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. Naive Clone 4 CD8 T cells proliferated levels were above 300 mg/dL in 2 consecutive measurements. under lymphopenic conditions in syngeneic recipients at a rate ‡The day of disease onset (d) is indicated when applicable. similar to that described for polyclonal naive CD8ϩ T cells [sup- porting information (SI) Fig. S1] (10). Proliferating cells acquired a phenotype resembling that of central memory T cells, CD44hi symptoms (Table 1). These results indicate that memory-like CD8ϩ CD122ϩ CD25Ϫ CD62Lhi, but not that of effector cells as they T cells require the participation of memory-like CD4ϩ T cells to failed to up-regulate CD25 (Fig. S1). However, proliferating CD8ϩ induce autoimmunity. T cells did display effector function, as demonstrated by their ability to produce IFN-␥, albeit to a lesser extent than effector cells (Fig. CD4؉ T Helper Cells Promote the Accumulation of Autoreactive CD8؉ S1). These results demonstrate that naive Clone 4 CD8ϩ T cells T Cells Under Lymphopenic Conditions. To shed light on the processes proliferate under lymphopenic conditions and differentiate into by means of which CD8ϩ and CD4ϩ T cells cooperate to induce memory-like T cells. autoimmune diabetes under lymphopenic conditions, we assessed Next, we sought to determine whether potentially autoreactive proliferation, phenotype, and gain of effector function of donor T memory-like CD8ϩ T cells, generated under lymphopenic condi- cells in lymphoid organs. The proliferation profiles, phenotype, and tions, are able to induce autoimmune diabetes. To this end, we functionality of HNT CD4ϩ T cells were not significantly different transferred naive Clone 4 CD8ϩ T cells into sublethally irradiated when injected alone or together with Clone 4 CD8ϩ T cells (Fig. InsHA transgenic mice, in which the target HA antigen of Clone S3). In contrast, the in vivo fate of Clone 4 CD8ϩ T cells was 4 cells is expressed at low levels in the beta cells of the pancreas. Our modulated by the presence of HNT CD4ϩ T cells as described results show that the transfer of varying numbers of naive Clone 4 below. CD8ϩ T cells (range, 2 to 10 ϫ 106) into irradiated InsHA mice was The 5- and 6-carboxy-fluorescein succinimidyl ester (CFSE) not sufficient to induce autoimmune diabetes during a 1-month profiles of the donor CD8ϩ T cell population revealed an extensive period (Table 1). These results strongly suggest that memory-like proliferation in spleen, LNs, and pancreatic (p)LN on transfer into CD8ϩ T cells are not in themselves sufficient to induce disease. irradiated hosts (Fig. 1A). These results, which very much resem- bled those obtained in wild type mice (Fig. S1), strongly suggest that Memory-Like CD8؉ and CD4؉ T Cells Cooperate to Induce Autoimmune most of the observed proliferation is due to the condition of Diabetes Under Lymphopenic Conditions. The important role of CD4 lymphopenia itself and not to the expression of the HA self-antigen help for CD8ϩ T cell responses, including responses to self-antigens, in the pancreas. In the presence of CD4ϩ T helper cells, there was has been well established (29–31). Therefore, it was important to an increased proliferation of CD8ϩ T cells, translating into a determine whether CD8ϩ T cell responsiveness to HA under significantly enhanced accumulation in the spleen and pLNs at day lymphopenic conditions would be modulated by the presence of 13 (Fig. 1). In the draining LNs of the pancreas, the HNT-promoted antigen-specific CD4ϩ T cells. Notably, coinjection of HA-specific enhanced proliferation of CD8ϩ T cells was already prominent at HNT CD4ϩ T cells together with Clone 4 CD8ϩ T cells does not day 7 (Fig. 1A), indicating that this effect may be related to the result in autoimmunity in lympho-replete InsHA mice (see Table presence of cross-presented HA antigen. ϩ ϩ 1 and ref. 29). In marked contrast, cotransfer of naive Clone 4 CD8 Because a modest increase in autoreactive CD8 T cell prolif- IMMUNOLOGY T cells with HNT CD4ϩ T cells, which also undergo lymphopenia- eration was not in itself likely to be the cause of disease, we analyzed induced proliferation and differentiation into memory-like cells the phenotype and functionality of proliferating Clone 4 cells. The (Fig. S2), results in the onset of diabetes in the vast majority of mice majority of un-helped CD8ϩ T cells displayed a typical central between day 11 and 20 after transfer (34 of 36 mice, Table 1). This memory phenotype (CD44hi CD25Ϫ CD62Lhi) in LNs, pLNs, and effect was not due to increased total T cell numbers, because even spleen at all time points tested (7, 10, and 13 days after transfer; Fig. at the lowest dose tested, 2 ϫ 106 Clone 4 ϩ 2 ϫ 106 HNT T cells, 2 and Fig. S4). In marked contrast, in the presence of HNT CD4ϩ 100% of mice became diabetic (Table 1). None of the mice that T cells, there was an important subpopulation of highly proliferating received 10 ϫ 106 Clone 4 CD8ϩ cells developed disease, and only CD62Llo CD25ϩ Clone 4 cells in the pLNs at days 10 and 13, but 2 of 10 mice that received 10 ϫ 106 HNT CD4ϩ cells showed any not in other LNs (Fig. 2 and Fig. S4). The generation of this effector

Le Saout et al. PNAS ͉ December 9, 2008 ͉ vol. 105 ͉ no. 49 ͉ 19415 Downloaded by guest on September 28, 2021 A pLN LN Spleen A pLN LN Spleen + + + + Cl 4 CD8 + Cl 4 CD8 + Cl 4 CD8 90 90 90 Cl 4 CD8 Cl 4 CD8 Cl 4 CD8 hi + HNT CD4+ + HNT CD4+ + HNT CD4+ 60 60 60 57±2 74±7 37±12 31±14 45±5 45±6 d 7 30 30 30 % CD44 0 0 0 49±5 77±7 52±1 76±1 47±6 76±1 50 50 50 d 13 * lo 40 40 40 * Cl 4 CD8+ 30 30 30 * + CFSE 20 20 20 Cl 4 CD8 + 10 10 10 + HNT CD4 % CD62L 0 0 0 ) 3 800 15 15 15 B * * + 600 10 * 10 10 Cl 4 CD8+ 5 5 5 400 Cl 4 CD8+ % CD25 + HNT CD4+ 0 0 0 200 * d 7 d 10 d 13 d 7 d 10 d 13 d 7 d 10 d 13 donor T cells (x10 + 0 CD8 pLN LN SP B pLN LN Spleen Cl 4 CD8+ Cl 4 CD8+ Cl 4 CD8+ ϩ Cl 4 CD8+ Cl 4 CD8+ Cl 4 CD8+ Fig. 1. Proliferation of Clone 4 CD8 T cells in irradiated InsHA mice is enhanced + HNT CD4+ + HNT CD4+ + HNT CD4+ by CD4 help. (A) Irradiated InsHA mice were injected with either 5 ϫ 106 CFSE- ϩ ϩ ϩ 20 44 24±5 26±5 25±5 20±2 labeled naive Clone 4 Thy1.1 CD8 T cells (Cl 4 CD8 ) or with a combination of d 7 6 ϩ ϩ 6 ϩ ϩ 5 ϫ 10 Clone 4 Thy1.1 CD8 T cells and 5 ϫ 10 HNT Thy1.1 CD4 T cells (HNT γ CD4ϩ). Mice were killed on days 7 or 13 after transfer and the levels of CFSE IFN-IFN- 10±2 21±3 40±1 45±3 29±4 47±3 fluorescence on gated CD8ϩ Thy1.1ϩ donor lymphocytes in pLNs, LNs, and spleen d 13 are shown. The mean percentages (Ϯ SD, n ϭ 3) of proliferating CD8ϩ donor T cells are indicated. (B) Total numbers of CD8ϩ donor T cells in these lymphoid CFSE organs were enumerated at day 13 posttransfer after the injection of Clone 4 CD8ϩ T cells alone (ᮀ) or together with HNT CD4ϩ T cells (■). Results are presented Fig. 2. Proliferating Clone 4 CD8ϩ T cells differentiate into effectors in irradi- as means ϮSD (nϭ3). Data of 1 representative experiment out of 4 are presented. ated InsHA mice in the presence of help. (A) Seven, 10, and 13 days after transfer, expression of CD44, CD62L, and CD25 on CD8ϩ donor T cells was assessed in pLN, LN, and spleen of mice from the groups described in Fig. 1 by flow cytometry. The phenotype subpopulation was associated with a 2-fold increase in percentages of CD44hi, CD62Llo, and CD25ϩ CD8ϩ donor T cells in InsHA mice ϩ ϩ effector function, as assessed by IFN-␥ production (Fig. 2B). injected with Clone 4 CD8 T cells alone (ᮀ) or together with HNT CD4 T cells (■) Ϯ ϭ Intriguingly, a subpopulation of highly proliferating CD62Llo Clone are presented as means SD (n 3–11). (B) On day 7 or 13 after transfer, cells from lymphoid organs were stimulated with Kd HA peptide. IFN-␥ production by 4 cells was also observed in the spleen of lymphopenic InsHA mice donor Clone 4 cells was assessed by intracellular staining and plots represent CFSE in the presence of helper cells, correlating with a 40% enhancement fluorescence versus IFN-␥ on gated CD8ϩ Thy1.1ϩ lymphocytes. The percentages in effector function (Fig. 2 and Fig. S4). However, these cells were of IFN-␥-producing CD8ϩ donor T cells are indicated as means Ϯ SD (n ϭ 3–5 per detected only at day 13 after transfer, a time point at which mice group). Background in nonstimulated controls was Ͻ1%. Data from 1 represen- were already sick for a few days (Fig. 2). Thus, these cells are likely tative experiment out of 4 are presented. a consequence of the autoreactive attack rather than the cause, whereas the earlier accumulation of CD62Llo cells in the pLNs and Fig. S3). No significant difference was observed in the pheno- probably reflects the generation of effector cells after antigen ϩ encounter in the presence of help. type or functionality of infiltrating donor CD4 T cells in the 2 conditions (data not shown). Together, our results indicate that ϩ ϩ CD4؉ T Helper Cells Promote the Migration of Autoreactive CD8؉ T CD4 T cells promote the differentiation of Clone 4 CD8 T cells Cells to the Pancreas Under Lymphopenic Conditions. Collectively, the into effector cells and the subsequent migration of these effectors results presented above point to the adoptively-transferred CD8ϩ T into the pancreas mediates diabetes in InsHA mice. cells as pathogenic effectors in our model. To corroborate this Antigen Specificity of CD4؉ T Helper Cells. To determine whether the hypothesis, we assessed the presence of donor T cells in the ϩ pancreas at different time points after transfer. Although we were CD4 T cell help necessary for the induction of autoimmunity not able to detect any infiltrating donor CD8ϩ T cells in the functions in an antigen-specific fashion, we adoptively transferred ϩ ϩ pancreas of InsHA mice that had received only Clone 4 T cells, they CD4 T cells with distinct TCR specificities. Specifically, CD4 T were easily detected in diabetic and even prediabetic InsHA mice, cells from DO11.10 transgenic mice, which express a TCR specific when administered together with HNT CD4ϩ T cells (Fig. 3A). for an I-Ad-restricted ovalbumin (OVA) epitope (32), and poly- ϩ Analyses of these infiltrating CD8ϩ T cells showed that only the clonal CD4 T cells from InsHA transgenic mice, which should be ϩ most highly proliferating cells had migrated to the site of antigen devoid of high-avidity HA-specific CD4 T cells (23), were co- ϩ expression, and these cells also had high effector potential (Fig. 3B). transferred with Clone 4 CD8 T cells into lymphopenic InsHA Phenotypic analyses of the infiltrating Clone 4 cells in prediabetic mice. Importantly, none of the mice coinjected with DO11.10 cells ϩ or recently diagnosed diabetic mice (approximately day 10 post T or polyclonal CD4 T cells from InsHA mice developed disease ϩ cell transfer) revealed that they were CD44hi CD62Llo CD25ϩ,a (Table 1). Phenotypic analyses of Clone 4 CD8 T cells transferred phenotype typical of effector cells. However, in mice that were with polyclonal CD4ϩ T cells revealed an absence of the CD62Llo hyperglycemic for 3 or 4 days and appeared sick (approximately day and CD25ϩ subpopulations that characterized the diabetic mice 13), the infiltrating Clone 4 CD8ϩ T cells were CD25Ϫ with a (Fig. 4). Also, Clone 4 CD8ϩ cells that proliferated in the presence phenotype resembling that of effector memory cells (Fig. 3C). In of nonspecific CD4ϩ T cells did not migrate to the pancreas (data contrast to CD8ϩ T cells, HNT CD4ϩ T cells infiltrated the not shown). The lack of helper activity by DO11.10 or InsHA CD4ϩ pancreas of InsHA mice even when injected alone (Fig. 3A). This T cells was not due to their inability to become activated, because infiltration was expected based on our previous observation that they underwent lymphopenia-induced proliferation and differen- proliferating HNT cells had a mixed effector-memory (CD62Llo)/ tiation in the irradiated InsHA recipient mice (Fig. 4 and data not central-memory (CD62Lhi) phenotype in lymphoid organs (Fig. S2 shown). These results demonstrate that the effect of helper T cells

19416 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0807743105 Le Saout et al. Downloaded by guest on September 28, 2021 pLN Spleen Cl 4 CD8+ Non KdHA A HNT CD4+ Cl 4 CD8+ B + HNT CD4+ stimulated stimulated Cl 4 CD8+ + Cl 4 CD8+ + Cl 4 CD8+ Cl 4 CD8+ polyclonal + polyclonal +

γ + HNT CD4 + HNT CD4 0 48 CD4+ CD4+ d 7 5 0 0 6 0 0

IFN- CD4+ donor CFSE T cells d 10 65 1 678 2069 0 2 C

Thy1.1 d 10

d 13 448 6 551 2376 0 4 CD8+ donor d 13 CD8 T cells 17 58 13 44 CD44 CD62L CD25 1 14 1 2 ϩ Fig. 3. Migration of Clone 4 CD8 T cells to the pancreas of irradiated InsHA CD25 CD62L mice depends on CD4 help. (A) Irradiated InsHA mice were injected with either CFSE 5 ϫ 106 CFSE-labeled naive HNT Thy1.1ϩ CD4ϩ T cells, 5 ϫ 106 CFSE-labeled naive Clone 4 Thy1.1ϩ CD8ϩ T cells, or a combination of 5 ϫ 106 Clone 4 CD8ϩ T cells and Fig. 4. Clone 4 CD8ϩ T cell-mediated autoimmunity in lymphopenic InsHA mice 5 ϫ 106 HNT CD4ϩ T cells. Mice were killed on days 7, 10, or 13 after transfer and depends on antigen-specific CD4 help. Irradiated InsHA mice were injected with the presence of CD8ϩ Thy1.1ϩ and CD4ϩ Thy1.1ϩ donor T cells in the pancreas was 3.5 ϫ 106 Thy1.1ϩ Clone 4 CD8ϩ T cells together with either 3.5 ϫ 106 InsHA evaluated by FACS. Numbers indicate FACS event counts in the depicted gates. (B) Thy1.1ϩ CD4ϩ T cells or 3.5 ϫ 106 HNT Thy1.1ϩ CD4ϩ T cells. Mice were killed on Cells from the pancreas were either stimulated with Kd HA peptide or left day 20 after transfer and proliferation of gated CD4ϩ Thy1.1ϩ and CD8ϩ Thy1.1ϩ untreated and IFN-␥ production was assessed by intracellular staining. Plots lymphocytes cells in pLN, and spleen was assessed as a function of CFSE fluores- represent CFSE fluorescence versus IFN-␥ on gated CD8ϩ Thy1.1ϩ lymphocytes. cence (Top). Expression of CD62L and CD25 on gated CD8ϩ Thy1.1ϩ lymphocytes The percentages of IFN-␥ secreting CD8ϩ donor T cells are indicated. (C) Expres- was assessed and the percentages of highly proliferating donor CD62Llo cells and sion of CD44, CD62L, and CD25 markers on gated CD8ϩ Thy1.1ϩ lymphocytes in CD25ϩ cells are indicated in each condition. Data from 1 representative experi- the pancreas is shown at days 10 and 13. Shaded histograms show isotype ment of 4 are presented. controls. Data from 1 representative experiment of 5 are presented.

liferating Clone 4 cells, in the presence of CD4ϩ T cells, migrated ϩ on the responsiveness of memory-like CD8 T cells is antigen to the pancreas and were also found in other lymphoid organs (Fig. dependent. 5). Therefore, antigen-specific CD4ϩ T helper cells enhanced the proliferation of memory-like CD8ϩ T cells in response to antigen CD4؉ T Helper Cells Promote the Generation of Effector CD8؉ T Cells cross-presentation and promoted their differentiation into effector Through Antigen Cross-Presentation and Their Migration to the Pan- cells capable of migrating to the site of antigen expression. creas. In InsHA mice, HA is cross-presented by professional APCs in the draining LNs of the pancreas (27). In the experiments Discussion described above (see Fig. 1), the CD4-enhanced proliferation of Understanding the checkpoints that prevent potentially autoreac- ϩ Clone 4 CD8 T cells was preferentially observed in pLNs. This tive T cells from mounting efficient anti-self responses is essential effect suggested that APCs may act as bridge between memory-like for the rational design of new immunotherapeutic strategies against ϩ ϩ CD4 and CD8 T cells. However, in those experiments, it was autoimmune diseases and cancer. Lymphopenia has been proposed difficult to specifically assess antigen-driven proliferation, because as a putative trigger of autoimmunity and, conversely, as a prom- lymphopenia-induced proliferation was extensive in all lymphoid ising tool for the development of anti-self tumor antigen T cell organs. To precisely investigate the role of CD4 help on the responses (1, 2, 33). It has long been known that the reduced ϩ recognition of the HA antigen by memory-like CD8 T cells, we numbers of regulatory cells associated with lymphopenia may designed double transfer experiments, in which homeostatic pro- promote autoimmunity (6, 7). The results that we present here liferation/differentiation was uncoupled from antigen recognition. demonstrate that, in this condition, additional mechanisms of InsHA mice were irradiated and half were injected with naive HNT peripheral tolerance, such as those mediated by self-antigen cross- CD4ϩ T cells. After 10 days, all mice were injected with lympho- presentation, are overcome. cytes from naive nonirradiated InsHA mice to ‘‘fill’’ the space and Collectively, our data point to the following sequence of events prevent any subsequent lymphopenia-induced proliferation. After in disease progression in InsHA mice. Potentially autoreactive resting for an additional 3 days, InsHA mice from both groups naive CD8ϩ T cells that reach secondary lymphoid organs in a received CFSE labeled memory-like Clone 4 CD8ϩ T cells that had lymphopenic environment proliferate and differentiate into central been generated by their prior transfer into irradiated BALB/c mice. memory-like T cells. In the absence of help, these memory-like At the time of secondary transfer, Clone 4 cells isolated from CD8ϩ T cells do not gain access to the pancreas, as shown by our irradiated BALB/c mice had a CD44hi CD25Ϫ CD62Lhi phenotype. inability to detect them in this organ at all tested time points. Five days later, InsHA mice were killed and proliferation of donor However, as they recirculate to the draining LNs of the pancreas, Clone 4 cells was assessed in the lymphoid organs. In the absence they do encounter HA ␤ cell antigen that is cross-presented by of help, memory-like Clone 4 CD8ϩ T cells proliferated exclusively APCs. In the presence of memory-like CD4ϩ T helper cells, this in the pLNs (Fig. 5A). Proliferation was slow and only a small encounter results in further proliferation and differentiation of the percentage of donor T cells had been activated after 5 days, likely CD8ϩ T cells into effector cytotoxic T lymphocytes (CTL) that are

reflecting the low amount of cross-presented antigen. Proliferating then able to migrate to the pancreas, inducing autoimmune diabe- IMMUNOLOGY T cells did not recirculate to other lymphoid organs or migrate to tes. It is important to note that the coinjection of HA-specific naive the pancreas (Fig. 5). Thus, memory-like Clone 4 cells behaved very CD8ϩ and CD4ϩ T cells into lymphoreplete InsHA mice does not much as naive cells undergoing deletional tolerance in the pLNs result in autoimmune diabetes, and naive CD8ϩ T cells do not (26). In marked contrast, CD4 help dramatically enhanced the differentiate into effectors after antigen encounter (29). This proliferation of memory-like Clone 4 CD8ϩ T cells in the pLNs of absence of reactivity is most likely due to the tolerization of both cell irradiated/‘‘refilled’’ InsHA mice (Fig. 5A). Also, CD4 help induced types through antigen cross-presentation (26, 29, 34). Therefore, the differentiation of highly proliferating memory-like CD8ϩ T cells under conditions of lymphopenia, the cooperation between CD4ϩ into effectors, as demonstrated by the dow-regulation of CD62L and CD8ϩ T cells bypasses one important checkpoint for autore- and the up-regulation of CD25 (Fig. 5C). Most importantly, pro- active T cells in the periphery, namely cross-tolerization.

Le Saout et al. PNAS ͉ December 9, 2008 ͉ vol. 105 ͉ no. 49 ͉ 19417 Downloaded by guest on September 28, 2021 HNT CD4ϩ T cells proliferating under lymphopenic conditions A pLN LN Spleen B Pancreas displayed a heterogeneous phenotype with the generation of both

InsHA 0 0 central and effector memory-like cells. Thus, it was not surprising Memory-like that they were able to migrate to the pancreas of InsHA mice even Cl 4 CD8+ when injected alone. We had shown that activated HNT cells InsHA Thy1.1 291 1265 + HNT CD4+ migrate to the pancreas, inducing peri-insulitis, but are not diabe- togenic in themselves (29). Indeed, NHT CD4ϩ T cells activated CFSECFSE CD8 under lymphopenic conditions also appear to be nondiabetogenic even if they are able to migrate to the pancreas. Although memory C ϩ InsHA HNT CD4 T cells do not induce diabetes, they could potentially Memory-like induce limited damage in the islets resulting in the release of HA Cl 4 CD8+ antigen. Larger amounts of available antigen could be the under- CD25 CD44

InsHA CD62L + HNT CD4+ lying mechanism by means of which they provide help and enhance the proliferation of CD8ϩ T cells. However, our data indicate that CFSECFSE antigen release is neither the only nor the main mechanism involved. We had shown that increasing amounts of antigen pre- ϩ Fig. 5. CD4 help enhances proliferation of memory-like Clone 4 CD8 T cells in sented by quiescent APCs enhances Clone 4 CD8ϩ T cell prolif- the pLNs of InsHA mice. (A) Irradiated InsHA mice were either injected with 5 ϫ 6 ϩ ϩ eration but not their differentiation into effectors (26). Notably, we 10 CFSE-labeled naive HNT Thy1.1 CD4 T cells or PBS. On day 10, both groups find that the main effect of helper cells on CD8ϩ T cells occurs in of mice were refilled with 100 ϫ 106 spleen and LN cells from unmanipulated InsHA mice. Three days later, the refilled InsHA mice received enriched CFSE- the pLNs and is antigen dependent. Also, we show that CD4 help labeled memory-like Clone 4 Thy1.1ϩ CD8ϩ T cells, generated by injection of naive promotes the conversion of a tolerogenic signal to a priming signal ϩ cells into irradiated BALB/c mice 13 days earlier. Five days after secondary transfer, for CD8 T cells, inducing not only enhanced proliferation but also InsHA mice were killed, and donor CD8ϩ Thy1.1ϩ lymphocytes from pLN, LN, and their differentiation into effectors, in the pLNs. This observation spleen were analyzed by FACS. Histograms represent CFSE fluorescence on gated strongly suggests an implication of the cross-presenting APCs on CD8ϩ Thy1.1ϩ lymphocytes. (B) The presence of CD8ϩ Thy1.1ϩ and CD8Ϫ Thy1.1ϩ this effect. These APCs may be activated by helper cells after donor T cells in the pancreas was evaluated by FACS. FACS event counts in the encounter (42–44) or mediate a direct effect of CD4ϩ on CD8ϩ T depicted gates are indicated. (C) Plots represent expression of CD44, CD62L, and ϩ ϩ cells responding to the same antigen (45–47). CD25 as a function of CFSE fluorescence on gated CD8 Thy1.1 lymphocytes in ϩ the pLN. Data from 1 representative experiment of 3 are presented. The molecular pathways by means of which CD4 T cells provide help for CD8ϩ T cell responses remain elusive. A major break- through was made when the role of the CD40/CD40L pathway in ϩ Memory-like Clone 4 CD8ϩ T cells, although they displayed the licensing of cross-presenting dendritic cells by CD4 T cells was shown (42–44). However, in certain models CD40 engagement considerable effector function, were not sufficient to induce auto- ϩ immunity under lymphopenic conditions on their own. This obser- cannot replace help and account for the full differentiation of CD8 vation is in agreement with a previous report, showing that con- T cells into effectors (29, 48). In agreement with these latter ventional antigen-driven memory Clone 4 CD8ϩ T cells are not able observations, we found no major role for CD40L in the help provided by HNT cells to memory-like Clone 4 CD8ϩ T cells (C.L. to induce disease in InsHA mice because they undergo deletional ϩ tolerance through antigen cross-presentation (35). Memory-like and J.H., unpublished results). The finding that CD4 help is Clone 4 CD8ϩ T cells likely follow the same fate in InsHA mice. partially or fully independent of CD40 engagement indicates the Our double transfer experiments showed that although these cells existence of additional, yet unknown, activation pathways. did undergo a few rounds of division, they did not accumulate as Our results emphasize the importance of memory-phenotype T they proliferated and did not migrate elsewhere in the lymphoid cells as targets for therapeutic intervention in autoimmunity and their potential implication in cancer immunotherapy. Cancer im- tissue or pancreas, suggesting that they died in situ after activation. ϩ ϩ Memory-like CD8ϩ T cells generated under lymphopenic con- munotherapy relies on the potential ability of CD8 and CD4 T ditions were modulated by memory-like CD4ϩ T helper cells, cells to eliminate tumor cells (37). Because most identified tumor- resulting in the generation of an efficient response against a associated antigens are self-proteins, responses against these anti- self-antigen. Although previous studies determined that help for gens are subject to the same constraints. Therefore, taking advan- CD8ϩ T cells responding to self-antigens could be provided by in tage of the lymphopenic environment ensuing after currently used vitro activated CD4ϩ T cells (29, 30), our data show that the conditioning therapies to optimize antitumor T cell responses is a ϩ promising strategy (33). Conditions favoring the generation and lymphopenic state itself drives the differentiation of CD4 T cells ϩ ϩ into memory-like cells fully capable of providing help. This obser- cooperation of memory-like CD8 and CD4 T cells may help to vation illustrates that autoimmunity can be induced without evok- overcome tolerance allowing the generation of efficient antitumor ing a requirement for external activation events. Initially, CD4 help responses. was thought to be required for the generation of primary CTL Materials and Methods responses (36). Today, it appears that this is the case only as regards Mice. BALB/c mice were purchased from Charles River and then bred at the responses generated under noninflammatory conditions, such as Institut de Ge´ne´ tique Mole´culaire de Montpellier. InsHA (23), Clone 4 TCR (24), for peptide immunization regimes or after cross-presentation of HNT TCR (25) (kindly provided by Linda A. Sherman, The Scripps Research Insti- tumor or self-antigens (29, 37, 38). More recently, it has been shown tute), and DO11.10 (a gift from Pascale Plence and Christian Jorgensen, Institut that even under inflammatory conditions, such as those generated National de la Sante´ et de la Recherche Me´dicale, Montpellier, France) (32), after viral infection, CD4ϩ T cells have an essential role in the transgenic mice lines had been backcrossed with BALB/c mice for at least 10 ϩ ϩ generation of functional memory CD8ϩ T cells (38–41). Interest- generations. Clone 4 and HNT mice were then crossed with BALB/c Thy1.1 / for ingly, in a recent report, Hamilton et al. (20) have shown that the 2 generations to achieve homozygosity for Thy1.1. All mice used in these studies generation of protective memory-like CD8ϩ T cells also requires were between 8 and 16 weeks of age. Mice were propagated and maintained ϩ under specific pathogen-free conditions at the animal facility of the Institut de the presence of polyclonal CD4 T cells during homeostatic Ge´ne´ tique Mole´culaire de Montpellier. All experimental procedures were ap- proliferation in the absence of cognate antigen. Here, we show that proved by the local animal facility Institutional Review Board. memory-like CD8ϩ T cells require antigen-specific CD4 help at the time of antigen encounter to differentiate into CTL and generate Preparation of CFSE Labeled Naive TCR Transgenic T Cells. CD8ϩ T cells from an efficient anti-self antigen response. Clone 4 TCR Thy1.1 transgenic mice were prepared from single cell LN suspensions

19418 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0807743105 Le Saout et al. Downloaded by guest on September 28, 2021 by magnetic depletion of non CD8ϩ T cells by using the T cell isolation kit from Diabetes Monitoring. Mice were monitored for diabetes by measuring blood Dynal, supplemented with an anti-CD4 mAb (clone 191.1), according to the glucose every 4 days for a maximum period of 30 days with a Glucomatic ESPRIT manufacturer’s instructions. CD4ϩ T cells from HNT Thy1.1, InsHA Thy1.1, and Apparatus (Bayer). In some instances, daily measurements were performed on DO11.10 transgenic mice were similarly prepared by using an anti-CD8 mAb urine samples with Multistix 8 SG strips (Bayer). Animals were considered diabetic (clone 169.4) for the depletion of CD8ϩ T cells. T cell purity was Ͼ90%. Purified T when glucose levels were Ͼ300 mg/dL during 2 consecutive measurements. cells (2 ϫ 107 cells per mL) were labeled with 5 ␮M CFSE (Molecular Probes) in PBS for 10 min at 37 °C. Flow Cytometry. Flow cytometry was performed essentially as described in ref. 26. For details, see SI Methods. Induction of Lymphopenia by Irradiation. BALB/c or InsHA mice were sublethaly irradiated (600 rads) by using a 60Cobalt source. Under these conditions, depletion Statistical Analyses. Statistical significance was determined by using a Student’s of host T cells was Ϸ90% at 24 h after irradiation and 70% at 7 days. Mice were t test with a 1-tailed distribution and 2-sample equal variance. Data were con- Ͻ used for adoptive transfer experiments 24 h after irradiation, unless otherwise sidered to be statistically different (*) for P 0.05. noted. ACKNOWLEDGMENTS. We thank Dr. Linda A. Sherman (The Scripps Research Institute, La Jolla, CA) for providing us with the HA transgenic mouse system and Adoptive Transfer. Irradiated or nonirradiated InsHA and BALB/c mice were critically reading the manuscript, Drs. Pascale Plence and Christian Jorgensen ϩ injected i.v. with either CFSE-labeled Clone 4 TCR Thy 1.1 CD8 T cells or CFSE- (Institut National de la Santé et de la Recherche Médicale U844, Montipellier, labeled HNT TCR Thy 1.1 CD4ϩ T cells in PBS. In other experiments, mice were France) for additional valuable mice, Dr. Thomas Stratmann for valuable discus- coinjected with equal numbers of CFSE-labeled Clone 4 TCR Thy 1.1 CD8ϩ T cells sions, Drs. Alfred Singer and Vale´rie Zimmermann for constructive comments on and CFSE-labeled HNT TCR Thy1.1, InsHA Thy1.1 or DO11.10 TCR CD4ϩ T cells. The the manuscript, the MRI-RIO imaging platform (GIS-IBISA, Languedoc-Roussillon) for flow cytometry experiments, the T&TA core facilities for animal experiments, numbers of injected cells are indicated for each experiment. and in particular Chantal Jacquet for excellent technical assistance with mice. C.L. For double transfer experiments, InsHA mice were irradiated, and 24h later was supported by the Ligue Contre le Cancer-Region Languedoc-Roussillon. N.T. half of the mice received HNT TCR Thy1.1 CD4ϩT cells. All mice were refilled with is supported by Institut National de la Sante´et de la Recherche Me´dicale and a a mixture of 100 ϫ 106 spleen and LN cells from nonirradiated InsHA mice 10 days Contrat d’interface with the Centre Hospitalier Universitaire de Amiens. J.H. is later. At day 13, the InsHA mice received secondary i.v. transfers of CFSE-labeled, supported by Institut National de la Sante´ et de la Recherche Me´dicale and a ϩ Contrat d’interface with the Centre Hospitalier Universitaire de Montellier. This enriched memory-like Clone 4 TCR Thy1.1 CD8 T cells. These latter cells were work was supported by grants from the European Community, Association pour obtained from the LNs and spleen of irradiated BALB/c mice that had been la Recherche contre le Cancer, the Association de langue franc¸aise pour l’e´tude du ϩ injected 13 days before with naive Clone 4 TCR Thy1.1 CD8 T cells. Cells recovered diabe`te et des maladies me´taboliques (J.H.), and the European Community from 1 single primary host were transferred into 1 secondary recipient mouse. Contract LSHC-CT-2005-018914 ‘‘ATTACK’’ (to N.T.).

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