We have developed a model for studying tolerance to persistent low dose antigen in vivo, which results in the generation of a large number of anergic (hyporesponsive) T cells. We call this state adaptive tolerance. We inject CD4+, antigen-specific T cells from a T cell receptor transgenic mouse on a Rag2-/- background (a monospecific T cell population) into a second transgenic mouse expressing the antigen in a lymphopenic background (no other T cells). Within 24 hours after transfer, the T cells are all activated by the antigen (as evidenced by an increase in size and expression of CD69), and proliferate extensively for several days, increasing in number about 100-fold. This expansion is followed by a deletional phase during which 50% of the cells disappear. Finally, the population reaches a steady state level in which the cells appear to be refractory to restimulation in vivo and in vitro. In this adaptive tolerant state, cytokine responses to high doses of antigen in vitro are inhibited 90%. However, in vivo bromodeoxyuridine labeling shows a slow T cell turnover of about 5% of the cells per day, and B cell help is still sufficient for the mice to eventually develop a mild form of chronic autoimmune arthritis or polychondritis depending on the antigen and the T cell specificity. The hyporesponsive state is reversible if the cells are transferred again into a second host not expressing the antigen. If the retransfer is into a host expressing the antigen, the T cells remain hyporesponsive and slowly decrease their IL-2 and IFN gamma production by another 6-10 fold over 3-4 weeks. This deeper state of anergy suggests that the tolerance process is adaptable to different levels. Finally, if the initial antigen-bearing host is T cell replete, i.e., contains a normal polyclonal T cell population, then the naive T cell response is blunted. The expansion phase is curtailed by 5 to 10 fold, the differentiation into effector T cells (producing IFN-gamma) does not occur, and the anergic T cells disappear with a half life of 9-12 days. As a consequence, the mice do not develop any autoimmune disease. In recent past the lab has focused on determining what causes the difference in the antigen-specific naive CD4+ T cell response when the cells are transferred into T cell-sufficient versus T cell-deficient antigen-bearing hosts. The difference must somehow be related to the presence of other T cells in the environment. The critical cells are peripheral alpha/beta T cells because adult thymectomy does not alter the full-host effects and introducing the TCR alpha KO mutation eliminates them all. To identify the inhibitory T cell, we first depleted the T cell-sufficient, host of CD25+ regulatory T cells by pretreatment with both anti-CD25 and anti-IL-2 mAbs. This had no impact on the blunted expansion or shortened half-life of a subsequently transferred population of antigen-specific, naive T cells. We then reconstituted T cell-deficient mice with normal CD4+/CD25+, CD4+/CD25-, or CD8+ T cells purified by flow cytometry. One to 7 days later, naive antigen-specific CD4+ T cells were injected and their expansion and longevity monitored. Both expanded polyclonal CD4+ T cell populations partially facilitated the loss of the antigen-specific T cells after the latter became anergic. This decrease from peak cell numbers occurred over a 3 week time period and was about 20 fold, compared to only a 2 fold loss in a host reconstituted with CD8+ T cells. Both CD25- and CD25+ T cells produced this effect, although CD25- T cells appeared to be more efficient. Since these experiments suggested that CD4+CD25- T cells could mediate at least one of the effects in a full host, we considered the possibility that the differences in outcome in the T cell-sufficient host were caused by T cell competition for nutrients, growth factors, or activation niches. To test this type of model we transferred the original antigen-specific T cells into a second TCR transgenic, Rag2-/- host specific for another antigen, but also expressing the first antigen. These hosts contain around 5 to 10 million naive T cells filling the space in lymph nodes and spleen. Despite this, the original naive antigen-specific T cells adoptively transferred into each such host expanded fully (100-fold), differentiated into IFN-gamma producers, entered the adaptive tolerant state, and persisted for months. This result was observed with 5 different CD4+ TCR transgenics. One of them was specific for an antigen presented by the same MHC molecule as that used by the first antigen. Thus, non-specific competition for things such as IL-7 would seem not to be responsible for the inhibitory effects of the polyclonal T cell population. We are currently carrying out experiments to try and clone the inhibitory CD4+ T cell population, to further determine its phenotype and characterize its mechanism of inhibition. In FY2009, polyclonal CD4+ T cells were plated at 100 cells per well from a V beta3 transgenic mouse crossed to a TCR alpha-deficient mouse. The cells were then stimulated with phorbol ester, ionomycin, and anti-CD28 monoclonal antibody, and after expansion to several million cells, tested for their ability to inhibit the survival of naive B10.A TCR-5C.C7 T cells transferred into an antigen-expressing lymphopenic host. A few populations were found to be greatly enriched for deletional activity, and RNA was isolated from them and their TCR alpha chains sequenced. In general there were between 10 and 20 unique alpha chain sequences per colony, and each of theses is now being introduced into V beta3 Rag2-/- bone marrow cultures using retroviral infection. These populations will then be used to create radiation-induced bone marrow chimeras expressing a unique alpha/beta TCR. We expect to be testing these chimeras for their deletor activity very shortly. In FY 2009 the lab also completed a series of experiments exploring the consequences of an allogeneic reaction in our adaptive tolerance model system. The 5C.C7 antigen-specific T cell receptor was found to be alloreactive toward the I-E molecule of the B10.S(9R) strain. When naive B10.A TCR-5C.C7 T cells from Rag2-deficient mice were transferred into B10.S(9R), CD3-epsilon KO mice, the T cells expanded rapidly to large numbers, although not as quickly as in our previous antigen-specific responses. Interestingly, the cells than slowly began to disappear, even though the hosts had no endogenous T cells. After elimination of this first cohort of T cells, a second graft of B10.A (H-2a) lymphoid tissue was rejected even more rapidly, consistent with the development of a hyper-acute antibody response. This correlated with the appearance of antibodies reactive against H-2a cells in the B10.S(9R) mice that had received the primary 5C.C7 graft. Serum from such mice was sufficient to transfer this rejection pattern to naive RAG2-deficient, B10.S(9R) mice, which were otherwise incapable of generating the hyper-acute response. Finally, we could tolerize the potential for such a response in B10.S(9R) mice, by pre-treating them with a single infusion of naive donor (H-2a) B cells prior to the introduction of the primary 5C.C7 graft. This treatment not only abrogated the development of a hyper-acute response, but also allowed the primary graft of H-2a T cells to survive in vivo for extended periods of time. Thus, we have developed a new mouse model that allows us to not only trace the development of B cell responses leading to a hyper-acute rejection, but also to validate potential therapeutic strategies aimed at tolerizing such a response.

Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Zip Code
Chappert, Pascal; Bouladoux, Nicolas; Naik, Shruti et al. (2013) Specific gut commensal flora locally alters T cell tuning to endogenous ligands. Immunity 38:1198-210
Schwartz, Ronald H (2012) Historical overview of immunological tolerance. Cold Spring Harb Perspect Biol 4:a006908
Singh, Nevil J; Bando, Jennifer K; Schwartz, Ronald H (2012) Subsets of nonclonal neighboring CD4+ T cells specifically regulate the frequency of individual antigen-reactive T cells. Immunity 37:735-46
Oh, Soyoung; Schwartz, Ronald H; Singh, Nevil J (2012) Development and tolerization of hyperacute rejection in a transgenic mouse graft versus host model. Transplantation 94:234-40
Steinert, Elizabeth M; Schwartz, Ronald H; Singh, Nevil J (2012) At low precursor frequencies, the T-cell response to chronic self-antigen results in anergy without deletion. Eur J Immunol 42:2875-80
Choi, Seeyoung; Schwartz, Ronald H (2011) Impairment of immunological synapse formation in adaptively tolerant T cells. J Immunol 187:805-16
Flomerfelt, Francis A; El Kassar, Nahed; Gurunathan, Chandra et al. (2010) Tbata modulates thymic stromal cell proliferation and thymus function. J Exp Med 207:2521-32
Chappert, Pascal; Schwartz, Ronald H (2010) Induction of T cell anergy: integration of environmental cues and infectious tolerance. Curr Opin Immunol 22:552-9
Johnson, Andy L; Aravind, L; Shulzhenko, Natalia et al. (2009) Themis is a member of a new metazoan gene family and is required for the completion of thymocyte positive selection. Nat Immunol 10:831-9