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Blood, 1 July 2006, Vol. 108, No. 1, pp. 270-277. Prepublished online as a Blood First Edition Paper on March 9, 2006; DOI 10.1182/blood-2006-01-0017.
IMMUNOBIOLOGY Self-recognition is crucial for maintaining the peripheral CD4+ T-cell pool in a nonlymphopenic environmentFrom the Institut National de la Santé et de la Recherche Médicale (INSERM) U561, Saint-Vincent-de-Paul Hospital, Paris, France; and the Department of Immunology, Cochin Institute, INSERM U567, Centre National de la Recherche Scientifique (CNRS) Unité Mixte de Recherche (UMR) 8104, René Descartes University, Cochin Hospital, Paris, France.
The role of self-recognition in the maintenance of the peripheral CD4+ T-cell pool has been extensively studied, but no clear answer has so far emerged. Indeed, in studies of the role of self-major histocompatibility complex (MHC) molecules in CD4+ T-cell survival, several parameters must be taken into account when interpreting the results: (1) in a lymphopenic environment, observations are biased by concomitant proliferation of T cells arising in MHC-expressing mice; (2) the peripheral T-cell compartment is qualitatively and quantitatively different in nonlymphopenic, normal, and MHC class II-deficient mice; and (3) in C57BL/6 A  -/- mice (traditionally considered MHC class II-deficient), the A chain and the E chain associate to form a hybrid AE MHC class II molecule. In light of these considerations, we revisited the role of interactions with MHC class II molecules in the survival of peripheral CD4+ T cells. We found that the answer to the question "is self-recognition required for CD4+ T cells to survive?" is not a simple yes or no. Indeed, although long-term survival of CD4+ T cells does not depend on self-recognition in lymphopenic mice, interactions with MHC class II molecules are required for maintaining the peripheral CD4+ T-cell pool in a nonlymphopenic environment. (Blood. 2006;108:270-277)
The role of self-peptide/self-major histocompatibility complex (MHC) class II molecule complexes in the survival of peripheral CD4+ T cells has been extensively studied, but no clear answer has yet emerged.1-12 Adoptive transfer of mature T cells, thymus grafting, and more elegant experimental systems in which MHC molecules are transiently expressed in the thymus have been used to address this issue. Based on differential CD4+ T-cell recovery from lymphopenic mice with and without MHC class II expression, numerous groups have proposed that the survival of peripheral CD4+ T cells depends on permanent interactions with MHC class II molecules expressed on peripheral antigen-presenting cells.1-5,8,10 However, these conclusions are undermined by the concomitant expansion of T cells arising in MHC-expressing lymphopenic mice.13,14 Indeed, few of these reports clearly distinguished between the effects of MHC recognition on T-cell survival from lymphopenia-induced proliferation of T cells in response to self-peptide/self-MHC molecule complexes, a phenomenon previously described in neonatal mice15,16 and after transfer into lymphopenic hosts.17 More recently, several groups have observed that, in a nonlymphopenic environment, the kinetics of CD4+ T-cell disappearance is independent of the ability of recipient mice to express MHC class II molecules.6,7 They concluded that the survival of peripheral CD4+ T cells does not depend on T-cell antigen receptor (TCR) signaling induced by recognition of self-peptide/self-MHC molecule complexes. However, these studies are also open to criticism, because the similar pattern of CD4+ T-cell decline in the different mice could occur for different reasons such as competition and replacement in normal recipients, and death due to the absence of TCR contact with self-MHC molecules in mice lacking MHC class II molecules.18
Another criticism applying to most studies of CD4+ T-cell survival is that nearly all groups working in this field have considered C57BL/6 A
Thus, in studies of the role of self-MHC molecules in CD4+ T-cell survival, one needs to take into account several parameters when interpreting the results: (1) in a lymphopenic environment, peripheral T-cell proliferation and survival may rely on different mechanisms; (2) the peripheral T-cell compartment is qualitatively and quantitatively different in nonlymphopenic, normal, and MHC class II-deficient mice; and (3) in C57BL/6 A
In light of these considerations, we decided: (1) to label purified CD4+ T cells with CFSE before transfer, in order to discriminate between survival and proliferation; (2) to conduct sequential transfer experiments in order to assess the role of defined T-cell competitors in CD4+ T-cell survival; and (3) to use mice in which neither conventional nor hybrid MHC class II molecules are expressed (MHC II In the present paper, we thus revisited the role of interactions with MHC class II molecules in the survival of peripheral CD4+ T cells. We found that the answer to the question "is selfrecognition required for CD4+ T cells to survive?" is not a simple yes or no. Indeed, the survival and proliferative capacities of transferred polyclonal CD4+ T cells differ strongly according to the ability of recipient mice to produce endogenous T cells (lymphopenic versus T-cell-containing mice) and to express MHC class II molecules.
Mice
C57BL/6 mice were obtained from Centre d'élevage Janvier (Le Genest Saint Isle, France), and C57BL/6 CD3 Adoptive transfer of T cells Lymph node cells were depleted of macrophages, granulocytes, and CD8+ and CD4+ T cells by incubating them first with anti-CD11b (Mac-1) antibody (Ab), anti-GR1 (8C5) Ab, and anti-CD8 (53-6.7) or CD4 (GK1.5) Ab, and then with magnetic beads coated with antirat Ab (Dynal, Great Neck, NY). B cells were removed by using magnetic beads coated with anti-mouse immunoglobulin (Ig) Ab (Dynal). In some experiments (Figure 3), purified CD4+ T cells from C57BL/6 mice were labeled with biotinylated anti-CD25 (clone PC61) and PE anti-CD44 (clone 1M7). CD4+ CD25-CD44- naive T cells were then purified by sorting in a FACSVantage flow cytometer (BD Biosciences, Mountain View, CA). Purified CD4+ T cells were injected intravenously into recipient mice. When indicated, CD4+ T cells were labeled with CFSE (Molecular Probes, Eugene, OR) before injection. In some other experiments (Figure 6), purified CD8+ or CD4+ T cells (50 x 106) were injected 3 days before the CFSE-labeled CD4+ T-cell cohort. In the experiment represented in Figure 7C, 7-week-old C57BL/6 mice were thymectomized or sham-thymectomized 35 days before CD4+ T-cell transfer. Cell-surface staining and flow cytometry
Spleen and lymph nodes were recovered, pooled for cell preparation, and analyzed at various times after CD4+ T-cell transfer. Lymph nodes and spleen were homogenized with a nylon cell strainer (Falcon, Franklin Lakes, NJ) in phosphate-buffered saline (PBS) + 5% fetal calf serum (FCS) + 0.2% NaN3, and then distributed in 96-well U-bottom microplates (6 x 106 cells per well). Staining was performed in ice for 30 minutes per step. Antibodies were purchased from BD Pharmingen (San Diego, CA) unless otherwise indicated. The following antibody combinations were used to characterize transferred CFSE-labeled lymph node CD4+ T cells: peridinin chlorophyll protein (PerCP) anti-CD4, and biotinylated anti-TCR Four-color immunofluorescence was analyzed by using a FACSCalibur cytometer (Becton Dickinson, San Jose, CA). List-mode data files were analyzed by using Cellquest software (Becton Dickinson).
Analysis of
Purified CD4+ T cells were washed twice with ice-cold PBS and placed in lysis buffer containing 1% NP-40, 10 mM Tris-HCl (pH 7.2), 140 mM NaCl, 2 mM EDTA, 5 mM iodoacetamide, 1 mM Na3 VO4, and protease inhibitors for 20 minutes on ice. Nuclear debris was removed and the resulting supernatants were collected. Protein was then eluted in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer and analyzed by immunoblotting. Two immunoblots were prepared with each supernatant; the first was developed with 4G10 Ab (a mouse Ab to phosphotyrosine; Upstate Biotechnology, Lake Placid, NY), and the second with 6B-10.2 Ab (a mouse Ab to In vivo CD8+ T-cell depletion Six-week-old C57BL/6 mice were injected intraperitoneally with 50 µg of purified anti-CD8 Ab (53-6.7) every 2 days for 4 weeks. We verified that such a treatment selectively depletes peripheral CD8+ T cells, without affecting thymic differentiation (data not shown).
Survival of CD4+ T cells in a lymphopenic environment does not depend on interactions with MHC class II molecules
In order to study the fate of CD4+ T cells transferred into lymphopenic mice as a function of the expression or nonexpression of MHC class II molecules, we injected CFSE-labeled purified CD4+ T cells into CD3
Absolute numbers of recovered CD4+ T cells were then calculated as a function of their CFSE labeling status (Figure 1B). In both recipients the absolute numbers of recovered CD4+ T cells increased with time and, as stated, this reflected the strong proliferation of a minority of injected cells (giving rise to CFSE- CD4+ T cells). More precisely, in CD3
The phenotype of CD4+ T cells recovered 28 days after transfer was then analyzed (Figure 1C). In both CD3  -/- mice and CD3-/- II / mice, CD44 up-regulation and TCR down-regulation were restricted to CFSE- CD4+ T cells. Thus, only the progeny of the few CD4+ T cells that strongly proliferate exhibit an effector-like phenotype. In contrast, the bulk of injected cells cycles slowly, survives, and retains a naive phenotype. Interestingly, in mice lacking MHC class II molecules, CD4 expression was up-regulated in both CFSE+ and CFSE- cells.
The kinetics of CFSE- CD4+ T-cell generation was different when CD4+ T cells were transferred into CD3 Generation of CFSE- CD4+ T cells in the absence of MHC class II expression is not related to preferential expansion of memory CD4+ T cells
In the absence of MHC class II molecule expression, the frequency of CD4+ T cells that are able to proliferate strongly in a lymphopenic environment is greatly reduced (Figure 2B). This lower frequency could be explained by the possibility that a proportion of both naive and memory CD4+ T cells undergo strong proliferation after transfer into MHC class II molecule-expressing mice, whereas only memory CD4+ T cells would expand strongly in CD3
To test this hypothesis, total CD4+ T cells and purified naive CD4+ T cells (CD44-CD25-CD4+ T cells) were injected into CD3 -/- mice and CD3-/-II/ mice (Figure 3A). The proliferation of injected cells was studied 14 days after transfer, before the occurrence of the wasting disease induced by transfer of naive CD4+ T cells alone.25 Independently of the ability of recipient mice to express MHC class II molecules, the transfer of both total and naive CD4+ T cells led to the generation of a subset of CFSE- CD4+ T cells (Figure 3B). No difference in the absolute numbers of recovered CFSE- CD4+ T cells was observed in CD3-/-II/ recipient mice whether total or naive CD4+ T cells were injected (Figure 3C). Thus, CFSE- CD4+ T-cell generation in CD3-/-II/ mice is not related to preferential expansion of memory CD4+ T cells. CFSE- CD4+ T-cell generation depends strictly on interactions with MHC molecules
Some naive CD4+ T cells were able to proliferate strongly and to convert to a memory-like phenotype in a lymphopenic environment, even in the absence of MHC class II molecule expression. We thus investigated whether T-cell receptor signaling was required for CFSE- CD4+ T-cell generation by measuring the phosphorylation status of TCR
Two days after transfer into CD3
Two months after transfer, the level of  phosphorylation in CD4+ T cells recovered from CD3-/-II/ mice had strongly increased, approaching the level found in CD4+ T cells freshly isolated from normal C57BL/6 mice. The high level of phosphorylation would reflect the phosphorylation status of this protein in the major subset of CD4+ T cells recovered at these late time-points (ie, CFSE- CD4+ T cells). These results point to the existence of an MHC molecule in CD3-/-II/ mice, with which some transferred CD4+ T cells need to interact in order to expand strongly.
Given the lack of MHC class II molecules in CD3
Survival of CD4+ T cells depends on interactions with MHC class II molecules in a nonlymphopenic environment Most CD4+ T cells that survive in a lymphopenic environment do so independently of interactions with MHC class II molecules (Figures 1-2). However, in lymphopenic conditions, donor CD4+ T cells do not have to compete with a full preformed pool of endogenous T cells, or with continuous thymic output. For these reasons, we chose to use endogenous T-cell-containing recipients to continue our study of the role of MHC class II molecules in the survival of peripheral CD4+ T cells.
CD4+ T cells from C57BL/6 CD45.1 mice were injected into MHC II
CD8+ T lymphocytes restrict the size of the peripheral CD4+ T-cell compartment
Normal mice contain both CD4+ and CD8+ T lymphocytes, whereas the peripheral T-cell compartment of MHC II
CD4+ T cells from C57BL/6 CD45.1 mice were transferred into CD3
We then examined whether the expansion of the minor CD4+ T-cell subset that generates CFSE- CD4+ T cells was modified in these conditions. As stated (Figures 3-4), CFSE- CD4+ T-cell generation depends strictly on interactions with MHC molecules (mainly MHC class II molecules in MHC class II-expressing mice, and MHC class I molecules in mice lacking MHC class II molecules). Accordingly, the proliferation and subsequent expansion of the minor CD4+ T-cell subset that produces CFSE- CD4+ T cells were both affected by the presence of preinjected CD4+ T cells in CD3
Finally, we studied the fate of the bulk of the second cohort of injected CD4+ T cells (CFSE+ CD45.1 CD4+). In CD3
Maintenance of the peripheral CD4+ T-cell pool seems to rely on different mechanisms in the presence and absence of CD8+ T cells. In the absence of CD8+ T cells, self-recognition is not required for the maintenance of CD4+ T-cell numbers. In contrast, interactions with MHC molecules become necessary to ensure the survival of CD4+ T cells in CD8+ T-cell-containing mice. The most logical explanation is that, in CD8+ T-cell-containing mice that lack MHC class II molecules, injected CD4+ T cells have to compete with preinjected CD8+ T cells for the soluble factor that permits their survival in a lymphopenic environment. We thus hypothesized that in MHC class II-expressing mice that do not contain CD8+ T cells, the 2 signals would synergize to maintain a larger CD4+ T-cell pool than in normal mice (CD8+ T-cell-containing MHC class II-expressing mice). To test this possibility, we estimated the size of the peripheral CD4+ T-cell compartment both in C57BL/6 mice lacking MHC class I molecules and therefore devoid of CD8+ T lymphocytes (C57BL/6 Continuous thymic output restricts the survival of peripheral CD4+ T cells in MHC class II molecule-expressing mice
Preinjected peripheral CD4+ or CD8+ T cells did not interfere with the survival of a cohort of CD4+ cells transferred into recipient mice expressing MHC class II molecules (Figure 6). By contrast, CD4+ T cells transferred into normal C57BL/6 mice disappeared with time (Figure 5). However, while preinjection can fill the peripheral T-cell compartment of a CD3
To test this possibilty, CD4+ T cells from C57BL/6 CD45.1 mice were transferred into thymectomized and sham-thymectomized C57BL/6 CD45.2 mice. The survival of CD45.1 CD4+ T cells was assessed 28 days after their transfer (Figure 7C). As expected, CD4+ T-cell survival was markedly enhanced in thymectomized mice, suggesting a role of thymic migrants in the continuous renewal of the peripheral CD4+ T-cell compartment. By contrast, the survival of transferred CD4+ T cells in MHC II / mice was not improved by thymectomy (data not shown).
In normal mice, the proliferation of transferred polyclonal CD4+ T cells is quite limited, whereas most CD4+ T cells proliferate after transfer into T-cell-free recipients. In these latter recipients, while most transferred CD4+ T cells undergo a limited number of divisions, some proliferate rapidly and expand strongly (leading to the generation of CFSE- CD4+ T cells), a process recently designated by Min et al as "spontaneous proliferation."22,23,26 Here we demonstrate that spontaneous proliferation of CD4+ T cells in a lymphopenic environment depends strictly on interactions with MHC molecules—mainly MHC class II molecules in MHC class II-expressing mice and MHC class I molecules in mice lacking MHC class II molecules. This requirement for interactions with MHC molecules may explain why, like antigen-driven proliferation, spontaneous proliferation of naive T cells in a lymphopenic environment is independent of interleukin-7 (IL-7),23 and results in their differentiation into memory cells.17 Indeed, strongly proliferating T cells in lymphopenic hosts share a similar gene-expression profile with conventional memory T cells.27 Although recent data suggest the involvement of enteric bacterial peptides in the spontaneous proliferation of naive T cells,24 a role for self-peptides in this process cannot be ruled out. If this is indeed the case, then spontaneous proliferation would correspond to an autoimmune process, potentially explaining why lymphopenic individuals are at much higher risk of developing certain autoimmune diseases.28-31
While a few clones rapidly proliferate and convert to a memory phenotype, the bulk of CD4+ T cells transferred into lymphopenic hosts (CFSE+ CD4+ T cells) proliferate slowly and retain a naive phenotype. The extent of this proliferation also depends on interactions with MHC class II molecules. Indeed, at all time-points after transfer, we found that CFSE+ CD4+ T cells proliferated more strongly in CD3 Regarding the biological significance of this slow cytokinemediated proliferation of T cells in a lymphopenic environment, most researchers consider it to be "homeostatic," because it would restore the peripheral T-cell compartment. In our hands, although CFSE+ CD4+ T cells cycled slowly, their absolute numbers did not increase with time, suggesting a counterbalancing effect due to death of proliferating cells. Furthermore, this slow proliferation is not required for T cells to maintain their numbers. Indeed, in lymphopenic mice lacking MHC class II molecules, although preinjected CD4+ T cells inhibited the slow proliferation of a second cohort of CD4+ T cells, they did not affect the survival of this cohort (Figure 6). Thus, the slow proliferation of naive T cells in a lymphopenic environment cannot fill the peripheral T-cell compartment and is not required for its maintenance. Slow proliferation of naive T cells in a lymphopenic environment does not therefore appear to be a homeostatic mechanism.
We found that long-term survival of CD4+ T cells did not depend on TCR signaling in lymphopenic mice. Indeed, despite the decrease in
In both normal mice and MHC II
Finally, as previously proposed by Tanchot et al38,39 for naive CD8+ T cells, we found that the disappearance of naive CD4+ T cells in normal C57BL/6 mice was due to their gradual replacement by new CD4+ thymic migrants. Indeed, transferred CD4+ T-cell numbers remained constant in thymectomized normal C57BL/6 mice while, importantly, they still failed to survive in thymectomized MHC II During thymic differentiation, recognition of self-peptide/self-MHC ligands and the resulting activation process (positive selection) are required for immature thymocytes to differentiate into fully mature lymphocytes.40,41 Thymocytes also require signals generated by receptor recognition of self-ligands for survival, maturation, and orientation toward the CD4+ or CD8+ lineage.42-44 Our results demonstrate that naive CD4+ T cells continue to interact with MHC molecules in the periphery, and that such interactions are required for their survival in a nonlymphopenic environment. Thus, in normal mice, intimacy between T cells and "self" is not restricted to the thymus. Our results strongly support a role of self-recognition in the maintenance of the peripheral CD4+ T-cell pool.
The authors thank B. Faideau and A. Le Campion for illuminating discussions.
Submitted January 3, 2006; accepted February 26, 2006.
Prepublished online as Blood First Edition Paper, March 9, 2006; DOI 10.1182/blood-2006-01-0017.
Supported by a PhD fellowship from Fondation pour la Recherche Médicale (B.M.). This work was supported by a grant from the French Research Ministry and by a grant for young investigators from the French National Research Agency (ANR).
An Inside Blood analysis of this article appears at the front of this issue.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Bruno Lucas, Department of Immunology, Cochin Institute, INSERM U567, CNRS UMR 8104, René Descartes University, Cochin Hospital, 75014 Paris, France; e-mail: lucas{at}paris5.inserm.fr.
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Abstract
Introduction
indicates C57BL/6; 
, MHC II
