Stimulation of human peripheral blood mononuclear cells with anti-CD3 monoclonal antibody vs IL2: disparate effects on T cell-dependent B cell differentiation despite similar effects on generation of unrestricted cytolytic activity.

Despite their each inducing MHC-unrestricted cytolytic activity in overnight PBMC cultures mediated predominantly by CD56+ non-T cells, anti-CD3 mAb and rIL2 induce diametrically opposite effects on subsequent polyclonal T cell-dependent B cell differentiation. When added to fresh autologous PBMC, irradiated anti-CD3-stimulated PBMC inhibit generation of Ig-secreting cells (IgSC) in the secondary cultures, whereas irradiated rIL2-stimulated PBMC enhance IgSC generation. Neither carryover of the respective stimuli nor quantitative differences in levels of cytolytic activity against Daudi cells, autologous PBMC, or autologous activated B cells can explain the dichotomous anti-CD3- vs rIL2-induced effects on B cell differentiation. For both anti-CD3- and rIL2-induced effects on B cell differentiation, CD56- cells, including CD4+ and CD8+ cells, play a more dominant role than they do in generation of MHC-unrestricted cytolytic activity. In addition, although rIL2-induced enhancement of IgSC generation is insensitive to monocyte depletion by plastic adherence or by treatment with leucine methyl ester, anti-CD3-induced inhibition of IgSC generation is highly sensitive to monocyte depletion, indicating that, at least for anti-CD3-induced inhibition, multiple cell populations are required to generate the functional effect. Taken together, these results indicate that differences in the means of generating in vitro tumoricidal activity may have profound ramifications for non-cytotoxic immune parameters, such as B cell differentiation. Not only might this be an important issue to address in adoptive immunotherapy protocols for cancer patients but also adoptive immunotherapy might be applicable to certain autoimmune disorders if the ability to inhibit B cell differentiation could be channeled against the pathogenic antibody-producing B cells.     

Responsiveness of systemic lupus erythematosus T cells to signals provided through LCA T200 (CD45) and T1 (CD5) antigens

It is currently unclear whether the T cell defective capacity to proliferate and to secrete interleukin-2 (IL-2) observed in systemic lupus erythematosus (SLE) reflects an intrinsic disorder of the T cell or defects secondary to a monocyte dysfunction. In order to clarify whether the disorder is intrinsic to the T cell, we have studied the proliferative capacity of cells highly depleted of monocytes, activated by Seph-CD3, as 'first signal,' and by monoclonal antibodies (MoAbs) CD45 and CD5 as 'second signal,' in 14 SLE patients. There were no significant differences between SLE patients and healthy volunteers in the response of the monocyte-depleted cells to Seph-CD3+CD45; Seph-CD3+CD5; Seph-CD3+IL-2; and Seph-CD3+phorbol myristate acetate (PMA). However, active SLE compared with non-active SLE had an impaired response of peripheral blood mononuclear cells (PBMC) to Seph-CD3 and to Seph-CD3+IL-2. The good responses obtained to second signals provided through CD45 and CD5 indicate that at least these mechanisms are not intrinsically impaired in SLE T cells. These findings, together with the abnormal response of PBMC suggest that a monocyte dysfunction plays an important role in SLE T cells hyporesponsiveness.     

Human CD4-reactive antibodies from sle patients induce reversible inhibition of polyclonal t lymphocyte proliferation

We report on isolation of human polyclonal CD4-reactive antibodies of IgM and/or IgG isotypes from several SLE patients. These antibodies bound specifically to CD4-expressing cell lines and to rCD4 in ELISA and immunoblots. Saturation of CD4-binding sites occurred at antibody concentrations between 5 and 15 μg/ml. Anti-CD4 antibodies, in a dose-dependent manner, suppressed the proliferative responses of human peripheral blood mononuclear cells (PBMC) to superantigens (Staphylococcal enterotoxins A and B), anti-CD3 antibodies, and mitogens (PWM and Con A, but not PHA). They could also inhibit the proliferation of highly purified human T cells induced by immobilized anti-CD3 antibodies. To promote their effects on T cells, human antiCD4 antibodies had to be present at lymphocyte cultures before or at the time of priming. There was no significant inhibition when antibodies were added more than 24 h following T cell activation. Substantial evidence that the immunosuppression induced by anti-CD4 antibodies was due to their direct effect on T cells was obtained. Downregulatory effect of anti-CD4 antibodies could be significantly reversed by addition of exogenous IL-2 and by preincubation with soluble recombinant (r)CD4. Interestingly, at least one affinity-purified anti-CD4 antibody could costimulate the T cell proliferation induced by superantigens or anti-CD3 antibodies, especially when used at subsaturating concentrations (1–4 μg/ml) and when added subsequently to the initiation of cultures.     

T Lymphocytes from Patients with Systemic Lupus Erythematosus Show Increased Response to Interleukin-2 after Costimulation with OKT3 Monoclonal Antibody and Phorbol Esters

In the present study we have examined the potential contribution of IL-2/IL-2R interactions in CD3-mediated responses by T lymphocytes from patients with systemic lupus erythematosus (SLE). T-cells from SLE patients showed normal IL-2 production when activated with OKT3 MAb and submitogenic concentrations of PMA, in cultures in which uptake of endogenous IL-2 was prevented by pretreatment with anti-Tac MAb. In contrast, PHA-induced IL-2 production was lower in patients under the same conditions. Under these stimulatory conditions the proportions of T-cells expressing IL-2R CD25 molecules was comparable in patients and controls. There was earlier and higher binding of exogenously added IL-2 in T lymphocytes from patients activated via the CD3 pathway. Furthermore, these cells responded to IL-2 with stronger proliferative responses than cells from control subjects. These findings may partly explain the increased proliferative responses of SLE T-cells when stimulated via the CD3 pathway.     

Herpesvirus saimiri-transformed CD8+ T cells as a tool to study Chediak-Higashi syndrome cytolytic lymphocytes

Cytolytic CD8+ T lymphocytes are the main cell type involved in the fatal lymphoproliferative-accelerated phase of the Chediak-Higashi syndrome (CHS). To generate a cellular tool to study the defects of this T cell subset in vitro, we have used Herpesvirus saimiri, a lymphotropic virus that transforms human T lymphocytes into extended growth and in addition, endows them with natural killer (NK) features. Transformed CHS CD8+ T cells were generated and characterized in comparison with healthy controls. The results showed that transformed CHS T cells maintained the defects described in primary CHS lymphocytes, such as giant secretory lysosomes and impaired NK and T cell receptor/CD3-induced, perforin-mediated cytolytic activity [which, however, could be restored after extended culture in the presence of interleukin-2 (IL-2)]. Upon activation with phorbol ester plus calcium ionophore or upon extended culture with IL-2, transformed CHS T cells showed normal, perforin-independent plasma membrane CD178/CD95L/FasL-mediated cytolytic activity but negligible secretion of microvesicle-bound CD95L. Transformed (and primary) CHS T cells were otherwise normal for cytolysis-independent activation functions, such as proliferation, surface expression of several activation markers including major histocompatibility complex class II, and cytokine or surface activation-marker induction. Therefore, the CHS protein [CHS1/LYST (for lysosomal traffic regulator)] can be dispensable for certain NK and T cell cytolytic activities of activated CHS CD8+ T lymphocytes, but it seems to be required for microvesicle secretion of CD95L. We conclude that transformed CHS T cells may be useful as a tool to study in vitro the relative role of CHS1/LYST in NK and T lymphocyte cytolysis and antigen presentation.     

Telomere shortening and decreased replicative potential, contrasted by continued proliferation of telomerase-positive CD8+CD28(lo) T cells in patients with systemic lupus erythematosus

To evaluate whether the immune system of systemic lupus erythematosus (SLE) patients shows features of premature aging, we compared telomere length and proliferative potential of SLE peripheral blood mononuclear cells (PBMC) (N = 90) to those of controls (N = 64). SLE samples showed accelerated loss of telomeric DNA (P = 0.00008) and higher levels of senescent (< or =5 kb) telomeric DNA (P = 0.00003). Viability cell counts and CFSE tracking in 6-week-old cell cultures indicated that SLE PBMC (CD8+ and CD4+ T cells) underwent fewer mitotic cycles and had shorter telomeres than controls (P = 0.04). However, a CD8(+)CD28(lo) T cell subset expanded preferentially in SLE-derived bulk cultures (P = 0.0009), preserved telomeric DNA (P = 0.01 vs entire CD8+), and displayed telomerase activity [2.1 telomerase arbitrary units (TAU) vs 0.5 TAU in CD8+CD28(hi) cells and 0.3 TAU in bulk PBMC; P = 0.05]. These T cell anomalies could be due to chronic in vivo stimulation of the immune system and may contribute to the immune dysregulation found in SLE.     

Vitamin D(3) and its synthetic analogs inhibit the spontaneous in vitro immunoglobulin production by SLE-derived PBMC

The production of high-affinity pathogenic autoantibodies in systemic lupus erythematosus (SLE) may result from aberrant immune regulation. Since 1,25 dihydroxy vitamin D(3) (1,25 D(3)) has immunoregulatory activity, we examined effects of 1,25 D(3) and its analogs HM, V, MC1288, and KH1060 on autoantibody production and proliferation of SLE PBMC. We found, in SLE, a higher percentage of T, B, and NK expressing vitamin D(3) receptors (VDRs) (P = 0.034, 0.006, 0.012, respectively). Incubating SLE PBMC with 1,25 D(3) compounds significantly reduced proliferation, polyclonal and anti-dsDNA IgG production, and the percentages of CD3(+)/DR(+) T and B (CD19(+)) cells, while elevating NK (CD16(+)) cells (P < 0.001). 1,25 D(3) analogs were more potent than the natural compound: KH1060 up-regulated CD14 expression by SLE monocytes (P < 0.001), inhibited polyclonal and anti-dsDNA IgG production by SLE-derived B lymphoblasts, and induced apoptosis of activated B lymphoblasts. These data suggest that 1,25 D(3) compounds can offer novel approaches to the clinical management of SLE.     

Expression of intracellular transforming growth factor-beta1 in CD4(+)CD25(+) cells in patients with systemic lupus erythematosus.

BACKGROUND AND PURPOSE: The CD4(+)CD25(+) regulatory T (Treg) cells exert immunoregulatory functions in various autoimmune diseases, in part through transforming growth factor-beta1 (TGF-beta1), and can be expanded by TGF-beta1 stimulation in normal subjects. This study aimed to examine intrinsic TGF-beta1 expression and the response to TGF-beta1 stimulation of this CD4(+)CD25(+) subset in patients with systemic lupus erythematosus (SLE). METHODS: Flow cytometry with multicolor staining of CD4(+), CD25(+), and TGF-beta1 was used to quantify the percentage of CD4(+)CD25(+) T cells in fresh peripheral blood and TGF-beta1-stimulated peripheral blood mononuclear cell (PBMC) cultures, and their corresponding intracellular TGF-beta1 expression. RESULTS: In fresh peripheral blood, we found that decreased percentages of CD4(+)CD25(+)/CD4(+) in SLE patients were associated with disease activity and renal involvement. Intracellular TGF-beta1 expression of CD4(+)CD25(+) cells was significantly elevated in SLE compared with matched controls (p<0.001). In addition, there was significant negative correlation between TGF-beta1 expression and percentage of CD4(+)CD25(+) cells present (r = -0.432, p=0.004). Nevertheless, in ex vivo unstimulated PBMC cultures, the percentage and intracellular TGF-beta1 expression of CD4(+)CD25(+) cells of SLE were normalized to the levels of the control group. In TGF-beta1-stimulated PBMC cultures, CD4(+)CD25(+) cells and their intracellular TGF-beta1 expression were significantly increased (p<0.001), both in SLE and controls. Moreover, the increments in the percentage of CD4(+)CD25(+) cells and intracellular TGF-beta1 expression by TGF-beta1 stimulation were comparable in SLE and controls, and were not significantly influenced by disease activity or renal involvement in SLE. CONCLUSIONS: CD4(+)CD25(+) cells were deficient in peripheral blood but not impaired either in intrinsic TGF-beta1 expression or in response to TGF-beta1 stimulation in patients with SLE. This study suggests that TGF-beta1, by inducing CD4(+)CD25(+) cells, has potential clinical application in treating SLE.     

The Stimulation of T Cells with Anti-CD2 Monoclonal Antibodies Facilitates the Induction of Polyclonal B-Cell Responses but does not Enhance the Activation of Antigen-Specific B Cells

In this report we compare the effect of stimulation of peripheral mononuclear cells (PBMC) by using two monoclonal antibodies (MoAb) directed against the CD2 receptor on T cells or by using autologous erythrocytes (E) which express on their surface lymphocyte function-associated antigen 3 (LFA3), a natural ligand for CD2. The addition of autologous erythrocytes to pokeweed mitogen (PWM)-stimulated PBMC results in the enhancement of polyclonal immunoglobulin synthesis and of antigen-specific B-cell responses. Because B cells lack the CD2 molecule, it can be concluded that their enhanced activity is a consequence of the delivery of activating signals by activated T lymphocytes. When PBMC cultures were stimulated with a pair of anti-CD2 MoAb (Leu5b and VIT13) we were able to induce polyclonal immunoglobulin synthesis, particularly IgM, in cultures supplemented with interleukin 2(IL-2). Specific responses to tetanus toxoid (TT) and keyhole limpet haemocyanin (KLH) were also enhanced by the addition of autologous E to PWM-stimulated PBMC. Significant anti-TT responses were observed in cultures stimulated with E + TT + IL-2. In contrast, stimulation of PBMC with VIT13 + Leu5b + IL-2 + antigen was not effective in inducing anti-TT antibody and only weakly effective in inducing anti-KLH antibodies. Replacing Leu5b by anti-CD3 had no effect on the induction of specific antibody responses; in contrast, replacement of Leu5b by E enhanced anti-TT antibody production while the effect on polyclonal production of IgM was minimal. Therefore, it appears that the signal delivered by the association of CD2 with LFA3 is a better potentiating signal for specific B-cell responses than the signal delivered by pairs of MoAb to different epitopes of CD2 or to CD2 and CD3 epitopes.     

Interleukin 4 (BSF-1) induces growth in resting murine CD8 T cells triggered via cross-linking of T3 cell surface structures

To analyze the role of interleukin 4 (IL4, BSF-1) during primary activation of resting (high-density) murine CD8 T cells, a model system was used which bypasses antigen-presenting cells by the use of anti-T3 monoclonal antibodies immobilized on Sepharose beads. In high, but not in low cell density cultures, IL4 alone induced cell growth. In low cell density cultures, further to T3 cross-linking a soluble macrophage product was required as co-stimulator to induce sensitivity to IL4. This co-stimulator activity was unrelated to recombinant (r)IL1, rIL6 and rTNF-α (tumor necrosis factor α). In primary CD8 T cell responses rIL4-driven growth was about half of that induced by rIL2, and not inhibitable by anti-IL 2 receptor antibodies. Higher concentrations of IL4 down-regulated cell proliferation. In the course of IL4-driven growth, the proliferating cells acquired sensitivitiy to the growth-promoting effect of IL2. Activated CD4 or CD8 T cells were found to be equally sensitive to the IL4 and IL2-driven growth pathway. Taken together, these results define a physiologic role of IL4 as growth factor during primary activation of resting CD8 T cells and thus extend the spectrum of target cells for IL4.     

两种多克隆刺激条件下人诱导性Treg表型的多样性变化

目的:研究人诱导性调节性T细胞(iTreg)细胞表型的多样性变化,并比较PMA/Ionomycin和PHA两种多克隆刺激对人iTreg的诱导的异同。方法:用Ficoll密度梯度离心分离出人PBMC,空白对照组直接检测,其余则在细胞培养液(非刺激对照组)、PMA/Ionomycin溶液(PMA/Ionomycin刺激组)或PHA溶液(PHA刺激组)中培养16小时,以流式细胞仪检测CD4+CD25+FoxP3+CD127-、CD4+CD25+FoxP3+IL-2-、CD4+CD25+FoxP3+IL-10+、CD4+CD25+FoxP3+TGF-β+和CD4+CD25+FoxP3+IFN-γ+的表达。结果:空白对照组显示,约4%的CD4+细胞表达CD4+CD25+FoxP3+CD127-,即nTreg。PBMC在细胞培养液培养16个小时,iTreg的表达无明显变化,但经多克隆刺激后,各不同细胞表型的iTreg的表达明显上升(均P<0.01),表明多克隆刺激可以明显诱导人iTreg的产生。PHA对IL-2-和TGF-β+iTreg的诱导比PMA/Ionomycin强(P<0.01),但仅PMA/Ionomycin可以诱导产生CD4+CD25+FoxP3+IFN-γ+iTreg,而PHA不能诱导。刺激后产生的CD4+FoxP3+IFN-γ+PBL表达与CD25水平呈逆相关。结论:多克隆刺激可以诱导产生人iTreg,PMA/Ionomycin和PHA对人iTreg诱导的机制和程度不一样。多克隆刺激后产生的不同细胞表型反映了人iTreg的多样性变化。     

Induction of cytolytic cells by pure recombinant human interleukin 2

Purified recombinant human interleukin 2, produced in Escherichia coli, was sufficient to generate cytolytic cells in concanavalin A-stimulated, T helper cell-depleted (Lyt-1.1-) or accessory cell-depleted (Ia-) murine spleen cell cultures. Moreover, recombinant interleukin 2 (rIL2) was able (even at 0.5 ng/ml) to induce cytolytic cells in undepleted murine spleen cell cultures and undepleted human peripheral blood lymphocyte cultures in the absence of a mitogen. Purified recombinant human interferon-gamma, produced in Chinese hamster ovary cells, did not induce either cytolytic activity or IL2 responsiveness in human peripheral blood lymphocyte cultures. Possibly, the rIL2-induced cytolytic cells are formed as a result of nonspecific secondary cytotoxic T lymphocyte activation.     

Abnormal production of B cell growth factor in patients with systemic lupus erythematosus.

In order to clarify the role of B cell growth factor (BCGF) in the pathogenesis of systemic lupus erythematosus (SLE), BCGF production by peripheral blood mononuclear cells (PBMC) and T cells was studied using a new bioassay for BCGF activity. For this purpose, we established an Epstein-Barr (EB) virus-transformed B cell line KS-3.F10 that proliferates only in response to two B cell-specific BCGF, low-mol. wt BCGF (LMW-BCGF) and high-mol. wt BCGF (HMW-BCGF). PBMC from active SLE patients produced less BCGF when stimulated with phytohaemagglutinin (PHA) compared with controls. The decreased BCGF production by PHA-stimulated PBMC from active SLE reverted to control values when SLE became inactive. However, PHA-stimulated T cells from active SLE patients produced more BCGF compared with controls, whereas those from inactive SLE showed normal BCGF production. Spontaneous BCGF production by T cells was not observed in active SLE patients. These findings suggest that decreased BCGF production by SLE PBMC is due to excessive BCGF consumption by B cells in vitro and that SLE T cells produce large amounts of BCGF with appropriate immune stimuli in vivo to promote polyclonal B cell activation.     

Lower GrB+ CD62Lhigh CD8 TCM effector lymphocyte response to influenza virus in older adults is associated with increased CD28null CD8 T lymphocytes

Older adults who are at risk of developing influenza illness, have a low level of influenza virus-stimulated cytotoxic T lymphocyte (CTL) activity as measured by an assay of granzyme B (GrB). The purpose of this study was to determine whether aging affected memory CTL populations identified by GrB expression in influenza virus-stimulated peripheral blood mononuclear cells (PBMC). The expression and activity of GrB increased with virus stimulation over 5 days of culture. Virus-specific CD8 effector T cells with the phenotype, GrB+ CD62L(high) CD8 T(CM), were found to be the source of the early CTL response to influenza virus. Comparing the CD8 T cell response in 5-day PBMC cultures of 161 adult subjects, the response of GrB+ CD62L(high) CD8 T(CM) lymphocytes in older individuals was significantly lower than in younger adults after viral stimulation (p<0.001). The increase in the proportion of CD28(null) CD8 T cells in fresh PBMC negatively correlated with the proportion GrB+ CD62L(high) CD8 T(CM) lymphocytes in virus-stimulated PBMC. Thus, the increase in CD28(null) CD8 T cells with age may contribute to the limited CTL response to influenza vaccination and diminished protection in older adults.     

Increased cytolytic T lymphocyte activity and decreased B7 responsiveness are associated with CD28 down-regulation on CD8+ T cells from HIV-infected subjects.

The CD28 receptor on CD4+ and CD8+ T cells interacts with B7 molecules on antigen-presenting cells (APC) to generate essential costimulatory signals. The cytolytic potential of CD8+ T cells could be linked to CD28 expression. Since HIV induces dysfunction of both CD4+ and CD8+ T cells, we evaluated CD28 expression and function in both subsets during HIV infection. CD28 expression on CD8+ T cells from HIV+ subjects was strongly reduced in a disease stage-related fashion. CD28- CD8+ T cells preferentially expressed CD57 and CD11b, but lacked CD26 and IL-2R alpha. The CD8+ T cells from the patients showed a significantly reduced proliferative response to co-stimulation with cell-bound anti-CD3 and B7. Nevertheless, when stimulated with plate-fixed anti-CD3, CD8+ T cells from HIV-infected subjects proliferated normally, and normal levels of IL-2R alpha and transferrin-receptor could be induced on CD28- CD8+ T cells from the patients. In addition, stimulation with plate-fixed anti-CD3 induced proliferative responses in highly purified CD28- CD8+ T cells from both HIV- and HIV+ persons. Furthermore, the increased cytotoxic activity of peripheral blood mononuclear cells (PBMC) from HIV+ subjects, measured in an anti-CD3 redirected assay, was predominantly exerted by CD28- CD57+ T cells. CD4+ T cells from the patients showed a slight but significant CD28 down-regulation and were slightly hyporesponsive to B7 co-stimulation. Decrease of CD28 on CD8+ T cells from HIV+ subjects is associated with an impaired response to co-stimulation via B7. CD28- CD8+ T cells from seropositives, however, are not completely inert, since they contain in vivo activated CTL and they can be additionally activated through a B7-independent stimulation.     

Regulatory T-cell expansion and function do not account for the impaired alloreactivity of ex vivo-expanded T cells

CD3- and CD28-activated T cells expanded for 12 days ex vivo to produce suicide gene-modified T cells are hyporesponsive to alloantigens. To investigate whether this impaired alloreactivity is a result of preferential expansion of regulatory T (Treg) cells, we compared peripheral blood mononuclear cells (PBMC) activated with CD3 and CD28 antibodies co-immobilized on beads and expanded for 12 days with interleukin (IL)-2 (Co(CD3/CD28) cells) to the respective unactivated PBMC in terms of proliferation, cytokine production, and expression of Treg markers [cytotoxic T-lymphocyte antigen 4 (CTLA4), glucocorticoid-induced tumour necrosis factor receptor (GITR) and forkhead box P3 (FoxP3)] after allostimulation. Alloreactive cells were identified by carboxyfluoresceine succinimidyl ester staining dilution. Alloreactive cells in Co(CD3/CD28) cells had a lower proliferative response and a lower potential for IL-2 and interferon-gamma secretion than did those in PBMC, demonstrating a functional impairment of alloreactive cells during ex vivo expansion. Expression of Treg markers transiently increased during ex vivo expansion and was unaffected by depletion of CD25(+) cells (containing Treg cells) before ex vivo PBMC expansion. Such prior CD25(+) depletion did not restore the alloreactivity of Co(CD3/CD28) cells. After allostimulation, expression of Treg markers was restricted to proliferative (alloreactive) cells among PBMC or Co(CD3/CD28) cells. Lastly, CD4(+) CD25(+) cells purified from Co(CD3/CD28) cells lacked suppressive activity when used as a third party, in contrast to CD4(+) CD25(+) cells purified from PBMC. In conclusion, the impaired alloreactivity of T cells expanded ex vivo is not a result of preferential Treg cell expansion and/or enhanced suppressive Treg activity.     

Human B cell differentiation induced by microbial superantigens: unselected peripheral blood lymphocytes secrete polyclonal immunoglobulin in response to Mycoplasma arthritidis mitogen.

Microbial superantigens (SA) activate a significant portion of the T cell repertoire based on their dual avidity for MHC class II antigens and T cell receptor (TCR) epitopes common to products of one or several TCR beta chain variable gene families. While SA that induce massive T cell proliferation and cytokine secretion have been implicated in clinical syndromes characterized by shock and generalized immunosuppression, SA activation of a more restricted T cell response may also have significant, perhaps immunostimulatory, effects on the immune system. To investigate this issue, we measured 3H-thymidine incorporation and polyclonal IgM and IgG secretion by normal human peripheral blood mononuclear cells (PBMC) cultured with a panel of microbial SA, including the Staphylococcus aureus-derived SA, SEA, SEB, SEC-1, SEC-2, SEC-3, SEE, TSST-1, and the Mycoplasma arthritidis-derived SA, MAM. The S. aureus-derived SA induce vigorous proliferation by PBMC, while optimal MAM-induced proliferation is significantly lower in magnitude. In all 12 subjects tested, mitogenic concentrations of MAM reproducibly stimulate unselected PBMC to secrete polyclonal IgM and IgG. In contrast, the S. aureus-derived SA induce Ig production only in cultures containing isolated B cell populations and either very low numbers of untreated autologous T cells, larger numbers of X-irradiated autologous T cells, or very low concentrations of the SA. No difference in the activation of helper (CD4) versus suppressor/cytotoxic (CD8) T cells by MAM and the S. aureus-derived SA was noted. Taken together, these data suggest that MAM's capacity to induce B cell differentiation correlates with its induction of a relatively weak proliferative response by unselected human T cells. MAM-like SA, when encountered in vivo, may result in a significant perturbation of the human immune system and potentially contribute to clinical syndromes characterized by immunostimulation and hypergammaglobulinemia.     

An inhibitor of cytotoxic functions produced by CD8+CD57+ T lymphocytes from patients suffering from AIDS and immunosuppressed bone marrow recipients

An inhibitor of the cytotoxic functions (ICF) mediated by human immunodeficiency virus (HIV)- or HLA-specific cytotoxic T lymphocytes, natural killer and lymphokine-activated killer (LAK) cells is secreted by CD8⁺CD57^? T lymphocytes, a subset expanded during infection with HIV and after bone marrow transplantation. We previously showed an apparent molecular mass of 20–30 kDa for this soluble glycosylated concanavalin A-binding inhibitor which is distinct from known cytokines. Here, we report a characterization of the mechanism of action of this CD8⁺CD57⁺ ICF. We show that the ICF-induced inhibition of LAK cell cytolytic activity is transient, with a spontaneous recovery of cytolytic potential after 18 h. When testing interactions of ICF with a large set of cytokines we found that the ICF-mediated inhibition of cytotoxic functions is antagonized by two cytokines: recombinant interleukin (rIL)-4 and recombinant interferon (rIFN)-γ. Finally, we show that ICF acts at the level of cytolytic effector cells, where it induces a significant increase of cyclic AMP (cAMP) level. In contrast, no modification of either cell surface antigen expression or of target/effector cell conjugate formation could be evidenced. Addition of rIL-4 and rIFN-γ reverses such an increase of cAMP levels and in parallel restores the cytolytic activity. Altogether, these data demonstrate that the glycoprotein ICF produced by CD8⁺CD57⁺ cells (1) inhibits cell-mediated cytotoxicity by sensitizing cytolytic effector cells to the cAMP pathway, and (2) is part of a cytokine network controlling cell-mediated cytotoxic functions.     

CD3+4-8-WT31-(T cell receptor gamma+) cells and other unusual phenotypes are frequently detected among spontaneously interleukin 2-responsive T lymphocytes present in the joint fluid in juvenile rheumatoid arthritis. A clonal analysis

T lymphocytes (E rosetting cells) isolated from the joint fluid of four patients with juvenile rheumatoid arthritis (JRA) were first analyzed for surface antigen expression. Approximately 15% of cells were CD25+ (interleukin, IL, 2 receptor positive), in addition, a remarkable proportion of cells expressed the CD2+3- phenotype. CD3+ cells outnumbered the sum of CD4+ and CD8+ cells as well as the cells reactive with the WT31 monoclonal antibody (which recognizes a framework determinant of the alpha/beta T cell receptor). Purified T cells were cloned under culture conditions (1% phytohemagglutinin, PHA plus IL2) which allow clonal expansion of most peripheral blood T lymphocytes. Under these conditions proliferating cells ranged from 25 to 65%; clones (derived from microcultures containing 0.5 or 0.25 cells/well) were tested for cytolytic activity against P815 cells (in the presence of PHA) or against the natural killer (NK)-sensitive K562 target cells. Fifty-four percent and 73% of clones obtained from the two patients with the polyarticular form of the disease displayed cytolytic activity in the lectin-dependent assay. Cytolytic clones were 22 and 29% in the two patients with single joint involvement. About half of all cytolytic clones displayed NK-like activity. Surface antigen analysis revealed that, in addition to conventional CD3+4+8- and CD3+4-8+, a noticeable fraction of clones (50/202) displayed unusual surface phenotypes. In particular, 33/50 coexpressed CD4 and CD8 antigens; 7/50 were CD2+3-4-8- and displayed NK-like activity; 10/50 expressed CD3 but lacked both CD4 and CD8 antigen and did not react with the WT31 monoclonal antibody. In order to allow selective growth of IL2-responsive cells, T lymphocytes were also cloned directly in IL2. As much as 57% of all clones thus obtained (48/84) displayed cytolytic activity. Moreover, about half expressed unusual surface phenotypes including CD2+3-4-8-, CD3+4+8+ and CD3+4-8-WT31-. Given the accumulation at the site of the joint involvement of unusual T cells, most of which displayed cytolytic activity and were likely to represent cells activated in vivo (IL2 responsive), one may speculate that these cells may be involved in the injury process.