Cytokine production by mature and immature CD4-CD8- T cells. Alpha beta-T cell receptor+ CD4-CD8- T cells produce IL-4.

This study follows our previous investigation describing the production of four cytokines (IL-2, IL-4, IFN-gamma, and TNF-alpha) by subsets of thymocytes defined by the expression of CD3, 4, 8, and 25. Here we investigate in greater detail subpopulations of CD4-CD8- double negative (DN) thymocytes. First we divided immature CD25-CD4-CD8-CD3- (CD25- triple negative) (TN) thymocytes into CD44+ and CD44- subsets. The CD44+ population includes very immature precursor T cells and produced high titers of IL-2, TNF-alpha, and IFN-gamma upon activation with calcium ionophore and phorbol ester. In contrast, the CD44- subset of CD25- TN thymocytes did not produce any of the cytokines studied under similar activation conditions. This observation indicates that the latter subset, which differentiates spontaneously in vitro into CD4+CD8+, already resembles CD4+CD8+ thymocytes (which do not produce any of the tested cytokines). We also subdivided the more mature CD3+ DN thymocytes into TCR-alpha beta- and TCR-gamma delta-bearing subsets. These cells produced cytokines upon activation with solid phase anti-CD3 mAb. gamma delta TCR+ DN thymocytes produced IL-2, IFN-gamma and TNF-alpha, whereas alpha beta TCR+ DN thymocytes produced IL-4, IFN-gamma, and TNF-alpha but not IL-2. We then studied alpha beta TCR+ DN T cells isolated from the spleen and found a similar cytokine production profile. Furthermore, splenic alpha beta TCR+ DN cells showed a TCR V beta gene expression profile reminiscent of alpha beta TCR+ DN thymocytes (predominant use of V beta 8.2). These observations suggest that at least some alpha beta TCR+ DN splenocytes are derived from alpha beta TCR+ DN thymocytes and also raises the possibility that these cells may play a role in the development of Th2 responses through their production of IL-4.     

Cutting edge: CD8+CD122+ regulatory T cells produce IL-10 to suppress IFN-gamma production and proliferation of CD8+ T cells

We recently identified CD8+CD122+ regulatory T cells that directly control CD8+ and CD4+ cells without intervention of APCs. In this study, we investigated the effector mechanism of CD8+CD122+ regulatory T cells by using an in vitro regulation system. The profile of cytokine expression revealed that IL-10 was predominantly produced by CD8+CD122+ cells, whereas other cytokines were similarly expressed in CD8+CD122+ cells and CD8+CD122- cells. Suppression of both proliferation and IFN-gamma production by CD8+CD122- cells by CD8+CD122+ cells was blocked by adding anti-IL-10 Ab to the culture but not by adding anti-TGF-beta Ab. When IL-10 was removed from the conditioned medium from CD8+CD122+ cells, the conditioned medium no longer showed regulatory activity. Finally, CD8+CD122+ cells from IL-10-deficient mice had no regulatory activity in vitro and reduced regulatory activity in vivo. Our results clearly indicate that IL-10 is produced by CD8+CD122+ cells and mediates the regulatory activity of these cells.     

Synergistic role of CD4+ and CD8+ T lymphocytes in IFN-gamma production and protective immunity induced by an attenuated Toxoplasma gondii vaccine.

BALB/c mice vaccinated with a temperature-sensitive mutant (TS-4) of Toxoplasma gondii develop complete resistance to lethal challenge with a highly virulent toxoplasma strain (RH). This immunity is known to be dependent on IFN-gamma synthesis. In vitro and in vivo T cell depletions were performed in order to identify the subsets responsible for both protective immunity and IFN-gamma production. When stimulated with crude tachyzoite Ag in vitro, CD4+ cells from vaccinated mice produced high levels of TH1 cytokines (IL-2 and IFN-gamma) but not TH2 cytokines (IL-4 and IL-5). CD8+ cells, in contrast, produced less IFN-gamma and no detectable IL-2. Nevertheless, they could be induced to synthesize IFN-gamma when exposed in culture to exogenous IL-2. In vivo treatment with anti-CD4 plus anti-CD8 or anti-IFN-gamma antibodies during challenge infection completely abrogated resistance to T. gondii. In contrast, treatment with anti-CD4 alone failed to reduce immunity, whereas anti-CD8 treatment partially decreased vaccine-induced resistance. These results suggest that although IFN-gamma and IL-2-producing CD4+ lymphocytes are induced by vaccination, IFN-gamma-producing CD8+ T cells are the major effectors of immunity in vivo. Nevertheless, CD4+ lymphocytes appear to play a synergistic role in vaccine-induced immunity, probably through the augmentation of IFN-gamma synthesis by the CD8+ effector cells. This hypothesis is supported by the observation that when giving during vaccination, as opposed to after challenge, anti-CD4 antibodies are capable of blocking protective immunity.     

Novel function for intestinal intraepithelial lymphocytes. Murine CD3+, gamma/delta TCR+ T cells produce IFN-gamma and IL-5.

Intestinal intraepithelial lymphocytes (IEL) from mice are greater than 80% CD3+ T cells and could be separated into four subsets according to expression of CD4 and CD8. In our studies designed to assess the functions of IEL, namely, cytokine production, it was important to initially characterize the various subsets of T cells that reside in IEL. The major subset was CD4-, CD8+ (75% of CD3+ T cells), which contained approximately 45 to 65% gamma/delta TCR+ and 35 to 45% alpha/beta TCR+ T cells. Approximately 7.5% of IEL T cells were CD4-, CD8- (double negative) and gamma/delta+ population. On the other hand, CD4+, CD8+ (double positive) and CD4+, CD8- fractions represented 10% and 7.5% of CD3+ T cells, respectively, which were all alpha/beta TCR+. Inasmuch as CD3+, CD4-, CD8+ T cells are a major subset of IEL which contain both gamma/delta TCR or alpha/beta TCR-bearing cells, the present study was focused on the capability of this subset of IEL T cells to produce the cytokines IFN-gamma and IL-5. Both gamma/delta TCR+ and alpha/beta TCR+ IEL spontaneously produced IFN-gamma and IL-5, although higher frequencies of cytokine spot-forming cells were associated with the alpha/beta TCR+ subset. Approximately 30% of CD8+, gamma/delta TCR+ cells produced both cytokines, whereas approximately 90% of alpha/beta TCR+ T cells produced either IFN-gamma or IL-5. Both gamma/delta TCR+ and alpha/beta TCR+ IEL possessed large quantities of cytokine-specific mRNA, clearly showing that these IEL were programmed for cytokine production. When IEL were activated with anti-gamma/delta or anti-CD8 antibodies, higher numbers of IFN-gamma and IL-5 spot-forming cells were noted. The present study has provided direct evidence that a major function of IEL involves cytokine production, and this is the first evidence that gamma/delta TCR+ cells in IEL possess the capability of producing both IL-5 and IFN-gamma.     

Development of CD8+ effector T cells is differentially regulated by IL-18 and IL-12

We investigated the effects of IL-18 on the development of CD8+ effector T cells in DBA/2 anti-BDF1 whole spleen cell MLC and compared the results with those of IL-12. Addition of IL-18 to the MLC resulted in a twofold increase in CD8/CD4 ratios compared with the control cultures when cells were expanded in IL-2-containing medium following MLC. Purified CD8+ T cells recovered from the IL-18-stimulated MLC produced 20- to 30-fold more IFN-gamma after secondary stimulation with C57BL/6 spleen cells or anti-CD3 mAb, and exhibited strong allospecific CTL activity. Neither IL-18 nor IL-18-supplemented culture supernatants from DBA/2 anti-BDF1 MLC induced type I CD8+ effector T cells when purified CD8+ T cells were used as responder cells in primary MLC. Furthermore, CD4+ T cell depletion from the responder cells abrogated the IL-18-induced increase in secondary IFN-gamma production by CD8+ T cells, suggesting that IL-18-induced type I effector CD8+ T cell development was CD4+ T cell dependent. In marked contrast, adding IL-12 to primary MLC decreased CD8/CD4 ratios by 50% and suppressed secondary IFN-gamma production and CTL activity by CD8+ T cells regardless of concentration, whereas Th1 development was promoted by IL-12. Moreover, both IL-12 and IL-18 efficiently induced type I CD8+ effector T cells in C57BL/6 anti-BDF1 MLC. These findings show that IL-18 plays an important role in the generation of type I CD8+ effector T cells, and further suggest that functional maturation of CD8+ T cells is differentially regulated by IL-18 and IL-12.     

T cell regulation of human B cell proliferation and differentiation. Regulatory influences of CD45R+ and CD45R- T4 cell subsets

The immunoregulatory functions of human T4 cell subpopulations defined by mAb to the CD45R molecule (2H4) were examined. Both CD45R- and CD45R+ T4 cells that had been treated with mitomycin C (CD45R- and CD45R+ T4-mito) provided help for the generation of Ig-secreting cells (ISC) in cultures stimulated by PWM or by immobilized mAb to CD3 (64.1). IL-2 enhanced the generation of ISC in PWM-stimulated cultures and in anti-CD3-stimulated cultures containing CD45R+ T4-mito. The generation of ISC was maximal in cultures containing anti-CD3-activated CD45R- T4-mito and was not increased by IL-2. By contrast, CD45R+ T4 cells that had not been treated with mitomycin C suppressed B cell responses in cultures stimulated with PWM or anti-CD3, whereas CD45R- T4 cells suppressed the generation of ISC only in cultures stimulated with anti-CD3. IL-2 enhanced suppression by anti-CD3, but not PWM, activated CD45R- T4 cells. Suppression by CD45R+ T4 cells was maximal and not increased by IL-2. CD45R+ T4-mito were more effective suppressor-inducers in PWM-stimulated cultures, promoting the differentiation of suppressor-effector cells from CD8+ T cells. However, both CD45R+ and CD45R- T4-mito exerted comparable suppressor-inducer function in anti-CD3-stimulated cultures. Moreover, in anti-CD3-stimulated cultures, T8 cells could function as both suppressor-effector cells and suppressor-inducer cells. One of the functions of suppressor-inducer cells in this system appeared to involve the production of IL-2. Thus, the addition of IL-2 facilitated the induction of suppressor-effector T8 cells by CD45R- T4-mito in PWM-stimulated cultures. Although IL-2 production by the T cell subsets varied widely depending on the nature of the stimulus, these differences could not entirely explain their capacity to function as helper cells, suppressor-effector cells or suppressor-inducer cells. These results indicate that both CD45R+ and CD45R- T4 cells can help or suppress B cell responses, as well as induce suppressor-effector T8 cells. Moreover, suppressor-inducer function of T cells is not limited to the T4 cell population, but rather can also be accomplished by T8 cells. The results indicate that both T4 cell subsets and T8 cells exert multiple regulatory effects on human B cell function, with the nature of the activating stimulus playing a major role in determining the functional capacity of various T cell subsets.     

Augmentation of effector CD8+ T cell generation with enhanced granzyme B expression by IL-27

IL-27 is a novel IL-12 family member that plays a role in the early regulation of Th1 initiation. We have recently demonstrated that IL-27 has a potent antitumor activity, which is mainly mediated through CD8+ T cells, and also has an adjuvant activity to induce epitope-specific CTL in vivo. In this study, we further investigated the in vitro effect of IL-27 on CD8+ T cells of mouse spleen cells. In a manner similar to CD4+ T cells, IL-27 activated STAT1, -2, -3, -4, and -5, and augmented the expression of T-bet, IL-12Rbeta2, and granzyme B, and slightly that of perforin in naive CD8+ T cells stimulated with anti-CD3. IL-27 induced synergistic IFN-gamma production with IL-12 and proliferation of naive CD8+ T cells. Moreover, IL-27 enhanced proliferation of CD4+ T cell-depleted spleen cells stimulated by allogeneic spleen cells and augmented the generation of CTL. In STAT1-deficient naive CD8+ T cells, IL-27-induced proliferation was not reduced, but synergistic IFN-gamma production with IL-12 was diminished with decreased expression of T-bet, IL-12Rbeta2, granzyme B, and perforin. In T-bet-deficient naive CD8+ T cells, IL-27-induced proliferation was hardly reduced, but synergistic IFN-gamma production with IL-12 was diminished with decreased expression of IL-12Rbeta2, granzyme B, and perforin. However, IL-27 still augmented the generation of CTL from T-bet-deficient CD4+ T cell-depleted spleen cells stimulated by allogeneic spleen cells with increased granzyme B expression. These results suggest that IL-27 directly acts on naive CD8+ T cells in T-bet-dependent and -independent manners and augments generation of CTL with enhanced granzyme B expression.     

Differential responses of invariant V alpha 24J alpha Q T cells and MHC class II-restricted CD4+ T cells to dexamethasone.

NK T cells are a T cell subset in the human that express an invariant alpha-chain (V alpha 24invt T cells). Because of the well-described immunomodulation by glucocorticoids on activation-induced cell death (AICD), the effects of dexamethasone and anti-CD3 stimulation on V alpha 24invt T cell clones and CD4+ T cell clones were investigated. Dexamethasone significantly enhanced anti-CD3-mediated proliferation of V alpha 24invt T cells, whereas CD4+ T cells were inhibited. Addition of neutralizing IL-2 Ab partially abrogated dexamethasone-induced potentiation of V alpha 24invt T cell proliferation, indicating a role for autocrine IL-2 production in corticosteroid-mediated proliferative augmentation. Dexamethasone treatment of anti-CD3-stimulated V alpha 24invt T cells did not synergize with anti-Fas blockade in enhancing proliferation or preventing AICD. The V alpha 24invt T cell response to dexamethasone was dependent on the TCR signal strength. In the presence of dexamethasone, lower doses of anti-CD3 inhibited proliferation of V alpha 24invt T cells and CD4+ T cells; at higher doses of anti-CD3, which caused inhibition of CD4+ T cells, the V alpha 24invt T cell clones proliferated and were rescued from AICD. These results demonstrate significant differences in TCR signal strength required between V alpha 24invt T cells and CD4+ cells, and suggest important immunomodulatory consequences for endogenous and exogenous corticosteroids in immune responses.     

Frequency analysis of lymphokine-secreting CD4+ and CD8+ T cells activated in a graft-versus-host reaction

Lymphokine secretion by in vivo-activated T cells was analyzed at the population and single-cell levels in lymphocytes from mice undergoing an acute allogeneic graft-vs-host reaction (GVHR). Three observations were made. First, constitutive lymphokine production by these cells was very low but could be dramatically up-regulated by TCR ligation. Thus, even when harvested at the peak of the GVHR, fewer than 0.1% of lymphocytes secreted detectable granulocyte-macrophage (GM)-CSF, IFN-gamma, or IL-3 in the first 24 h in vitro, and average production of these lymphokines in bulk cultures was less than 10(-5) U/cell. However, when cultured for 24 h with anti-CD3 antibody under conditions which activated less than 0.1% of normal cells, about 30% of GVHR T cells secreted GM-CSF, IFN-gamma, and/or IL-3, and average production levels were increased by 10(3)- to 10(4)-fold. Together with evidence that host alloantigen-induced lymphokine secretion was 10 to 100 times lower than the anti-CD3 response, these data suggest that physiologic lymphokine synthesis by most T cells is low (less than 10(-18) mol of IL-3 per cell) but can be raised above the threshold of detection by TCR cross-linking. Second, individual GVHR lymphocytes varied markedly in their total and relative production of different lymphokines in response to anti-CD3 stimulation, with some cells secreting IL-3 alone, some secreting IL-3 accompanied by other lymphokines (GM-CSF and/or IFN-gamma), and some secreting other lymphokines without detectable IL-3. Finally, both CD4+ and CD8+ T cells from GVHR mice responded to anti-CD3 antibody by secreting IL-3 and other lymphokines: purified CD4+ cells contained an average of 16% and CD8+ cells an average of 10% anti-CD3-inducible lymphokine-secreting cells. By contrast, only 2 to 3% of cells of either subset formed clones in cultures with host allogeneic cells and IL-2, suggesting that clonogenic alloreactive cells were a minority of the T cells activated in the GVHR.     

4-1BB ligand induces cell division, sustains survival, and enhances effector function of CD4 and CD8 T cells with similar efficacy

A costimulatory member of the TNFR family, 4-1BB, is expressed on activated T cells. Although some reports have suggested that 4-1BB is primarily involved in CD8 T cell activation, in this report we demonstrate that both CD4 and CD8 T cells respond to 4-1BB ligand (4-1BBL) with similar efficacy. CD4 and CD8 TCR transgenic T cells up-regulate 4-1BB, OX40, and CD27 and respond to 4-1BBL-mediated costimulation during a primary response to peptide Ag. 4-1BBL enhanced proliferation, cytokine production, and CTL effector function of TCR transgenic T cells. To compare CD4 vs CD8 responses to 4-1BBL under similar conditions of antigenic stimulation, we performed MLRs with purified CD4 or CD8 responders from CD28(+/+) and CD28(-/-) mice. We found that CD8 T cells produced IL-2 and IFN-gamma in a 4-1BBL-dependent manner, whereas under the same conditions the CD4 T cells produced IL-2 and IL-4. 4-1BBL promoted survival of CD4 and CD8 T cells, particularly at late stages of the MLR. CD4 and CD8 T cells both responded to anti-CD3 plus s4-1BBL with a similar cytokine profile as observed in the MLR. CD4 and CD8 T cells exhibited enhanced proliferation and earlier cell division when stimulated with anti-CD3 plus anti-CD28 compared with anti-CD3 plus 4-1BBL, and both subsets responded comparably to anti-CD3 plus 4-1BBL. These data support the idea that CD28 plays a primary role in initial T cell expansion, whereas 4-1BB/4-1BBL sustains both CD4 and CD8 T cell responses, as well as enhances cell division and T cell effector function.     

Testosterone acts directly on CD4+ T lymphocytes to increase IL-10 production

Males are less susceptible than females to experimental autoimmune encephalomyelitis and many other autoimmune diseases. Gender differences in cytokine production have been observed in splenocytes of experimental autoimmune encephalomyelitis mice stimulated with myelin proteins and may underlie gender differences in susceptibility. As these differences should not be limited to responses specific for myelin proteins, gender differences in cytokine production upon stimulation with Ab to CD3 were examined, and the mechanisms were delineated. Splenocytes from male mice stimulated with Ab to CD3 produced more IL-10 and IL-4 and less IL-12 than those from female mice. Furthermore, splenocytes from dihydrotestosterone (DHT)-treated female mice produced more IL-10 and less IL-12 than those from placebo-treated female mice, whereas there was no difference in IL-4. IL-12 knockout mice were then used to determine whether changes in IL-10 production were mediated directly by testosterone vs indirectly by changes in IL-12. The results of these experiments favored the first hypothesis, because DHT treatment of female IL-12 knockout mice increased IL-10 production. To begin to delineate the mechanism by which DHT may be acting, the cellular source of IL-10 was determined. At both the RNA and protein levels, IL-10 was produced primarily by CD4+ T lymphocytes. CD4+ T lymphocytes were then shown to express the androgen receptor, raising the possibility that testosterone acts directly on CD4+ T lymphocytes to increase IL-10 production. In vitro experiments demonstrated increased IL-10 production following treatment of CD4+ T lymphocytes with DHT. Thus, testosterone can act directly via androgen receptors on CD4+ T lymphocytes to increase IL-10 gene expression.     

Induction of high level IL-2 production in CD4+8- T helper lymphocytes requires post-thymic development.

Triggering of the CD3:TCR complex by optimal concentrations of anti-CD3, anti-TCR beta-chain, and allogeneic stimulator cells induced dramatically higher levels (fivefold for anti-CD3, greater than 10-fold for anti-TCR beta-chain, 84-fold for alloantigen) of IL-2 production in spleen CD4+8- T cells than their thymic counterparts, despite comparable levels of CD3 and TCR beta-chain expression. The nature of the reduced IL-2 production was examined by analysis of anti-CD3-induced IL-2 production at the single cell level. The frequency of IL-2-producing cells in spleen CD4+8- T cells (40.0%) was approximately threefold that of thymus CD4+8- T cells (14.5%). Furthermore, the average IL-2 levels among positive IL-2 producers was also approximately threefold higher in spleen CD4+8- T cells than their thymic counterparts. Adoptive transfer of purified Thy-1.2+ CD4+8- T cells into Thy-1.1-congenic hosts provided a physiologic and histocompatible system that enabled identification of transferred donor (Thy-1.2+) among a sea of host (Thy-1.2-) CD4+ T cells, whose immune function with respect to IL-2 inducibility was examined after isolation by electronic cell sorting. Donor CD4+ T cells thus isolated from host spleen shortly (1 day) after i.v. transfer of thymus CD4+8- T cells were similar to freshly isolated thymus CD4+8- T cells in that they both produced little IL-2 in response to anti-CD3. However, by day 3 post-transfer, IL-2 production by donor CD4+8- T cells had more than doubled and by day 8, they produced IL-2 levels comparable to those of host spleen CD4+8- T cells. A similar acquisition of high level IL-2 inducibility in thymus CD4+8- T cells upon i.v. transfer into Thy-1.1-congenic hosts was also observed using allogeneic cells as the stimulus of IL-2 production. When thymus CD4+8- T cells were intra-thymically transferred into Thy-1.1-congenic hosts, those donor cells that emigrated to the periphery became high IL-2 producers in a time-dependent manner, whereas those that remained inside the thymus showed no signs of up-regulation in IL-2 inducibility. Intrathymic transfer of CD4-8- thymocytes revealed that the most recent thymic emigrant CD4+8- T cells contained few IL-2-producing cells and were not functionally mature with respect to high level IL-2 inducibility.     

Analysis of the functional capabilities of CD3+CD4-CD8- and CD3+CD4+CD8+ human T cell clones

The functional capabilities of human peripheral blood CD3+CD4-CD8- and CD3+CD4+CD8+ T cell clones were examined. The clones were generated by culturing purified populations of CD3+CD4-CD8- and CD3+CD4+CD8+ T cells at limiting dilution (0.3 cell/well) in the presence of PHA, rIL-2, and irradiated PBMC as feeders. Twelve CD3+CD4-CD8- and 5 CD3+CD4+CD8+ clones were generated. Clonality was documented by analyzing TCR gamma- and beta-chain rearrangement patterns. All CD3+CD4-CD8- clones were stained by the TCR-delta 1 mAb that identifies a framework epitope of the TCR delta-chain, but not by mAb WT31 that identifies the TCR-alpha beta on mature T cells. In contrast, the CD3+CD4+CD8+ clones were all stained by WT31 and not by TCR-delta 1. All 17 clones were screened for various functional activities. Each secreted IL-2, IFN-gamma, and lymphotoxin/TNF-like factors when stimulated with immobilized mAb to CD3 (64.1), albeit in varying quantities. These clones secreted far less IL-2 and IFN-gamma than CD3+CD4+CD8- or CD3+CD4-CD8+ alpha beta expressing clones, but comparable amounts of lymphotoxin/TNF. All clones also functioned as MHC-unrestricted cytotoxic cells. This activity was comparable to that mediated by the CD3+CD4+CD8- or CD3+CD4-CD8+ alpha beta clones. Nine of 12 CD3+CD4-CD8- and 4 of 5 CD3+CD4+CD8+ clones were able to support B cell differentiation when activated by immobilized anti-CD3, but usually not as effectively as the CD3+CD4+CD8- or CD3+CD4-CD8+ alpha beta clones. The differences in the functional capabilities of the various clones could not be accounted for by alterations in the signaling capacity of the CD3 molecular complex as mAb to CD3 induced comparable increases in intracellular free calcium in each clone examined. When clones were stimulated with PWM, each suppressed B cell differentiation supported by mitomycin C-treated fresh CD4+ T lymphocytes. Suppression was dependent on the number of clone cells added to culture, but could be observed with as few as 12,500 cells per microtiter well. Phenotypic analysis of the clones revealed that all expressed CD29, CD11b, and the NKH1 surface Ag. These results demonstrate that the CD3+CD4-CD8- and CD3+CD4+CD8+ T cell clones exhibit many of the functional characteristics of mature T cells, although they produce IL-2 and IFN-gamma and provide help for B cell differentiation less effectively than CD3+CD4+CD8- and CD3+CD4-CD8+ alpha beta T cell clones.     

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.     

Anti-CD3 + IL-2-stimulated murine killer cells. In vitro generation and in vivo antitumor activity

The growth, phenotype, in vitro cytolytic characteristics, and in vivo antitumor activity of murine splenocytes stimulated with anti-murine CD3 mAb in combination with IL-2 as compared with IL-2 alone was investigated. When cultured for 12 days with anti-CD3 mAb + IL-2, murine splenocytes increased 100- to 4000-fold in number compared with only 6- to 20-fold for cultures stimulated with IL-2 alone. Anti-CD3 mAb + IL-2 activated cultures developed high lymphokine-activated killer activity against NK-resistant targets including the P815 mastocytoma cell line and fresh MCA 106 sarcoma. Peak cytotoxicity on a per cell basis developed by day 8 after anti-CD3 mAb + IL-2 activation. A large proportion of the total cytolytic activity of long term anti-CD3 mAb + IL-2-stimulated cultures was related to the presence of anti-CD3 in the assay, indicating enhancement of cytotoxicity by activated CD3+ T cells. Phenotypic analysis indicated that anti-CD3 mAb + IL-2-stimulated cultures contained heterogeneous populations of T cells with increased percentages of both CD4+ and CD8+ phenotypes compared with cultures stimulated with IL-2 alone. Anti-CD3 mAb + IL-2-stimulated cells were tested for their in vivo antitumor activity by using C57BL/6 mice bearing MCA 106 sarcoma pulmonary metastases. IL-2-activated murine killer cells were given in combination with in vivo IL-2 and indomethacin, the latter of which was shown to potentiate the antitumor effect of IL-2. When given on day 5 after tumor inoculation, cell doses as low as 5 x 10(6) anti-CD3 mAb + IL-2-stimulated cells per mouse significantly reduced the number of pulmonary metastases (p less than 0.005). Thus, activation with the combination of anti-CD3 mAb + IL-2 produces rapidly expanding cultures of cytolytic cells with demonstrated in vivo antitumor efficacy.     

CD8+ T cells can be primed in vitro to produce IL-4.

IL-4 production by T lymphocytes from naive mice in response to stimulation by plate-bound anti-CD3 is concentrated among CD4+ T cells. In vitro stimulation of lymph node T cells with anti-CD3 plus IL-2 and IL-4 strikingly increases the frequency of cells that produce IL-4 in response to subsequent stimulation with anti-CD3 plus IL-2. Separation of these primed cell populations into CD4+ and CD8+ T cell by cell sorting reveals that the frequency of IL-4-producing cells in both population is similar. Verification that CD8+ T cells produce IL-4 is provided by the capacity of anti-IL-4 mAb to inhibit the response of the indicator cell line to the growth factor produced by the primed cells and by detection of IL-4 by an IL-4-specific ELISA. The in vitro "priming" of CD8+ T cells to produce IL-4 is not dependent on the presence of CD4+ T cells because highly purified CD8+ T cells can be stimulated to develop into cells capable of producing IL-4 by culture with plate-bound anti-CD3 plus IL-2 and IL-4.     

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.