IL-4 synthesis by in vivo primed keyhole limpet hemocyanin-specific CD4+ T cells. I. Influence of antigen concentration and antigen-presenting cell type.

The development of IL-4 synthesis is a critical step in the regulation of immune responses. Our studies focused on the production of IL-4 by CD4+ T cells taken from mice primed with the Ag keyhole limpet hemocyanin (KLH). In vitro stimulation of such CD4+ T cells with KLH resulted in little or no IL-4 production in the first 24 h of stimulation, indicating that little IL-4 synthesis persists in vivo after immunization. However, IL-4 was generated later at 24 to 96 h of in vitro stimulation, indicating that the potential to produce IL-4 was retained by the KLH-primed CD4+ T cells, but that in vitro maturation of the T cells was required before initiation of IL-4 production. The amount of IL-4 produced in vitro by KLH-primed T cells from BALB/c mice was influenced by several factors. First, stimulation of KLH-primed CD4+ T cells with higher in vitro concentrations of KLH resulted in more IL-4 synthesis, but this was accompanied by more IFN-gamma as well. Second, primed CD4+ T cells from lymph nodes (axillary and popliteal) produced significantly more IL-4 than primed splenic T cells. Third, when primed B cells were utilized to present low concentrations of KLH to the T cells, IL-4 but not IFN-gamma was produced. In contrast, use of splenic adherent cells resulted in IFN-gamma but not IL-4 synthesis. These restricted patterns of lymphokine synthesis, however, were observed only with low concentrations of KLH. Fourth, the amount of IL-4 produced and its regulation by the presence of IFN-gamma differed among mouse strains, in that BALB/c T cells produced much more IL-4 than H-2 identical DBA/2 T cells. Our results characterizing the APC and Ag dose requirements for IL-4 synthesis in KLH-primed T cells from different strains of mice are consistent with previous observations that distinct strains of mice differ in the type of immune response generated against different pathogens, and with the concept that low Ag concentrations preferentially result in high levels of IgE synthesis, which is absolutely dependent on IL-4 production.     

Functions of rat CD4+ T cell subsets defined by CD45RB: CD45RB- cells have a much stronger response to recall antigens, whereas polyclonally activated cells of both subsets are equally efficient producers of IFN in the presence of exogenous IL-2.

CD4+45RB- rat T cells were shown to respond strongly to recall antigens and produce IFN and TNF after polyclonal activation. Compared to CD4+45RB- cells, CD4+45RB+ cells showed a very weak response to recall antigens but produced higher amounts of IFN and TNF after polyclonal activation. Addition of rIL-2 reduced the difference between the subsets with respect to the level of IFN produced at 48 and 72 hr after activation, but did not influence the level of TNF production. The CD4+45RB- cells clearly showed a faster response to polyclonal activation than that of CD4+45RB+ cells detected as an earlier IFN production and CD25 expression. The earlier IFN production by the CD45RB- population could not only be explained by their faster production of IL-2, since the difference persisted when rIL-2 was added to both populations at the beginning of culture. We conclude that the CD4+45RB- rat T cell population resemble the CD4+45RA-0+ human T cell subset with respect to a good responsiveness to recall antigen and efficient production of IFN. However, the CD4+45RB+ rat T cell subset functionally differs from the CD4+45RA+0- human T cell subset. We suggest that the CD4+45RB+ subset comprises a major CD4+45RA+B+0- and a minor CD4+4+45A-B+0+ T cell subpopulation, the latter possibly mediating a response to recall antigen and the production of IFN.     

Rapid response to Con A by CD4+CD45R- rat memory lymphocytes as compared to CD4+CD45R+ lymphocytes

Functionally distinct subpopulations within the CD4+ subset of T lymphocytes have been described in man, rat, and mouse. In the rat different functions have been assigned to CD45R+ and CD45R- T helper cells. The CD45R+ in contrast to the CD45R- T helper cells have been reported to produce IL-2 and to proliferate well in response both to Con A and in MLR. In the present investigation the kinetics of the response to Con A by the CD45R+ and CD45R- rat T helper subsets have been analyzed. We confirm a strong proliferative response to Con A by CD4+CD45R+ rat T lymphocytes and also that they are the best IL-2 producers. We further demonstrate that CD4+CD45R- cells also produce IL-2, although in order to appreciate this production quantitatively by assays of the culture supernatants it was necessary to block IL-2 absorption by IL-2 receptor (IL-2R) antibodies. This blockage was of importance also in comparisons of the two subsets, since they showed different kinetics of IL-2R appearance. It is demonstrated that the CD4+CD45R- cells respond more rapidly to Con A than the CD4+CD45R+ cells as reflected by phenotypic conversion, IL-2 production, and proliferation. The fast response of the CD4+CD45R- T subset shown in the present study of rat cells and analogous studies of human cells suggests that the memory compartment of T cells besides other characteristics also has the capacity for a more rapid response than naive lymphocytes.     

Recruitment and activation of tumor-specific immune T cells in situ. CD8+ cells predominate the secondary response in sponge matrices and exert both delayed-type hypersensitivity-like and cytotoxic T lymphocyte activity

This study analyzes the involvement of CD4+ and CD8+ T cells in a secondary cellular immune response to the highly metastatic murine lymphoma ESb in situ. This tumor line expresses tumor-associated transplantation Ag which can induce protective immunity in vivo and specific CTL in vitro. In tumor-immune mice the injection of a tumor vaccine (x-irradiated ESb tumor cells) into s.c. implanted vascularized sponges resulted in the generation of a specific secondary immune response characterized by massive leukocyte recruitment and generation of strong CTL activity at the restimulation site. During the antitumor immune response the CD4+:CD8+ T cell ratio decreased significantly and specifically in the restimulated sponges. Depletion of CD8+ but not CD4+ T cells from the tumor immune mice before restimulation significantly reduced the delayed-type hypersensitivity-like response and totally blocked the generation of tumor-specific CTL activity in situ. Only a minority of the CD8+ immune T cells which predominated the secondary response in situ expressed IL-2R and lymph node homing receptors as detected by the mAb MEL-14.     

Suppressor effector function of CD4+CD25+ immunoregulatory T cells is antigen nonspecific

CD4+CD25+ T cells represent a unique population of "professional" suppressor T cells that prevent induction of organ-specific autoimmune disease. In vitro, CD4+CD25+ cells were anergic to simulation via the TCR and when cultured with CD4+CD25- cells, markedly suppressed polyclonal T cell proliferation by specifically inhibiting the production of IL-2. Suppression was cytokine independent, cell contact dependent, and required activation of the suppressors via their TCR. Further characterization of the CD4+CD25+ population demonstrated that they do not contain memory or activated T cells and that they act through an APC-independent mechanism. CD4+CD25+ T cells isolated from TCR transgenic (Tg) mice inhibited responses of CD4+CD25- Tg T cells to the same Ag, but also inhibited the Ag-specific responses of Tg cells specific for a distinct Ag. Suppression required that both peptide/MHC complexes be present in the same culture, but the Ags could be presented by two distinct populations of APC. When CD4+CD25+ T cells were cultured with anti-CD3 and IL-2, they expanded, remained anergic, and in the absence of restimulation via their TCR, suppressed Ag-specific responses of CD4+CD25- T cells from multiple TCR transgenics. Collectively, these data demonstrate that CD4+CD25+ T cells require activation via their TCR to become suppressive, but once activated, their suppressor effector function is completely nonspecific. The cell surface molecules involved in this T-T interaction remain to be characterized.     

CD4+ T cell subsets. Lymphokine secretion of memory cells and of effector cells that develop from precursors in vitro

We have studied the properties of several developmentally defined subpopulations of CD4+ T cells from normal animals which can be stimulated to secrete lymphokines. We find that the Th cells responsible for direct secretion of lymphokines after stimulation are from a resting, very long lived subpopulation of CD4+ T cells which persists for over 25 wk after adult thymectomy. These T cells are depleted by in vivo administration of antithymocyte serum and they are enriched among T cells which express high levels of Pgp-1. This phenotype suggests that the T cells responsible are most likely memory T cells which have resulted from antigen exposure in vivo. T cells in this subset secrete predominantly IL-2 with small quantities of IL-3, granulocyte/macrophage CSF, and IFN-gamma. In contrast, the CD4+ T cells which require in vitro culture and restimulation before they develop into an effector population with the ability to secrete lymphokines after restimulation, differ dramatically by most of these criteria. The precursors we study are resting Th cells which are considerably shorter lived after adult thymectomy (5 to 10 wk) and resistant to the same doses of antithymocyte serum which deplete the putative memory population. We hypothesize that this precursor population represents naive helper cells which have not yet encountered Ag. The effectors derived from such precursors can be stimulated to secrete high levels of both Th cell types 1 and 2 lymphokines (IFN-gamma, IL-4, IL-5, granulocyte/macrophage CSF, and IL-3). Generation of effectors requires proliferation and differentiation events which occur during a mandatory culture with lymphokines and antigen presenting cells for 3 to 4 days. We discuss the striking phenotypic and functional differences among these subpopulations of helper cells--the precursor population and the two types--memory and cultured effector Th which secrete lymphokines. We also discuss the relationship of these populations to CD4+ T cell subsets defined by other studies of patterns of lymphokine secretion and by cell surface phenotype.     

Generation propagation, and targeting of human CD4+ helper/killer T cells induced by anti-CD3 monoclonal antibody plus recombinant IL-2. An efficient strategy for adoptive tumor immunotherapy.

We developed a culture system for the rapid generation of CD4+ T cells that have both helper and killer functions. CD4+ T cells isolated from human PBL did not proliferate or develop significant cytotoxicity when treated with rIL-2 because of the lack of p75 IL-2R expression. However, culture of isolated CD4+ T cells with immobilized anti-CD3 mAb plus rIL-2 resulted in a marked proliferation (500-fold increase in 14 days) of CD4+ T cells. The proliferating CD4+ T cells produced IL-2 (92 U/ml) and showed strong cytotoxicity against OKT3 hybridoma cells and Daudi, K562, and U937 tumor cells in an anti-CD3 mAb-dependent manner. The CD4+ T cells contained significant amounts of cytolytic granule-related proteins such as serine esterase and perforin. Activated CD4+ helper/killer cells can be generated from both healthy donors and tumor patients and can be propagated in vitro for 14 to 35 days by biweekly restimulation with immobilized anti-CD3 mAb plus rIL-2. This culture yielded about 20,000-fold increase in cell number after a 21-day culture. Bispecific antibody containing anti-CD3 and anti-glioma Fab components enhanced the cytotoxicity of activated CD4+ helper/killer cells against IMR32 glioma cells. Moreover, the activated CD4+ helper/killer cells showed both helper and antitumor activity in vivo and prevented growth of anti-CD3 hybridoma cells in nude mice whether or not IL-2 was administered. These results indicate that anti-CD3 mAb plus IL-2-activated CD4+ helper/killer cells may provide an effective strategy for adoptive tumor immunotherapy of cancer.     

Heterogeneous mechanisms of human cytotoxic T lymphocyte generation. I. Differential helper cell requirement for the generation of cytotoxic effector cells from CD8+ precursor subpopulations.

The subpopulations of CD8+ T cells defined by CD45RA Ag expression have been hypothesized to represent cells varying in their relative maturation along a common, activation-dependent differentiation pathway. Previous studies have shown that both the CD8+CD45RA+ and CD8+CD45RA- subsets contain precursor cells capable of developing into alloreactive CTL. In the current study, we have examined the mechanisms involved in the generation of CTL effector cells from these two CD8+ subsets. Purified CD8+CD45RA+ or CD8+CD45RA- cells were stimulated with allogeneic non-T cells, either alone or in the presence of CD4+ Th cells. Although the generation of CTL from CD8+CD45RA- precursor cells consistently required the presence of CD4+ Th cells, cytotoxic effector cells could be generated from CD8+CD45RA+ precursor cells in the absence of CD4+ cells. Several lines of evidence indicated that the helper cell-independent generation of cytotoxic effector cells from CD8+CD45RA+ precursors resulted from the unique ability of this subset to produce and use IL-2 in an autocrine fashion: 1) exogenous IL-2 could replace the effects of CD4+ helper cells for either CD8+ subset; 2) the helper cell-independent functional maturation of CD8+CD45RA+ cells could be blocked by anti-CD25 or anti-IL-2 antibodies; and 3) CD8+CD45RA+ cells produced IL-2 after activation with allogeneic cells. The finding that precursors for helper cell-independent CTL generation are restricted to the CD8+CD45RA+ subset suggests that this capability may vary as a function of the maturation of CD8+ cells.     

Both naive and memory T cells can provide help for human IgE production, but with different cytokine requirements.

Most in vitro systems for the induction of IgE production by human B cells require both IL-4 and the presence of T cells. Little is known about the mechanism of T cell help or the ability of different T cell subsets to provide this helper activity. In the present study we demonstrate that, in the presence of exogenous IL-4, anti-CD3 stimulated naive T cells (CD4+CD45RA+) are potent helper cells for human IgE production. In their presence, as little as 750 autologous B cells can produce up to 100 ng/ml IgE. This response was found over a broad range of anti-CD3 concentrations. IgE helper activity by naive T cells was inhibited by IL-2. Under all conditions tested, naive T cells were unable to provide help for IgM production. This is in contrast to activated memory T cells (CD4+CD45RO+), which are very efficient helper cells for IgM or IgE production, provided that IL-2 or IL-2 plus IL-4 are present respectively.     

Relative resistance in the development of T cell anergy in CD4+ T cells from simian immunodeficiency virus disease-resistant sooty mangabeys

Despite high viral loads, T cells from sooty mangabey (SM) monkeys that are naturally infected with SIV but remain clinically asymptomatic, proliferate and demonstrate normal Ag-specific memory recall CD4(+) T cell responses. In contrast, CD4(+) T cells from rhesus macaques (RM) experimentally infected with SIV lose Ag-specific memory recall responses and develop immunological anergy. To elucidate the mechanisms for these distinct outcomes of lentiviral infection, highly enriched alloreactive CD4(+) T cells from humans, RM, and SM were anergized by TCR-only stimulation (signal 1 alone) and subsequently challenged with anti-CD3/anti-CD28 Abs (signals 1 + 2). Whereas alloreactive CD4(+)T cells from humans and RM became anergized, surprisingly, CD4(+) T cells from SM showed marked proliferation and IL-2 synthesis after restimulation. This resistance to undergo anergy was not secondary to a global deficiency in anergy induction of CD4(+) T cells from SM since incubation of CD4(+) T cells with anti-CD3 alone in the presence of rapamycin readily induced anergy in these cells. The resistance to undergo anergy was reasoned to be due to the ability of CD4(+) T cells from SM to synthesize IL-2 when incubated with anti-CD3 alone. Analysis of phosphorylated kinases involved in T cell activation showed that the activation of CD4(+) T cells by signal 1 in SM elicited a pattern of response that required both signals 1 + 2 in humans and RM. This function of CD4(+) T cells from SM may contribute to the resistance of this species to SIV-induced disease.     

CD127+CCR5+CD38+++ CD4+ Th1 effector cells are an early component of the primary immune response to vaccinia virus and precede development of interleukin-2+ memory CD4+ T cells

The stages of development of human antigen-specific CD4+ T cells responding to viral infection and their differentiation into long-term memory cells are not well understood. The inoculation of healthy adults with vaccinia virus presents an opportunity to study these events intensively. Between days 11 and 14 postinoculation, there was a peak of proliferating CCR5+CD38+++ CD4+ effector cells which contained the cytotoxic granule marker T-cell intracellular antigen 1 and included gamma interferon (IFN-gamma)-producing vaccinia virus-specific CD4+ T cells. The majority of these initial vaccinia virus-specific CD4+ T cells were CD127+ and produced interleukin-2 (IL-2) but not CTLA-4 in response to restimulation in vitro. Between days 14 and 21, there was a switch from IFN-gamma and IL-2 coexpression to IL-2 production only, coinciding with a resting phenotype and an increased in vitro proliferation response. The early CCR5+CD38+++ vaccinia virus-specific CD4+ T cells were similar to our previous observations of human immunodeficiency virus (HIV)-specific CD4+ T cells in primary HIV type 1 (HIV-1) infection, but the vaccinia virus-specific cells expressed much more CD127 and IL-2 than we previously found in their HIV-specific counterparts. The current study provides important information on the differentiation of IL-2+ vaccinia virus-specific memory cells, allowing further study of antiviral effector CD4+ T cells in healthy adults and their dysfunction in HIV-1 infection.     

The in vivo elimination of CD4+ T cells prevents the induction but not the expression of carrier-induced epitopic suppression.

Injection of mice with an immunogenic dose of carrier (keyhole limpet hemocyanin (KLH)) followed by immunization with hapten-carrier conjugate (TNP-KLH) selectively suppresses anti-hapten antibody response. In this study, the cellular basis of this epitopic suppression and also of the suppression induced by a high dose of carrier were analyzed by in vivo depletion of CD4+ or CD8+ T cell subsets by using mAb. The mAb treatments were performed either at the time of carrier priming or at the time of hapten-carrier immunization. The elimination of CD8+ T cells has not modified the anti-carrier antibody response, whether this treatment was performed at the time of KLH-priming or during TNP-KLH immunization. Moreover, the in vivo treatment with the anti-CD8 mAb did not modify the carrier-induced epitopic suppression induced either by a low immunogenic dose of KLH or by a high dose of this Ag. The elimination of CD4+ T cells at the time of KLH immunization has prevented the induction of a memory response to KLH, clearly establishing that CD4+ T cells are essential in memory B cell development to T-dependent Ag. Moreover, this treatment has totally abrogated the epitopic suppression induced either by low or high dosages of KLH. In contrast, the in vivo elimination of CD4+ T cells after carrier immunization did not abolish the secondary anti-carrier antibody response and did not prevent the expression of epitopic suppression. These data indicate that primed CD4+ T cells are required neither for memory B cell expression nor for the expression of suppression. Finally, once induced, the suppression can be evidenced after in vivo depletion of both primed CD4+ and CD8+ T cells. These data support the view that epitopic suppression is induced through the expansion of carrier-specific B cells and resulted from intramolecular antigenic competition between hapten and carrier epitopes.     

Transforming growth factor-beta enhances the in vivo effector function and memory phenotype of antigen-specific T helper cells in experimental autoimmune encephalomyelitis.

Transforming growth factor-beta (TGF-beta) had a profound effect on the in vitro phenotypic development of Ag-activated Th cells and enhanced the in vivo effector function of these cells upon adoptive transfer. Previous studies have shown that there are two types of Th cell populations found in unimmunized animals, naive helper cells, which are short-lived and express low levels of CD44 and high levels of CD45R and Mel-14, and memory helper cells, which have a long life span and express high levels of CD44 and low levels of CD45R and Mel-14. Culturing of Ag-specific murine Th cell lines and clones in the presence of TGF-beta greatly enhanced both the memory phenotype of the cultured cells and the effector function upon adoptive transfer in experimental autoimmune encephalomyelitis. Histologic evaluation of spinal cords from recipients receiving passively transferred murine T cell lines cultured with TGF-beta revealed large demyelinated plaques (multiple sclerosis-like) that were not present in animals receiving cells cultured with Ag alone. TGF-beta also enhanced the capability of myelin basic protein-specific Lewis rat T cell lines to transfer experimental autoimmune encephalomyelitis and potentiated a purified protein derivative-specific rat helper cell line to transfer delayed type hypersensitivity. Thus, the effects of TGF-beta did not appear to be limited by species specificity, Ag specificity, or in vivo T cell function. This is the first study showing that TGF-beta can potentiate the development and maintenance of the Th cell memory phenotype in vitro and enhance their in vivo effector function in an animal disease model.     

The functionally distinct subpopulations of human CD4+ helper/inducer T lymphocytes defined by anti-CD45R antibodies derive sequentially from a differentiation pathway that is regulated by activation-dependent post-thymic differentiation

The CD4+ helper/inducer T cell population is comprised of functionally distinct subsets identifiable by the HB11 (anti-CD45R) mAb. We have previously shown that the cells that provide help for antibody production express the CD4+CD45R- phenotype. In contrast, CD4+ CD45R+ cells have minimal, if any, helper cell functions; rather, these cells function as inducers of Ts cell activity. The lineal relationship of these phenotypically and functionally distinct CD4+ subsets is unknown. In the present studies, we have examined the hypothesis that the CD4+ subpopulations identifiable with anti-CD45R antibodies represent "virgin" or "memory" T cells sequentially derived from a common differentiation pathway but differing in their relative maturation. When freshly purified cells were tested, CD4+ CD45R+ cells had no Th cell function. However, after in vitro activation with PHA and propagation in IL-2, CD4+CD45R+ cells acquired the ability to provide help for antibody production. Moreover, this functional acquisition by these cells was accompanied by their conversion to the CD4+CD45R- phenotype. Analyses of the activation, growth kinetics, and functional dose-response characteristics of CD4+CD45R+ and CD4+CD45R- cells demonstrated that our findings did not result from the selective growth of CD4+ CD45R- cells contaminating the CD4+CD45R+ preparations. Thus, these data demonstrate that the "helper" and "suppressor-inducer" subsets of CD4+ cells identified by anti-CD45R antibodies are not comprised of fully mature, phenotypically and functionally stable T cells. Rather, these CD4+ subsets appear to represent cells at different maturational stages of an activation-dependent, post-thymic differentiation pathway.     

IL-2 is required for the activation of memory CD8+ T cells via antigen cross-presentation

Dendritic cells (DCs) are capable of capturing exogenous Ag for the generation of MHC class I/peptide complexes. For efficient activation of memory CD8(+) T cells to occur via a cross-presentation pathway, DCs must receive helper signals from CD4(+) T cells. Using an in vitro system that reflects physiologic recall memory responses, we have evaluated signals that influence helper-dependent cross-priming, while focusing on the source and cellular target of such effector molecules. Concerning the interaction between CD4(+) T cells and DCs, we tested the hypothesis that CD40 engagement on DCs is critical for IL-12p70 (IL-12) production and subsequent stimulation of IFN-gamma release by CD8(+) T cells. Although CD40 engagement on DCs, or addition of exogenous IL-12 are both sufficient to overcome the lack of help, neither is essential. We next evaluated cytokines and chemokines produced during CD4(+) T cell/DC cross talk and observed high levels of IL-2 produced within the first 18-24 h of Ag-specific T cell engagement. Functional studies using blocking Abs to CD25 completely abrogated IFN-gamma production by the CD8(+) T cells. Although required, addition of exogenous IL-2 did not itself confer signals sufficient to overcome the lack of CD4(+) T cell help. Thus, these data support a combined role for Ag-specific, cognate interactions at the CD4(+) T cell/DC as well as the DC/CD8(+) T cell interface, with the helper effect mediated by soluble noncognate signals.     

Alloantigen-specific T suppressor-inducer and T suppressor-effector cells can be activated despite blocking the IL-2 receptor

To determine IL-2 requirement for activation of suppressor cells, PBMC were primed in one-way MLR in the presence of 10 micrograms/ml anti-IL-2R beta-chain antibody 2A3 (CD25) or control antibody, then irradiated and added as regulators in a fresh MLR. Cells primed in the presence of antibody 2A3 suppressed the proliferative response to fresh autologous lymphocytes to specific alloantigen but had no effect on the response to cells from third party donors. Priming in the presence of an antibody of irrelevant specificity induced only limited suppressor activity. Activated suppressor cells did not show cytolytic activity specific for the stimulators when tested at the time of the suppressor cell assay. To identify the subset(s) responsible for suppression, cells primed in the presence of antibody 2A3 were separated into CD4+/CD45RA+, CD4+/CD45RA-, and CD8+ subsets, which were irradiated and then tested. The suppressive activity was found predominantly in the CD4+/CD45RA+ subset, whereas CD8+ cells had some activity and CD4+/CD45RA- cells had none. No subset suppressed the response of autologous cells to third-party cells. When primed CD4+/CD45RA+ cells were cocultured with fresh autologous lymphocytes depleted of CD8+ cells, no suppression was observed, indicating that, although the CD4+/CD45RA+ cells can function as inducers of suppressors, they cannot function as suppressor-effectors. Conversely, CD8+ cells activated in MLR in the presence of 2A3 caused suppression, regardless of whether the fresh autologous responder population contained CD8+ cells. CD4+/CD45RA+ and CD8+ subsets isolated after priming in the presence of 2A3 also demonstrated Ag-specific suppression in the generation of cytotoxic T lymphocytes whereas CD4+/CD45RA- cells had no activity. Our data are consistent with the model that suppression of alloreactivity requires the cooperation of two types of cells, a CD4+/CD45RA+ suppressor-inducer and a CD8+ suppressor-effector population. Activated Tsi and fresh Tse or activated Tse alone can suppress lymphocyte proliferation and generation of CTL in response to specific Ag. Activation of Ag-specific T suppressor-inducer and T suppressor-effector cells appears to be relatively IL-2 independent and presumably require one or more other growth factors.     

CD28, IL-2-independent costimulatory pathways for CD8 T lymphocyte activation.

We investigate, here, the mechanism of the costimulatory signals for CD8 T cell activation and confirm that costimulation signals via CD28 do not appear to be required to initiate proliferation, but provide survival signals for CD8 T cells activated by TCR ligation. We show also that IL-6 and TNF-alpha can provide alternative costimulatory survival signals. IL-6 and TNF-alpha costimulate naive CD8 T cells cultured on plate-bound anti-CD3 in the absence of CD28 ligation. They act directly on sorted CD8-positive T cells. They also costimulate naive CD8 T cells from Rag-2-deficient mice, bearing transgenic TCRs for HY, which lack memory cells, a potential source of IL-2 secretion upon activation. IL-6 and TNF-alpha provide costimulation to naive CD8 T cells from CD28, IL-2, or IL-2Ralpha-deficient mice, and thus function in the absence of the B7-CD28 and IL-2 costimulatory pathways. The CD8 T cell generated via the anti-CD3 plus IL-6 and TNF-alpha pathway have effector function in that they express strong cytolytic activity on Ag-specific targets. They secrete only very small amounts of any of the cytokines tested upon restimulation with peptide-loaded APC. The ability of the naive CD8 T cells to respond to TCR ligation and costimulatory signals from IL-6 and TNF-alpha provides a novel pathway that can substitute for signals from CD4 helper cells or professional APC. This may be significant in the response to viral Ags, which can be potentially expressed on the surface of any class I MHC-expressing cell.     

IL-2/anti-IL-2 antibody complex enhances vaccine-mediated antigen-specific CD8+ T cell responses and increases the ratio of effector/memory CD8+ T cells to regulatory T cells

IL-2 is well described as a cytokine with two markedly distinct functionalities: as a necessary signal during CD4(+) and CD8(+) T cell activation/expansion and as an essential cytokine for the maintenance of CD4(+)CD25(+)FoxP3(+) T cells (regulatory T (T(REG)) cells) during homeostasis. In this study we demonstrate for the first time that, compared with the use of IL-2 alone, a complex of IL-2 and anti-IL-2 Ab (IL-2 complex) enhances the effectiveness of a viral vaccine in a mouse model with known Ag specificity. IL-2 complex led to an increase in the number of Ag-specific effector/memory CD8(+) T cells, cytokine production, and CTL lysis following Ag-specific restimulation in a vaccination setting. Our results further demonstrate that this effect is temporary and declines over the course of a few days after the IL-2 complex treatment cycle. Moreover, in contrast to the use of IL-2 alone, IL-2 complex greatly increased the ratio of effector/memory CD8(+) T cells to T(REG) cells. This phenomenon can thus potentially be used in the enhancement of immune responses to vaccination.     

IL-10 is produced by subsets of human CD4+ T cell clones and peripheral blood T cells.

Murine IL-10 has been reported originally to be produced by the Th2 subset of CD4+ T cell clones. In this study, we demonstrate that human IL-10 is produced by Th0, Th1-, and Th2-like CD4+ T cell clones after both Ag-specific and polyclonal activation. In purified peripheral blood T cells, low, but significant, levels of IL-10 were found to be produced by the CD4+CD45RA+ population, whereas CD4+CD45RA- "memory" cells secreted 5- to 20-fold higher levels of IL-10. In addition, IL-10 was produced by activated CD8+ peripheral blood T cells. Optimal induction of IL-10 was observed after activation by specific Ag and by the combination of anti-CD3 mAb and the phorbol ester tetradecanoyl phorbol acetate, whereas the combination of calcium ionophore A23187 and 12-O-tetradecanoylphorbol-13-acetate acetate was a poor inducer of IL-10 production. Kinetic studies indicated that IL-10 was produced relatively late as compared with other cytokines. Maximal IL-10 mRNA expression in CD4+ T cell clones and purified peripheral blood T cells was obtained after 24 h, whereas maximal IL-10 protein synthesis occurred between 24 h and 48 h after activation. No differences were observed in the kinetics of IL-10 production among Th0, Th1-, and Th2-like subsets of CD4+ T cell clones. The results indicate a regulatory role for IL-10 in later phases of the immune response.     

Antigen-specific T cell activation and proliferation during oral tolerance induction

One of several routes of achieving immunologic tolerance is through functional inactivation of Ag-specific T cells. Oral administration of Ag can allow survival of the Ag-specific T cells that are functionally anergic. The aim of this study was to investigate whether functional inactivation of Ag-specific T cells is directed through an activation process and to further define the differentiative pathways and functional characteristics of anergic T cells. Mice were transplanted with OVA-specific TCR-transgenic T cells and either fed OVA or immunized s.c. with the OVA peptide 323-339 in CFA. OVA-specific T cells from OVA-fed mice were unresponsive to restimulation in vitro within 48-72 h after treatment. In vivo, however, T cell proliferation was detected by 5, 6-carboxy-succinimidyl-fluoresceine-ester intensity changes in OVA-specific T cells. The mesenteric lymph nodes (LNs) from OVA-fed mice more frequently contained OVA-specific dividing cells in vivo than those in the peripheral LNs, and the reciprocal was observed following s.c. immunization of the OVA peptide in CFA. The induction of anergy in OVA-fed mice was accompanied by rapid up-regulation of CD69 and CTLA-4, later down-regulation of CD45RB on OVA-specific T cells, and a marked decrease in T cell secretion of IL-2, IL-10, and IFN-gamma after OVA restimulation in vitro. Results from this study indicate that the inductive phase of oral tolerance is preceded by Ag-specific T cell activation in vivo, proliferation in the regional draining LNs, and differentiation into a memory-like state. These results indicate that Ag-directed differentiation occurs as a part of T cell tolerance through anergy.