Interaction between cytokines and 8-mercaptoguanosine in humoral immunity: synergy with interferon

In previous studies it has been demonstrated that a T cell-like differentiation signal is transmitted by C8-substituted guanine ribonucleosides such as 8-mercaptoguanosine (8MGuo) to antigen-stimulated B cells. A large subset of potentially reactive B cells remains unresponsive to antigen even in the presence of signals provided by these nucleosides except when this signal is preceded by a soluble activity present in mixed lymphocyte culture supernatants. Studies with purified preparations of interleukin (IL)-1, IL-2, IL-3, granulocyte-macrophage colony stimulating factor, B cell stimulatory factor 1 (IL-4), and B cell growth factor II (IL-5) indicated that none of these activities is capable of synergizing with 8MGuo to augment B cell responsiveness to antigen. Therefore, supernatants from a number of cloned cell lines were examined for activity that could synergize with 8MGuo, in order to determine the cellular source of this activity. Soluble products secreted by cloned 24/G1 T cells act synergistically with 8MGuo to evoke enhanced antibody responses to specific antigen in populations of purified B cells. Because concanavalin (Con) A-activated 24/G1 cells produce large quantities of interferon-gamma (IFN-gamma), the possibility that interferons might mediate synergy with 8MGuo was investigated. Purified murine IFN-gamma is unable to interact synergistically with 8MGuo; moreover, treatment of active 24/G1 supernatants with monoclonal anti-IFN-gamma antibodies or at pH 2 fails to abrogate their ability to synergize. In contrast to IFN-gamma, when B cells were supplemented with either IFN-alpha or IFN-beta, antigen-dependent synergy with 8MGuo was observed. However, abrogation of IFN-alpha and IFN-beta activity with specific antibodies fails to interfere with synergy between 8MGuo and mixed lymphocyte culture or Con A supernatants. Therefore, it appears that although IFN-alpha and IFN-beta are not responsible for the synergizing activity present in activated T cell supernatants, they nonetheless represent a previously unrecognized source of synergizing activity.     

Lymphokine-like activity of 8-mercaptoguanosine: induction of T and B cell differentiation

Lymphocyte proliferation and differentiation result from ordered cellular interactions governed by soluble products (lymphokines). Dissecting the individual steps in these processes has been difficult, due to a paucity of pure lymphokines. Recently, it was reported that the derivatized ribonucleoside 8-mercaptoguanosine (8MGuo) has both mitogenic and differentiative effects on murine B cells. In the present studies, we tested 8MGuo for its ability to stimulate both B and T cell responses. In contrast to the murine studies, 8MGuo does not stimulate rat B cells to proliferate and, when tested for B cell growth factor-like activity, no stimulation was observed. The addition of 8MGuo (0.5 to 1 mM final concentration) to mitogen-stimulated B cells led to a marked increase in IgM and a modest increase in IgG secretion. When mixed with conditioned medium, 8MGuo acted synergistically in stimulating secretion of both isotypes, arguing that 8MGuo has both B cell-differentiating factor-mu (BCDF-mu) and BCDF-gamma activity. 8MGuo had no IL 2-like activity when tested on a mouse IL 2-dependent cell line, and no IL 1-like activity on addition to mouse thymocytes with or without submitogenic doses of lectin. However, when added to cultures of murine allogeneic cells in which the stimulating cell populations had been heat-inactivated, 8MGuo induced the generation of specific allogeneic cytotoxic T lymphocytes. Together, these results suggest that a simple derivatized nucleoside can induce both T and B cell differentiation without concomitant proliferation, and thus represent a unique probe for studying events in lymphocyte differentiation.     

B cell depletion delays collagen-induced arthritis in mice: arthritis induction requires synergy between humoral and cell-mediated immunity

Rheumatoid arthritis is a systemic autoimmune disease. B cells are likely to play a critical role in arthritis pathogenesis, although it is unclear whether they are necessary for disease induction, autoantibody production, or disease progression. To assess the role of B cells in inflammatory arthritis, B cells were depleted using mouse anti-mouse CD20 mAbs in a mouse model of collagen-induced arthritis. CD20 mAbs effectively depleted mature B cells from adult DBA-1 mice. When B cells were depleted using CD20 mAbs before collagen immunization, there was a delay in disease onset and autoantibody production, with significantly diminished severity of arthritis both clinically and histologically. B cell depletion further delayed disease onset if initiated before, as well as after, collagen immunization. However, in both cases, the eventual reappearance of peripheral B cells triggered autoantibody production and the subsequent development of arthritis in collagen-sensitized mice. By contrast, B cell depletion after collagen immunizations did not have a significant effect on arthritis progression or severity. Thus, disease symptoms were only induced when peripheral B cells and their autoantibody products were present in collagen-immunized mice, documenting a critical role for B cells during the elicitation phase of collagen-induced arthritis. These studies suggest that B cell depletion strategies will be most effective when initiated early in the development of inflammatory arthritis, with sustained B cell depletion required to inhibit the production of isotype-switched pathogenic Abs and the evolution of joint inflammation and destruction.     

Dissociation of inductive from differentiative signals transmitted by C8-substituted guanine ribonucleosides to B cells from SJL mice

A hitherto unknown defect in the immune responsiveness of B lymphocytes from SJL mice has enabled us to distinguish two qualitatively distinct classes of signal delivered to B cells by C8-substituted guanine ribonucleosides. This defect renders B cells from SJL mice unresponsive to the inductive (early acting) signal of 8-mercaptoguanosine (8MGuo) that culminates in mitogenesis and nonspecific secretion of immunoglobulin. Unresponsiveness is not attributable to a shift in either the dose-response or kinetic profiles, nor can the presence of suppressor cells be demonstrated. In striking contrast, however, SJL B cells exhibit normal responsiveness to the differentiative (T cell-like, or late acting) signal provided by the substituted nucleoside. This signal enables SJL B cells, depleted of T cells, to respond to T cell-dependent antigens, and synergizes with T cell-derived lymphokines. These data suggest 1) that nonspecific secretion of immunoglobulin is dependent on both inductive and differentiative signals, 2) that antigen alone can supply an effective inductive signal for antigen-specific responses, and 3) that the SJL mouse will provide a useful model for selective study of inductive vs differentiative events.     

Restoration of in vitro responsiveness of xid B cells to TNP-Ficoll by 8-mercaptoguanosine

8-Mercaptoguanosine (8sGuo) has been reported to enhance responses of normal mice to the type 2 antigen trinitrophenol (TNP)-Ficoll. In this report, we demonstrate that this immune adjuvant restores the immune responsiveness of B cells from mice with the x-linked immune defect (xid), which are nonresponsive to the type 2 antigen TNP-Ficoll. The data demonstrate that TNP-Ficoll, which by itself cannot stimulate anti-TNP responses in CBA/N mice, is able to initiate the initial steps of cell activation in xid B cells and render them sensitive to the subsequent differentiative effects of 8sGuo. We propose that the unresponsiveness of xid B cells to type 2 antigens reflects not the inability of these antigens to stimulate xid B cells from G0 to G1, but rather the inability of these antigen-activated cells to respond to a second signal to which these immune defective B cells are poorly responsive and can be substituted for by 8sGuo.     

Enhancement of the human antibody response by C8-substituted guanine ribonucleosides in synergy with interleukin 2

The antigen-specific primary antibody response of human lymphocytes in vitro was studied with respect to dependency upon the lymphokine interleukin 2 (IL 2) and its subsequent modulation by C8-substituted guanine ribonucleosides. The specific response to sheep erythrocytes was shown to be dependent on the presence of IL 2 in culture. However, addition of optimal concentrations of the nucleoside, 7-methyl-8-oxoguanosine (7m8oGuo), to cultures containing antigen and IL 2 resulted in marked amplification of the underlying antibody response. This synergistic effect between 7m8oGuo and IL 2 was antigen dependent and could not be accounted for by summation of the independent antigen-specific and nonspecific (polyclonal) components. That IL 2 itself was in fact responsible for both the specific response to antigen and the synergistic interaction with 7m8oGuo was confirmed in experiments with purified IL 2 produced by recombinant DNA technology. The response to antigen was enhanced by 7m8oGuo in a dose-dependent fashion. The results of kinetic studies demonstrated that this nucleoside is fully effective within the context of an ongoing immune response, because addition of 7m8oGuo could be delayed up to 3 days of the 6-day culture period without loss of subsequent immunoenhancement. Lymphocyte populations largely depleted of T cells were capable of mounting vigorous responses to antigen in the presence of 7m8oGuo so long as IL 2, either partially purified or purified recombinant material, was added to culture.     

Critical synergy of CD30 and OX40 signals in CD4 T cell homeostasis and Th1 immunity to Salmonella

CD30 and OX40 (CD134) are members of the TNFR superfamily expressed on activated CD4 T cells, and mice deficient in both these molecules harbor a striking defect in the capacity to mount CD4 T cell-dependent memory Ab responses. This article shows that these mice also fail to control Salmonella infection because both CD30 and OX40 signals are required for the survival but not commitment of CD4 Th1 cells. These signals are also needed for the survival of CD4 T cells activated in a lymphopenic environment. Finally, Salmonella and lymphopenia are shown to act synergistically in selectively depleting CD4 T cells deficient in OX40 and CD30. Collectively these findings identify a novel mechanism by which Th1 responses are sustained.     

Regulation of B cell activation by prostaglandins: cell cycle-specific effects on activation by anti-immunoglobulin and 8-mercaptoguanosine

The current studies were undertaken to explore the regulatory effects of macrophages and their soluble products on B cell activation in defined medium by surface membrane-directed mitogens (anti-Ig, LPS) and by intracellular mitogens (8-mercaptoguanosine, [8MGuo]). Supplementation of macrophage-depleted B cell cultures with adherent cells enhanced the response to anti-Ig but depressed the response to 8MGuo. These changes could be eliminated by adding indomethacin to B cell cultures containing supplemental macrophages. Moreover, they could be reproduced by adding exogenous prostaglandins (PGE1, PGE2) but not other macrophage products to cultured B cells. Prostaglandins regulate B cell function (i.e., immunoglobulin secretion) in the same manner as they do mitogenesis. Thus, the polyclonal response to LPS is enhanced, whereas that to 8MGuo is inhibited. We showed previously that anti-Ig acts on a B cell subpopulation distinct from that stimulated by 8MGuo. Moreover, when addition of prostaglandin is delayed for more than 24 hr, the effect on the anti-Ig response changes from enhancement to inhibition and the effect on 8MGuo is lost, suggesting that in the course of activation the cell progresses through a series of cell cycle-specific regulatory states. Additionally, the mitogenic effects of 8MGuo appear to involve larger, cycling cells more than smaller cells. In concert, these data suggest a model for regulation of the B cell cycle in which prostaglandins, whose secretion is elicited by many surface-directed B cell stimuli, enhance the entry of cells into the cell cycle and subsequently regulate their passage through the cycle.     

T8 cell regulation of human B cell responsiveness: regulatory influences of CD45RA+ and CD45RA- T8 cell subsets

The immunoregulatory functions of human T8 cell subpopulations defined by mAb to the CD45RA molecule (2H4) were examined. Both CD45RA+ and CD45RA- T8 cells that had been treated with mitomycin C provided help for the production of immunoglobulins by B cells in cultures stimulated with immobilized mAb to CD3 (64.1). In contrast, both CD45RA+ and CD45RA- T8 cells that had not been treated with mitomycin C suppressed B cell responses in anti-CD3-stimulated cultures, although CD45RA+ T8 cells were more effective in this regard. Interleukin 2 (IL2) enhanced suppression by anti-CD3-activated CD45RA- T8 cells, whereas suppression by CD45RA+ T8 cells was almost maximal and not as much increased by IL2. The differentiation into suppressor-effector cells in this system appeared to involve the production of IL2, but not the production of interferon (INF)-gamma. Thus, CD45RA+ T8 cells produced higher amounts of IL2 but lower amounts of IFN-gamma than CD45RA- T8 cells in anti-CD3-stimulated cultures. Moreover, addition of mAb to the p55 component of IL2 receptor (anti-Tac) inhibited the generation of suppressor activity from CD45RA+ and CD45RA- T8 cells. The pattern and magnitude of suppression of B cell responses by CD45RA+ and CD45RA- T4 cells were similar to that by CD45RA+ and CD45RA- T8 cells in this system. Finally, preactivated CD45RA+ T8 cells that had lost CD45RA expression suppressed the B cell responses as effectively as fresh CD45RA+ T8 cells. The results indicate that both CD45RA+ and CD45RA- T8 cells can help or suppress B cell responses. More importantly, the data suggest that the suppressor-effector function of human T cells may rather be related with the stages of the post-thymic differentiation as evidenced by the expression of the CD45RA molecule than represent the fully differentiated T cell subsets, such as T4 and T8 cells. In addition, the CD45RA molecule appeared not to be involved in the suppressor-effector function, but to determine the stage of post-thymic differentiation.     

Role of salvage and phosphorylation in the immunostimulatory activity of C8-substituted guanine ribonucleosides

Lymphokine-like activity and selective stimulation of B cell growth is exerted by a group of synthetic ribonucleosides derivatized at C8 and exemplified by 8-bromoguanosine (8BrGuo), 8-mercaptoguanosine, and 7-methyl 8-oxoguanosine. However, relatively little is known about their molecular mechanism of action. Like naturally occurring nucleosides, 8BrGuo is taken up into lymphocytes by a process of facilitated diffusion. Naturally occurring nucleosides are then reclaimed by a well characterized salvage pathway, involving sequential phosphorolysis and phosphoribosylation. The studies reported in this communication demonstrate that, in contrast to naturally occurring nucleosides, 8BrGuo is not a substrate for salvage by purine nucleoside phosphorylase. The base that would be produced by putative phosphorolysis, 8-bromoguanine, is biologically inactive and is not a substrate for hypoxanthine-guanine phosphoribosyl-transferase. Accordingly, inhibitors of purine nucleoside phosphorylase-mediated salvage fail to inhibit nucleoside-induced immunostimulation selectively. Examination of the metabolism of 8BrGuo provides no direct evidence that 8BrGuo is phosphorylated by B lymphocytes. Direct enzymatic phosphorylation does not seem to be essential to the mechanism of action of the nucleoside insofar as competitive inhibition of deoxycytidine kinase (an enzyme that directly phosphorylates purines as well as pyrimidines) or of deoxyguanosine kinase fails to inhibit 8BrGuo stimulation selectively. Moreover, studies with synthetic nucleosides in which 3' and/or 5' hydroxyl groups were irreversibly blocked, precluding their phosphorylation, demonstrated that immunobiologic activity can occur in the absence of 3' and/or 5' phosphorylation. Finally, experiments with radiolabeled nucleosides provide no evidence to support the hypothesis that they are incorporated into cellular nucleic acid. These data, together with previous studies, suggest that it is the unmetabolized nucleoside that is active and, as such, is most likely to act in a regulatory capacity.     

Dendritic cells charged with apoptotic tumor cells induce long-lived protective CD4+ and CD8+ T cell immunity against B16 melanoma

Dendritic cells (DCs) are potent APCs and attractive vectors for cancer immunotherapy. Using the B16 melanoma, a poorly immunogenic experimental tumor that expresses low levels of MHC class I products, we investigated whether DCs loaded ex vivo with apoptotic tumor cells could elicit combined CD4(+) and CD8(+) T cell dependent, long term immunity following injection into mice. The bone marrow-derived DCs underwent maturation during overnight coculture with apoptotic melanoma cells. Following injection, DCs migrated to the draining lymph nodes comparably to control DCs at a level corresponding to approximately 0.5% of the injected inoculum. Mice vaccinated with tumor-loaded DCs were protected against an intracutaneous challenge with B16, with 80% of the mice remaining tumor-free 12 wk after challenge. CD4(+) and CD8(+) T cells were efficiently primed in vaccinated animals, as evidenced by IFN-gamma secretion after in vitro stimulation with DCs loaded with apoptotic B16 or DCs pulsed with the naturally expressed melanoma Ag, tyrosinase-related protein 2. In addition, B16 melanoma cells were recognized by immune CD8(+) T cells in vitro, and cytolytic activity against tyrosinase-related protein 2(180-188)-pulsed target cells was observed in vivo. When either CD4(+) or CD8(+) T cells were depleted at the time of challenge, the protection was completely abrogated. Mice receiving a tumor challenge 10 wk after vaccination were also protected, consistent with the induction of tumor-specific memory. Therefore, DCs loaded with cells undergoing apoptotic death can prime melanoma-specific helper and CTLs and provide long term protection against a poorly immunogenic tumor in mice.     

Targeted disruption of SMAD3 results in impaired mucosal immunity and diminished T cell responsiveness to TGF-beta

SMAD3 is one of the intracellular mediators that transduces signals from transforming growth factor-beta (TGF-beta) and activin receptors. We show that SMAD3 mutant mice generated by gene targeting die between 1 and 8 months due to a primary defect in immune function. Symptomatic mice exhibit thymic involution, enlarged lymph nodes, and formation of bacterial abscesses adjacent to mucosal surfaces. Mutant T cells exhibit an activated phenotype in vivo, and are not inhibited by TGF-beta1 in vitro. Mutant neutrophils are also impaired in their chemotactic response toward TGF-beta. Chronic intestinal inflammation is infrequently associated with colonic adenocarcinoma in mice older than 6 months of age. These data suggest that SMAD3 has an important role in TGF-beta-mediated regulation of T cell activation and mucosal immunity, and that the loss of these functions is responsible for chronic infection and the lethality of Smad3-null mice.     

Active transformation of tolerogenic to immunogenic signals in T and B cells by 8-bromoguanosine

The injection of deaggregated human gamma-globulin (DHGG) into A/J mice results in the establishment of a state of unresponsiveness to subsequent challenge with immunogenic aggregated human gamma-globulin (AHGG). Administration of the B cell activator 8-bromoguanosine (8BrGuo) 3 hr after administration of DHGG converts the tolerogen to an immunogen and results in an antibody response of even greater magnitude than the primary response elicited by AHGG alone. Adoptive transfer studies with separated populations of T and B cells demonstrated that although transformation of the tolerogenic signal to an immunogenic signal involves effects of 8BrGuo on both T cells and B cells, the major effect appears to be activation of antigen-specific T cells that would otherwise become tolerant. Modulation of T cell tolerance could conceivably be mediated either by direct or indirect mechanisms. Interestingly, optimal responsiveness of B cells from animals treated with DHGG and 8BrGuo is not a T cell-independent event, but requires antigen-reactive T cells. 8BrGuo is not able to override unresponsiveness when given 10 to 20 days after tolerance induction, at a time point when both T and B lymphocytes are tolerant. However, when given at day 60, when T cells (but not B cells) remain tolerant to this antigen, the nucleoside is able to terminate the tolerant state prematurely, possibly by providing an alternate T helper-like signal directly to B cells or by recruiting nonspecific functional T helper cells.     

T4 cell activation by immobilized phytohemagglutinin: differential capacity to induce IL-2 responsiveness and IL-2 production

Soluble mitogens, such as PHA induce accessory cell (AC)-dependent T cell proliferation. One function of the AC is to create a stimulatory matrix. Therefore, experiments were carried out to determine whether PHA immobilized onto microtiter plates could stimulate T cells in the absence of AC. Peripheral blood T4 cells were cultured under limiting dilution conditions with either soluble or immobilized PHA with or without rIL-1 beta, rIL-2, r-TNF-alpha, an anti-CD28 mAb (9.3), or irradiated EBV-transformed B cells as AC. The frequency of proliferating T4 cells was assessed by examining wells microscopically, and the frequency of T4 cells producing IL-2 was assessed by examining the ability of supernatants to support CTLL-2 proliferation. The percentage of T4 cells growing and producing IL-2 was determined by a maximum likelihood procedure. Immobilized, but not soluble, PHA induced a mean of 20.0 +/- 2.6% of T4 cells to grow in the complete absence of AC in medium supplemented with rIL-2. Whereas rIL-1 beta, rTNF-alpha, and 9.3 were unable to support T4 cell growth in the absence of rIL-2, each enhanced the percentage of T4 cells responding to immobilized PHA in the presence of rIL-2. In contrast, both soluble and immobilized PHA were unable to induce T4 cell IL-2 production in the absence of AC, even when cultures were supplemented with rIL-1 beta or 9.3. In the presence of AC, a small percentage of T4 cells (5.4 to 11.7%) was stimulated to produce detectable amounts of IL-2 by either immobilized or soluble PHA. Moreover, in the presence of AC, a very small population (approximately 1%) of PHA-stimulated T4 cells proliferated without supplemental rIL-2. The data indicate that a matrix of immobilized PHA is sufficient for some T4 cells to be activated to respond to IL-2, whereas others require additional signals provided by rIL-1 beta, rTNF alpha, 9.3, or AC. In contrast, neither soluble nor immobilized PHA is sufficient to induce T cell IL-2 production. This response requires signals provided by intact AC.     

Ageing, autoimmunity and arthritis: Senescence of the B cell compartment – implications for humoral immunity

Immunosenescence is associated with a decline in both T and B lymphocyte function. Although aged individuals have normal numbers of B cells in the periphery and are capable of mounting robust humoral responses, the antibodies produced are generally of lower affinity and are less protective than those produced by young animals. Here we review multiple studies that address the mechanisms that contribute to this decline. Taken together, these studies suggest that age-associated loss of the ability to generate protective humoral immunity results in part from reduced B lymphopoiesis. As the output of new, na?ve B cells declines, homeostatic pressures presumably force the filling of the peripheral B cell pool by long-lived antigen-experienced cells. Because the antibody repertoire of these cells is restricted by previous antigenic experience, they make poor quality responses to new immunologic insults.     

Ageing, autoimmunity and arthritis: senescence of the B cell compartment - implications for humoral immunity

Immunosenescence is associated with a decline in both T and B lymphocyte function. Although aged individuals have normal numbers of B cells in the periphery and are capable of mounting robust humoral responses, the antibodies produced are generally of lower affinity and are less protective than those produced by young animals. Here we review multiple studies that address the mechanisms that contribute to this decline. Taken together, these studies suggest that age-associated loss of the ability to generate protective humoral immunity results in part from reduced B lymphopoiesis. As the output of new, na?ve B cells declines, homeostatic pressures presumably force the filling of the peripheral B cell pool by long-lived antigen-experienced cells. Because the antibody repertoire of these cells is restricted by previous antigenic experience, they make poor quality responses to new immunologic insults.     

Antigen chemically coupled to the surface of liposomes are cross-presented to CD8+ T cells and induce potent antitumor immunity

We have previously demonstrated that liposomes with differential lipid components display differential adjuvant effects when Ags are chemically coupled to their surfaces. In the present study, Ag presentation of liposome-coupled OVA was investigated in vitro, and it was found that OVA coupled to liposomes made using unsaturated fatty acid was presented to both CD4+ and CD8+ T cells, whereas OVA coupled to liposomes made using saturated fatty acid was presented only to CD4+ T cells. Confocal laser scanning microscopic analysis demonstrated that a portion of the OVA coupled to liposomes made using unsaturated, but not saturated fatty acid, received processing beyond the MHC class II compartment, suggesting that the degradation of OVA might occur in the cytosol, and that the peptides generated in this manner would be presented to CD8+ T cells via MHC class I. The ability to induce cross-presentation of an Ag coupled to liposomes consisting of unsaturated fatty acid was further confirmed by in vivo induction of CTL and by the induction of tumor eradication in mice; E.G7 tumors in mice that received combined inoculation with OVA(257-264)-liposome conjugates, CpG, and anti-IL-10 mAbs were completely eradicated. In those mice, the frequency of CD8+ T cells reactive with OVA(257-264) peptides in the context of H-2K(b) was significantly increased. These results suggested that, by choosing lipid components for liposomes, surface-coupled liposomal Ags might be applicable for the development of tumor vaccines to present tumor Ags to APCs and induce antitumor responses.     

Regulation of human B cell responsiveness by CD8+ T cells: differential effects of stimulation with monoclonal antibodies to CD3 and pokeweed mitogen

Previous studies have shown that human CD8-positive T cells activated by immobilized mAb to the CD3 complex have the capacity to support the generation of Ig secreting cells (ISC). The experiments reported here were undertaken to examine the nature of CD8+ T cell helper function in greater detail. CD8+ T cells that had been treated with mitomycin C (CD8+ mito) and stimulated by immobilized mAb to CD3 (64.1) provided help for the generation of ISC from resting B cells. By contrast, CD8+ mito did not support the generation of ISC in cultures stimulated by pokeweed mitogen (PWM). This could not be explained by differences in the production of IL2, since PWM and anti-CD3 induced comparable amounts of IL2 from CD8+ mito. In anti-CD3-stimulated cultures, CD8+ mito supported the generation of larger numbers of ISC when B cells were also activated with Staphylococcus aureus (SA). By contrast, in PWM-stimulated cultures, CD8+ mito did not provide help for SA-activated B cells. Rather, PWM-stimulated CD8+ mito appeared to suppress the generation of ISC induced by PWM-activated CD4+ mito or by SA + IL2, whereas anti-CD3-stimulated CD8+ mito did not. Only control CD8+ T cells, which were able to proliferate, exerted suppressive effects in anti-CD3-stimulated cultures. Examination of the functional capacities of a battery of CD8+ T cell clones indicated that the same clonal population of CD8+ cells could provide help or suppress responses when stimulated with anti-CD3 or PWM, respectively. The functional activities of CD8+ clones differed from those of fresh CD8+ cells. Thus, anti-CD3-stimulated CD8+ clones provided help for B cells regardless of whether they were treated with mitomycin C. Moreover, PWM stimulated suppression by CD8+ clones was abrogated by treating the clones with radiation or mitomycin C. These results indicate that helper T cell function is not limited to the CD4+ T cell population, but that help can also be provided by appropriately stimulated CD8+ T cells. Taken together, these results indicate that CD8+ T cells are not limited in their capacity to regulate B cell responses, but rather can provide positive or negative influences depending on the nature of the activating stimulus.     

Interplay between T cell receptor binding kinetics and the level of cognate peptide presented by major histocompatibility complexes governs CD8+ T cell responsiveness

Through a rational design approach, we generated a panel of HLA-A*0201/NY-ESO-1(157-165)-specific T cell receptors (TCR) with increasing affinities of up to 150-fold from the wild-type TCR. Using these TCR variants which extend just beyond the natural affinity range, along with an extreme supraphysiologic one having 1400-fold enhanced affinity, and a low-binding one, we sought to determine the effect of TCR binding properties along with cognate peptide concentration on CD8(+) T cell responsiveness. Major histocompatibility complexes (MHC) expressed on the surface of various antigen presenting cells were peptide-pulsed and used to stimulate human CD8(+) T cells expressing the different TCR via lentiviral transduction. At intermediate peptide concentration we measured maximum cytokine/chemokine secretion, cytotoxicity, and Ca(2+) flux for CD8(+) T cells expressing TCR within a dissociation constant (K(D)) range of ～1-5 μM. Under these same conditions there was a gradual attenuation in activity for supraphysiologic affinity TCR with K(D) < ～1 μM, irrespective of CD8 co-engagement and of half-life (t(1/2) = ln 2/k(off)) values. With increased peptide concentration, however, the activity levels of CD8(+) T cells expressing supraphysiologic affinity TCR were gradually restored. Together our data support the productive hit rate model of T cell activation arguing that it is not the absolute number of TCR/pMHC complexes formed at equilibrium, but rather their productive turnover, that controls levels of biological activity. Our findings have important implications for various immunotherapies under development such as adoptive cell transfer of TCR-engineered CD8(+) T cells, as well as for peptide vaccination strategies.