Sarcoidosis is not associated with a generalized defect in T cell suppressor function

In pulmonary sarcoidosis, the marked expansion of CD4+ (helper/inducer) T cells in the alveolar structures of the lung is maintained by local IL-2 release by activated CD4+ HLA-DR+ T cells without concomitant expansion and activation of CD8+ (suppressor/cytotoxic) T cells, suggesting that sarcoid may be associated with a generalized abnormality of CD8+ T cells. Consistent with this concept, evaluation of the expression of the IL-2R on fresh lung T cells from individuals with active sarcoidosis demonstrated that 7 +/- 1% of sarcoid lung CD4+ T cells are spontaneously expressing the IL-2R compared with only 1 +/- 1% lung CD8+ T cells (p less than 0.01). However, stimulation of purified sarcoid blood CD8+ T cells with the anti-T3/TCR complex mAb OKT3 was followed by the normal expression of IL-2R (p greater than 0.1) and proliferation (p greater than 0.1). In addition, lung sarcoid CD8+ T cells responded to OKT3 similarly to normal lung CD8+ T cells and to autologous blood CD8+ T cells as regards expression of IL-2R (p greater than 0.1) and proliferation (p greater than 0.1). Finally, using CD4+ cells activated with allogenic Ag to induce, in coculture, fresh autologous CD8+ cells to suppress proliferation of fresh autologous CD4+ cells to the same Ag, sarcoid CD8+ T cells suppressed CD4+ cell proliferation in a normal fashion (p greater than 0.1). These results demonstrate that sarcoid CD8+ (suppressor/cytotoxic) T cells are competent to respond to a proliferation signal normally and can be induced to normally suppress CD4+ T cell proliferation to Ag, suggesting that the expansion of activated CD4+ T cells in pulmonary sarcoidosis is not due to a generalized abnormality of CD8+ T cells or of their suppressor T cell function.     

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.     

IL-2-R beta expression and function within resting CD8+ T cells preferentially segregate with the CD45R0+ subset.

Human peripheral blood CD8+ T cells constitutively express a low level of IL-2-R beta chains which were shown in this study to be preferentially carried by the CD45R0+ subset. Such receptors can transduce signals for in vitro IL-2-induced cytolytic function and for the initiation of soluble anti-CD3 and IL-2-induced cell proliferation. Using these stimulation models, a comparison was made between the responsiveness of resting, small CD45R0+ and CD45RA+ subpopulations of CD8+ T cells, both of them being isolated by negative selection and rigorously depleted of monocytes and of IL-2-inducible non-MHC-restricted CTL. Strong proliferation was induced in CD8+/CD45R0+ cells in response to IL-2 and soluble anti-CD3 (each of these stimuli being by itself ineffective), while in contrast, CD8+/CD45RA+ cells manifested, in this system, little reactivity. Accordingly, no conversion to the CD45R0 phenotype occurred in single stained CD45RA+ T cells following their incubation with the stimuli. A similar restriction of reactivity to CD8+/CD45R0+ T cells was observed with respect to IL-2-induced targetable T cell cytotoxicity. The CTL activity induced by IL-2 alone occurred without cell division. In contrast, the additional increase in CTL activity occurring upon the synergistic actions of anti-CD3 mAb and IL-2 coincided with intense cell proliferation, with no generation of LAK activity. The inhibition exerted by anti-IL-2-R beta mAb in the cytolytic and the proliferative activities induced by these stimuli in resting CD8+/CD45R0+ T cells emphasizes the importance of constitutive IL-2-R beta chains in the biology of these cells.     

Modulation by IL-2 of CD70 and CD27 expression on CD8+ T cells: importance for the therapeutic effectiveness of cell transfer immunotherapy

Proper T cell function relies on the integration of signals delivered by Ag, cytokine, and costimulatory receptors. In this study, the interactions between IL-2, CD27, and its ligand CD70 and their effects on human T cell function were examined. Unstimulated CD8(+) T cells expressed relatively low levels of CD70 and high levels of CD27. Incubation in vitro with high doses of IL-2 (3,000 IU/ml) or administration of IL-2 in vivo resulted in substantial up-regulation of CD70 expression and the concomitant loss of cell surface CD27 expression on CD8(+) cells. Withdrawal of IL-2 from activated CD8(+) T cells that had been maintained in IL-2 resulted in a reversal of the expression of these two markers, whereas reciprocal changes were seen following treatment of PBMCs with IL-2. The proliferation observed in cells stimulated with IL-2 primarily occurred in a subset of the CD70(+)CD8(+) T cells that up-regulated IL-2 receptor expression but did not occur in CD70(-)CD8(+) T cells. Blocking CD70 resulted in a significant reduction of T cell proliferation induced by high-dose IL-2, indicating that the interaction of CD70 with CD27 played a direct role in T cell activation mediated by IL-2. Finally, studies conducted on tumor-infiltrating lymphocyte (TIL) samples that were administered to melanoma patients indicated that the size of the pool of CD27(+)CD8(+) T cells in bulk TILs was highly associated (p = 0.004) with the ability of these TILs to mediate tumor regression following adoptive transfer.     

Murine lymphocytes exhibit heterogeneity in their proliferative responsiveness to calcium ionophore.

The calcium ionophore, A23187, when used alone was found to induce proliferation of murine T cells, at concentrations of 0.5-1 mM. This response required the presence of syngeneic splenic adherant cells (SAC) as a source of accessory cells. Interestingly, only CD4+ T cells but not CD8+ T cells or B cells responded to the calcium ionophore by proliferation. The inability of CD8+ T cells or B cells to respond was not related to decreased elevation in the intracellular ionized calcium [Ca2+]i concentration induced by the ionophore, because activated CD4+ T, CD8+ T and B cells all exhibited similar elevation in [Ca2+]i. The inability of CD8+ T cells to respond to calcium ionophore was probably due to insufficient production of autocrine growth factors, such as IL-2, inasmuch as the addition of exogenous IL-2 could completely restore the CD8+ T cell responsiveness. Also, exogenous rIL-1 could partially restore purified T cell response to calcium ionophore, whereas, rIL-6 failed to do so. IL-2, but not IL-4, acted as an autocrine growth factor for T cells responding to the calcium ionophore in the presence of SAC, since, antibodies against IL-2 or IL-2 receptor (IL-2R) but not against IL-4, could inhibit the T cell proliferation. Furthermore, exogenous rIL-2 but not rIL-4 supported the proliferation of T cells to calcium ionophore in the absence of accessory cells. Our results suggest that murine lymphocytes exhibit heterogeneity in their proliferative responsiveness to calcium ionophore and that this may not depend on the early activation signal such as the elevation in [Ca2+]i) induced by the ionophore but may depend on subsequent signals which regulate endogenous growth factor production.     

Stimulation via the CD3 and CD28 molecules induces responsiveness to IL-4 in CD4+CD29+CD45R- memory T lymphocytes

Although both IL-2 and IL-4 can promote the growth of activated T cells, IL-4 appears to selectively promote the growth of those helper/inducer and cytolytic T cells which have been activated via their CD3/TCR complex. The present study examines the participation of CD28 and certain other T cell-surface molecules in inducing T cell responsiveness to IL-4. Purified small high density T cells were cultured in the absence of accessory cells with various soluble anti-human T cell mAb with or without soluble anti-CD3 mAb and their responsiveness to IL-4 was studied. None of the soluble anti-T cell mAb alone was able to induce T cell proliferation in response to IL-4. A combination of soluble anti-CD3 with anti-CD28 mAb but not with mAb directed at the CD2, CD5, CD7, CD11a/CD18, or class I MHC molecules induced T cell proliferation in response to IL-4. Anti-CD2 and anti-CD5 mAb enhanced and anti-CD18 mAb inhibited this anti-CD3 + anti-CD28 mAb-induced T cell response to IL-4. In addition, anti-CD2 in combination with anti-CD3 and anti-CD28 mAb induced modest levels of T cell proliferation even in the absence of exogenous cytokines. IL-1, IL-6, and TNF were each unable to replace either anti-CD3 or anti-CD28 mAb in the induction of T cell responsiveness to IL-4, but both IL-1 and TNF enhanced this response. The anti-CD3 + anti-CD28 mAb-induced response to IL-4 was exhibited only by cells within the CD4+CD29+CD45R- memory T subpopulation, and not by CD8+ or CD4+CD45R+ naive T cells. When individually cross-linked with goat anti-mouse IgG antibody immobilized on plastic surface, only anti-CD3 and anti-CD28 mAb were able to induce T cell proliferation. These results indicate that the CD3 and CD28 molecules play a crucial role in inducing T cell responsiveness to IL-4 and that the CD2, CD5, and CD11a/CD18 molecules influence this process.     

Interleukin 2 and interleukin 10 function synergistically to promote CD8+ T cell cytotoxicity,which is suppressed by regulatory T cells in breast cancer

The precise role of interleukin (IL)-10 in breast cancer is not clear. Previous studies suggested a tumor-promoting role of IL-10 in breast cancer, whereas recent discoveries that IL-10 activated and expanded tumor-resident CD8⁺ T cells challenged the traditional view. Here, we investigated the role of IL-10 in HLA-A2-positive breast cancer patients with Grade III, Stage IIA or IIB in-situ and invasive ductal carcinoma, and compared it with that of IL-2, the canonical CD8⁺ T cell growth factor. We first observed that breast cancer patients presented higher serum levels of IL-2 and IL-10 than healthy controls. Upon prolonged TCR stimulation, peripheral blood CD8⁺ T cells from breast cancer patients tended to undergo apoptosis, which could be prevented by the addition of IL-2 and/or IL-10. The cytotoxicity of TCR-activated CD8⁺ T cells was also enhanced by exogenous IL-2 and/or IL-10. Interestingly, IL-2 and IL-10 demonstrated synergistic effects, since the enhancement in CD8⁺ T cell function when both cytokines were added was greater than the sum of the improvements mediated by each individual cytokine. IL-10 by itself could not promote the proliferation of CD8⁺ T cells but could significantly enhance IL-2-mediated promotion of CD8⁺ T cell proliferation. In addition, the cytotoxicity of tumor-infiltrating CD8⁺ T cells in breast tumor was elevated when both IL-2 and IL-10 were present but not when either one was absent. This synergistic effect was stopped by CD4⁺CD25⁺ regulatory T cells (Treg), which depleted IL-2 in a cell number-dependent manner. Together, these results demonstrated that IL-2 and IL-10 could work synergistically to improve the survival, proliferation, and cytotoxicity of activated CD8⁺ T cells, an effect suppressible by CD4⁺CD25⁺ Treg cells.     

Independent regulation of IFN-gamma and tumor necrosis factor by IL-1 in human T helper cells

The lymphokines IL-2 and IL-4 promoted the growth of human PHA-triggered T cells, but only IL-2 induced the production of IFN-gamma and TNF. The addition of purified monocytes strongly enhanced the production of IFN-gamma in IL-2-stimulated T cell cultures but did not influence the production of TNF or the level of T cell proliferation. The addition of IL-1 to T cells activated by PHA and optimal concentrations of IL-2 resulted in a strong induction of IFN-gamma production but had no influence on TNF production or T cell proliferation. IL-6 did not influence IFN-gamma or TNF production or T cell proliferation induced by PHA-IL-2 and did not modulate IL-1-induced IFN-gamma production. The production of IFN-gamma by CD4+ 45R+ Th cells was strongly enhanced by IL-1, whereas CD8+ T cells were less responsive to IL-1 and CD4+ 45R+ T cells were unresponsive to IL-1. We demonstrate, at the clonal level, that the optimal production of IFN-gamma by human Th cells requires both IL-1 and IL-2, whereas the production of TNF and T cell proliferation are induced by IL-2 alone. We suggest that IL-1 acts as a second signal for IFN-gamma production and that it may have an important function in regulating the pattern of lymphokines produced by T cell subsets during activation.     

Intrinsic IL-21 signaling is critical for CD8 T cell survival and memory formation in response to vaccinia viral infection

CD4 T cell help plays an important role in promoting CD8 T cell immunity to pathogens. In models of infection with vaccinia virus (VV) and Listeria monocytogenes, CD4 T cell help is critical for the survival of activated CD8 T cells during both the primary and memory recall responses. Still unclear, however, is how CD4 T cell help promotes CD8 T cell survival. In this study, we first showed that CD4 T cell help for the CD8 T cell response to VV infection was mediated by IL-21, a cytokine produced predominantly by activated CD4 T cells, and that direct action of IL-21 on CD8 T cells was critical for the VV-specific CD8 T cell response in vivo. We next demonstrated that this intrinsic IL-21 signaling was essential for the survival of activated CD8 T cells and the generation of long-lived memory cells. We further revealed that IL-21 promoted CD8 T cell survival in a mechanism dependent on activation of the STAT1 and STAT3 pathways and subsequent upregulation of the prosurvival molecules Bcl-2 and Bcl-x(L). These results identify a critical role for intrinsic IL-21 signaling in CD8 T cell responses to an acute viral infection in vivo and may help design effective vaccine strategies.     

Pathogen-specific CD8 T cell responses are directly inhibited by IL-10

Regulation of CD8 T cell expansion and contraction is essential for successful immune defense against intracellular pathogens. IL-10 is a regulatory cytokine that can restrict T cell responses by inhibiting APC functions. IL-10, however, can also have direct effects on T cells. Although blockade or genetic deletion of IL-10 enhances T cell-mediated resistance to infections, the extent to which IL-10 limits in vivo APC function or T cell activation/proliferation remains unknown. Herein, we demonstrate that primary and memory CD8 T cell responses following Listeria monocytogenes infection are enhanced by the absence of IL-10. Surface expression of the IL-10R is transiently up-regulated on CD8 T cells following activation, suggesting that activated T cells can respond to IL-10 directly. Consistent with this notion, CD8 T cells lacking IL-10R2 underwent greater expansion than wild-type T cells upon L. monocytogenes infection. The absence of IL-10R2 on APCs, in contrast, did not enhance T cell responses following infection. Our studies demonstrate that IL-10 produced during bacterial infection directly limits expansion of pathogen-specific CD8 T cells and reveal an extrinsic regulatory mechanism that modulates the magnitude of memory T cell responses.     

Disparate in vitro and in vivo requirements for IL-2 during antigen-independent CD8 T cell expansion

Transient TCR stimulation induces multiple rounds of CD8 T cell division without further requirement for Ag. The mechanism driving Ag-independent proliferation, however, remains unclear. In this study, we show that the initial duration of TCR stimulation positively correlates with the number of divisions that CD8 T cells subsequently undergo. We find that increased periods of Ag stimulation result in enhanced CD25 up-regulation and greater IL-2 production by CD8 T cells. Depletion of IL-2 from T cell cultures with specific Abs dramatically impairs programmed proliferation. Consistent with this result, IL-2-deficient T cells undergo markedly attenuated Ag-independent proliferation in vitro. Although IL-2 production by stimulated CD8 T cells appears to be essential for in vitro proliferation, upon transfer into recipient mice, IL-2-deficient CD8 T cells undergo extensive proliferation in vivo after transient stimulation. Furthermore, the extent of in vivo proliferation correlates with the duration of in vitro Ag stimulation. These results indicate that the requirements for autocrine IL-2 production by CD8 T cells differs between in vitro and in vivo conditions and suggests that factors in addition to IL-2 can support Ag-independent CD8 T cell proliferation.     

Different effects of IL-2 addition or antibody crosslinking on T-cell subset stimulation by CD3 antibodies

The effect of exogenous recombinant interleukin-2 (IL-2) or of antibody crosslinking on the activation of human T-cell subsets by IgG2a (OKT3/BMA030), IgG1 (Leu4 and UCHT1), or IgG2b (BMA031) anti-T3 antibodies (CD3) was investigated. In so-called nonresponder cultures as well as in monocyte-depleted cell cultures addition of IL-2 increased the CD3-induced activation and proliferation of T4 and T8 cell subsets. Relatively more T8 than T4 cells were stimulated by antibody binding and IL-2. Crosslinking the cell-bound CD3 antibodies by plastic bound goat anti-mouse antibodies activated both T-cell subsets optimally and increased the IL-2 production of the IgG1-CD3 stimulated cultures. The data show that T cells (T8 greater than T4) can be stimulated by CD3 antibody binding and IL-2, but that crosslinking the cell-bound CD3 antibodies is crucial for optimal T4 cell stimulation and IL-2 production.     

High IFN-gamma production of individual CD8 T lymphocytes is controlled by CD152 (CTLA-4)

CD8 T cell expansion and cytokine production is needed to generate an effective defense against viral invasion of the host. These features of CD8 T lymphocytes are regulated, especially during primary responses, by positive and negative costimulation. We show in this study that surface expression of CD152 is highly up-regulated on activated CD8 T lymphocytes during primary immune responses, suggesting a prominent regulatory role. Indeed, production of the proinflammatory cytokine IFN-gamma, but not TNF-alpha, by CD8 T cells was inhibited by CD152 engagement. The inhibition was regulated independent of proliferation and IL-2 production, but dependent on the quality of the TCR signaling. We show that signals induced by CD152 on activated CD8 T lymphocytes reduce the frequency of IFN-gamma(high)-expressing cells. Our data also show that in activated CD8 T cells, the CD152-mediated inhibition of cytokine production is more pronounced than inhibition of their proliferation.     

Analysis of the mechanisms of T cell-dependent polyclonal activation of human B cells. Induction of human B cell responses by fixed activated T cells.

Coculture of resting human B cells with T cells stimulated with immobilized mAb to the CD3 molecular complex induces polyclonal activation and the production of Ig of all isotypes. The current experiments were carried out to determine the nature of the signals provided to B cells by the anti-CD3-activated T cells. For these experiments, fresh T cells or T cell clones were activated with immobilized mAb to CD3 and then fixed with 1% paraformaldehyde. Upon coculture, the fixed activated T cells or T cell clones induced B cell RNA synthesis and IL-2R expression, but only minimal DNA synthesis and no Ig production. Induction of B cell RNA synthesis by fixed activated T cells was not inhibited by mAb to the alpha-chain of the IL-2R, and was not enhanced by supplementing cultures with IL-2, IL-4, IL-6, or supernatants of mitogen-activated T cells. Upon the addition of IL-2, but not IL-4 or IL-6, to cultures of B cells and fixed activated T cells, sustained proliferation was noted along with the production of Ig. Control fixed T cells or T cell clones did not induce any of these responses. The presence of cycloheximide or cyclosporin A during the activation with anti-CD3 prevented T cells from developing the capacity to provide help for B cells. The use of mAb to a variety of cell surface molecules indicated that several T cell surface molecules including CD11a/CD18, CD44, CD54, and class I MHC molecules are involved in the induction of B cell responses. Among the mAb that inhibited B cell DNA synthesis and/or Ig production, only mAb to CD11a, CD18, or CD54 inhibited initial B cell activation as assessed by RNA synthesis. Even though mAB to CD11a/CD18 inhibited the capacity of fixed activated T cells to induce B cell responses, the finding that fixed activated CD18 deficit clones provided help for B cells indicated that expression of the beta 2 family of integrins by T cells was not necessary. These results indicate that activated T cells acquire the capacity to stimulate B cells polyclonally and induce cytokine responsiveness, proliferation, and Ig production by utilization of a variety of surface molecules. Moreover, these results indicate that the initial activation of the B cell is independent of the metabolic activity of the T cell and the production of cytokines.     

Regulation of human lymphocyte proliferation by a heterodimeric cytokine, IL-12 (cytotoxic lymphocyte maturation factor).

IL-12 is a heterodimeric cytokine that was identified on the basis of its ability to synergize with IL-2 in the induction of cytotoxic effector cells and was originally called cytotoxic lymphocyte maturation factor (CLMF). IL-12 was also found to stimulate the proliferation of PHA-activated lymphoblasts which were greater than 90% CD3+ T cells. In this report we further characterize the effects of IL-12 on lymphocyte proliferation. Studies with purified subpopulations of PHA-activated lymphoblasts and with cloned lines of human T cells indicated that IL-12 caused the proliferation of activated T cells of both the CD4+ and CD8+ subsets. This effect of IL-12 was independent of IL-2 because it was not blocked by antibodies to either IL-2 or IL-2R. The maximum proliferation induced by IL-12 was 31 to 72% of the maximum caused by IL-2; however, IL-12 was active at a lower effective concentration (EC50 = 8.5 +/- 1.3 pM) than IL-2 (EC50 = 52 +/- 8 pM). Combination of suboptimal amounts of IL-12 and IL-2 resulted in additive proliferation, up to the maximum induced by IL-2 alone. IL-12 also caused the proliferation of lymphocytes activated by culture with IL-2 for 6 to 12 days. CD56+ NK cells were among the IL-12-responsive cells in the IL-2-activated lymphocyte population. Unlike IL-2 or IL-7, IL-12 caused little or no proliferation of resting peripheral blood mononuclear cells (PBMC). In this regard, IL-12 was similar to IL-4. However, IL-12 could enhance the proliferation of resting PBMC caused by suboptimal amounts of IL-2, whereas IL-4 inhibited IL-2-induced PBMC proliferation. Thus, IL-12 is a growth factor for activated human T cells and NK cells; however, its spectrum of lymphocyte growth-promoting properties is distinct from that of IL-2, IL-4, or IL-7.     

Monoclonal antibody against the human peripheral lymph node homing receptor homologue (Leu 8) inhibits B cell differentiation but not B cell proliferation.

Previous studies have shown that a subpopulation of circulating human B cells expresses the Leu 8 peripheral lymph node homing receptor homologue and that these B cells are capable of producing Ig in response to staphylococcus A Cowan I (SAC). In the present study the effect of a signal delivered via the Leu 8 molecule (using anti-Leu 8 mAb) on B cells was examined. Initially, it was shown that immobilized anti-Leu 8 suppressed IgM and IgG secretion of B cells activated by SAC + IL-2 but not that by PWM-prestimulated B cells or B cells stimulated with PWM in the presence of CD4+, Leu 8- T cells (a source of helper cells). It was also shown that anti-Leu 8 did not suppress SAC + IL-2-stimulated B cell proliferation or expression of IL-2R alpha-chain or c-myc mRNA in B cells. The addition of T cells, monocytes, purified IL-2, rIL-1, rIL-6, or human B cell growth factor did not overcome the inhibitory effect of anti-Leu 8 on SAC-stimulated B cell Ig production, and the inhibitory effect of anti-Leu 8 was not blocked by anti-TGF-beta. Finally, inhibition of B cell differentiation occurred even when anti-Leu 8 was added up to 72 hrs after initiation of cell culture. Thus, anti-Leu 8 is unique among inhibitors of B cell function in that it can down-regulate immunoglobulin synthesis without affecting B cell proliferation. These findings suggest that a natural ligand for Leu 8 could affect not only homing of B cells, but also B cell differentiation.     

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.     

T cell-dependent activation of B cell proliferation and differentiation by immobilized monoclonal antibodies to CD3

The capacity of mAb directed at the CD3 molecular complex (64.1) to induce T cell-dependent B cell proliferation and differentiation was examined. Coculture of B cells with mitomycin C-treated T4 cells (T4 mito) stimulated by immobilized 64.1 resulted in marked B cell proliferation and Ig-secreting cells (ISC) generation in the absence of any additional stimulation. The magnitude of the B cell responses induced by immobilized 64.1-stimulated T4 mito was far greater than that induced by other stimuli, such as Staphylococcus aureus plus factors produced by mitogen-activated T cells, PWM, or soluble 64.1. The induction of maximal B cell responsiveness required direct contact between activated T cells and responding B cells. Of note, immobilized 64.1 also induced B cell proliferation and ISC generation in the presence of mitomycin C-treated T8 cells. By contrast, immobilized 64.1 stimulated T4 or T8 cells that had not been treated with mitomycin C induced very modest ISC generation and suppressed B cell responses supported by T4 mito even in the presence of exogenous IL-2 or factors produced by mitogen-activated T cells. The interactions between T and B cells in these cultures not only induced B cell responses, but also enhanced the production of IL-2 by activated T cells. Increased IL-2 production was facilitated when culture conditions afforded the opportunity for contact between B cells and activated T cells. These results indicate that the establishment of interactions between B cells and anti-CD3-stimulated T4 or T8 cells provides all of the signals necessary for proliferation and differentiation of B cells without other stimuli and also augments the production of lymphokines by the activated T cells. The data emphasize the role of Ag-nonspecific interactions between B cells and T cells in promoting polyclonal responses of both cell types.     

Activation of CD 4-, CD 8- thymocytes with IL 4 vs IL 1 + IL 2

Thymocytes from C57BL/6 mice were highly purified to obtain the CD 4-, CD 8- subpopulation which constitutes only 5% of all thymocytes. Substantial proliferation was induced in vitro with either IL-1 + IL-2 or with IL-4 in the presence of PMA. IL-1 and IL-2 synergized in inducing proliferation of these purified CD 4-, CD 8- thymocytes whereas neither synergized with IL-4. In order to determine whether stimulation with IL-1 + IL-2 acted via IL-4 or vice versa, cultures were treated reciprocally with affinity-purified anti-IL-2 or anti-IL-4 antibodies. Cultures with IL-4 were inhibited by anti-IL-4 but were unaffected by anti-IL-2. The CD 4-, CD 8- thymocytes cultured with IL-1 + IL-2 + anti-IL-2 were inhibited to baseline IL-1 stimulation. At low concentrations of IL-1 (1 U/ml) and IL-2 (100 U/ml), anti-IL-4 had no effect, whereas at higher levels of IL-1 (2 U/ml IL-1), and 100 or 200 U/ml IL-2, anti-IL-4 significantly reduced DNA synthesis. This result suggests that at higher concentrations the combination of IL-1 + IL-2 can induce cells to produce IL-4 which then contributes to overall proliferation. When CD 4-, CD 8- thymocytes were cultured with the low doses of IL-1 + IL-2 for 72 h, 62% expressed cell surface T3 complex (vs 11% at initiation) and 27% were F23.1+ (vs 5% at initiation). In contrast, culture with IL-4 led to no increase in numbers of T3+ cells and none were F23.1+; however, there was coexpression of Thy1 and 6B2 on 20% of cells at the end of culture (vs 4% at initiation). Thus, IL-1 + IL-2 causes expansion of a CD 4-, CD 8- thymocyte population expressing the alpha, beta-T cell receptor, whereas IL-4 induces cells to express a phenotype present in small numbers in the periphery of normal mice and in larger numbers in mice bearing the lpr gene.