Heterogeneity of helper/inducer T lymphocytes. II. Effects of interleukin 4- and interleukin 2-producing T cell clones on resting B lymphocytes

To compare the helper function of murine T cell clones that secrete IL-2 and IFN-gamma (Th1 cells) or IL-4 and IL-5 (Th2), purified resting B cells were stimulated with F(ab')2 rabbit anti-mouse Ig (RAMG) and rabbit Ig-specific, class II MHC-restricted cloned T cells belonging to the two subsets. Both Th2 clones examined induced strong proliferative responses of B cells in the presence of RAMG, as well as the secretion of IgM and IgG1 antibodies. In contrast, the Th1 clones tested failed to stimulate B cell growth or antibody secretion. Th2-mediated B cell activation was dependent on IL-4 and IL-5, and was also inhibited by IFN-gamma or IFN-gamma produced by Th1 cells present in the same cultures. However, the failure of Th1 cells to help resting B cells could not be reversed with neutralizing anti-IFN-gamma antibody. In addition to this inhibitory effect, IFN-gamma was required for the secretion of IgG2a antibody, particularly when B cells were stimulated with polyclonal activators such as LPS. Finally, both sets of T cell clones secreted lymphokines when stimulated with purified B cells and RAMG. These experiments demonstrate that T cells that differ in lymphokine production also differ in their ability to help B cells as a result of cognate interactions at low concentrations of antigens. Moreover, IL-4, IL-5, and IFN-gamma serve different roles in the T cell-dependent proliferative and differentiative responses of resting B lymphocytes.     

Clonal analysis of functionally distinct human CD4+ T cell subsets

A large number of CD4+ T cell clones, obtained from peripheral blood T lymphocytes by direct limiting dilution, allowed us to address the question whether functional heterogeneity exists within the human CD4+ T cell subset. Cytotoxic capacity of cloned T cells was analyzed with the use of anti-CD3 antibodies and target cells bearing FcR for murine IgG. 6 of 12 CD4+ clones obtained were able to lyse Daudi or P815 cells in the presence of anti-CD3 antibodies. The remaining six CD4+ T cell clones tested did not display anti-CD3-mediated cytotoxic activity and did not acquire this cytotoxic capacity during a culture period of 20 wk. In the absence of anti-CD3 mAb, no lytic activity against Daudi, P815, and K562 target cells was observed under normal culture conditions. Phenotypic analysis of these two distinct types of CD4+ T cells did not reveal differences with regard to reactivity with CDw29 (4B4) and CD45R (2H4) mAbs that have been described to recognize antigens associated with helper suppressor/inducer (respectively) CD4+ cells. The CD4+ clones without anti-CD3-mediated cytotoxic activities (Th2) consistently showed a high expression level of CD28 antigens, whereas the cytotoxic clones (Th1) expressed low amounts of CD28. Th1 CD4+ clones did produce IL-2, IFN-gamma, and TNF-alpha/beta, whereas the Th2 T cell clones produced minimal amounts of IL-2 and only low levels of INF-gamma and TNF-alpha/beta in response to anti-CD3 mAbs and PMA. Although not all CD4+ clones did release IL-4, there was no correlation with cytotoxic activity. Moreover, as compared with the Th1 CD4+ clones, Th2 CD4+ T cell clones proliferated moderately in response to immobilized anti-CD3 mAbs. However, proliferation reached the level of the cytotoxic clones when anti-CD28 mABs were present during culture. Both CD4+ subsets provided help for B cell differentiation upon stimulation with anti-CD3 mAbs. Our data suggest that the human CD4+ subset, in analogy to the murine system, comprises two functionally distinct T cell subpopulations, both of which are able to exert helper activity for polyclonal B cell differentiation, but which differ in cytotoxic capacity, lymphokine production, and requirements for proliferation. A function for these two types of T cells in the immune response is discussed.     

Cholera toxin discriminates between T helper 1 and 2 cells in T cell receptor-mediated activation: role of cAMP in T cell proliferation

CD4+ T helper (Th) clones can be divided into interleukin 2 (IL-2)-secreting Th1 and IL-4-secreting Th2 cells. We show in the present report that these two Th subsets have different activation requirements for lymphokine production and proliferation: namely, cholera toxin (CT) as well as forskolin inhibit T cell receptor (TCR)-mediated IL-2 production and proliferation in Th1 cells, while the same reagents fail to block IL-4 production and proliferation in Th2 cells. In addition, CT and forskolin differentially influence the proto-oncogene mRNA expression in Th1 vs. Th2 cells after stimulation with Con A. Since both reagents lead to elevated levels of intracellular cAMP, it is likely that Th1 and Th2 cells differ in their sensitivity to an increase in cAMP. Our results indicate that the two Th subsets use different transmission signal pathways upon TCR-mediated activation.     

Protease inhibitors selectively block T cell receptor-triggered programmed cell death in a murine T cell hybridoma and activated peripheral T cells

The hypothesis that cytoplasmic proteases play a functional role in programmed cell death was tested by examining the effect of protease inhibitors on the T cell receptor-mediated death of the 2B4 murine T cell hybridoma and activated T cells. The cysteine protease inhibitors trans-epoxysuccininyl-L-leucylamido-(4-guanidino) butane (E-64) and leupeptin, the calpain selective inhibitor acetyl-leucyl-leucyl- normethional, and the serine protease inhibitors diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride, all showed dose- dependent blocking of the 2B4 death response triggered by the T cell receptor complex and by anti-Thy-1. These protease inhibitors enhanced rather than inhibited IL-2 secretion triggered by T cell receptor cross- linking, showing that they did not act by preventing signal transduction. Growth inhibition induced by cross-linking the 2B4 T cell receptor, measured by inhibition of thymidine incorporation, was not generally blocked by these protease inhibitors. All five of these protease inhibitors enhanced rather than blocked 2B4 cell death triggered by dexamethasone, an agent previously shown to have a death pathway antagonistic with that of the TCR. 2B4 cytolysis by the cytotoxic agents staphylococcal alpha-toxin and dodecyl imidazole, and that caused by hypotonic conditions, was not significantly affected by the five protease inhibitors tested. The selected protease inhibitors blocked both the apoptotic nuclear morphology changes and DNA fragmentation induced by T cell receptor cross-linking, and enhanced both these properties induced by dexamethasone in 2B4 cells. The T cell receptor-induced death of activated murine lymph node T cells and human peripheral blood CD4+ T cells was blocked by both cysteine and serine protease inhibitors, showing that the protease-dependent death pathway also operates in these systems.     

Specific inhibition of lymphokine biosynthesis and autocrine growth using antisense oligonucleotides in Th1 and Th2 helper T cell clones

T helper cells have recently been divided into two subsets. The Th1 subset secretes and responds to IL-2 in an autocrine manner. The Th2 subset upon mitogen or antigen stimulation releases IL-4. Here we describe a novel technology that allowed us to confirm this distinction. We have used synthetic oligonucleotides complementary to the 5' end of mouse IL-2 and IL-4 to specifically block the biosynthesis of IL-2 or IL-4 in two murine helper T cell clones from the Th1 or Th2 subset. We show that the antisense IL-2 oligonucleotide inhibited the proliferation of the Th1 clone and had no effect on the Th2 clone. In parallel experiments, the antisense IL-4 oligonucleotide blocked the proliferation of the Th2 clone and not the proliferation of the Th1 clone. The inhibition was significantly reversed in both cases by the addition of the relevant lymphokine (IL-2 in the case of the Th1 clone, IL-4 in the case of the Th2 clone). Northern analysis, using cDNA probes specific for the two lymphokines, showed a decrease in the steady-state level of the relevant lymphokine mRNA, suggesting the specific degradation of the mRNA by an RNase H-like enzymatic activity. This strategy, which allows the specific blockade of the biosynthesis of a lymphokine, could be useful for future studies on the role of each T helper subset in physiological immune responses.     

CD28-induced costimulation of T helper type 2 cells mediated by induction of responsiveness to interleukin 4

Type 1 and type 2 cloned T helper (Th) cells are believed to require different antigen-presenting cell (APC)-derived costimuli for proliferation. In the case of Th1-cloned T cells, CD28 signaling costimulates production of autocrine interleukin 2 (IL-2). Th2 cells produce their autocrine growth factor, IL-4, without costimulation, but require APC-derived costimuli, or IL-1, to respond to IL-4. Here we demonstrate that engagement of CD28 on Th2 cells with anti-CD28 antibody or with APC-associated B7 costimulates Th2 responsiveness to IL-4 but does not affect IL-4 or IL-2 production by Th2 cells. Costimulation of Th2 cells via CD28 appears to involve the induction of IL-1 production by Th2 cells, which acts in an autocrine fashion to induce IL-4 responsiveness. These results suggest that CD28-induced costimulation plays an important role in responses mediated by both types of Th cells.     

Heterogeneity of autoreactive T cell clones specific for the E2 component of the pyruvate dehydrogenase complex in primary biliary cirrhosis

The extraordinary specificity of bile duct destruction in primary biliary cirrhosis (PBC) and the presence of T cell infiltrates in the portal tracts have suggested that biliary epithelial cells are the targets of an autoimmune response. The immunodominant antimitochondrial response in patients with PBC is directed against the E2 component of pyruvate dehydrogenase (PDC-E2). Hitherto, there have only been limited reports on the characterization and V beta usage of PDC-E2-specific cloned T cell lines. In this study, we examined peripheral blood mononuclear cells (PBMC) for their reactivity to the entire PDC complex as well as to the E1- and E2-specific components. We also examined the phenotype, lymphokine profile, and V beta usage of PDC-specific T cell clones isolated from cellular infiltrates from the livers of PBC patients. We report that PBMC from 16/19 patients with PBC, but not 12 control patients, respond to the PDC-E2 subunit. Interestingly, this response was directed to the inner and/or the outer lipoyl domains, despite the serologic observation that the autoantibody response is directed predominantly to the inner lipoyl domain. Additionally, lymphokine analysis of interleukin (IL) 2/IL-4/interferon gamma production from individual liver-derived autoantigen-specific T cell clones suggests that both T helper cell Th1- and Th2-like clones are present in the liver. Moreover, there was considerable heterogeneity in the T cell receptor for antigen (TCR) V beta usage of these antigen- specific autoreactive T cell clones. This is in contrast to murine studies in which animals are induced to develop autoimmunity by specific immunization and have an extremely limited T cell V beta repertoire. Thus, our data suggest that in human organ-specific autoimmune diseases, such as PBC, the TCR V beta repertoire is heterogenous.     

Expression of specific cytolytic activity by H-2I region-restricted, influenza virus-specific T lymphocyte clones

Among murine class II major histocompatibility complex (MHC)-restricted cytotoxic T lymphocyte (CTL) clones specific for type A influenza virus, we have identified both noncytolytic clones and clones exhibiting H-2 I region-restricted cytolytic activity. After appropriate antigenic stimulation, both cytolytic and noncytolytic clones proliferated in the absence of exogenous interleukin 2. All of the clones possess the Thy-1.2+, Lyt-1+2-, L3T4+ phenotype. The class II MHC restriction of viral recognition by the CTL clones was mapped by proliferation using recombinant mouse strains and by inhibition of cytotoxic activity with monoclonal antibodies directed to class II MHC products and L3T4a. The restriction specificity of two CTL clones was unambiguously assigned to the E beta d chain by using L cell transfectant lines expressing E alpha kE beta d or E alpha kE beta k gene products. Analysis of the viral specificity of the cloned lines revealed subtype-specific and crossreactive patterns of viral antigen recognition; the pattern of viral antigen specificity exhibited by each clone in proliferation and cell-mediated cytotoxicity was identical. Each CTL clone also demonstrated antigen-dependent release of helper factor(s) that promoted in vitro primary anti-SRBC responses. Finally, the cytotoxic effector function of the class II MHC-restricted CTL clones was mediated by direct lysis of virus-infected cells, and not by secretion of a cytolytic lymphokine.     

Regulation of T lymphocyte proliferation. Interleukin 2-mediated induction of c-myb gene expression is dependent on T lymphocyte activation state

We previously reported that with time, after antigenic stimulation of antigen-regulated murine T lymphocyte clones, total IL-2-R expression decayed 10-50-fold, commensurate with a decline in the ability of the cells to proliferate to IL-2. However, late after antigenic stimulation, when the cells were refractory to the IL-2-proliferative stimulus, high levels of high affinity IL-2-R remained. In this report we further explore the basis of unresponsiveness to IL-2 in the quiescent clones. We show that the proto-oncogene c-myc is induced in the late cell population by IL-2 to comparable levels observed early after antigen stimulation. IL-2-dependent c-myb induction, however, is seen only early after activation but not in the late-activated population. Analysis of the IL-2-dependent expression of c-myb mRNA with time after antigenic stimulation showed that steadystate c-myb expression declines dramatically with kinetics closely paralleling a decay in IL-2-dependent proliferative ability. In contrast, steadystate c-myc expression remains high throughout this period. Expression of c-myb is critical for proliferation of these cells since antisense oligodeoxy-nucleotide to c-myb can inhibit their IL-2-dependent proliferation. We present evidence for a pathway of c-myb induction via the TCR that is independent of the IL-2/IL-2-R interaction. In addition, the inhibition of IL-2-R-induced c-myb expression by 2-aminopurine and enhanced induction of c-myb via the TCR demonstrate that TCR activation and IL-2-R activation lead to induction of c-myb by different mechanisms.     

Heterogeneity of helper/inducer T lymphocytes. I. Lymphokine production and lymphokine responsiveness

Antigen-specific, Ia-restricted helper/inducer T lymphocytes consist of subsets that can be distinguished by lymphokine secretion. One, called Th1, secretes IL-2 and the other, termed Th2, produces BSF-1/IL-4 in response to stimulation by lectin or antigen receptor signals, and each uses the respective lymphokine as its autocrine growth factor. Cloned lines representing Th2 cells proliferate in response to both IL-2 and their autocrine lymphokine, BSF-1/IL-4, but this proliferation is dependent on the synergistic costimulator activity of the monokine, IL-1. In contrast, Th1 clones proliferate only in response to IL-2, are unresponsive to BSF-1/IL-4, and their growth is unaffected by IL-1. These response patterns are not attributable to variations in culture conditions but apparently reflect intrinsic properties of the two T cell subsets. Moreover, the unresponsiveness of Th1 cells to BSF-1/IL-4 may be related to lower levels of expression of surface receptors for this lymphokine. These results may explain the observed heterogeneity among bulk populations of T cells in terms of lymphokine responsiveness and requirement for accessory factors (costimulators). In addition, our findings suggest that IL-2, unlike BSF-1/IL-4, is a fully competent growth factor that is potentially involved in antigen-independent expansion of bystander T cells present at sites of immune stimulation.     

T cell receptor ligation induces interleukin (IL) 2R beta chain expression in rat CD4,8 double positive thymocytes, initiating an IL-2- dependent differentiation pathway of CD8 alpha+/beta- T cells

The role of interleukin (IL)2 in intrathymic T cell development is highly controversial, and nothing is known about IL-2R expression on thymocytes of the T cell receptor (TCR) alpha/beta lineage undergoing TCR-driven differentiation events. We analyze here IL-2R alpha and beta mRNA expression in an in vitro system where newly generated rat CD4,8 double positive (DP) thymocytes respond to TCR ligation plus IL-2 (but not to either stimulus alone) with rapid differentiation to functional CD8 single positive T cells (Hunig, T., and R. Mitnacht. 1991. J. Exp. Med. 173:561). TCR ligation induced expression of IL-2R beta (but not alpha) chain mRNA in DP thymocytes. Addition of IL-2 then lead to functional maturation and expression of the IL-2R alpha chain. To investigate if the CD8 T cells generated via this IL-2R beta-driven pathway in vitro correspond to the bulk of CD8 T cells seeding peripheral lymphoid organs in vivo, we compared their phenotype to that of lymph node CD8 T cells. Surprisingly, analysis of CD8 cell surface expression using a novel anti-CD8 monoclonal antibody specific for the alpha/beta heterodimeric isoform, and of CD8 alpha and beta chain mRNA revealed that T cells generated by TCR ligation plus IL-2 resemble thymus-independent rather than thymus-derived CD8 cells in that they express CD8 alpha without beta chains. These findings demonstrate that TCR crosslinking induces functional IL-2R on immature DP rat thymocytes. In addition, they show that at least in vitro, CD8 alpha/alpha T cells are generated from TCR-stimulated DP thymocytes (which express the CD8 alpha/beta in the heterodimeric isoform) along an IL-2-driven pathway of T cell differentiation.     

Selective activation of gamma/delta + T cell clones by single anti-CD2 antibodies

The CD2 antigen is the target for an "alternative" T cell activation pathway. Numerous studies have demonstrated that pairs of monoclonal antibodies (mAbs) directed toward two different epitopes are required for activation of T cell receptor (TCR)-alpha/beta + T cells via CD2. We have now explored the activation of human TCR-gamma/delta + T cell clones by a panel of anti-CD2 mAbs directed against the sheep erythrocyte-binding (T11.1) epitope of CD2. Seven of seven gamma/delta + clones expressing different molecular forms of the TCR-gamma/delta responded to stimulation by a single anti-CD2 mAb (OKT11, 9E8, BW0110, M-T910) with IL-2 secretion and/or proliferation. Immobilization of anti-CD2 mAbs in microculture plates was essential for activation of gamma/delta + clones, which occurred in the absence of feeder cells. In addition to interleukin 2 (IL-2) production and proliferation, anti-CD2 mAbs also triggered cytotoxic effector activity in gamma/delta + clones as measured against FcR+ P815 target cells. In contrast to gamma/delta + clones (but in line with established data), none of five CD4+ or CD8+ TCR-alpha/beta + clones were activated by any of the tested individual anti-CD2 mAbs. Taken together, our results reveal a striking difference between cloned gamma/delta + and alpha/beta + T cells in that gamma/delta + T cells are selectively activated by a single anti-CD2 (T11.1) mAb, without need for the simultaneous signal of a second anti-CD2 mAb directed against another (T11.2 or T11.3) CD2 epitope.     

B7 and interleukin 12 cooperate for proliferation and interferon gamma production by mouse T helper clones that are unresponsive to B7 costimulation

We have previously shown that dendritic cells isolated after overnight culture, which can express B7 and are potent stimulators of naive T cell proliferation, are relatively poor at inducing the proliferation of a panel of murine T helper 1 (Th1) clones. Maximal stimulation of Th1 clones was achieved using unseparated splenic antigen presenting cells (APC). An explanation for these findings is provided in the present study where we show that FcR+ L cells transfected with B7 stimulate minimal proliferation of Th1 clones in response to anti-CD3 antibodies, in contrast to induction of significant proliferation of naive T cells. However, addition of interleukin 12 (IL-12) to cultures of Th1 cells stimulated with anti-CD3 and FcR+ B7 transfectants resulted in a very pronounced increase in proliferation and interferon gamma (IFN-gamma) production. Exogenous IL-12 did not affect the B7- induced proliferation of naive T cells. This showed that whereas costimulatory signals delivered via B7-CD28 interaction are sufficient to induce significant proliferation of naive T cells activated through occupancy of the T cell receptor, Th1 T cell clones require cooperative costimulation by B7 and IL-12. This costimulation was shown to be specific by inhibition of proliferation and IFN-gamma production using chimeric soluble cytolytic T lymphocyte-associated antigen 4-human IgG1Fc (CTLA4-Ig) and anti-IL-12 antibodies. Furthermore, the significant antigen specific proliferation and IFN-gamma production by Th1 clones observed when splenocytes were used as APC was almost completely abrogated using CTLA4-Ig and anti-IL-12 antibodies. Thus two costimulatory signals, B7 and IL-12, account for the ability of splenic APC to induce maximal stimulation of Th1 clones. IL-10 downregulates the expression of IL-12 by IFN-gamma-stimulated macrophages and this may account largely for t the ability of IL-10 to inhibit APC function of splenic and macrophage APC for the induction of Th1 cell proliferation and IFN-gamma production. Indeed we show that IL-12 can overcome the inhibitory effect of IL-10 for the APC-dependent induction of proliferation and IFN-gamma production by Th1 clones. These results suggest that proliferation by terminally differentiated Th1 clones, in contrast to naive T cells, requires stimulation via membrane-bound B7 and a cytokine, IL-12. It is possible that these signals may result in the activation of unresponsive T cells during an inflammatory response. IL-10, by its role in regulating such innate inflammatory responses, may thus help to maintain these T cells in an unresponsive state.     

Induction of interleukin 12 responsiveness is impaired in anergic T lymphocytes

The cytokine, interleukin 12 (IL-12), stimulates both natural killer cells and T cells to proliferate and to secrete interferon gamma (IFN- gamma). The T cell proliferative response to IL-12 must be induced and is evident after T cell receptor-mediated stimulation. As reported here, tolerant CD4+ T cells and clones, that are anergic for IL-2 production, are also anergic for induction of the proliferative response to IL-12. Murine T helper 1 clones tolerized in vitro, as well as anergic CD4+ T cells isolated from mice tolerized to the Mls-1a antigen (Ag) in vivo, demonstrated defective induction of proliferation to IL-12 upon restimulation with Ag. IL-12-enhanced production of IFN- gamma was observed in both control and anergic cells after Ag/antigen- presenting cell (APC) activation, although total IFN-gamma secretion by anergic cells was less than that produced by control cells, even in the presence of IL-12. These data indicate that T cell clonal anergy results in profound inhibition of proliferative responses, since the autocrine growth factor, IL-2, is not produced, and the APC-derived cytokine, IL-12, is not an effective stimulus for anergic T cell proliferation.     

Clonal analysis of cytotoxic and regulatory T cell responses against human melanoma

T cell-mediated immune response against autologous melanoma cells was analyzed, at population and clonal levels, in 31 patients with recurrent and/or metastatic disease. Fresh PBL and lymph node lymphocytes (LNL) from melanoma-involved nodes were not cytotoxic against the respective melanoma cells. When activated in in vitro coculture (IVC) against the autologous melanoma cells in the presence of IL-2, a majority of the activated PBL and LNL became cytotoxic against the autologous targets. The activated effector cells were cloned in limiting dilution microcultures, and growing clones were phenotypically defined and were functionally characterized for cytotoxicity and for potential regulatory function. Functional T cell clones were obtained from 15 of 31 cases. Of these, CTL responses exhibiting cytotoxicity restricted against the autologous melanoma were seen in four cases. All four CTL clones were CD3+, CD8+, and CD4-. Three of these four CTL clones were studied extensively. All three of these CTL clones expressed MHC class I-restricted cytotoxicity. mAb anti-CD3 blocked cytotoxicity in two and enhanced cytotoxicity in the other. Neither autologous sera nor autologous nonactivated fresh PBL modulated the cytotoxic functions of the CTL clones at the effector phase. T cell lines exhibiting regulatory function were obtained in 11 cases. The regulatory T cell lines were CD3+, CD4+, and CD8-. In three cases CD4+ clones amplified the cytotoxic response in the PBL in coculture, while in eight other cases the T cell lines downregulated the cytotoxic responses. Such T cell-mediated down-regulations were either restricted to the autologous system, induced by D/DR antigens expressed by the autologous or allogeneic melanoma cells, or induced by stimulus other than D/DR antigens. Taken together, these findings clearly demonstrate the existence of T cell-mediated cytotoxic and regulatory responses against human melanoma.     

Dysregulated T cell expression of TIM3 in multiple sclerosis

T cell immunoglobulin- and mucin domain-containing molecule (TIM)3 is a T helper cell (Th)1-associated cell surface molecule that regulates Th1 responses and promotes tolerance in mice, but its expression and function in human T cells is unknown. We generated 104 T cell clones from the cerebrospinal fluid (CSF) of six patients with multiple sclerosis (MS) (n = 72) and four control subjects (n = 32) and assessed their cytokine profiles and expression levels of TIM3 and related molecules. MS CSF clones secreted higher amounts of interferon (IFN)-gamma than did those from control subjects, but paradoxically expressed lower levels of TIM3 and T-bet. Interleukin 12-mediated polarization of CSF clones induced substantially higher amounts of IFN-gamma secretion but lower levels of TIM3 in MS clones relative to control clones, demonstrating that TIM3 expression is dysregulated in MS CSF clones. Reduced levels of TIM3 on MS CSF clones correlated with resistance to tolerance induced by costimulatory blockade. Finally, reduction of TIM3 on ex vivo CD4+ T cells using small interfering (si)RNA enhanced proliferation and IFN-gamma secretion, directly demonstrating that TIM3 expression on human T cells regulates proliferation and IFN-gamma secretion. Failure to up-regulate T cell expression of TIM3 in inflammatory sites may represent a novel, intrinsic defect that contributes to the pathogenesis of MS and other human autoimmune diseases.