Resistance to Apoptosis and Elevated Expression of Bcl-2 in Clonally Expanded CD4  CD28  T Cells from Rheumatoid Arthritis Patients 1 Michael Schirmer, Abbe N. Vallejo, Cornelia M. Weyand, and Jo ¨rg J. Goronzy 2 Patients with rheumatoid arthritis have a subset of CD4  T lymphocytes that are characterized by a defect in CD28 expression. CD4  CD28  T cells frequently undergo clonal expansion in vivo. These clonotypes include autoreactive cells and persist over many years. The clonogenic potential and longevity of these T cells could be related to an altered response to apoptosis-inducing signals. To explore this possibility, CD4  CD28  T cell lines and clones were examined for their response pattern to stimuli inducing physiologic cell death. CD4  CD28  T cells were found to be resistant to apoptosis upon withdrawal of the growth factor, IL-2. To examine whether the altered sensitivity to this apoptotic signal was correlated with the expression of proteins of the bcl-2 family, the expression of bcl-2, bcl-x, and bax proteins was determined. CD28  and CD28  CD4  T cells could not be distinguished by the levels of bax or bcl-x L protein; however, CD4  CD28  T cells expressed higher amounts of bcl-2 protein than did CD4  CD28  T cells. The increased bcl-2 expression in CD4  CD28  T cells was relatively independent of signals provided by exogenous IL-2. In CD28-deficient CD4  T cells, bcl-2 was not significantly up-regulated by the addition of exogenous IL-2 and was maintained despite IL-2 withdrawal, as opposed to CD28-expressing CD4  T cells. We propose that CD4  CD28  T cells are characterized by a dysregulation of the survival protein, bcl-2, which may favor the clonal outgrowth of autoreactive T cells and thus contribute to the pathogenesis of rheumatoid arthritis. The Journal of Immunology, 1998, 161: 1018 –1025. M echanisms maintaining immune homeostasis are es- sential for the normal functioning of the immune sys- tem. Ag-reactive responses are generally associated with the proliferation of Ag-specific T and B cells, which then differentiate into effector and memory cells (1). This oligoclonal expansion is counterbalanced by apoptotic cell death (2, 3). The survival of effector lymphocytes is tightly regulated, as demon- strated by the stringent control of the global size of the lymphocyte compartment. While apparently effective control mechanisms exist to prevent the clonal outgrowth of single T and B cell specificities, the system allows for the survival of certain effector cells to es- tablish T cell memory. Abnormal survival of lymphocytes can con- tribute to the development of lymphoid malignancies and may be related to altered immune responses in autoimmune diseases, but the mechanisms that allow for T cell survival vs cell death are only partially understood. Two fundamentally different mechanisms have been implicated in controlling cell survival in the immune system (4 –7). Like many other cell types, lymphocytes can un- dergo programmed cell death if growth factors are withdrawn dur- ing proliferation. In addition, T cell activation can directly induce apoptosis. Activation-induced cell death (AICD) 3 appears to be particularly important in controlling clonal size after repeated stimulation (3). Despite extensive apoptotic death of lymphocytes after a pri- mary response, some cells survive and differentiate into memory cells. Mechanisms underlying the survival of memory cells have been difficult to address because no convincing phenotypic differ- ences have been described that distinguish effector and memory T cells. In addition, memory T cells appear to be able to revert to a naive phenotype (8). Members of the bcl-2 family have been im- plicated in the survival of activated T cells following an immune response. However, the role of these survival genes in T cell lon- gevity is undetermined (9 –11). We have recently made the observation that most patients with rheumatoid arthritis (RA) carry, in the peripheral blood, clonally expanded CD4 + T cells that have proliferated to a large clonal size (12). In some patients, single clonotypes can represent as much as 1% of all CD4 + T lymphocytes. The expansion of such clonotypes is not a transient phenomenon. Expanded clonotypes persist over years and can be found in similar frequencies in blood samples taken as many as 5 years apart (13). The longevity of these T cell clones, as well as their clonal size, suggests that these T cells, in contrast to normal recently activated effector cells, must have de- veloped mechanisms to prolong survival. One of these mecha- nisms could be chronic antigenic stimulation. Indeed, we have shown that in vivo expanded T cell clones isolated from RA pa- tients recognize self Ags expressed on blood-derived adherent cells (14). The majority of the expanded clonotypes in the periph- eral blood do not express activation markers, suggesting that con- tinuous activation alone is not sufficient to explain the extent of the Department of Medicine, Division of Rheumatology, Mayo Clinic and Foundation, Rochester, MN 55905 Received for publication September 15, 1997. Accepted for publication March 9, 1998. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This study was supported by grants from the National Institutes of Health (RO1 AR41974 and RO1 AR42527) and the National Arthritis Foundation (AF#16). Mi- chael Schirmer was supported by Schroedinger Grant J01194 of the Austrian Re- search Fund. 2 Address correspondence and reprint requests to Dr. J.J. Goronzy, 401 Guggenheim Building, Mayo Clinic, Rochester, MN 55905. E-mail address: goronzy.jorg@mayo.edu 3 Abbreviations used in this paper: AICD, activation-induced cell death; rhIL-2, re- combinant human IL-2; 7-AAD, 7-amino actinomycin D; ADU, arbitrary density units; RA, rheumatoid arthritis; PE, phycoerythrin; CD40L, CD40 ligand. Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00