Selective expansion of memory CD4(+) T cells by mitogenic human CD28 generates inflammatory cytokines and regulatory T cells

Costimulatory signals are important for development of effector and regulatory T cells. In this case, CD28 signaling is usually considered inert in the absence of signaling through the TCR. By contrast, mitogenic rat CD28 mAb reportedly expand regulatory T cells without TCR stimulation. We found that a commercially available human CD28 mAb (ANC28) stimulated PBMC without TCR co-ligation or cross-linking; ANC28 selectively expanded CD4(+)CD25(+)FOXP3(-) (Teff) and CD4(+)CD25(+)FOXP3(+) (Treg) cells. ANC28 stimulated the CD45RO(+) CD4(+) (memory) population, whereas CD45RA(+)CD4(+) (naive) cells did not respond. ANC28 also induced inflammatory cytokines. Treg induced by ANC28 retain the Treg phenotype longer than costimulated Treg. Treg induced by ANC28 suppressed CD25(-) T cells through a contact-dependent mechanism. Purity influenced the response of CD4(+)CD25(+ )cells because bead-purified CD4(+)CD25(+ )cells (85-90% pure) responded strongly to ANC28, whereas 98% pure FACS-sorted CD4(+)CD25(bright) (Treg) did not respond. Purified CD4(+)CD25(int) cells responded similarly to the bead-purified CD4(+)CD25(+) cells. Thus, pre-activated CD4(+) T cells (CD25(int)) respond to ANC28 rather than Treg (CD25(bright)). The ability of ANC28 to expand both effectors producing inflammatory cytokines as well as suppressive regulatory T cells might be useful for ex vivo expansion of therapeutic T cells.     

Impaired interleukin-2 synthesis and T cell proliferation following antibody-mediated CD3 and CD2 or CD28 cross-linking in trans: evidence that T cell activation requires the engagement of costimulatory molecules within the immunological synapse

Although the T cell costimulatory molecules CD2 and CD28 are enriched within the immunological synapse (IS), it has been suggested that costimulatory molecules need not be localized to the contact site between a T cell and an antigen-presenting cell (APC) in order to costimulate T cell activation. To determine whether CD2 or CD28 engagement outside of the IS is sufficient to costimulate T cell activation, we compared mouse T cell responses to anti-CD3 and anti-CD2 monoclonal antibodies (mAbs) or anti-CD3 and anti-CD28 mAbs immobilized on the same, i.e., in cis, or on different, i.e., in trans, 10 micron polystyrene microspheres. In comparison to T cells that were stimulated with co-immobilized anti-CD3 and anti-CD2 or anti-CD28 mAbs, DNA synthesis, interleukin (IL)-2 production, and cellular proliferation were all severely impaired following T cell stimulation with anti-CD3 and anti-CD2 mAbs or anti-CD3 and anti-CD28 mAbs on different microspheres. Deficient cellular proliferation and IL-2 synthesis by T cells that experienced CD3 and CD2 or CD28 cross-linking in trans provides evidence that costimulatory molecules must function in the context of the IS for optimal T cell activation.     

Antibody-mediated signaling through PD-1 costimulates T cells and enhances CD28-dependent proliferation

Programmed death-1 (PD-1, CD279) is a molecule expressed on activated T, B and myeloid cells. The role of the interaction of PD-1 ligands (PD-L1 and PD-L2) with PD-1 receptor and the type of signals (costimulatory or inhibitory) that are delivered is a subject of intense debate. Our study has characterized two monoclonal antibodies (mAb) against murine PD-1, termed clone 1H10 and clone 4F10, that recognized different epitopes from that of anti-PD-1, clone J43. We showed that neither of them inhibited anti-CD3-mediated proliferation, but 1H10 mAb induced direct T cell proliferation in the absence of any other stimulus. Moreover, PD-1 engagement with 1H10 mAb costimulated anti-CD3-mediated proliferation and enhanced anti-CD3/CD28 proliferation on both CD4+ and CD8+ T cells in the low range of anti-CD3 concentrations. Anti-PD-1-mediated proliferation induced with 1H10 mAb was also observed in vivo on CD4+ and CD8+ T cells, when CFSE-labeled splenocytes were adoptively transferred to irradiated syngeneic and allogeneic recipients. Overall, our data indicate that PD-1 might not only deliver negative signals to T cells upon interaction through one of its ligands, PD-L1 as reported, but also could costimulate T cells, suggesting a dual potential functional activity of the extracellular domains of this receptor.     

B7-1-dependent co-stimulation results in qualitatively and quantitatively different responses by CD4+ and CD8+ T cells

To characterize better the co-stimulatory activity of native B7-1 in the absence of other receptor/ligand interactions that might contribute to the response, B7-1 was purified by monoclonal antibody (mAb) affinity chromatography. Immobilization of purified B7-1 with anti-T cell receptor (TCR) mAb on cell-sized latex microspheres provided an effective stimulus for activation of both CD4⁺ and CD8⁺ T cells as measured by proliferation, development of effector function, and changes in motility and adhesion. The CD4⁺ T cell response was prolonged and resulted in efficient interleukin-2 production and clonal expansion. In contrast, CD8⁺ responses were transient. Proliferation and clonal expansion peaked on days 3 and 4, coincident with maximal expression of lytic effector function, and the cells then died. These results demonstrate that B7-1 mediated co-stimulation is sufficient for the induction of effector function in both helper and cytotoxic T cell precursors, but suggest that B7-1 co-stimulation is not sufficient to sustain helper-independent CD8⁺ CTL responses. When the dose responses of CD4⁺ and CD8⁺ T cells to B7-1 were compared, CD8⁺ T cells were found to require higher densities of B7-1 to attain an equivalent level of activation, suggesting that the level of expression of B7-1 by APC may influence the development of helper or CTL responses. Finally, in contrast to results obtained by others with B7-1 transfectants, purified B7-1 did not provide co-stimulation when presented on a surface separate from the TCR stimulus.     

T cell activation by antigens on human melanoma cells co-stimulation by B7-1 is neither sufficient nor necessary to stimulate IL-2 secretion by melanoma-specific T cell clones in vitro

B7-1 expression, induced by transfection in poorly Immunogenicmurine tumours, was shown to elicit a T cell-mediated rejectionof these tumours and further active immunity against the nontransfectedtumour. We therefore asked to what level similarly induced expressionof B7 on human melanoma cells would affect the antigen-dependentresponses of tumour-specific T cell clones in vitro. Data presentedshow that B7-1 expression by melanoma lines: (i) significantlyinduced, or improved, an IL-2-dependent proliferative responseof such clones to the antigen; (ii) increased the amount ofIL-2 produced by two clones in response to the parental non-transfectedtumour cells;and (iii) increased the TNF responses of all theCD4⁺ clones. However, despite these clear co-stimulatory effectson antigen-induced responses of all T cell clones, which demonstratedaneffective interaction of the B7-1 transfected molecule withone or the other of its counter-receptors expressed on T cellclones, B7 co-stimulation did not correct the defect of IL-2secretion exhibited by many of these clones in response to invitro antigen presentation by melanoma cells. We further showthat defective IL-2 secretion in response to melanoma antigenswas not due to a T cell clone refractoriness induced by theculture, since one of these clones could be induced to secreteIL-2 by an antigen-expressing melanoma line, upon increasedlymphocyte function associated antigen-3expression induced bygene transfectlon. Together these data suggest that defectiveIL-2 secretion by many tumour-infiltrating lymphocytes clonesin response to antigen presentation by melanoma cells in vitrois not exclusively due to the inability of these cells to providean appropriate co-stimulation through the B7-1 molecule.     

CD154-CD40-independent up-regulation of B7-2 on splenic antigen-presenting cells and efficient T cell priming by staphylococcal enterotoxin A

It has been demonstrated that in vivo T cell priming requires CD154-CD40 interaction, which is suggested to be critical in the induction of co-stimulatory activities on antigen-presenting cells (APC). In the current study, we demonstrate that in vivo administration of a high dose of a superantigen, staphylococcal enterotoxin A (SEA), could up-regulate B7-2 on most splenic APC independently of the CD154-CD40 interaction, followed by efficient expansion of SEA-reactive V(beta)3(+) T cells in CD154- or CD40-deficient mice. However, the CD154-CD40 interaction may be involved in SEA-mediated T cell activation, since a contribution of the CD154-CD40 interaction was observed when a lower dose of SEA was injected. CD154-independent T cell priming by SEA appeared also independent of the TRANCE-RANK pathway, which was shown to be capable of mediating CD154-independent activation of naive T cells during the infection of some viruses. These results indicate that SEA, which provokes rapid and efficient T cell responses without adjuvant, could utilize multiple CD154/TRANCE-independent pathways, to prime T cells.     

Expression and co-stimulatory function of B7-2 on murine CD4+ T cells

Co-stimulatory signals mediated by the interaction of B7-1/B7-2 with CD28 are important for the activation of CD4⁺ T cells stimulated with antigen on antigen-presenting cells. There are controversies about the expression and function of B7-1/B7-2 on CD4⁺ T cells. The aim of this study was to analyze the expression of B7-1/B7-2 on naive and memory CD4⁺ T cells and the co-stimulatory function in the activation of naive CD4⁺ T cells stimulated by TCR ligation. Present results indicate that memory CD4⁺ T cells express B7-2 molecules on their surface, whereas naive CD4⁺ T cells do not. Neither memory nor naive CD4⁺ T cells expressed B7-1 molecule on their surface, although B7-1 mRNA was faintly expressed in memory T cells. B7-2 molecules expressed on memory T cells co-stimulated CD4⁺ naive T cells stimulated with plate-coated anti-CD3 to produce IL-2. Naive CD4⁺ T cells were shown to express B7-2 after co-stimulation with B7-2 and TCR ligation, because the naive T cells stimulated with anti-CD3 and B7-2CHO expressed B7-2 on their surface, although it remained to be studied whether the co-stimulation with B7-2 directly induced B7-2 expression on naive T cells. Our present results indicate that memory CD4⁺ T cells play some role in the activation of naive CD4⁺ T cells through the co-stimulation with B7-2 molecules.     

CD28 ligands CD80 (B7-1) and CD86 (B7-2) induce long-term autocrine growth of CD4+ T cells and induce similar patterns of cytokine secretion in vitro

The interaction of CD28 and its ligands is critical for antigen-inducedT cell activation. Recent studies have demonstrated the existenceof at least two members of the B7 receptor family. In this report,the co-stimulatory signals provided by CD80 (B7-1) or CD86 (B7-2)were compared to CD28 ligation by mAb. We demonstrate that thekinetics of induction of T cell proliferation after anti-CD3stimulation was similar regardless of the form of co-stimulation.Similarly, B7-1 and B7-2 could both maintain long-term expansionof CD4 cells. The co-stimulatory effects of both B7-1 and B7-2were dependent on CD28 cross-linking, based on complete inhibitionof proliferation by CD28 antibody Fab fragments. Co-stimulationwith B7-1 and B7-2 induced high levels of cytokine secretionby resting T cells, and the effects of B7-1 and B7-2 could notbe distinguished. This conclusion is based on analysis of theinitial activation of CD28⁺ T cells. as well as T cell subpopulationsconsisting of CD4⁺ and CD8⁺ T cells. Both B7-1 and B7-2 couldelicit IL-4 secretion from CD4⁺ T cells while anti-CD28 antibodyinduced substantially less IL-4 secretion. Furthermore, bothB7-1 and B7-2 could stimulate high levels of IFN- and IL-4 fromCD4⁺CD45RO⁺ cells, while neither B7 receptor could co-stimulateIFN- and IL-4 secretion from CD4⁺CD45RA⁺ T cells. B7-1 and B7-2could, however, co-stimulate CD4⁺CD45RA⁺ T cells to secreteIL-2. By contrast, when previously activated T cells were tested,re-stimulation of CD4⁺ T cell blasts with B7-1 or B7-2 resultedin higher secretion of IL-4 and IL-5 than anti-CD28, while re-stimulationwith anti-CD28 antibody maintained a higher level of secretionof IL-2 and IFN- than B7-1 or B7-2. These observations may haveimportant implications because they suggest that the mannerof CD28 ligation can be a critical determinant in the developmentof cytokine secretion that corresponds to T_h1- and T_h2-likepatterns of differentiation. Together these observations suggestthat there are no Intrinsic differences between B7-1 and B7-2in their ability to co-stimulate the populations of cells thatwe have tested.     

CXCR5+ CCR7- CD8 T cells are early effector memory cells that infiltrate tonsil B cell follicles

Naive and central memory CD8 T cells use CCR7 to recirculate through T cell zones of secondary lymphoid organs where they can encounter antigen. Here we describe a subset of human CD8 T cells expressing CXCR5 which enables homing in response to CXCL13 produced within B cell follicles. CXCR5+ CD8 T cells were found in tonsil B cell follicles, and isolated cells migrated towards CXCL13 in vitro. They expressed CD27, CD28, CD45RO, CD69, and were CD7low, and produced IFN-gamma and granzyme A but lacked perforin, a functional profile suggesting that these cells are early effector memory cells in the context of contemporary T cell differentiation models. Receptors important in the interaction with B cells, including CD70, OX40 and ICOS, were induced upon activation, and CXCR5+ CD8 T cells could to some extent support survival and IgG production in tonsil B cells. Furthermore, CXCR5+ CD8 T cells expressed CCR5 but no CCR7, suggesting a migration pattern distinct from that of follicular CD4 T cells. The finding that a subset of early effector memory CD8 T cells use CXCR5 to locate to B cell follicles indicates that MHC class I-restricted CD8 T cells are part of the follicular T cell population.     

IL-4 influences the differentiation and the susceptibility to activation-induced cell death of human naive CD8+ T cells

It is now well established that the cytokine environment influences the activation, differentiation, proliferation and death of T lymphocytes during the primary response to antigen. Using an in vitro model, we investigated the influence of IL-4, added at the onset of TCR stimulation, on phenotypic and functional markers of naive CD8+ T cell activation including the up-regulation of activation markers, proliferation as well as the susceptibility to activation-induced cell death (AICD). We report that IL-4, unlike IL-2 added at the onset of repeated TCR stimulation of naive CD8+ T cells prevents AICD, in part due to its ability to maintain the level of the survival-related protein Bcl-2. Moreover, TCR-triggered activation of naive CD8+ T cells in the presence of IL-4 leads to the development of a CD8+ T cell subset that proliferates normally, but which fails to exhibit characteristic activation parameters such as the up-regulation of CD25 and Granzyme B. Taken together, these results demonstrate that exposure to IL-4 during primary activation influences CD8+ T cell differentiation by inducing the development of a sub-population of AICD-resistant, proliferation-competent cells that do not show some of the typical features of CD8+ T cell activation.     

Engagement of the CD4 receptor inhibits the interleukin-2-dependent proliferation of human T cells transformed by Herpesvirus saimiri

Infection with Herpesvirus saimiri, a tumor virus of non-human primates, transformed human CD4⁺ T cell clones to permanent interleukin (IL)-2-dependent growth without need for restimulation with antigen and accessory cells. The IL-2-dependent proliferation of these cells was dramatically inhibited by soluble anti-CD4 whole antibodies, F(ab′)₂ and Fab fragments, and also by gp 120 of human immunodeficiency virus. The inhibition was not due to cell death and could be overcome by high concentrations of exogenous IL-2. Cell surface expression of CD4, and to a lesser degree the density of the IL-2 receptor α chain, were reduced upon anti-CD4 treatment. After long lasting (>12h) incubation with anti-CD4, abundance and activity of CD4-bound p56^lck were diminished while the free fraction of p56^lck remained unchanged. Since IL-2 binding to its receptor activated only the CD4-bound fraction of p56^lck, the IL-2-induced p56^lck activity was diminished after long-term CD4 ligation. Taken together, our results suggest a cross talk between CD4- and IL-2 receptor-mediated signaling via p56^lck.     

B7 2 (CD86) is essential for the development of IL-4-producing T cells

The CD28/CTLA-4 ligands, B7–1 (CD80) and B7–2 (CD86),provide a co-stimulatory signal necessary for optimal T cellactivation. We have examined the effect of blocking B7–1and B7–2 in an in vitro system using ovalbumin-specificT cells from ß TCR-transgenic mice. This system allowedus to examine the interaction of B7 co-stimulators on physiologicantigen-presenting cells (APC) with antigen-specific T helperprecursor (T_hp) cells. We report that blocking T_hp/B7–1or B7–2 interactions in a primary response differentiallyaffects the cytokine profile observed in a secondary stimulation,even in the absence of additional anti-B7 antibody. Engagementof B7–2 in the primary stimulation was found to be essentialfor production of the T_h2 cytokine, IL-4, but not the T_h1 cytokines,IL-2 and IFN-, in a secondary stimulation. Conversely, inclusionof the anti-B7–1 mAb in cultures using highly purifiednaive T cells increased levels of IL-4 and significantly depressedlevels of IFN-, upon re-stimulation. The effect of the anti-B7–2mAb in reducing IL-4 production could be overcome by the additionof recombinant IL-4 in the primary stimulation. The effectsof the anti-B7–2 mAb appear to be due to blocking andnot cross-linking, as F(ab) fragments mimicked the intact antibody.Taken together, our data demonstrate that the interaction betweenThp and B7–2 favors the development of T_h2 cells.     

The role of CD2/LFA-3 interaction in antigen- and mitogen-induced activation of human T cells

Binding of LFA-3 to the T cell surface receptor CD2 promotes intercellular adhesion and is costimulatory with anti-CD2 mAbs in 'alternative pathway' activation of T cells. Since all AC-dependent systems of T cell activation are inhibited by anti-LFA-3 mAb, it was asked whether in mitogen- and antigen-induced activation of human T cells, the function of CD2/LFA-3 interaction involves signalling beyond its function in promoting intercellular adhesion. In order to selectively block and reconstitute CD2/LFA-3 interaction while leaving other AC functions available, the response of unseparated PBMC to various T cell mitogens and to allogeneic cells was blocked by a newly developed mAb (G26) to human LFA-3. Addition of purified T11TS, the sheep form of LFA-3 that binds to human CD2 but is not recognized by mAb G26, restored the T cell response to PHA-P but not to ConA, surface aldehydes, anti-CD3 mAb, or allogeneic cells. In addition, purified resting human T cells which were unresponsive to stimulation by lectins or anti-CD3 mAbs were activated by PHA-P in the presence of purified T11TS, demonstrating that provision of LFA-3 is a sufficient accessory cell function in the activation of human T cells by this mitogen. Again, the responses to ConA, cell surface aldehydes, or soluble anti-CD3 mAb were not restored by T11TS. T cell activation by PHA-P, but not by the other polyclonal T-cell activators studied thus seems to be mechanistically similar to 'alternative pathway' activation induced by anti-CD2 mAb in that the costimulatory effect of LFA-3 is independent of its prescence on an accessory cell membrane.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of CD28-B7.1 and B7.2 functional interaction in resting human T cells: Phosphatidylinositol 3-kinase association to CD28 and cytokine production

CD28 is a 44-kDa homodimer present on T cells providing an important costimulatory signal for T cell proliferation, cytokine production and cytokine receptor expression. CD28 activation is mediated by interaction with its counter-receptors, B7.1/CD80 and B7.2/B70/CD86. The biochemical basis of these costimulatory signals are still poorly understood, particularly in resting T cells. However, various biochemical pathways such as tyrosine phosphorylation, phospholipase C, sphingomyelinase and phosphatidylinositol 3-kinase (PI3-K) activation have been reported to play a role in CD28 signaling in tumor T cell lines and CD28-transfected cells or pre-activated T cells. In addition, recent reports propose that CD28-B7.1 and B7.2 interaction could be involved in the production of Th1 and Th2 cytokines, respectively, but the putative biochemical basis for these different functions is still unknown. We have analyzed the functional and molecular consequences of CD28 activation by B7.1 and B7.2 in human resting T cells. We demonstrate in this report that both CD28-B7.1 and CD28-B7.2 interactions induce the association of PI3-K to CD28 in the CD4 subpopulation, whereas it was barely detectable in CD8 cells. This association involves the binding of the src homology domain 2 (SH2) of p85 to tyrosine-phosphorylated CD28 and does not require pre-activation by CD3-T cell receptor. Worthmannin, a specific inhibitor of PI3-K enzymatic activity within the nanomolar range also inhibits the interleukin-2 production induced by costimulation mediated by either the B7.1- and B7.2-transfected cells or CD28 monoclonal antibodies. The only slight difference between B7.1 and B7.2 costimulation is the IC₅₀ of wortmannin being 25 and 110 nM, respectively, which could suggest differences in their activation of the T cell PI3-K.     

Expression and function of B7-1 (CD80) and B7-2 (CD86) on human epidermal Langerhans cells

In addition to T cell receptor triggering, activation of T cells requires co-stimulatory signals that have been shown to be mainly initiated through CD28. We analyzed the expression and function of the two ligands for CD28, B7-1 (CD80) and B7-2 (CD86), on human Langerhans cells (LC), the antigen-presenting cells from epidermis. Human LC freshly isolated from epidermis (fLC) expressed significant level of B7-2, which was increased upon a short culture in vitro. In contrast, B7-1 was undetectable on fLC but appeared at the cell surface after a 3-day culture in vitro. Pre-incubation of 18-h cultured LC with anti-B7-2 monoclonal antibodies (mAb) was sufficient to abrogate the binding of CTLA4-Ig fusion protein, while a combination of both mAb against B7-1 and B7-2 was necessary to obtain a complete inhibition of CTLA4-Ig binding on 3-day cultured LC, showing the absence of a third CTLA4 ligand. The function of B7-1 and B7-2 on human LC has been analyzed by adding mAb at the beginning of mixed epidermal cell lymphocyte reactions. Anti-B7-2 mAb and CTLA4-Ig, but not anti-B7-1 mAb, strongly inhibited allogeneic, as well as recall antigen-induced T cell proliferation supported by fLC or 3-day cultured LC. Collectively, these results demonstrate that B7-2 is the major ligand for CD28/CTLA4 at the LC surface and that it plays a crucial role in human LC co-stimulatory function with little, if any, dependence on B7-1 expression.