Activation Signal Induces the Expression of B Cell-Specific CD45R Epitope (6B2) on Murine T Cells

Tlymphocytes express multiple forms of the leukocyte-common antigen CD45, transcribed by alternative usage of leukocyte-common antigen exon 4 6. The various isoforms of CD45R expressed differentially on T cells are involved in different stages of development and activation. The monoclonal antibody (MoAb) RA3-6B2 is established as a B cell-type isoform (B220)-specific marker. However, it reacts with certain activated T cells although the relationship between 6B2 expression and T-cell activation is unclear. We have examined the 6B2 expression on activated T cells and found that concanavalin A, anti-CD3 antibody and staphylococcal enterotoxin B (SEB) induced 6B2 expression on T cells. The expression was found on both CD4⁺ and CD8⁺ T cells and also was induced by SEB in vivo predominantly on CD8⁺ T cells. The 6B2⁺ T cells are IL-2R⁺ and blasted cells according to flow cytometry analysis. Therefore, the 6B2⁺ T cells are supposed to be in an activated stage. Enzymatic analysis demonstrated that trypsin treatment decreased the 6B2 expression, whereas neuraminidase increased the intensity on activated T cells. Neither endo-D or endo-H have any effect on the expression and there are no differences, in the results of immunoprecipitation and RT-PCR analysis, between control T cells and activated T celts. Taken together, the 6B2 epitope is presumed to be the product of CD45R modification and is expressed on activated T cells. These results illustrate a novel classification of a T-cell subpopuiation bearing a 6B2 epitope.     

Differential effects of B7-1 and B7-2 on the costimulation of mouse nonspecific cytotoxic T lymphocyte development in response to anti-CD3 antibody

Despite extensive study, the relative contribution of B7-1 and B7-2 molecules to the costimulation of cytotoxic T lymphocyte (CTL) activation remains controversial. We used blocking mAbs to B7-1 and B7-2 molecules to determine the role of these B7 family members in the in vitro induction of mouse nonspecific CTL in response to soluble anti-CD3 mAb. Optimal induction of anti-CD3-activated killer-T (AK-T) cells was found to require interactions with B7-2 on residual accessory cells in nylon wool-nonadherent spleen cell preparations during the first 12 h of culture in the presence of anti-CD3 mAb. Because B7-1 is not expressed at high enough levels on residual accessory cells in primary T cell cultures to be an effective ligand for CD28, we used LPS-stimulated B cells, which express substantial B7-1, in addition to B7-2, to determine the contribution of B7-1 to AK-T cell development. Compared with B7-2, the contribution of B7-1 to the costimulation of AK-T cells in this system was modest because anti-B7-1 mAb had only a minimal inhibitory effect on the generation of cytotoxicity, whereas anti-B7-2 mAb strongly inhibited AK-T cell development. Anti-CD3-induced cytotoxicity of T cells from CD4 knockout mice and CD4-depleted nylon wool-nonadherent spleen cells from wild-type mice was inhibited by anti-B7-2 mAb, implying that B7-2 is able to bind directly to CD28 on CD8+ T cells and costimulate their activation. B7-1 blockade, on the other hand, did not affect the costimulation of CD8+ T cells. Blockade of B7-2/ CD28 interactions with anti-B7-2 mAb strongly inhibited granzyme B, but not perforin or Fas ligand gene expression, suggesting an explanation for the inhibitory effect of anti-B7-2 mAb on AK-T cell development. These data indicate that B7-2 is superior to B7-1 as a costimulator of mouse AK-T cell induction.     

Age-related defects in CD2 receptor-induced activation in human T-cell subsets.

It is well documented that the proliferative capacity of T cells declines with advancing age. There are, however, conflicting data as to the role of the accessory cell and whether or not this loss in responsiveness extends to all T-cell stimuli and to all T cells. We report here on the capacity of subpopulations of peripheral blood CD4+ T cells from the healthy aged to proliferate in response to anti-CD2 receptor-induced activation in the complete absence of accessory cells by using various exogenous cofactors as second signals. These costimulatory factors included phorbol 12-myristate 13-acetate (PMA), interleukin (IL)-1, IL-2, IL-6 and IL-7 and the monoclonal antibodies, anti-CD28 and anti-CD44. Under these conditions, the proliferative responsiveness of CD4+CD45RO+ T cells from the aged was found to be comparable to young control cells for all stimuli tested, except anti-CD2 plus IL-7. This suggests that signal transduction pathways involving CD2, except IL-7-mediated events, are essentially intact in 'old' memory CD4+ T cells. On the other hand, several cofactors, namely IL-2, IL-6, IL-7 and to a lesser extent IL-1 beta and PMA, failed to support adequately CD2-induced activation in 'old' CD4+CD45RA+ T cells suggesting severe and multiple signalling deficiencies in this subset.     

T Helper-Independent Activation of Human CD8+ Cells: the Role of CD28 Costimulation

The concept that activation of MHC class I-restricted CD8⁺ cells entirely depends on help from MHC class II-restricted CD4⁺ T cells has recently been supplemented with an alternative model in which CD8⁺ cells can directly be activated by MHC class I-expressing professional antigen-presenting cells (APC), which are able to deliver an accessory signal. The authors analysed the role of CD28-mediated costimulation for T helper cell-independent activation of purified human CD8⁺ T cells in two different in vitro models. Freshly isolated CD8⁺ cells could be activated (proliferation, IL-2 production and cytotoxic activity) by anti-CD3-presenting FcγR⁺ mouse cells transfected with the human CD28 ligand, CD80, as the only accessory signal. On the other hand, activation of CD8⁺ cells by allogeneic MHC class I on EBV-transformed B cells, which express two different CD28 ligands, CD80 and CD86, also proceeded very efficiently (proliferation, cytotoxic activity and CD25 expression), but was either not, or only partially, blocked by anti-CD80 and anti-CD86 MoAb or CTLA-4Ig. This indicates that other costimulatory signals are also effective, and that CD28 triggering is not absolutely required for initial T-cell activation. CsA and CD80/CD86-blocking agents were synergistic in completely inhibiting activation of CD8⁺ cells in the MLR with allogeneic B-cell lines. This combination also induced non-responsiveness of CD8⁺ cells upon restimulation in the absence of blocking agents. Therefore, although professional APC can apparently provide multiple costimulatory signals for direct activation of CD8⁺ T cells, the signal derived from CD80/CD86 is unique in providing CsA-resistance.     

An optimized method for the functional analysis of human regulatory T cells

Naturally occurring regulatory T cells (Treg) suppress the activation of antigen-responsive T cells in a cell contact-dependent manner. In order to investigate the impact of soluble mediators and receptor-ligand interactions on the interplay between naive T cells and Treg, a reproducible suppressor cell assay which functions in the absence of additional feeder cells or antigen-presenting cells is mandatory. Here, we describe such a method which is suited to study the modulation of responder T cell/Treg interactions in vitro. Treg were isolated from negatively purified total human CD4+ T cells by positive selection using anti-CD25 monoclonal antibody (MoAb)-coated Dynabeads followed by a detachment step. The remaining CD4+ CD25- responder T cells were cocultured with CD4+ CD25+ Treg in the presence of T-cell Activation/Expansion Beads from Miltenyi Biotec pre-coated with anti-CD3 plus anti-CD28 monoclonal antibody (MoAb). The optimal concentration for coating was 5 microg/ml for both MoAb. At this concentration, strong proliferation of responder T cells was elicited which was almost completely suppressed by Treg at 1:1 cell ratios. When higher concentrations of anti-CD3/anti-CD28 MoAb were used for coating, Treg also showed some degree of proliferation. The optimized suppressor assay proved to be highly reproducible and was used here to confirm the partial or complete reversal of Treg-mediated T-cell suppression by some cytokines (IL-2, IL-15), soluble IL-6 receptor/IL-6 fusion protein and recombinant GITR-ligand. Furthermore, our data confirm that Treg do not need other cell types to suppress proliferation of CD4+ CD25- responder T cells.     

B7.2-Ig fusion proteins enhance IL-4-dependent differentiation of tumor-sensitized CD8+ cytotoxic T lymphocyte precursors

The B7/CD28 co-stimulatory pathway plays a critical role in T cell activation and differentiation. Our previous study demonstrated that administration of B7.2-Ig fusion proteins to tumor-bearing mice elicits IL-4-dependent, CD8+ T cell-mediated tumor regression. Here, we investigated whether B7.2-Ig stimulation of tumor-sensitized CD8+ CTL precursors during in vitro antigen re-sensitization actually results in their differentiation into mature CTLs and if so, whether such a process depends on IL-4 signals. Splenocytes from tumor-sensitized (tumor-bearing or tumor-immunized) mice exhibited low levels of anti-tumor CTL responses upon culturing alone, but induced strikingly enhanced CTL responses when stimulated in vitro with B7.2-Ig fusion proteins. Because CTLs were not generated from normal splenocytes even by B7.2-Ig stimulation, the expression of the B7.2-Ig effect required the in vivo tumor sensitization of CD8+ CTL precursors. Administration of anti-CD4 or anti-CD40 ligand (CD40L) to mice before tumor sensitization resulted in almost complete inhibition of CTL responses generated in the subsequent culture containing B7.2-Ig. In contrast, anti-IL-4 did not influence in vivo tumor sensitization required for CTL induction. However, B7.2-Ig stimulation of tumor-sensitized splenocytes enhanced IL-4 production and neutralization of this IL-4 with anti-IL-4 potently down-regulated CTL responses. These results indicate that B7.2-Ig enhances IL-4-dependent differentiation of anti-tumor CD8+ CTL precursors that can be sensitized in vivo depending on collaboration with CD4+ T cells involving CD40L function.     

Interleukin-12 can replace CD28-dependent T-cell costimulation during nonspecific cytotoxic T lymphocyte induction by anti-CD3 antibody

Cytotoxic T lymphocyte (CTL) development is regulated closely by an intricate series of signals provided by the T-cell receptor/CD3 complex, cytokines, and costimulatory ligand/receptor systems. In this study, we have explored the role of interleukin (IL)-12 and CD28 in mouse CTL development. Activation of T cells with anti-CD3 monoclonal antibody (mAb) in the presence of anti-CD86 mAb, which prevents CD28-CD86 interaction, led to decreased production of type 1 (IL-2, interferon-gamma) and type 2 (IL-4, IL-6, IL-10) cytokines, as well as diminished expression of granzyme B (Gzm B) and reduced cytotoxic effector function. Cytolytic activity in T-cell cultures that were activated in the presence of anti-CD86-blocking mAb alone or in combination with anti-CD80 mAb could be restored by the addition of exogenous IL-12 at initiation of culture. The ability of IL-12 to substitute for CD28-costimulatory signaling during CTL development was found to be dependent on the presence of IL-2 rather than interferon-gamma. IL-2 is required for IL-12Rbeta2 expression by T cells activated in the presence of anti-CD86 mAb. Moreover, IL-12Rbeta2 expression by T cells activated in the presence of anti-CD86 mAb is enhanced by IL-12. We, therefore, conclude that the ability of IL-12 to substitute for CD28-costimulatory signaling during CTL development is a result of the interaction of IL-12 with IL-12Rbeta2 induced by low levels of IL-2 synthesized by T cells activated in a CD28-independent manner.     

Effect of 12 Neutralizing Anti-Cytokine Antibodies on In Vitro Activation of B-Cells. Interleukin-12 is Required by B1a but not B2 Cells

Normal human peripheral blood mononuclear cells, depleted of most monocytes and virtually all CD8-positive cells, were stimulated in vitro with pokeweed mitogen plus Staphylococcus aureus Cowan I in the presence or absence of variousneutralizing anti-cytokine antibodies. Numbers of CD5⁺ and CD5⁻ immunoglobulin-secreting cells were determined using the protein A haemolytic plaque assay after labelling B1a cells with anti-CD5-coated beads. Antibodiesagainst IL-2, IL-5 and IL-10 had little or no effect on plaque-forming cell (PFC) induction; anti-IL-6, -TNFα and -TGFβ enhanced PFC induction; anti-IL-1α, -IL-1β, -IL-4, -IFNγ and -IL-13 suppressed PFC induction. B1a and B2 cells were equally affected by cytokine deprivation using these 11 neutralizing antibodies. In contrast, neutralizing anti-IL-12 suppressed induction of CD5⁺ but not CD5⁻ PFC. Furthermore, recombinant IL-12, if added during thefirst 48 h of culture, enhanced CD5⁺ PFC induction while marginally suppressing (IgG-) or not affecting (IgA-, IgM-) induction of CD5⁻ PFC. IL-12 did not preferentially increase survival in culture of B1a cells norinduce expression of CD5 on B2-cells. Further studies are required to determine whether manipulation of B1a and B2 subsets in vivo using IL-12 could be achieved in clinical situations where imbalances in the two populations have beenobserved.     

Massive production of Th2 cytokines by human CD4+ effector T cells transiently expressing the natural killer cell marker CD57/HNK1.

We have reported previously that uncommitted human CD4+ CD45RO- T cells default to the T-helper type 1 (Th1) pathway, if they are costimulated by anti-CD3 plus anti-CD28 monoclonal antibodies (mAb). In contrast, 5% of the uncommitted T cells differentiate into Th2 cells, if they are stimulated by anti-CD28 plus interleukin-2 (IL-2) in the absence of T-cell receptor (TCR) signals. The anti-CD28/IL-2-induced proliferation (and the resulting Th2 commitment) was not affected by neutralizing anti-IL-4 mAb, suggesting a non-conventional IL-4-independent Th2 differentiation pathway. Here we report that the respective CD4+ Th2 cells (but not the Th1 cells) coexpressed the natural killer (NK) cell marker HNK1/CD57. Expression of CD57 on Th2 cells required CD28 stimulation, and was suppressed by CD3/TCR signals. However, Th2 effector cells displayed a TCR V beta-chain usage comparable to that of committed Th1 cells (with V beta 8 dominating). Our data suggest that expression of CD57 on human CD4 T cells may be associated with defined stages of Th2 cell activation/differentiation, and may not necessarily characterize a separate T-cell lineage. The induction of cytokine production and B-cell helper function in both Th1 and Th2 populations required CD3/TCR signalling in costimulation with anti-CD28 or IL-2. Importantly, anti-CD28/IL-2-primed Th2 cells readily secreted IL-4 and induced IgE production by surface IgE- B cells in response to the first TCR signal and independent of previous contact with IL-4. Therefore, CD4+ CD57+ T cells responded comparably to murine CD4+ NK1.1+ T cells, which are critical for the development of Th2/IgE immune responses in vivo. The possible role of human CD4+ CD57/HNK1+ Th2-like cells in cancer, infection and allergy is discussed.     

Anti CD5 Sustains the Proliferative Response of IgM-Activated Human CD5+B Cells

The CD5 molecule is expressed by most T cells but it is present on a minor B cell subset. Whilst several studies have provided information on the physiological role of T cell CD5, the functional role of CD5 on B lymphocytes remains unclear. To address this question, tonsillar CD5⁺ B cells were sorted by dual-colour fluorescence and FACS. Sorted cells were stimulated with polyclonal anti-IgM antibodies (Ab), and monoclonal (MoAb) F(ab')₂ fragments of anti-CD5. Proliferative responses were evaluated by enumeration of Ki-67 positive cells using quantitative flow cytometry. Co-stimulation with anti-CD5 MoAb for 3 days did not affect the anti-IgM and IL2-induced proliferation of CD5⁺ B cells. This was seen under conditions where the anti-CD5 was soluble, adsorbed to the microwells or cross-linked by anti-mouse antibodies. Fewer CD25⁺ cells were detected, however, in the presence of anti-CD5. In contrast, the proliferative response of CD5⁺ B cells prestimulated for 3 days with IL-2 and anti-IgM, was sustained in a further 3-day culture period when anti-CD5 was added. It is concluded that CD5 occupancy might provide an additional signal to activated CD5⁺ B cells favouring their proliferation and differentiation into autoantibody secreting cells.     

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.     

Control of T-cell activation by CD4+ CD25+ suppressor T cells

Summary: Depletion of the minor (∼10%) subpopulation of CD4⁺ T cells that co-expresses CD25 (interleukin (IL)-2 receptor α-chain) by thymectomy of neonates on the third day of life or by treatment of adult CD4⁺ T cells with anti-CD25 and complement results in the development of organ-specific autoimmunity. Autoimmune disease can be prevented by reconstitution of the animals with CD4⁺ CD25⁺ cells. CD4⁺ CD25⁺-mediated protection of autoimmune gastritis does not require the suppressor cytokines IL-4, IL-10, or transforming growth factor (TGF)-β. Mice that express a transgenic T-cell receptor (TCR) derived from a thymectomized newborn that recognizes the gastric parietal cell antigen H/K ATPase all develop severe autoimmune gastritis very early in life. CD4⁺ CD25⁺ T cells are also powerful suppressors of the activation of both CD4⁺ and CD8⁺ T cells in vitro . Suppression is mediated by a cell contact-dependent, cytokine-independent T–T interaction. Activation of CD4⁺ CD25⁺ via their TCR generates suppressor effector cells that are capable of non-specifically suppressing the activation of any CD4⁺ or CD8⁺ T cell. Activation of suppressor effector function is independent of co-stimulation mediated by CD28/CTLA-4 interactions with CD80/CD86. We propose that CD4⁺ CD25⁺ T cells recognize organ-specific antigens, are recruited to sites of autoimmune damage where they are activated by their target antigen, and then physically interact with autoreactive CD4⁺ or CD8⁺ effector cells to suppress the development of autoimmune disease.     

Hyper IgE in stimulatory graft-versus-host disease: role of interleukin-4.

Intravenous injection of 2 x 10(8) DBA/2 spleen cells into adult intact (C57BL/6 x DBA/2) F1 mice results in a stimulatory graft-versus-host reaction (GVHR) linked to the recognition by donor CD4+ T cells of Ia alloantigens on host B cells. In the experiments presented here, we found that this GVHR is associated with a major increase in IgE serum levels which was already present 7 days after the cell transfer. At 6 weeks, mean IgE levels were more than 200-fold above the control values. Host B cells were responsible for the hypersecretion of IgE in stimulatory GVHR since it was also observed when the DBA/2 donor inoculum was depleted of B cells but not when the F1 recipients were irradiated. The induction of IgE secretion required donor CD4+ T cells as treatment of the donor inoculum with lytic anti-CD4 monoclonal antibody (MoAb) completely prevented the occurrence of the hyper IgE whereas depletion of CD8+ cells had no influence on this parameter. The role played by interleukin-4 (IL-4) in this model was analysed in vivo by the administration of the 11B11 anti-IL-4 rat MoAb (total dose 36 mg) during the first 12 days following induction of stimulatory GVHR by 8 x 10(7) DBA/2 spleen cells. This treatment completely prevented the development of hyper IgE whereas the administration of a control rat MoAb had no significant effect. We conclude that stimulatory GVHR in mice is associated with a major increase in serum IgE which is mediated by IL-4.     

Isotype-specific cross-linking of select human Fc gamma R isoforms triggers release of IL-6.

Anti-CD3 MoAbs are widely used in T cell activation studies, and are effective in immunosuppressive therapy. We used a panel of mouse (m) anti-CD3 switch variant MoAbs of five different isotypes to study IL-6 release from accessory cells. Incubation of human (h) mononuclear cells with anti-CD3 MoAbs resulted in increased IL-6 levels with MoAbs of mIgG1 and mIgG2a isotypes, with no effect of mIgG2b or mIgA. This suggested involvement of IgG Fc receptors (Fc gamma R) in triggering IL-6 production. To evaluate the role of different Fc gamma R molecules individually we used a panel of hFc gamma R-transfected mouse fibroblasts, and Jurkat T cells as a model. IL-6 secretion by CD32 transfectants expressing the hFc gamma RIIa high-responder (HR) allelic form was triggered by mIgG1 anti-CD3 MoAb, with no effect of four other isotypes. None of the anti-CD3 MoAbs induced IL-6 secretion by CD32 transfectants expressing either a variant of this receptor, containing only a single intracellular amino acid (CT-), the hFc gamma RIIa low-responder (LR) allelic form, or hFc gamma RIIb1. hFc gamma RI (CD64) transfectants exhibited IL-6 production after incubation with mIgG2a anti-CD3 MoAb, and to a lesser extent with mIgG2b, and mIgG1 MoAb. Indirect involvement of T cells in triggering IL-6 secretion could be excluded by experiments in which transfectants were cultured with immobilized anti-CD3 MoAb. These data indicate that cross-linking of either hFc gamma RI, or hFc gamma RIIaHR by appropriate anti-CD3 MoAbs triggers IL-6 production of accessory cells, and not T cells. This may also take place in vivo during immunosuppressive therapy with anti-CD3 MoAbs, and related antibody-mediated immune responses.     

Priming of T-Cell Responses in Mice by Porins of Salmonella typhimurium

An imbalance in signals delivered to T cells via T-cell receptor and accessory molecules can lead to anergy, apoptosis, or both. In the present study we have demonstrated that Salmonella typhimurium infection in mice leads to a progressive loss of CD4⁺ T helper (Th) cell population, abnormal T-cell death by apoptosis and loss of accessory molecules (B7 and intracellular adhesion molecule-1) on macrophages. Quantification of interleukin-2 (IL-2), interleukin-4 (IL-4) and interferon-γ (IFN-γ) secretion revealed a Th2-type of response in lymphocytes isolated from spleen. However, preimmunization of mice with porins resulted in an increased CD4⁺ Th cell population and accessory molecules on the surface of macrophages. Quantification of cytokines revealed a Th1-type of response. We conclude that preimmunization of mice with porins provides a microenvironment in which a well-balanced accessory molecule and cytokine network is established, which results in the prevention of cell death by apoptosis.     

Signalling via CD28 of human naive neonatal T lymphocytes.

Accessory molecules play a crucial role in the development of the T cell response to antigenic challenge. We have examined the role of CD28 in modulating the 'naive' neonatal T cell response to anti-CD2-mediated activation. To compare the role of CD28, neonatal and adult T cells were stimulated with a pair of mitogenic anti-CD2 antibodies in the presence or absence of anti-CD28 MoAb. With anti-CD2 alone, neonatal T cells proliferated slightly but produced no detectable IL-2, whereas adult T cells proliferated vigorously, with significant IL-2 production. Costimulation with anti-CD28 MoAb greatly enhanced the proliferative response of neonatal T cells to levels equivalent to those of adult T cells, whereas adult T cells showed only slight increases. Although IL-2 secretion was increased in the presence of anti-CD28 MoAb, neonatal T cell IL-2 production remained lower than in adults. In contrast, enhancement of IL-2 mRNA expression in neonates was similar to adult levels. Anti-CD28 MoAb costimulation increased NF kappa B levels in neonates, albeit to levels lower than that of adults. The cellular mechanism governing the diminished proliferative response of neonatal T lymphocytes to anti-CD2 may therefore be due to decreased NF kappa B induction, reduced IL-2 mRNA expression and deficient IL-2 production. Although anti-CD28 MoAb costimulation enhances all of the above signals, NF kappa B and IL-2 levels remain lower than in adults, suggesting the need for further activation requirements in the neonate.     

Signal requirements for activation of leukaemic T cells from a chronic lymphocytic leukaemia (T-CLL).

In order to define the signal requirements for leukaemic T cell activation, the proliferation and interleukin-2 (IL-2) production of peripheral lymphocytes from a patient with a HTLV-I-, CD4+, CD45RA+ CD45RO+ CD25- T-CLL were evaluated after the delivery of different stimuli. Unlike resting CD4+ normal T lymphocytes that can be activated only by a two-signal stimulation, T-CLL cells proliferated and released IL-2 in response to a pair of anti-CD2 monoclonal antibodies (MoAbs) or concanavalin A (Con A) in the absence of both accessory cells (AC) and phorbol myristate acetate (PMA). The two stimuli were also able to induce CD25 expression within 12-20 h on the majority of T-CLL cells. A response to anti-CD3 and anti-CD28 MoAbs was detected only in the presence of PMA, similar to that observed in normal resting T lymphocytes matched for phenotype. Both Con A- and CD2-induced proliferation were strongly inhibited by the addition of anti-CD25 MoAb. Furthermore, T-CLL lymphocytes acquired anti-tumour lytic activity after culture in the presence of PMA and ionomycin. We conclude that HTLV1- CD25- T-CLL can be characterized not only by morphological and phenotypical studies but also on the basis of signal requirements for cell activation.     

Regulation of the IL-10 production by human T cells

Interleukin (IL)-10, an immunomodulatory cytokine predominantly produced by monocytes/macrophages and T cells, inhibits several functions of dendritic cells (DC), monocytes and T cells including their cytokine production, but it stimulates B cell immunoglobulin (Ig) production and cytotoxic T lymphocyte (CTL) generation. A precise knowledge of the mechanisms that control the IL-10 production is therefore highly important for understanding the immunoregulation. The IL-10 production was studied in cultures of freshly isolated human T cells. A rise in intracellular calcium as well as the common gamma-chain containing cytokine receptor triggering or CD28 triggering were found to be important signals for IL-10 induction. CD80, CD58, rIL-12 and rIFN-alpha all had efficacious and independent costimulatory activities on the IL-10 production, while PGE2 was inhibitory. Dependence on autocrine IL-2 signalling was shown by the effects of anti-IL-2 and anti-IL-2R monoclonal antibodies (MoAb), but the IL-10 production proceeded partly IL-2-independent when CD80 provided costimulation. Sensitivity to inhibition by CsA was not removed by CD80 or CD58 costimulation and/or by addition of rIL-12 or rIFN-alpha, pointing to the absolute requirement for calcineurin activity. These data reveal important differences in the regulatory pathways between IL-10 (a cytokine-inhibitory interleukin) and IL-2 (a cytokine-inducing interleukin), which can potentially be exploited therapeutically. The fact that CsA blocks the production of IL-10, which itself has important immunosuppressive properties, should be taken into account in defining immunosuppressive treatment schedules which include the use of CsA.     

Regulation of 4-1BB expression by cell-cell interactions and the cytokines,interleukin-2 and interleukin-4

4-1BB expression increased gradually following T cell activation, and by day 3 post-stimulation with immobilized anti-CD3 (anti-CD3i) or concanavalin A (Con A), splenic T cells were routinely 35–45% 4-1BB⁺ by flow cytometric analysis. 4-1BB was expressed on activated CD8⁺, CD4⁺, CD28⁺ and CD45RB⁺ T cells. Optimal 4-1BB expression was seen by day 6 post-stimulation and was cell density dependent. When T cells were cultured for 6 days at 1 × 10⁶/well in a 24-well plate with anti-CD3i, 82% of the cells were 4-1BB⁺. In contrast, at lower cell densities (4 × 10⁵, 2 × 10⁵ and 1 × 10⁵), optimal 4-1BB expression was observed only if the cultures were supplemented with recombinant interleukin-2 (IL-2) or recombinant IL-4 (IL-4). In agreement, with these results, modes of inducing endogenous IL-2 production such as cross-linking the costimulatory molecule, CD28, or the addition of syngeneic accessory cells to T cells activated with anti-CD3i, resulted in high levels of 4-1BB expression. The addition of interleukin-1α(IL-1α) or interferon-γ (IFN-γ) did not increase 4-1BB expression on anti-CD3i-activated T cells. In addition, if T cells were incubated with IL-2, IL-4, IL-1α, IFN-γ or anti-CD28 alone, no 4-1BB expression was induced. T cells activated with soluble anti-CD3 (anti-CD3s) in the presence of IL-2, IL-4, or accessory cells, did not express higher levels of 4-1BB on their cell surface. These data suggest that initial events crucial for efficient T cell activation, such as signals delivered through the T cell receptor/CD3 complex and the CD28 molecule, are instrumental in regulating subsequent 4-1BB expression.