone marrow engraftment then led to peripheral deletion of mAb-blockaded, clonotype⁺ CD8⁺ T cells.     

Application of central immunologic concepts to cancer: Helping T cells and B cells become intolerant of tumors

CD4‐mediated T‐cell help in the activation of CD8⁺ T cells and B cells, through linked‐recognition of antigenic determinants, is a long‐standing concept foundational to our understanding of immunity (presence of help) versus tolerance (lack of help). Surprisingly, this function of CD4⁺ T cells has not been extensively examined as a means to overcome immune tolerance of the self‐antigens made by tumor cells. Hesitation to employ this powerful mechanism may be due to the potential to cause unwanted autoimmune pathology. In this issue of the European Journal of Immunology, Snook et al. [Eur. J. Immunol. 2014. 44: 1956–1966] identify a state of split tolerance, showing that CD4⁺ T cells specific for a number of tumor‐associated self‐antigens are robustly tolerant, while their CD8⁺ T‐cell and B‐cell counterparts are far less tolerant. Furthermore, the authors demonstrate that provision of linked foreign helper epitopes, such as influenza hemagglutinin, substantially enhances both CD8⁺ T‐cell and B‐cell responses to tumor self‐antigens without causing any overt autoimmune pathology. These findings provide a strong rationale to employ foreign helper epitopes in cancer vaccines and highlight the need to fully explore therapeutic strategies that are based on well‐established immunologic concepts.     

Activation and 'deletion' of self-reactive mature and immature T cells during ontogeny of Mls-1a mice: implications for neonatal tolerance induction

We assessed the kinetics of V_ß6⁺ T cell eliminationin the lymph nodes and thymus during Mls-1^a mouse ontogeny.Our results suggest that induction of tolerance to Mls-1^a antigensinvolves mechanisms other than clonal deletion of immature Tcells in the thymus. Mature CD4⁺CD8^– (CD4SP) T cells wereaffected by Mls-1^a antigens earlier than immature thymocytepopulations. Up to 2 weeks after birth, reduced frequenciesof V_ß6⁺ T cells were detected only in CD4SP cellsfrom the thymus and lymph nodes, and generation of CD4SP cellsin the thymus was blocked at least 1 week earlier than thatof their CD4⁺CD8^loTCR^hl immature precursors. The number of V_ß6⁺CD4SPT cells increased during the first 2 weeks of life and remainedconstant thereafter. We thus found no evidence of deletion ofmature V_ß6⁺CD4SP T cells, as the reduced frequenciesin adult mice can be attributed to the dilution of previouslygenerated cells in lymphoid organs of growing mice, which increasein cellulartty after birth. V_ß6⁺CD4⁺ T cells wereactivated in vivo shortly after birth, as shown by a selectiveincrease in IL-2 receptor a chain expression in the thymus andlymph nodes from day 0 to day 2 after birth. It is thereforelikely that endogenous expression of Mls-1^a antigen shortlyafter birth activates V_ß6⁺CD4SP T cells and rendersthem anergic. This process of tolerance induction may precedethe clonal deletion of immature T cells in the thymus, describedin the adult mouse.