Retroviruses as tools to study the immune system.

Retrovirus-based vectors provide an efficient means to introduce and express genes in cells of the immune system and have become a popular tool to study immune function. They are easy to manipulate and provide stable, long-term gene expression because they integrate into the genome. Current retroviral vectors do have limitations that affect their usefulness in certain applications. However, recent advances suggest a number of ways in which these vectors might be improved to extend their utility in immunological research.

Retroviral expression in embryonic stem cells and hematopoietic stem cells.

Achieving long-term retroviral expression in primary cells has been problematic. De novo DNA methylation of infecting proviruses has been proposed as a major cause of this transcriptional repression. Here we report the development of a mouse stem cell virus (MSCV) long terminal repeat-based retroviral vector that is expressed in both embryonic stem (ES) cells and hematopoietic stem (HS) cells. Infected HS cells and their differentiated descendants maintained long-term and stable retroviral expression after serial adoptive transfers. In addition, retrovirally infected ES cells showed detectable expression level of the green fluorescent protein (GFP). Moreover, GFP expression of integrated proviruses was maintained after in vitro differentiation of infected ES cells. Long-term passage of infected ES cells resulted in methylation-mediated silencing, while short-term expression was methylation independent. Tissues of transgenic animals, which we derived from ES cells carrying the MSCV-based provirus, did not express GFP. However, treatment with the demethylating agent 5-azadeoxycytidine reactivated the silent provirus, demonstrating that DNA methylation is involved in the maintenance of retroviral repression. Our results indicate that retroviral expression in ES cells is repressed by methylation-dependent as well as methylation-independent mechanisms.

Uncoupling IL-2 signals that regulate T cell proliferation, survival, and Fas-mediated activation-induced cell death.

IL-2 is an important growth and survival factor for T lymphocytes but also sensitizes these cells to Fas-mediated activation-induced cell death (AICD). The molecular basis of these different effects of IL-2 was studied by introducing wild-type and mutant forms of the IL-2 receptor beta (IL-2Rbeta) chain that lacked specific signaling capacities into receptor-deficient T cells by retroviral gene transfer. Activation of Stat5 by IL-2 was found to be involved in T cell proliferation and promoted Fas ligand (FasL) expression and AICD. T cell survival was dependent on a receptor region that activated Akt and the expression of Bcl-2. Thus, distinct IL-2Rbeta chain signaling modules regulate T cell fate by stimulating growth and survival or by promoting apoptosis.

Genetic models of abnormal apoptosis in lymphocytes.

Apoptosis is a critical mechanism for regulating cell numbers during development, normal responses to hormones and other stimuli, and immune and inflammatory reactions. Recent advances in defining the biochemical mechanisms of cell death, and the development of animal models with isolated defects in cell death pathways, have led to an increasing appreciation of the pathophysiologic importance of lymphocyte apoptosis. In this article, we review our current understanding of the pathways and roles of apoptosis in lymphocytes, with an emphasis on transgenic and knockout models. We also summarize the relevance of these animal models to human diseases.

Autoimmunity as a consequence of retrovirus-mediated expression of C-FLIP in lymphocytes.

The induction of apoptosis by death receptors serves to regulate immune responses by eliminating unwanted and harmful cells. Mature lymphocytes express FLICE inhibitory proteins (FLIPs) that block death receptor-induced cell death. Here, we show that both B and T cells downregulate c-FLIP upon activation in vitro. Retrovirus-mediated expression of c-FLIP blocks Fas-induced apoptosis of activated lymphocytes but does not affect cell death resulting from cytokine withdrawal. In vivo, c-FLIP expression results in defective superantigen-mediated elimination of T cells, the accumulation of activated B cells, the production of autoantibodies, and the development of autoimmune disease. No effect was seen on negative selection of thyomocytes. These results suggest that activation-dependent downregulation of c-FLIP renders mature lymphocytes sensitive to death receptor-mediated apoptosis and is required to maintain self-tolerance.

Mechanisms of peripheral T cell tolerance.

Peripheral tolerance to self proteins is induced because these antigens are presented to T lymphocytes under conditions that do not allow effective immune responses to develop, or because the responses of the specific T cells are tightly regulated. The two principal mechanisms of peripheral tolerance are activation-induced cell death (AICD) and anergy. In CD4+ T lymphocytes, AICD is induced by repeated stimulation, with high levels of interleukin (IL)-2 production. Under these conditions, the T cells co-express Fas (CD95) and Fas ligand (FasL), and engagement of Fas triggers apoptotic death of the T cells. Mice with defects in Fas, FasL, IL-2R alpha or beta chain exhibit defects in AICD and develop autoimmune disease. The induction of T cell anergy is dependent on the recognition of B7 co-stimulators by the inhibitory T cell counter-receptor, CTLA-4. Failure of anergy is the likely basis for the fatal autoimmune disease of CTLA-4 knockout mice. The single-gene defects that result in autoimmunity are all defects in lymphocyte regulation, indicating that tolerance is often maintained by the control of lymphocyte responses to self antigen. The existence of distinct pathways of T cell tolerance suggest that different types of antigens induce tolerance by distinct mechanisms.

Biochemical mechanisms of IL-2-regulated Fas-mediated T cell apoptosis.

Activation-induced cell death (AICD) of lymphocytes is an important mechanism of self-tolerance. In CD4+ T cells, AICD is mediated by the Fas pathway and is enhanced by IL-2. To define the mechanisms of this pro-apoptotic action of IL-2, we analyzed CD4+ T cells from wild-type and IL-2-/- mice expressing a transgenic T cell receptor. T cells become sensitive to AICD after activation by antigen and IL-2. IL-2 increases transcription and surface expression of Fas ligand (FasL) and suppresses transcription and expression of FLIP, the inhibitor of apoptosis. The ability of IL-2 to enhance expression of a pro-apoptotic molecule, FasL, and to suppress an inhibitor of Fas signaling, FLIP, likely accounts for the role of this cytokine in potentiating T cell apoptosis.

Homeostasis and self-tolerance in the immune system: turning lymphocytes off.

The immune system responds in a regulated fashion to microbes and eliminates them, but it does not respond to self-antigens. Several regulatory mechanisms function to terminate responses to foreign antigens, returning the immune system to a basal state after the antigen has been cleared, and to maintain unresponsiveness, or tolerance, to self-antigens. Here, recent advances in understanding of the molecular bases and physiologic roles of the mechanisms of immune homeostasis are examined.

The Fas/Fas ligand pathway and Bcl-2 regulate T cell responses to model self and foreign antigens.

We have examined the role of Fas and Bcl-2 in T cell survival and responses to antigen in vivo using T cells that express a transgenic antigen receptor specific for hen egg lysozyme (HEL) and that either lack functional Fas or Fas ligand (FasL) or overexpress Bcl-2 as a transgene. HEL-specific, Bcl-2-transgenic T cells showed prolonged responses to immunization with cognate peptide but were eliminated rapidly when exposed to HEL expressed systemically as a self antigen. In contrast, Fas- and FasL-defective T cells did not display exaggerated responses to immunization with HEL peptide, but did show increased expansion and survival in response to systemic self antigen and were able to activate anti-HEL (self) antibody-forming cells. Thus, Bcl-2 and Fas play different roles in the regulation of T cell responses to antigen in vivo and in self tolerance.

Functional roles of Fas and Bcl-2-regulated apoptosis of T lymphocytes.

Apoptotic cell death is an important mechanism for maintaining homeostasis in the immune system and for regulating the fates of lymphocytes following encounters with self and foreign Ags. To study the physiologic roles of the proapoptotic Fas pathway and the antiapoptotic protein, Bcl-2, in T cell maturation and homeostasis, a TCR transgene has been bred into mice lacking functional Fas and mice that express Bcl-2 constitutively. In vitro, Fas-deficient T cells are resistant to activation-induced cell death, whereas Bcl-2-overexpressing T cells are resistant to death induced by withdrawal of growth factors. In vivo, Bcl-2-overexpressing mice accumulate T cells in the thymus and peripheral lymphoid tissues in the absence of Ag, but these cells are deleted normally after Ag administration. In contrast, Fas-deficient mature T cells are present in normal numbers in the absence of Ag, but are resistant to Ag-induced deletion. Both Fas-deficient and Bcl-2 overexpressing thymocytes are deleted when exposed to transgene-encoded circulating self Ag, indicating that the pathways of apoptosis controlled by these proteins are not critical for negative selection of developing thymocytes. Moreover, deficiency of Fas, but not Bcl-2 overexpression, results in the accumulation of autoreactive T cells in peripheral lymphoid tissues. These results demonstrate that Fas and Bcl-2 regulate different pathways of apoptosis that may serve distinct functions in lymphocyte homeostasis and in the maintenance of T cell tolerance.

Role of interleukin 12 and costimulators in T cell anergy in vivo.

The induction of T cell anergy in vivo is thought to result from antigen recognition in the absence of co-stimulation and inflammation, and is associated with a block in T cell proliferation and Th1 differentiation. Here we have examined the role of interleukin (IL)-12, a potent inducer of Th1 responses, in regulating this process. T cell tolerance was induced by the administration of protein antigen without adjuvant in normal mice, and in recipients of adoptively transferred T cells from T cell receptor transgenic mice. The administration of IL-12 at the time of tolerance induction stimulates Th1 differentiation, but does not promote antigen-specific T cell proliferation. Conversely, inhibiting CTLA-4 engagement during anergy induction reverses the block in T cell proliferation, but does not promote full Th1 differentiation. T cells exposed to tolerogenic antigen in the presence of both IL-12 and anti-CTLA-4 antibody are not anergized, and behave identically to T cells which have encountered immunogenic antigen. These results suggest that two processes contribute to the induction of anergy in vivo; CTLA-4 engagement, which leads to a block in the ability of T cells to proliferate to antigen, and the absence of a prototypic inflammatory cytokine, IL-12, which prevents the differentiation of T cells into Th1 effector cells. The combination of IL-12 and anti-CTLA-4 antibody is sufficient to convert a normally tolerogenic stimulus to an immunogenic one.

Functional responses and apoptosis of CD25 (IL-2R alpha)-deficient T cells expressing a transgenic antigen receptor.

IL-2 was initially defined as a T lymphocyte growth factor, but recent studies have provided evidence that it may also play a role in regulating T cell differentiation, apoptosis, and tolerance. To examine the contribution of IL-2 to these processes, we have bred a class II-restricted TCR transgene into mice deficient in the alpha-chain of the IL-2R, CD25. We show that in response to Ag, T cells from these mice are unable to use IL-2 and, as a result, are less efficient at traversing the cell cycle, and proliferate less than wild-type cells. Furthermore, CD25 -/- T cells exhibit reduced survival in vitro, even in the presence of costimulatory signals. IL-4 and IL-15, a cytokine related to IL-2, enhance the survival and Ag-induced proliferation of CD25 -/- T cells. Activated CD25 -/- T cells are resistant to Fas-mediated activation-induced cell death (AICD), and this defect cannot be corrected by other cytokines. Therefore, IL-2 plays a unique role in regulating AICD, but has redundant roles in T cell survival and proliferation in vitro. The failure of AICD observed with CD25 -/- T cells may explain the unexpected observation that deficiency of IL-2 or of the alpha- or beta-chain of the IL-2R results not in immunodeficiency, but in autoimmune disease.

Induction of peripheral T cell tolerance in vivo requires CTLA-4 engagement.

Studies of T cell anergy in vitro have led to the widely accepted view that anergy is induced by T cell antigen recognition without costimulation. We show that the induction of T cell anergy in vivo is due to an abortive T cell response that requires recognition of B7 molecules, since blocking B7 maintains T cells in an unactivated but functionally competent state. Furthermore, the induction of anergy is prevented by blocking CTLA-4, the inhibitory T cell receptor for B7 molecules. Thus, in vivo T cell anergy may be induced not because of a lack of costimulation, but as a result of specific recognition of B7 molecules by CTLA-4. In contrast, blocking CD28 on T cells prevents priming but not the induction of tolerance. Therefore, the outcome of antigen recognition by T cells is determined by the interaction of CD28 or CTLA-4 on the T cells with B7 molecules.

Functional consequences of dysregulated B7-1 (CD80) and B7-2 (CD86) expression in B or T lymphocytes of transgenic mice.

T cell activation and tolerance are regulated by interactions between CD28 or CTLA-4 on T cells and B7 costimulatory molecules on APCs. We have generated transgenic mouse strains that constitutively express B7-1 (CD80) at high levels on B cells or T cells or express B7-2 (CD86) on T lymphocytes to examine the consequences of dysregulated B7 expression on T cell responses. The transgene-derived B7 molecules are functional, because B7-1 transgenic B cells are more efficient APCs than are wild-type B cells, and the activation of B7 transgenic T cells is less dependent on exogenous costimulation than that of wild-type T cells. In vivo, constitutive expression of B7 molecules leads to the elimination of immature B cells. The expression of B7 molecules on thymocytes results in the down-regulation of CD28 expression. However, B7 transgenic mice have normal numbers of mature lymphocytes and mount normal T cell responses following immunization with protein Ag. Neither anergy induction nor superantigen-mediated deletion of T cells is altered by the dysregulated expression of B7-1 or B7-2 on B or T lymphocytes in these transgenic strains. Therefore, functionally significant levels of B7 expressed constitutively on mature lymphocytes are not, by themselves, sufficient to abrogate T cell tolerance or induce autoimmune disease.

Role of Fas-mediated cell death in the regulation of immune responses.

Interaction of the cell-surface molecule Fas (CD95 APO-1) with its specific ligand triggers apoptotic death of T and B lymphocytes. This pathway is important for eliminating self-reactive lymphocytes and thus preventing autoimmunity. Fas is also involved in controlling injurious lymphocyte reactions in immunologically 'privileged' tissues, and may provide a strategy for reducing graft rejection.

The roles of costimulation and Fas in T cell apoptosis and peripheral tolerance.

Using cells from TCR transgenic mice that do or do not express Fas, we show that there are two mechanistically distinct forms of apoptosis in CD4+ T cells. Naive T cells undergo apoptosis if cultured in the absence of antigen or costimulation. This form of programmed cell death (PCD) is not dependent on Fas, and is prevented by CD28-mediated signals, which lead to the secretion of growth factors and the expression of survival genes, such as bcl-xL. Recently activated T cells undergo apoptotic death upon repeated stimulation. This activation-induced cell death (AICD) is mediated by Fas, but is independent of costimulation and is not prevented by IL-2 or bcl-xL. Finally, we show that peripheral tolerance may be induced in vivo independent of Fas-mediated cell death.

Differentiation and tolerance of CD4+ T lymphocytes.

The development of effector and memory populations of T lymphocytes is determined by antigen-induced growth and differentiation of naive T cells, and it is regulated by antigen-induced functional tolerance and cell death. CD4+ helper T lymphocytes that vary in their profiles of cytokine production and in effector functions also show distinct responses to antigens and co-stimulatory signals, and they differ in their sensitivity to tolerance induction. Thus, stimuli that trigger T cell growth and differentiation, as well as mechanisms that inhibit T cell expansion, determine both the magnitude and the nature of T cell-dependent immune responses to protein antigens.

A negative regulatory function of B7 revealed in B7-1 transgenic mice.

To analyze the functions of T cell costimulators in vivo, we have constructed a transgenic mouse strain that constitutively expresses murine B7-1 on mature B cells. Antibody responses to T-dependent hapten-protein conjugates and serum immunoglobulin levels are markedly depressed in B7-1 transgenic mice. This immune deficiency is not due to an intrinsic B cell defect, as antibody responses to T-independent hapten conjugates are normal. Furthermore, treatment with anti-B7-1 restores the capacity of transgenic mice to respond to hapten-protein conjugates, demonstrating that the deficient antibody responses are directly attributable to the expression of B7-1. These results suggest that the temporally regulated expression of costimulators such as B7-1 may contribute to either initiation or down-regulation (feedback inhibition) of T-dependent immune responses in vivo, and that the inhibitory function is dominant in transgenic mice that constitutively express high levels of this costimulator.

Serum lipids and apoproteins in students whose parents suffered prematurely from a myocardial infarction.

Lipids and apoproteins as well as other coronary risk factors were measured in offspring of patients who suffered from a myocardial infarction before the age of 50 years; the results are compared with the results of a control group matched for age and sex. Significant differences were observed in the apoprotein A1 level, in the protein/fat ratios of high- and low-density lipoproteins, and in smoking habits. In a multivariate analysis, the offspring group was found to be different from the control group in nonhigh-density lipoprotein cholesterol/apoprotein B ratio, high-density lipoprotein cholesterol/apoprotein A1 ratio, smoking habits, apoprotein A1, and apoprotein A2. By means of these variables a total of 85% of all subjects could be correctly classified. We conclude that as early as age 21 years the offspring of patients with premature coronary heart disease differ from matched control subjects in lipoprotein measurements and in smoking habits.