Development of T cell-mediated immunity after autologous stem cell transplantation: prolonged impairment of antigen-stimulated production of gamma-interferon.

The conditioning regimens for autologous SCT (auto-SCT) lead to impairment of the immune system and concomitant increase in susceptibility to infections. We studied the recovery of cellular immunity by in vitro analysis of T-cell proliferation and cytokine production profiles during the first 15 months after auto-SCT in patients with multiple myeloma and non-Hodgkin's lymphoma. PBMC were collected at 6, 9 and 15 months after transplantation and stimulated with a combination of CD2 and CD28 monoclonal antibodies, with PHA or with tetanus toxoid as recall antigen. A multiplex enzyme linked immunoassay was used to determine levels of Th1 cytokines IL-2, IFN-gamma and tumour-necrosis factor-alpha (TNF-alpha), Th2 cytokines IL-4, IL-5 and IL-13, the regulatory cytokine IL-10 and the proinflammatory cytokines IL-1alpha, IL-1beta, IL-6 and the chemokine IL-8. T-cell proliferation progressively increased from 6 to 15 months after auto-SCT. Overall, cytokine production increased after auto-SCT. Production of Th2 cytokines IL-5 and IL-13 was superior to production of Th1 cytokines IFN-gamma and TNF-alpha. We hypothesize that prolonged impairment of IFN-gamma production might contribute to the relatively high incidence of viral infections after auto-SCT.

The Noxa/Mcl-1 axis regulates susceptibility to apoptosis under glucose limitation in dividing T cells.

Throughout lymphocyte development, cellular persistence and expansion are tightly regulated by survival and apoptosis. Within the Bcl-2 family, distinct apoptogenic BH3-only members like Bid, Bim, and Puma appear to function in specific cell death pathways. We found that naive human T cells after mitogenic activation, apart from expected protective Bcl-2 members, also rapidly upregulate the BH3-only protein Noxa in a p53-independent fashion. The specific role of Noxa became apparent during glucose limitation and involves interaction with the labile Bcl-2 homolog Mcl-1. Knockdown of Noxa or Mcl-1 results in protection or susceptibility, respectively, to apoptosis induced by glucose deprivation. Declining Mcl-1 levels and apoptosis induction are inversely correlated to Noxa levels and prevented by readdition of glucose. We propose that the Noxa/Mcl-1 axis is an apoptosis rheostat in dividing cells, in a selective pathway that functions to restrain lymphocyte expansion and can be triggered by glucose deprivation.

LFA-1-dependent HuR nuclear export and cytokine mRNA stabilization in T cell activation.

Lymphokine gene expression is a precisely regulated process in T cell-mediated immune responses. In this study we demonstrate that engagement of the beta(2) integrin LFA-1 in human peripheral T cells markedly extends the half-life of TNF-alpha, GM-CSF, and IL-3 mRNA, as well as a chimeric beta-globin mRNA reporter construct containing a strongly destabilizing class II AU-rich element from the GM-CSF mRNA 3'-untranslated region. This integrin-enhanced mRNA stability leads to augmented protein production, as determined by TNF-alpha ELISPOT assays. Furthermore, T cell stimulation by LFA-1 promotes rapid nuclear-to-cytoplasmic translocation of the mRNA-stabilizing protein HuR, which in turn is capable of binding an AU-rich element sequence in vitro. Abrogation of HuR function by use of inhibitory peptides, or marked reduction of HuR levels by RNA interference, prevents LFA-1 engagement-mediated stabilization of T cell TNF-alpha or IFN-gamma transcripts, respectively. Thus, HuR-mediated mRNA stabilization, stimulated by integrin engagement and controlled at the level of HuR nuclear export, is critically involved in T cell activation.

Condensation of the plasma membrane at the site of T lymphocyte activation.

After activation, T lymphocytes restructure their cell surface to form membrane domains at T cell receptor (TCR)-signaling foci and immunological synapses (ISs). To address whether these rearrangements involve alteration in the structure of the plasma membrane bilayer, we used the fluorescent probe Laurdan to visualize its lipid order. We observed a condensation of the plasma membrane at TCR activation sites. The formation of ordered domains depends on the presence of the transmembrane protein linker for the activation of T cells and Src kinase activity. Moreover, these ordered domains are stabilized by the actin cytoskeleton. Membrane condensation occurs upon TCR stimulation alone but is prolonged by CD28 costimulation with TCR. In ISs, which are formed by conjugates of TCR transgenic T lymphocytes and cognate antigen-presenting cells, similar condensed membrane phases form first in central regions and later at the periphery of synapses. The formation of condensed membrane domains at T cell activation sites biophysically reflects membrane raft accumulation, which has potential implications for signaling at ISs.

Cutting edge: silencing suppressor of cytokine signaling 3 expression in dendritic cells turns CD28-Ig from immune adjuvant to suppressant.

CTLA-4-Ig and CD28-Ig are both agonist ligands of B7 coreceptor molecules on mouse dendritic cells (DCs), yet they bias the downstream response in opposite directions, and CTLA-4-Ig promotes tolerance, whereas CD28-Ig favors the onset of immunity. Although B7 engagement by either ligand leads to a mixed cytokine response, a dominant IL-6 production in response to CD28-Ig prevents the IFN-gamma-driven induction of immunosuppressive tryptophan catabolism mediated by IDO. In the present study, we show that silencing the expression of suppressor of cytokine signaling 3 (SOCS3) in DCs by RNA interference renders CD28-Ig capable of activating IDO, likely as a result of unrestrained IFN-gamma signaling and IFN-gamma-like actions of IL-6. Thus, in the absence of SOCS3, CD28-Ig becomes immunosuppressive and mimics the action of CTLA-4-Ig on tryptophan catabolism.

The CD28 peptidemimic can induce mixed chimerism and prolong the survival of cardiac allografts.

Costimulatory blockade with CD28 peptidemimic (CD28PM, CD28 PM was synthesized by solid phase synthetic methods) prolongs cardiac allograft survival in mice, but has not reliably induced tolerance when used alone. In the current studies, we evaluated the effect of adding B7 blockade to a chimerism inducing nonmyeloablative regimen in mice and observed a significant improvement of donor bone marrow cells (BMC) engraftment, which had been associated with mixed chimerism and long-term survival of cardiac allografts. The mixed lymphocyte reaction (MLR) and the ear pinna cardiac transplantation model were performed to evaluate the effects of CD28PM in induction of specific immune hypo-response and extension of allograft survival. The expressed rates of B7.1 and B7.2 on the C57BL/6 splenocytes were 56.25% and 20.52%, respectively. The specific hypo-response status was established after immunization with CD28PM pre-treated donor splenocytes and the average inhibition rate was only 43% compared with normal control. Subsequently, a total number of 2 x 10(7) bone marrow cells per mouse were implanted to the recipients. The allogenic chimerism was obviously observed with the rate as high as 8.84% (mean) at the time point of day 14. During the first 50 days post bone marrow transfusion (BMT) the chimerism rate declined stepwise. But from 50 to 100 days, the chimerism rate sustained in a range of 3.35% to 4.6%. The results of transplantation experiments showed the survival of allgenic cardiac grafts were maintained over 100 days in recipients. Thus, donor BMC engraftment with mixed chimerism appears essential for induction of allograft tolerance using this conditioning regimen. Mixed chimerism approach, by the addition of CD28-B7 costimulatory blockade with CD28PM, has been shown to establish mixed chimerism and induce cardiac allograft tolerance in mice.

Conversion of CTLA-4 from inhibitor to activator of T cells with a bispecific tandem single-chain Fv ligand.

Abs or their recombinant fragments against surface receptors of the Ig superfamily can induce or block the receptors' native function depending on whether they induce or prevent the assembly of signalosomes on their cytoplasmic tails. In this study, we introduce a novel paradigm based on the observation that a bispecific tandem single-chain variable region fragment ligand of CTLA-4 by itself converts this inhibitory receptor into an activating receptor for primary human T lymphocytes. This reversal of function results from increased recruitment of the serine/threonine phosphatase 2A to the cytoplasmic tail of CTLA-4, consistent with a role of this phosphatase in the regulation of CTLA-4 function, and assembly of a distinct signalosome that activates an lck-dependent signaling cascade and induces IL-2 production. Our data demonstrate that the cytoplasmic domain of CTLA-4 has an inherent plasticity for signaling that can be exploited therapeutically with recombinant ligands for this receptor.

Transient and selective NF-kappa B p65 serine 536 phosphorylation induced by T cell costimulation is mediated by I kappa B kinase beta and controls the kinetics of p65 nuclear import.

Full transcriptional activity of the nuclear, DNA-bound form of NF-kappaB requires additional posttranslational modifications. In this study, we systematically mapped the T cell costimulation-induced phosphorylation sites within the C-terminal half of the strongly trans-activating NF-kappaB p65 subunit and identified serine 536 as the main phosphorylation site. The transient kinetics of serine 536 phosphorylation paralleled the kinetics of IkappaBalpha and IkappaB kinase (IKK) phosphorylation and also mirrored the principle of T cell costimulation. The TCR-induced pathway leading to serine 536 phosphorylation is regulated by the kinases Cot (Tpl2), receptor interacting protein, protein kinase Ctheta, and NF-kappaB-inducing kinase, but is independent from the phosphatidylinositol 3-kinase/Akt signaling pathway. Loss-of-function and gain-of-function experiments showed phosphorylation of p65 serine 536 by IKKbeta, but not by IKKalpha. Phosphorylation occurs within the cytoplasmic and intact NF-kappaB/IkappaBalpha complex and requires prior phosphorylation of IkappaBalpha at serines 32 and 36. Reconstitution of p65(-/-) cells either with wild-type p65 or a p65 mutant containing a serine to alanine mutation revealed the importance of this phosphorylation site for cytosolic IkappaBalpha localization and the kinetics of p65 nuclear import.

Cathepsin-dependent apoptosis triggered by supraoptimal activation of T lymphocytes: a possible mechanism of high dose tolerance.

High doses of Ag can paradoxically suppress immune responses in vivo. This Ag-specific unresponsiveness (termed high dose tolerance) involves extrathymic mechanisms in mature T lymphocytes. To investigate these mechanisms, we used the in vitro model of PBL activated with anti-CD3 or PHA. In these conditions, increasing mitogen concentrations resulted in a reduction of the proliferative response, associated with an increased percentage of apoptotic cells. Apoptosis did not require prior exposure to IL-2, it was not the consequence of CD178/CD95 or TNF/TNFR interactions, and was therefore clearly distinct from activation-induced cell death. Although the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk) decreased DNA fragmentation, cytochrome c release and caspase-9 and caspase-3 activation were not implicated, suggesting that this apoptosis did not primarily involve the intrinsic mitochondrial pathway. E64d, a cysteine protease inhibitor, as well as specific inhibitors of cathepsin B and cathepsin L conferred protection. We further demonstrated that cathepsin B and cathepsin L were released from the lysosomes and catalytically active in the cytosol. Release of cathepsin B and cathepsin L was the consequence of lysosomal membrane permeabilization without complete disruption of the cytosol-lysosome pH gradient. These results demonstrate a role for cathepsins in supraoptimal activation-induced apoptosis in vitro and suggest their possible participation in high dose tolerance in vivo.

Induction of lysosomal and plasma membrane-bound sialidases in human T-cells via T-cell receptor.

Among the three isoenzymes of neuraminidase (Neu) or sialidase, Neu-1 has been suggested to be induced by cell activation and to be involved in IL (interleukin)-4 biosynthesis in murine T-cells. In the present study, we found that antigen-induced airway eosinophilia, a typical response dependent on Th2 (T-helper cell type 2) cytokines, as well as mRNA expression of Th2 cytokines, including IL-4, are suppressed in Neu-1-deficient mice, thereby demonstrating the in vivo role of murine Neu-1 in regulation of Th2 cytokines. To elucidate the roles of various sialidases in human T-cell activation, we investigated their tissue distribution, gene induction and function. Neu-1 is the predominant isoenzyme at the mRNA level in most tissues and cells in both mice and humans, including T-cells. T-cells also have significant levels of Neu-3 mRNAs, albeit much lower than those of Neu-1, whereas the levels of Neu-2 mRNAs are minimal. In human T-cells, both Neu-1 and Neu-3 mRNAs are significantly induced by T-cell-receptor stimulation, as is sialidase activity against 4-methylumbelliferyl- N -acetylneuramic acid (a substrate for both Neu-1 and Neu-3) and the ganglioside G(D1a) [NeuAcalpha2-3Galbeta1-3GalNAcbeta1-4(NeuAcalpha2-3)Galbeta1-4Glcbeta1-cer] (a substrate for Neu-3, but not for Neu-1). The expression of the two sialidase genes may be under differential regulation. Western blot analysis and enzymic comparison with recombinant sialidases have revealed that Neu-3 is induced as a major isoform in activated cells. The induction of Neu-1 and Neu-3 in T-cells is unique. In human monocytes and neutrophils stimulated with various agents, the only observation of sialidase induction has been by IL-1 in neutrophils. Functionally, a major difference has been observed in Jurkat T-cell lines over-expressing Neu-1- and Neu-3. Upon T-cell receptor stimulation, IL-2, interferon-gamma, IL-4 and IL-13 are induced in the Neu-1 line, whereas in the Neu-3 line the same cytokines are induced, with the exception of IL-4. Taken together, these results suggest an important immunoregulatory role for both Neu-1 and Neu-3 in humans.

EphA3 is induced by CD28 and IGF-1 and regulates cell adhesion.

Stimulation of CD28 alone has been shown to regulate cytokine gene transcription and expression of the type 1 insulin-like growth factor receptor (IGF-1R) in lymphocytes. In this study, the ephrin receptor tyrosine kinase ephA3, was identified as a new CD28-responsive gene in Jurkat cells by using a human cytokine/receptor array. EphA3 was not detected in normal peripheral T cells, in any subset of thymus-derived developing T cells, or in Hodgkin's lymphoma. However, contrary to previous findings, EphA3 was detected in a panel of T-cell lymphomas. Stimulation of Jurkat cells with ephrin-A5 resulted in loss of cell adhesion to fibronectin and recruitment of the adapter protein CrkII to EphA3. Interestingly, EphA3 expression in CD28-stimulated Jurkat cells was enhanced by IGF-1 or by overexpression of the IGF-1R, and was suppressed by anti-IGF-1R blocking antibodies. The data suggest that CD28- and IGF-1-regulated expression of EphA3 is associated with adherence and that it may be involved in the motility of malignant T cells.

A failure to repair self-proteins leads to T cell hyperproliferation and autoantibody production.

It is clear that many factors can perturb T cell homeostasis that is critical in the maintenance of immune tolerance. Defects in the molecules that regulate homeostasis can lead to autoimmune pathology. This simple immunologic concept is complicated by the fact that many self-proteins undergo spontaneous posttranslational modifications that affect their biological functions. This is the case in the spontaneous conversion of aspartyl residues to isoaspartyl residues, a modification occurring at physiological pH and under conditions of cell stress and aging. We have examined the effect of isoaspartyl modifications on the effector functions of T lymphocytes in vivo using mice lacking the isoaspartyl repair enzyme protein carboxyl methyltransferase (PCMT). PCMT(-/-) CD4(+) T cells exhibit increased proliferation in response to mitogen and Ag receptor stimulation as compared with wild-type CD4(+) T cells. Hyperproliferation is marked by increased phosphorylation of members of both the TCR and CD28 signaling pathways. Wild-type mice reconstituted with PCMT(-/-) bone marrow develop high titers of anti-DNA autoantibodies and kidney pathology typical of that found in systemic lupus erythematosus. These observations, coupled with the fact that humans have polymorphisms in the pcmt gene, suggest that isoaspartyl self-proteins may alter the maintenance of peripheral immune tolerance.

The flotillins are integral membrane proteins in lipid rafts that contain TCR-associated signaling components: implications for T-cell activation.

Lipid rafts play an important role in signal integration and cellular activation by the T-cell antigen receptor (TCR). We demonstrate that flotillin-1 and flotillin-2 are important structural raft components, which redistribute to the site of TCR engagement. An antibody to flotillin-1 was able to immobilize other TCR-associated raft components. Although rafts purified from unstimulated cells demonstrated abundant Lck but inabundant LAT, rafts from stimulated cells include an abundance of both components. This suggests dynamic changes in lipid raft composition during CD3/CD28 costimulation. Stimulation of primary human CD4(+) T cells leads to increased GM1 and flotillin-1 expression in the surface membrane, where these components colocalize. This may reconstitute new signaling complexes required for T-cell activation. Altered lipid raft composition and function may play a role in the decline of antigen responsiveness in senescent T cells. In this regard, we observed an increase in the raft-associated gangliolipid, GM1, in resting human CD4(+) and CD8(+) lymphocytes with aging.

Incomplete activation of CD4 T cells by antigen-presenting transitional immature B cells: implications for peripheral B and T cell responsiveness.

B cells leave the bone marrow as transitional B cells. Transitional B cells represent a target of negative selection and peripheral tolerance, both of which are abrogated in vitro by mediators of T cell help. In vitro, transitional and mature B cells differ in their responses to B cell receptor ligation. Whereas mature B cells up-regulate the T cell costimulatory molecule CD86 (B7.2) and are activated, transitional B cells do not and undergo apoptosis. The ability of transitional B cells to process and present Ag to CD4 T cells and to elicit protective signals in the absence of CD86 up-regulation was investigated. We report that transitional B cells can process and present Ag as peptide:MHC class II complexes. However, their ability to activate T cells and elicit help signals from CD4-expressing Th cells was compromised compared with mature B cells, unless exogenous T cell costimulation was provided. A stringent requirement for CD28 costimulation was not evident in interactions between transitional B cells and preactivated CD4-expressing T cells, indicating that T cells involved in vivo in an ongoing immune response might rescue Ag-specific transitional B cells from negative selection. These data suggest that during an immune response, immature B cells may be able to sustain the responses of preactivated CD4(+) T cells, while being unable to initiate activation of naive T cells. Furthermore, the ability of preactivated, but not naive T cells to provide survival signals to B cell receptor-engaged transitional immature B cells argues that these B cells may be directed toward activation rather than negative selection when encountering Ag in the context of a pre-existing immune response.

Induction of CD4+ T cell apoptosis as a consequence of impaired cytoskeletal rearrangement in UVB-irradiated dendritic cells.

Low dose UVB irradiation of dendritic cells (DC) dose-dependently decreases their allostimulatory capacity and inhibits alloreactive T cell proliferation. The reduction of the stimulatory capacity is not associated with a perturbation of CD28 costimulation. To examine the underlying mechanism, cell cycle analysis of T cells from cocultures with UVB-irradiated DC (UVB-DC) was performed, revealing no cell cycle arrest, but an increased number of apoptotic T cells in sub-G(0) phase. We confirmed T cells to undergo apoptosis after coincubation with UVB-DC by TUNEL staining and DNA laddering. To analyze whether T cell apoptosis requires the Fas/Fas ligand (FasL) pathway, MLRs were performed with Fas-, FasL-deficient, and wild-type DC and T cells. No differences were found on comparison of wild-type DC with Fas-/FasL-deficient DC or T cells. Likewise, addition of a neutralizing anti-TNF-alpha mAb to cocultures could not overcome inhibition of T cell proliferation by UVB-DC, excluding involvement of the TNF-alpha/TNF-alphaR pathway. FACS analysis of CD69 and CD25 revealed no up-regulation on T cells cocultured with UVB-DC, suggesting a perturbation of early T cell activation. Analysis of UVB-DC by confocal microscopy demonstrated impaired filamentous actin bundling, a process critical for T cell stimulation. To investigate the functional relevance of these observations, time lapse video microscopy was performed. Indeed, calcium signaling in CD4(+) T cells was significantly diminished after interaction with UVB-DC. In conclusion, UVBR of DC impairs their cytoskeletal rearrangement and induces apoptosis in CD4(+) T cells by disruption of early DC-T cell interaction, resulting in a reduced Ca(2+) influx in T cells.

The cyclic adenosine 5'-monophosphate response element modulator suppresses IL-2 production in stimulated T cells by a chromatin-dependent mechanism.

The production of IL-2 is tightly controlled by several transcription factors that bind to the IL-2 promoter. The cAMP response element modulator (CREM) is known to form complexes with CREB and bind to the -180 site of the IL-2 promoter in anergic and in systemic lupus erythematosus T cells. In this study we show that CREM is transcriptionally induced in T cells following stimulation through CD3 and CD28, binds to the IL-2 promoter in vivo, and suppresses IL-2 production. Transfection of an antisense CREM plasmid into T cells blocked the expression and binding of CREM to the IL-2 promoter and the decrease of IL-2 production, which follows the early increase after T cell stimulation with CD3 and CD28. In addition, as assessed by chromatin immunoprecipitation experiments, antisense CREM prevented the binding of protein 300 and cAMP response element binding protein and promoted the acetylation of histones. Antisense CREM also enhanced the accessibility of the IL-2 promoter to endonucleases and prevented the condensation of chromatin in vivo. Our data suggest that upon T cell activation, CREM gradually replaces phosphorylated CREB at the -180 site of the IL-2 promoter. CREM, in turn, binds protein 300 and cAMP response element binding protein, but CREM is unable to activate its histone acetyltransferase activity, which results in condensation of chromatin and down-regulation of IL-2 production.

c-Rel is required for chromatin remodeling across the IL-2 gene promoter.

IL-2 gene transcription occurs in an activation-dependent manner in T cells responding to TCR and CD28 activation. One of the critical events leading to increased IL-2 transcription is an alteration in chromatin structure across the 300-bp promoter region of the gene. We initially showed that IL-2 gene transcription in CD4(+) primary T cells is dependent on the NF-kappaB family member, c-Rel, but not RelA. We found that c-Rel is essential for global changes in chromatin structure across the 300-bp IL-2 promoter in response to CD3/CD28 in primary CD4(+) T cells, but not in response to pharmacological signals, paralleling the requirement for c-Rel in IL-2 mRNA and protein accumulation. Interestingly, measurement of activation-induced localized accessibility changes using restriction enzyme digestion revealed that accessibility close to the c-Rel binding site in the CD28RR region of the promoter is specifically dependent on c-Rel. In contrast, restriction enzyme sites located at a distance from the CD28RR behave independently of c-Rel. These results suggest a nonredundant role for c-Rel in generating a correctly remodeled chromatin state across the IL-2 promoter and imply that the strength of the signal determines the requirement for c-Rel.

Src homology 2 domain-containing leukocyte phosphoprotein of 76 kDa and phospholipase C gamma 1 are required for NF-kappa B activation and lipid raft recruitment of protein kinase C theta induced by T cell costimulation.

The NF-kappaB activation pathway induced by T cell costimulation uses various molecules including Vav1 and protein kinase C (PKC)theta. Because Vav1 inducibly associates with further proteins including phospholipase C (PLC)gamma1 and Src homology 2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76), we investigated their role for NF-kappaB activation in Jurkat leukemia T cell lines deficient for expression of these two proteins. Cells lacking SLP-76 or PLCgamma1 failed to activate NF-kappaB in response to T cell costimulation. In contrast, replenishment of SLP-76 or PLCgamma1 expression restored CD3/CD28-induced IkappaB kinase (IKK) activity as well as NF-kappaB DNA binding and transactivation. PKCtheta activated NF-kappaB in SLP-76- and PLCgamma1-deficient cells, showing that PKCtheta is acting further downstream. In contrast, Vav1-induced NF-kappaB activation was normal in SLP-76(-) cells, but absent in PLCgamma1(-) cells. CD3/CD28-stimulated recruitment of PKCtheta and IKKgamma to lipid rafts was lost in SLP-76- or PLCgamma1-negative cells, while translocation of Vav1 remained unaffected. Accordingly, recruitment of PKCtheta to the immunological synapse strictly relied on the presence of SLP-76 and PLCgamma1, but synapse translocation of Vav1 identified in this study was independent from both proteins. These results show the importance of SLP-76 and PLCgamma1 for NF-kappaB activation and raft translocation of PKCtheta and IKKgamma.

IL-2 receptor blockade inhibits late, but not early, IFN-gamma and CD40 ligand expression in human T cells: disruption of both IL-12-dependent and -independent pathways of IFN-gamma production.

mAbs directed against the alpha-chain (Tac/CD25) of the IL-2R are an emerging therapy in both transplantation and autoimmune disease. However, the mechanisms underlying their therapeutic efficacy have not been fully elucidated. Therefore, we examined the effect of IL-2R blockade on Th1 and Th2 cytokine production from human PBMC. Addition of a humanized anti-Tac Ab (HAT) to activated PBMC cultures inhibited IFN-gamma production from CD4 and CD8 T cells by 80-90%. HAT partially inhibited production of TNF-alpha and completely inhibited production of IL-4, IL-5, and IL-10. Furthermore, IL-12, a central regulatory cytokine that induces IFN-gamma, was undetectable in treated cultures. As T cell-dependent induction of IL-12 is regulated via CD40/CD40 ligand (CD40L) interactions, we examined the effect of HAT on CD40L expression. We found CD40L expression to be biphasic with an early (6 h) peak that is CD28/IL-2-independent, but a later peak (48 h) being CD28/IL-2-dependent and inhibited by HAT. Similarly, IFN-gamma production at 6 h was CD28/IL-2-independent but CD28/IL-2-dependent and inhibited by HAT at 48 h. Nonetheless, addition of rCD40L or exogenous IL-12 to HAT-treated cultures could not restore IFN-gamma production. The IFN-gamma deficit in such cultures appears to be due to a direct inhibition by HAT of IL-12-independent IFN-gamma production from T cells rather than altered expression of either the IL-12Rbeta1 or IL-12Rbeta2 chains. These data demonstrate that IL-2 plays a critical role in the regulation of Th1 and Th2 responses and impacts both IL-12-dependent and -independent IFN-gamma production.

Differential effect of CD28 versus B7 blockade on direct pathway of allorecognition and self-restricted responses.

Immunosuppression with B7 antagonists might have 2 opposite effects: reducing T-cell costimulation through CD28 but also preventing CTLA-4 from transmitting its negative regulatory signal. We therefore hypothesized that a selective blockade of CD28 might be qualitatively different from blocking B7. It was previously reported that CD28 modulation prolongs allograft survival in the rat and reverses induction of experimental autoimmune encephalomyelitis in mice. However, whether CD28 or B7 blockade results in similar immunosuppression on alloimmune and self-restricted responses to soluble antigens has not yet been investigated. Here, we addressed this issue in vitro with antagonist anti-CD28 Fab fragments and in vivo using the modulating anti-rat JJ319 monoclonal antibody. As in the inhibition of B7 with CTLA4 immunoglobulin, anti-CD28 Fab fragments inhibited allogenic T-cell proliferation in mixed cultures. In vivo modulation of CD28 blocked the expansion of alloreactive T cells and promoted their apoptosis. In contrast, selective blockade of CD28 did not modify T-cell proliferative responses and antibody production to soluble antigens, whereas blocking B7 with CTLA4 immunoglobulin did. Our data show that blocking CD28, while leaving CTLA4-B7 interactions undisturbed, inhibits alloreactive CD4+ T-cell expansion but does not modify the response to nominal antigens presented in the context of a self-major histocompatibility complex. That B7 engagement is needed for self-restricted responses whereas engagement of CD28 is not essential adds to the suggestion that another unidentified ligand of B7 might deliver a costimulatory signal in the absence of CD28.