Memory-like CD8؉ and CD4؉ T cells cooperate to break peripheral tolerance under lymphopenic conditions Cecile Le Saout, Sandie Mennechet, Naomi Taylor, and Javier Hernandez1 Institut de Ge´ne´ tique Mole´culaire de Montpellier Unite´Mixte de Recherche 5535, Centre National de la Recherche Scientifique, Université de Montpellier 1 and 2, 34293 Montpellier, Cedex 5, France Edited by N. Avrion Mitchison, University College London, London, United Kingdom, and approved October 20, 2008 (received for review August 10, 2008) The onset of autoimmunity in experimental rodent models and established (9). In the case of naive T cells, T cell antigen receptor patients frequently correlates with a lymphopenic state. In this (TCR) interactions with MHC/self-peptide complexes (those that condition, the immune system has evolved compensatory homeo- mediate positive selection) and the IL-7 cytokine appear to be static mechanisms that induce quiescent naive T cells to proliferate required for this expansion (10–15). Recently, an IL-7 independent and differentiate into memory-like lymphocytes even in the apparent form of lymphopenia-driven proliferation has also been described absence of antigenic stimulation. Because memory T cells have less (16). However, in all these cases, proliferating cells differentiate and stringent requirements for activation than naive cells, we hypothe- acquire a memory-like phenotype and the ability to rapidly secrete sized that autoreactive T cells that arrive to secondary lymphoid effector cytokines (12, 17–19). Although memory-like T cells have organs in a lymphopenic environment could differentiate and bypass never been activated by cognate antigen, do not pass through an the mechanisms of peripheral tolerance such as those mediated by effector phase, and, as such, cannot be considered to be ‘‘true’’ self-antigen cross-presentation. Here, we show that lymphopenia- memory cells, recent reports demonstrate that they are functionally driven proliferation and differentiation of potentially autoreactive indistinguishable from memory cells (20). Therefore, it has been -CD8؉ T cells into memory-like cells is not sufficient to induce self- hypothesized that the proliferation and differentiation of poten reactivity against a pancreatic antigen. Induction of an organ-specific tially autoreactive T cells may result in the induction of autoim- .autoimmunity required antigen-specific CD4؉ T cell help. Notably, we munity in a lymphopenic environment (21). Indeed, the group of N (found that this function could be accomplished by memory-like CD4؉ Sarvetnik elegantly demonstrated that nonobese diabetic (NOD T cells generated in vivo through lymphopenia-induced proliferation. mice are mildly lymphopenic and that there is a strong correlation These helper cells promoted the further differentiation of memory- between lymphopenia-induced proliferation of autoreactive T cells ؉ like CD8 T cells into effectors in response to antigen cross-presen- and the onset of diabetes (22). Autoreactive CD8ϩ and CD4ϩ T tation, resulting in their migration to the tissue of antigen expression cells displayed a memory phenotype, with CD8ϩ T cells having a where autoimmunity ensued. Thus, the cooperation of self-reactive major role in the pathogenesis of the disease (22). ؉ ؉ memory-like CD4 and CD8 T cells under lymphopenic conditions The ensemble of these observations points to lymphopenia as a overcomes cross-tolerance resulting in autoimmunity. trigger of autoimmunity and to memory-like T cells, generated through homeostatic mechanisms, as pathogenic effectors. How- autoimmunity ͉ T cell help ͉ T cell homeostasis ever, in complex models such as NOD mice, multiple genetic and environmental factors contribute to disease. Therefore, it is not utoimmunity has been paradoxically associated with lym- known whether potentially autoreactive memory-like T cells are in Aphopenia, a state in which reduced numbers of circulating themselves sufficient to induce organ-specific autoimmunity under lymphocytes are present, in a number of experimental rodent lymphopenic conditions and whether CD8ϩ and CD4ϩ T cells models and patients (1, 2). In humans, the association of T cell cooperate in this task. Also, the mechanisms by means of which lymphopenia with autoimmunity has been described in patients lymphopenia-induced proliferation and differentiation of poten- with Sjogren’s syndrome, rheumatoid arthritis, systemic lupus er- tially autoreactive T cells interfere with the normal mechanisms of ithematosus, and Crohn’s disease, among others (2). Lymphopenia peripheral self-tolerance have not been determined. To address may also occur after viral infections, as has been described in the these issues, we have used a well-characterized transgenic murine case of HIV infection. In AIDS patients who undergo highly active system consisting of 3 different mouse lines: InsHA mice express antiretroviral therapy, the frequency of HIV-associated immune the hemagglutinin (HA) of the influenza virus under the control of reconstitution inflammatory syndrome, a phenomenon that shares the rat insulin promoter, driving its expression in the beta cells of many characteristics with autoimmune processes, and rheumatoid the pancreas (23). Clone 4 TCR and HNT TCR transgenics express arthritis and type 1 diabetes, is increased (3, 4). Importantly, the HA-specific MHC class I and class II-restricted TCRs, respectively association between lymphopenia and the exacerbation of anti-self (24, 25). After transfer into InsHA mice, naive Clone 4 CD8ϩ T responses has recently been exploited to augment the responsive- cells recirculate through secondary lymphoid organs, but are unable ness of T lymphocytes against cancer. Specifically, adoptive T cell to reach peripheral tissues and, hence, cannot directly enter in immunotherapy targeting self-tumor antigens has been enhanced contact with pancreatic islets (26, 27). Under these conditions, the by prior lymphodepletion of melanoma patients (5). Studies in murine models have been instrumental in furthering our understanding of the link between lymphopenia and the onset Author contributions: C.L.S., N.T., and J.H. designed research; C.L.S. and S.M. performed of autoimmunity. Indeed, thymectomy in neonatal mice and the research; C.L.S., S.M., N.T., and J.H. analyzed data; and C.L.S., N.T., and J.H. wrote the paper. transfer of low numbers of lymphocytes into irradiated, SCID or The authors declare no conflict of interest. RAGϪ/Ϫ mice result in autoimmune processes (1). This type of This article is a PNAS Direct Submission. studies served to unveil the role of regulatory lymphocytes in Freely available online through the PNAS open access option. peripheral tolerance (6, 7). However, an absence of regulatory T 1To whom correspondence should be addressed. E-mail: [email protected]. cells is not the sole factor triggering autoimmunity (8). Under This article contains supporting information online at www.pnas.org/cgi/content/full/ lymphopenic conditions, the expansion of residual conventional T 0807743105/DCSupplemental. cells, in the apparent absence of antigenic stimulation, is well © 2008 by The National Academy of Sciences of the USA 19414–19419 ͉ PNAS ͉ December 9, 2008 ͉ vol. 105 ͉ no. 49 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0807743105 Downloaded by guest on September 28, 2021 transferred CD8ϩ lymphocytes encounter HA antigen only in the Table 1. CD8؉ and CD4؉ T cells cooperate to induce draining lymph nodes (LNs) of the pancreas cross-presented by autoimmunity under lymphopenic conditions antigen presenting cells (APCs) (26, 27). Cross-presentation is a Number Diabetes Diabetes process by means of which professional APCs, in particular den- Cell transfer* of cells incidence† onset‡ dritic cells, acquire exogenous antigens, transport them to the lymphoid organs, and present them to T cells (28). In InsHA mice, Recipient, irradiated InsHA ϩ the encounter between cross-presenting APCs and HA-specific T Clone 4 CD8 2 ϫ 106 0/11 — ϫ 6 cells results in tolerance, a process that has been termed cross- 3.5 10 0/6 — 5 ϫ 106 1/21 d 12 tolerance, characterized by the abortive activation of the Clone 4 ϫ 6 ϩ 7 10 0/5 — CD8 T cells and their subsequent apoptosis (26). ϫ 6 ϩ 10 10 0/6 — Here, we show that the cotransfer of naive Clone 4 CD8 T cells HNT CD4ϩ 2 ϫ 106 0/10 — ϩ together with naive HNT CD4 T helper cells into lympho-replete 3.5 ϫ 106 1/8 d 12 InsHA mice does not result in autoimmunity. This absence of 5 ϫ 106 0/18 — reactivity is because the CD8ϩ T cells are tolerized through antigen 7 ϫ 106 0/5 — cross-presentation even in the presence of helper cells (29). Nota- 10 ϫ 106 2/10 d 21 ϩ ϩ bly, when InsHA mice are rendered lymphopenic the same poten- Clone 4 CD8 ϩ HNT CD4 2 ϫ 106 ϩ 2 ϫ 106 7/7 d 14 Ϯ 4 ϫ 6 ϩ ϫ 6 Ϯ tially autoreactive CD8ϩ and CD4ϩ T cells differentiate into 3.5 10 3.5 10 13/15 d 14 3 5 ϫ 106 ϩ 5 ϫ 106 14/14 d 11 Ϯ 1 memory-like cells and cooperate to induce autoimmune diabetes ϩ ϩ Clone 4 CD8 ϩ DO11.10 CD4 3.5 ϫ 106 ϩ 3.5 ϫ 106 0/11 — overcoming cross-tolerance. Clone 4 CD8ϩ ϩ InsHA CD4ϩ 3.5 ϫ 106 ϩ 3.5 ϫ 106 0/8 — Recipient, nonirradiated InsHA Results Clone 4 CD8ϩ ϩ HNT CD4ϩ 5 ϫ 106 ϩ 5 ϫ 106 0/8 — Memory-Like CD8؉ T Cells Are Not Sufficient to Induce Autoimmune ϩ ϩ Diabetes Under Lymphopenic Conditions. To assess whether HA- *Purified transgenic CD8 and/or CD4 T cells, as indicated, were injected into specific Clone 4 CD8ϩ T cells undergo lymphopenia-induced either irradiated or nonirradiated InsHA mice. †The onset of autoimmunity was evaluated by measuring blood glucose levels. proliferation, these cells were adoptively transferred into sublethally ϩ Mice were followed over a 1 month period and were considered diabetic when irradiated BALB/c mice.