Scientific Report 2005

Immunology

Genes and Genetics of Systemic Autoimmunity and T-Cell Homeostasis in Autoimmunity and Cancer

A.N. Theofilopoulos, D.H. Kono, R. Baccala, R. Chintalapati, R. Gonzalez-Quintial, M.K. Haraldsson, C.A. Louis-Dit-Sully, K.M. Pollard,* J. Schettini

* Department of Molecular and Experimental Medicine, Scripps Research

Our main interests are identifying predisposing loci and genes in murine models of systemic autoimmunity, clarifying the role of type I interferons in systemic lupus erythematosus (SLE), determining why activated/memory phenotype T cells accumulate in SLE and why cyclin-dependent kinase inhibitors are increased in these cells, and characterizing factors that influence acute homeostatic proliferation of T-cell subsets and the relevance of this process in autoimmunity and cancer.

Genetic Basis of Systemic Autoimmunity

Susceptibility to SLE is in large part determined by genetic predisposition. Thus, defining the specific genes and how certain alterations lead to autoimmunity should yield new insights into the pathogenesis of SLE and facilitate the development of innovative approaches to disease management. Because of the complexity of defining susceptibility genes in humans, we use both spontaneous and induced models of SLE in well-characterized inbred mouse strains. Previously, we identified loci that predispose mice to spontaneous manifestations of SLE in NZB, NZW, BXSB, MRL-Fas^lpr, and C57BL/6-Fas^lpr strains and a DBA/2 locus associated with resistance to mercury-induced autoimmunity.

Currently, we are identifying the underlying genes and their specific roles in autoimmunity for 4 loci: Lbw2, Lbw5, Lmb3, and Hmr1. Lbw2 is a locus on chromosome 4 in NZB mice that promotes spontaneous activation of B cells, production of autoantibodies, glomerulonephritis, and autoimmune hemolytic anemia. The Lbw2 locus appears to contain at least 3 subloci that affect different component phenotypes mapped to this interval. Lbw5 is a recessive locus on chromosome 7 in NZW mice that enhances production of IgG autoantibodies, glomerulonephritis, and autoimmune hemolytic anemia. Further mapping of Lbw5 suggests that this interval contains as least 2 subloci.

The dominant MRL Lmb3 is also a locus on chromosome 7, but it occurs at a different, more distal location than does Lbw5. Lmb3 congenic MRL-Fas^lpr mice containing an introgressed chromosome 7 fragment of C57BL/6 have marked reductions in lymphoproliferation, production of autoantibodies, glomerulonephritis, and early mortality. This locus was recently mapped to a 0.8 Mb-sized interval, and a likely candidate gene with a functional mutation has been identified. The Hmr1 locus on chromosome 1 does not confer resistance to deposition of glomerular immune complexes in congenic NZB and SJL mice that have the DBA/2 Hmr1 interval, suggesting that epistatic interactions with other DBA/2 resistance genes are required. In support of this notion, the reciprocal congenic DBA/2 mice with the NZB Hmr1 region are susceptible to mercury-induced autoimmunity. We are mapping the number and location of the various subloci and are identifying and characterizing possible genes in the reduced intervals.

Type I Interferons in SLE

Type I interferons (IFN-αβ) are highly pleiotropic cytokines that affect both innate and adaptive immune responses. Long-standing observations indicate the central role of these cytokines in the pathogenesis of SLE in humans and in animal models. In our studies of SLE-prone NZB mice that lack the common receptor for IFN-αβ, we clearly delineated the pathogenic role of these effector molecules. Compared with mice that had the receptor, mice that lacked the receptor had significant decreases in humoral, cellular, and histologic characteristics of SLE and increases in survival.

Several questions remain unanswered, however, particularly about the mechanisms associated with endogenous stimuli for production of IFN-αβ. We are determining the efficacy of a nonviral vector that encodes the IFN-αβ–binding IFNAR2 chain fused to the Fc fragment of IgG1 in inhibiting disease when applied either at the early (prophylactic) stage or the late (therapeutic) stage of the disease in various models of spontaneous SLE. The efficacy of this approach will provide the basis for translating these results to similar contemplated efforts for treatment of SLE in humans.

We are also interested in differentiating the effects of IFN-α from those of IFN-β and in defining the postulated central role of plasmacytoid dendritic cells as the major producers of IFN-αβ in this disease. Most importantly, in collaboration with B. Beutler and K. Hoebe, Department of Immunology, we are creating congenic SLE mice that lack adaptor molecules involved in the production of IFN-αβ mediated by the Toll-like receptors. We are also concentrating on identifying the exact nature of the endogenous (self) factors that stimulate production of IFN-αβ, particularly the roles of apoptotic materials and immune complexes composed of autoantibodies and particles containing DNA and/or RNA.

Cyclin-Dependent Kinase Inhibitors in Systemic Autoimmunity

In recent studies, we focused on the role of the cell-cycle inhibitor p21 in normal immune responses and autoimmunity. The cell cycle, which plays a critical role in determining both the fate and the differentiation of cells, is highly regulated by complexes of proteins, including cyclin, cyclin-dependent kinases, and cyclin-dependent kinase inhibitors, that are themselves controlled by external stimuli via a number of signaling pathways. Previously, in SLE-prone BXSB mice, we found that high levels of certain cyclin-dependent kinase inhibitors, such as p21, p18, and p27, are present in activated/memory (CD44^hi) phenotype CD4⁺ T cells, a population commonly increased in SLE. Therefore, we hypothesized that repeated stimulation of T cells reactive to self-antigens might lead to a state similar to “replicative senescence,” in which T cells are no longer cycling but are resistant to apoptosis, accumulate, and transcribe autoimmune-promoting proinflammatory cytokines. In support of this notion, we found that male BXSB mice lacking p21 had a marked reduction in SLE-like disease associated with both enhanced apoptosis of T and B lymphocytes and significant decreases in the number of activated/memory CD4⁺ T cells. Recently, we created diabetes-susceptible nonobese diabetic mice that lacked the gene for p21. In sharp contrast to the situation in SLE-prone mice, p21 deficiency had no effect on the development and severity of diabetes, indicating that p21 plays different roles in systemic and organ-specific diseases. Currently, we are addressing the role of p21 in other SLE-prone strains, immune responses to foreign antigens and viral infection, and the observed reduction in Fas-mediated apoptosis.

Homeostatic T-Cell Proliferation in Autoimmunity and Cancer

Homeostasis is defined as the ability of a biological system to maintain its internal equilibrium by adjusting critical physiologic properties. Recent studies have largely defined the factors that control homeostasis of naive and memory T cells under states in which the number of lymphocytes is sufficient or is markedly reduced (lymphopenia). Of particular relevance to autoimmunity is the phenomenon termed “acute homeostatic T-cell proliferation,” which signifies proliferation of the remaining T cells after a lymphopenia-inducing event (e.g., treatment with cytotoxic drugs, viral infection) to reestablish a pool with normal numbers of lymphocytes. Efficient acute homeostatic proliferation appears to be based on recognition of self-peptide–MHC complexes and signaling by trophic cytokines, such as IL-7 and IL-15.We recently hypothesized that such lymphopenia-mediated T-cell proliferation may be a contributing factor to autoimmunity, and we have discussed several examples in the literature in which lymphopenia was paradoxically associated with autoimmune phenomena. Our recent experiments in SLE-prone mice that lack the gene for the α-chain of the T-cell receptor and thus lack
T cells, provided evidence that homeostatic proliferation of syngeneic cells in this empty environment can recapitulate an SLE-like disease. Others have shown that increased proliferation but inefficient survival of T cells can lead to lymphopenia and can be a contributing factor in the organ-specific autoimmune disease of nonobese diabetic mice.

Thus, the perplexing association of lymphopenia with autoimmunity might be explained on the basis of compensatory self-mediated homeostatic proliferation of T cells. Overall, we postulate that in normal mice, the rare occurrence of lymphopenia and physiologic proliferation of a polyclonal T-cell population containing few (if any) autoreactive cells will be a physiologic process without pathologic consequences. Similarly, in animals that have more autoreactive T cells, a rare occurrence of homeostatic proliferation of T cells most likely will be innocuous. In contrast, in animals predisposed to autoimmunity, lymphopenia might contribute to the initiation and/or progression of recurrent or chronic disease (Fig. 1).

Fig. 1. Postulated mechanisms for recurrent lymphopenia-induced expansion of autoreactive T cells and autoimmunity. In animals with a normal genetic background, the rare occurrence of lymphopenia leads to homeostatic proliferation (HP) and survival of diverse T cells (white areas, nonautoreactive; black areas, autoreactive) without predominance of the few potentially autoreactive clones in the periphery. Similarly, in animals with a genetic predisposition to autoimmunity (autoimmune background), which have a higher frequency of potentially autoreactive T cells, the rare occurrence of homeostatic proliferation might not lead to autoimmunity because the frequency of the autoreactive cells remains low and other requirements are absent. By contrast, in animals with a genetic predisposition to autoimmunity in which lymphopenia occurs recurrently or chronically, proliferation and selection of autoreactive T cells together with other factors such as adequate antigen presentation and costimulation might lead to autoimmunity.

T cells that express γδ T-cell receptors constitute a considerable fraction of lymphocytes in secondary lymphoid organs and blood and predominate in the mucosa and epithelia of various tissues. Considerable evidence indicates that γδ T cells have important immunologic functions, including antitumor activities, and may contribute to the pathogenesis of autoimmune diseases. Among subsets of T cells, γδ T cells uniquely have a tissue distribution based on their antigen receptors, but what defines the preferential homing and homeostasis of these cells is unknown. To address this question, we studied the resources that control the homeostasis of γδ T cells in secondary lymphoid organs.

We found that γδ and αβ T cells are controlled by partially overlapping resources, because lymphopenia-induced acute homeostatic proliferation of γδ T cells was inhibited by an intact αβ T-cell compartment and both γδ and αβ T cells were dependent on IL-7 and IL-15. Significantly, acute homeostatic proliferation of γδ T cells also required depletion of γδ cells. Thus, homeostasis of γδ T cells is maintained by trophic cytokines commonly used by other types of lymphoid cells and by additional, as yet unidentified, γδ-specific ligands.

Efforts to develop effective antitumor immunotherapies are hampered by the difficulty of overcoming tolerance against tumor antigens, which in most instances are normal gene products that are overexpressed, preferentially expressed, or reexpressed in cancer cells. Because lymphopenia-induced homeostatic proliferation of T cells is mediated by recognition of self-peptide–MHC complexes and because the expanded cells acquire some effector functions, we hypothesized that lymphopenia-induced homeostatic proliferation could

be used to break tolerance against tumor antigens. Our earlier studies with W. Dummer, Genentech, Inc., South San Francisco, California, A.G. Niethammer and R. Reisfeld, Department of Immunology, in mouse models of melanoma and colon carcinoma indicated that availability of tumor antigens during homeostatic proliferation of T cells indeed leads to effective antitumor autoimmunity with specificity and memory. We hypothesize that this effect is mediated by a reduction in the activation threshold of low-affinity tumor-specific T cells, leading to preferential engagement and proliferation of the cells in the presence of a high concentration of tumor antigens.We are further defining the parameters of this approach, particularly its efficacy in the treatment of primary and metastatic tumors with different immunologic and histologic characteristics. Our emphasis is on mechanistic issues, such as the exact process by which tolerance is broken, modes of antigen presentation (direct vs indirect), the efficacy of refined T-cell subsets, and the potentiating effects of appropriate vaccines and cytokines. Overall, we think that because of its simplicity, this approach will have considerable application in the treatment of malignant neoplasms in humans because it relies on conventional lymphopenia-inducing cancer therapies, tumor-specific vaccination at the early phases of lymphopenia, and, optimally, infusion of autologous lymphocytes.

Publications

Baccala, R., Gonzalez-Quintial, R., Dummer, W., Theofilopoulos, A.N. Tumor immunity via homeostatic T cell proliferation: mechanistic aspects and clinical perspectives. Springer Semin. Immunopathol. 27:75, 2005

Baccala, R., Kono, D.H., Theofilopoulos, A.N. Interferons as pathogenic effectors in autoimmunity. Immunol. Rev. 204:9, 2005.

Baccala, R., Theofilopoulos, A.N. The new paradigm of T-cell homeostatic proliferation-induced autoimmunity. Trends Immunol. 26:5, 2005.

Baccala, R., Witherden, D., Gonzalez-Quintial, R., Dummer, W., Surh, C.D., Havran, W.L., Theofilopoulos, A.N. γδ T cell homeostasis is controlled by IL-7 and IL-15 together with subset-specific factors. J. Immunol. 174:4606, 2005.

Haraldsson, M.K., dela Paz, N.G., Kuan, J.G., Gilkeson, G.S., Theofilopoulos, A.N., Kono, D.H. Autoimmune alterations induced by the New Zealand Black Lbw2 locus in BWF1 mice. J Immunol. 174:5065, 2005.

Kono, D.H., Theofilopoulos, A.N. Genetics of autoantibody production in mouse models of lupus. In: Autoantibodies and Autoimmunity. Pollard, K.M. (Ed.). Wiley-VCH, New York, in press.

Pollard, K.M., Arnush, M., Hultman, P., Kono, D.H. Costimulation requirements of induced murine systemic autoimmune disease. J. Immunol. 173:5880, 2004.

Pollard, K.M., Hultman, P., Arnush, M., Hildebrand, J.A., Kono, D.H. Immunology and genetics of xenobiotic-induced autoimmunity. In: From Animal Models to Human Genetics: Research on the Induction and Pathogenicity of Autoantibodies. Conrad, K., et al. (Eds.). Pabst Science Publishers, Lengerich, Germany, 2004, p. 130.

Theofilopoulos, A.N., Baccala, R., Beutler, B., Kono, D.H. Type I interferons (αβ) in immunity and autoimmunity. Annu. Rev. Immunol. 23:307, 2005.