Cutting Edge: Responder T cells regulate human DR+ effector regulatory T cell activity via granzyme B.

MHC class II expression identifies an effector subset of human CD4(+)CD25(high)FoxP3(high) natural regulatory T cells (DR(+) Tregs) that induces more rapid suppression and exhibits higher FoxP3 expression than the remaining Treg population. Although Tregs are known to be highly sensitive to apoptosis, in this study we demonstrate that this sensitivity is primarily a feature of DR(+) Tregs. Granzyme B (GzmB) is strongly expressed by nonregulatory responder CD4 T cells, whereas effector DR(+) Tregs express little GzmB. Strong TCR stimulation markedly increases the expression of GzmB in all dividing responder CD4 T cells and mitigates the suppression by DR(+) Tregs. DR(+) Treg suppressive activity reemerges if GzmB is neutralized. We show that responder cells actively kill effector Tregs by producing GzmB in response to strong TCR stimulation. Thus, the production of GzmB by strongly activated CD4 T cells represents a mechanism by which CD4 T cells resist Treg suppression.

Myocardial biopsy based classification and treatment in patients with dilated cardiomyopathy.

BACKGROUND: We investigated whether myocardial biopsy analysis for inflammation and viruses correlates with outcome in dilated cardiomyopathy. METHODS: Myocardial biopsies of 82 patients were analyzed for HLAI, HLAII, CD54, CD2, CD68 and entero-/adenovirus. Ejection fraction was determined by left ventriculography. NYHA classification, electrocardiogram (ECG) and echocardiography were analyzed at first admission and for follow up. Patients were attributed to three groups: (A) no inflammation/no virus (B) inflammation/no virus (C) virus with/without inflammation. Patients not responding to conventional treatment of heart failure received interferon beta1b (group C) or prednisolone (group B). Median follow up was 7 months (group A), 11 months (group B) and 14.5 months (group C). RESULTS: Thirty nine patients (48%) belonged to group A, 33 patients (40%) to group B, 10 patients (12%) to group C. Only enterovirus was detected. Ejection fraction at admission was worse for group B compared to group A (p=0.003). Groups A and B improved for echocardiography and NYHA (p< or =0.001). Group C improved for echocardiography only (p=0.031). Group B showed a better outcome for echocardiography (p=0.014) and NYHA (p=0.023) than group A. CONCLUSIONS: Inflammatory cardiomyopathy shows the best outcome. Antiinflammatory or antiviral treatment may be an option in patients not responding to conventional therapy.

Leishmania donovani: effect of therapy on expression of CD2 antigen and secretion of macrophage migration inhibition factor by T-cells in patients with visceral leishmaniasis.

Visceral leishmaniasis (VL) commonly known as Kala-azar in India is one of the several clinically important infections, where Th1 sub-population of CD4+ T-cells, despite a pre-requisite, fails to express macrophage migration inhibition factor (MIF) and interferon-gamma which both activate the macrophage and coordinate the immune response to intra-cellular Leishmania sp. Expression of CD2 receptors before and after antileishmanial therapy on CD4+ T-cells of VL patients and their corresponding effect on MIF were examined. Before treatment the number of T-cells expressing CD2 was low which incorporated insignificant MIF response. The immunological reconstitution was, however, observed after treatment as manifested through upregulation of CD2+ T-cells with pronounced MIF generation response. The study, therefore, identifies a possible role of CD2 antigen in immunity to VL.

Treatment targeted to cell surface epitopes.

Expression of a variety of surface epitopes is a characteristic feature of immune cells. Receptors and adhesion molecules are the most predominant ones. It is also characteristic that epitope expression is modulated during cellular activation. In inflammatory skin diseases these structures can be used to define not only the type of cell but also their activity status. The availability of monoclonal antibodies and fusion proteins enabled to target cellular surface epitopes in order to modulate the cellular function as a principle of treatment. In psoriasis receptor-targeted therapy has been developed and tested in a considerable number of clinical trials. However, these approaches revealed that not all the strategies are equally effective. In this review the development of receptor-targeted treatment for skin disorders, mainly psoriasis, is described. Clinical as well as experimental data obtained with the various compounds employed are discussed with regard to clinical efficacy, safety and tolerability.

Alefacept (Biogen).

Alefacept (B-9273) is an LFA-3-Ig fusion protein CD2 antagonist under development by Biogen for the potential treatment of autoimmune diseases, including psoriasis and transplant rejection [270267]. It is in phase III trials for psoriasis [349467]. In October 2000, the company reported that Amevive was on track for regulatory filing in the second half of 2001 [385250], with a possible launch in the second half of 2002 [395628]. The company began a pivotal phase III trial in the US in December 1999, involving patients with chronic plaque psoriasis [349467]. A second phase III trial has also been initiated [362199], [374040]. Results from both trials are expected in mid-2001 [396544]. In April 2001, SalomonSmithBarney confirmed that a regulatory filing was expected in Europe and the US in the second half of 2001 and stated that alefacept would be critical to the future earnings growth of the company [407796]. In June 1999, Merrill Lynch estimated product launch in 2001 [327145], [344773]. Sales in 2001 and 2002 were anticipated to be US $20 million and US $100 million, respectively [327145].

Effect of mycophenolate mofetil in heart transplantation.

OBJECTIVE: To study the effect of mycophenolate mofetil (MMF), a new immunosuppressive drug that acts by inhibiting de novo pathways of purine synthesis, and rabbit antithymocyte globulin (RATG) on the lymphocyte subpopulation after heart transplantation. DESIGN: A review of clinical and laboratory records. SETTING: The Montreal Heart Institute. PATIENTS: Thirty-one patients who underwent heart transplantation. In 9 patients, neoral cyclosporine, prednisone and azathioprine were administered (group 1). In 14 patients RATG was added during the first 3 postoperative days (group 2) and in 8 patients RATG and combination immunosuppression was given, but MMF was used instead of azathioprine (group 3). The demographic characteristics of donors and recipients were similar among the 3 groups. MAIN OUTCOME MEASURES: The proportion of CD2, CD4 and CD8 receptor-positive lymphocytes, expressed as a mean (and standard deviation) percentage of the total lymphocyte population, measured at 7, 15 and 30 days and 6 months after transplantation. RESULTS: At 7 days after transplantation, CD2 lymphocytes averaged 55% (18%), 16% (15%) and 14% (11%) in groups 1, 2 and 3 respectively (p < 0.05), CD4 averaged 36% (11%), 9% (12%) and 7% (8%) in groups 1, 2 and 3 (p < 0.05), and CD8 averaged 14% (6%), 4% (3%) and 4% (3%) in groups 1, 2 and 3 (p < 0.05). At 15 days after transplantation CD2 averaged 69% (10%), 42% (16%) and 47% (20%) in groups 1, 2 and 3 respectively (p < 0.05), and CD8 averaged 16% (7%), 16% (6%) and 19% (7%) (p = NS). At 30 days after transplantation the percentages of CD2, CD4 and CD8 lymphocytes were similar among the groups. The freedom rate from acute rejection averaged 22% (14%), 9% (8%) and 50% (18%) (p < 0.05) in groups 1, 2 and 3 at 6 months after transplantation, and the freedom rate from infection averaged 56% (17%), 36% (13%) and 38% (17%) for the 3 groups at this time period (p = NS). CONCLUSIONS: A short course of RATG causes severe, transitory depletion of CD2, CD4 and CD8 lymphocyte subpopulations. MMF decreases the incidence of early acute rejection after heart transplantation without affecting the lymphocyte subpopulation when compared with azathioprine.

The experimental (in vitro) and clinical (in vivo) immunosuppressive effects of a rat IgG2b anti-human CD2 mAb, LO-CD2a/BTI-322.

BACKGROUND: CD2 is a cell surface glycoprotein expressed on most human T cells and natural killer (NK) cells, working as a cell adhesion and costimulatory molecule. The aim of this paper is to analyze the mechanism of action of a rat IgG2b anti-human CD2 monoclonal antibody (mAb) (LO-CD2a/BTI-322 mAb), which is a potent immunosuppressive agent and inducer of cell death. In vivo, this mAb is able to prevent or treat kidney allograft rejection. METHODS: The mechanisms by which the LO-CD2a/BTI-322 mAb is able to induce inhibition of cell activation and cell death were analyzed by mixed lymphocyte reactions and by flow cytometry. After in vivo treatment, levels of circulating mAb were measured by ELISA as well as anti-rat immunization and cytokine release. RESULTS: We show that the inhibition of cell activation induced by LO-CD2a/BTI-322 mAb after allogeneic or OKT3 stimulation is due to an Fcgamma receptor-dependent CD2 down-modulation and to T-cell depletion through an antibody-dependent cell-mediated cytotoxicity mechanism mediated by NK cells or activated monocytes. Peripheral T- and NK-cell depletion was observed after in vivo treatment with LO-CD2a/BTI322. Cytokine release (TNFalpha) was correlated with some side effects, but only after the first injection, and the effects were never severe or life threatening. CONCLUSION: The correlation between the in vitro and in vivo data suggests that T-cell depletion, especially of activated cells, and inhibition of cell activation after CD2 down-modulation are the main mechanisms of action of the LO-CD2a/BTI-322 mAb.

Caspase-independent cell death induced by anti-CD2 or staurosporine in activated human peripheral T lymphocytes.

We examined the effects of the cell-permeable, broad spectrum peptide caspase inhibitors, benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethyl ketone (Z-VAD.fmk), and BOC-Asp(OMe)-fluoromethyl ketone (BOC-D.fmk), on apoptosis induced by anti-CD2, anti-Fas, and the protein kinase inhibitor staurosporine in activated human peripheral T lymphocytes. We monitored ultrastructural, flow cytometric, and biochemical apoptotic changes, including externalization of phosphatidylserine, cleavage of poly(ADP-ribose) polymerase (PARP) and lamins, activation of caspase-3 and caspase-7, decrease in mitochondrial membrane potential, and DNA fragmentation. Z-VAD.fmk and BOC-D.fmk completely inhibited all the biochemical and ultrastructural changes of apoptosis in anti-Fas-treated cells. In marked contrast, neither Z-VAD.fmk nor BOC-D.fmk inhibited CD2- or staurosporine-mediated cell shrinkage, dilatation of the endoplasmic reticulum (seen in anti-CD2-treated cells), externalization of phosphatidylserine, and loss of mitochondrial membrane potential that accompanied cell death. However, these inhibitors did inhibit the cleavage of PARP and lamins and the formation of hypodiploid cells, and partially inhibited chromatin condensation. These results demonstrate that in activated T cells, anti-CD2 and staurosporine induce a caspase-independent cell death pathway that exhibits prominent cytoplasmic features of apoptosis. However, caspase activation is required for the proteolytic degradation of nuclear substrates such as PARP and lamins together with the DNA fragmentation and extreme chromatin condensation that occur in apoptotic cells.

Thiol-mediated inhibition of FAS and CD2 apoptotic signaling in activated human peripheral T cells.

Fas and CD2 receptors can transduce apoptotic signals through two independent biochemical pathways. In this study, we first evaluated the role of intracellular GSH in these signaling pathways by inducing variations in the GSH pool of activated peripheral T lymphocytes. Increasing the concentration of intracellular GSH by means of N-acetyl-L-cysteine (NAC) and GSH ethyl ester (OEt) resulted in total protection against cell death, while inhibiting GSH synthesis with buthionine sulfoximine (BSO) greatly enhanced cell sensitivity to Fas and CD2 apoptotic signaling. The protection exerted by NAC and GSH OEt was essentially based on their capacity to establish an intracellular reducing environment as it still occurred in BSO-treated cells. Thiol-containing compounds (cysteine, captopril, D-penicillamine and 2-mercaptoethanol) inhibited apoptosis while a series of non-thiol antioxidants (including catalase and vitamin E) failed to do so, suggesting that protection was secondary to thiols/disulfides exchange reactions at the level of cysteine residues in proteins and not to detoxification of reactive oxygen intermediates. This conclusion was further supported by the finding that no enhanced generation of O.-2 and H2O2 could be detected in cells experiencing early stages of apoptosis such as a decreased concentration of intracellular GSH and cell shrinkage. Also, protection occurred in the presence of protein synthesis inhibitors, indicating that it was due to post-translational sulfhydryl redox regulation of critical molecules involved in the apoptotic cascade. These data suggest that GSH, the most abundant intracellular thiol antioxidant, may be important in counteracting Fas- and CD2-mediated apoptosis of T lymphocytes.

Dexamethasone modulates CD2 expression.

Glucocorticoid hormones (GCs) are able to modulate leukocyte activity. We studied the effect of dexamethasone (DEX) on the expression of CD2, an adhesion molecule involved in T-lymphocyte homing and activation. Results of flow cytometry analysis and immunoprecipitation with anti-CD2 monoclonal antibodies (mAbs) indicated that in vitro treatment with DEX augments CD2 expression in transformed T-cell lines. This effect correlated with a rapid increase in the mRNA and was inhibited by actinomycin-D (AD). The DEX-induced CD2 augmentation was transient, peaked at days 1-2 and returned to the levels of untreated controls at days 3-4. It was a dose-dependent phenomenon, mediated by the GC receptor (GCR), because it was inhibited by the GCR antagonist RU486, and was not induced by other steroids such as testosterone and progesterone. This CD2 modulation could presumably contribute to GC-induced effects on T-cell activity.

Blockade of multiple costimulatory receptors induces hyporesponsiveness: inhibition of CD2 plus CD28 pathways.

T-lymphocyte activation requires engagement of the T cell receptor with antigen-major histocompatibility complex, and simultaneous ligation of costimulatory pathways via the lymphocyte receptors CD2 and CD28/ CTLA4. Anti-CD2 monoclonal antibody (mAb) blocks the interaction of the antigen-presenting cell receptor CD48 with its ligand CD2, whereas CTLA4Ig binds with high affinity to the antigen-presenting cell ligands B7-1 and B7-2, blocking their interaction with CD28/CTLA4. We tested the immunosuppressive effects of simultaneously blocking both costimulatory pathways. Using donor C57BL/6J (H2b) hearts transplanted to CBA/J (H2k) recipients, anti-CD2 mAb plus CTLA4Ig administered at the time of transplantation prolonged cardiac allograft mean survival time to >120 days compared with untreated controls (12.2+/-0.5 days, P<0.01), anti-CD2 mAb alone (24.8+/-1.0 days, P<0.01), or CTLA4Ig alone (55.0+/-2.0 days, P<0.01). Retransplantation of these recipients with donor-specific and third-party grafts demonstrated that hyporesponsiveness and tolerance were achieved. In vitro stimulation of lymphocytes from tolerant recipients with donor-specific alloantigen resulted in normal cytotoxic T lymphocyte and mixed lymphocyte reaction responses, showing that clonal deletion or anergy did not occur, but that graft adaptation or suppression likely helped to maintain long-term graft survival. In vitro combinations of anti-CD2 mAb and CTLA4Ig suppressed the generation of allogeneic cytotoxic T lymphocytes (58%) and the mixed lymphocyte reaction (36%); CTLA4Ig was more effective in this regard and the two agents were not synergistic. Anti-CD2 mAb and CTLA4Ig suppressed mitogen-driven proliferation in differential fashions, suggesting that they affected independent signaling pathways. Anti-CD2 mAb and CTLA4Ig also inhibited interleukin (IL)-2, IL-4, and IL-2 receptor (CD25). These data indicate that anti-CD2 mAb plus CTLA4Ig induces hyporesponsiveness and tolerance. The mechanism is likely related to the initial disruption of independent pathways of T-lymphocyte activation leading to antigen-specific long-term graft survival.

CD2 ligation abrogates antigen-independent apoptosis in B cells.

We have described recently the prevention of apoptosis by CD2-soluble CD48 interaction on antigen B cell receptor occupancy. Here, we show that CD2 ligation is also able to interfere with B cell receptor-independent apoptosis pathways such as spontaneous death in spleen B cells or serum deprivation and hydrogen peroxide exposure in the BAL-17 cell line. In all cases, CD2 ligation induces a signal that prevents the downregulation of Bcl-2 expression. The specific CD2 signal pathway involved in this phenomenon is still unknown. As reported, CD2 did not appear to induce Ca2+ mobilization, phosphatidylinositol turnover, or PKC translocation in B cells. Nevertheless, we show that CD2 receptor ligation is coupled to the tyrosine phosphorylation pathway in B cells. These observations indicate that CD2 is functionally able to trigger at least an early signal that could play a role in apoptosis blockage B cells in addition to the adhesion one. The results suggest the participation of cellular membrane receptors other that CD40 in apoptosis rescue, not only in the antigen-dependent but also in the antigen-independent phases of B cell lymphopoiesis.

HTLV-I-induced T-cell activation.

Infection by the human T-cell lymphotropic virus type I (HTLV-I) causes T-cell activation by at least two separate mechanisms. One mechanism involves activation of the T cells harboring the virus and is exemplified by in vivo infected nonimmortalized T-cell clones that display a prolonged state of activation. This HTLV-I-induced T-cell activation is inhibited by rapamycin, a drug that inhibits p70 S6-kinase and blocks cell cycle in G1, but is not inhibited by FK506 or cyclosporin A, both of which inhibit interleukin-2 (IL-2) production. The phenotype of this pathway is consistent with an hyperactive IL-2R pathway or CD28 pathway, indicating that HTLV-I may contribute a costimulatory signal to the infected T cell. As a separate mechanism, HTLV-I-infected T cells can induce activation of uninfected T cells via T-T-cell interaction mediated by the LFA-3-CD2 pathway. This may induce IL-2 production from the uninfected T cells, leading to a more generalized activation of the immune system that potentially could provide a basis for some of the diseases associated with HTLV-I. Moreover, this THTLV-I-T-cell interaction could explain the spontaneous proliferation observed in patients with HTLV-I-associated myelopathy/tropical spastic paraparesis.

Analysis of CD2 and TCR-beta gene expression in Jurkat cell mutants suggests a cis regulation of gene transcription.

Thirty CD2- J32 stable clones, derived by mutagenesis and subsequent immunoselection with anti-CD2 Ab, were used to study the regulation of CD2 and TCR gene expression. Analysis of RNA expression revealed that the loss of surface expression of CD2 was due to a lack of expression of CD2 mRNA and was associated with a lack of expression of VDJ TCR-beta transcripts in 12 of these mutants, sparing the expression of DJ TCR-beta, TCR-alpha, CD3 gamma, delta, epsilon, and zeta RNA. The expression of other differentiation molecules was unaffected, except for CD1, CD4, and CD5, which were either decreased or absent in most of these mutants. A gain in the expression of TCR-gamma transcripts was observed in each of these mutants, while, as expected, no TCR-gamma transcripts were detected in wild-type J32 cells. Several mutants were able to use the human CD2 enhancer and the murine TCR-beta enhancer and promoter to activate transcription from reporter genes in the context of heterologous promoters, indicating that the mutation(s) does not affect transcription pathways. Consistent with this finding is the adequate expression in these mutants of several lineage-specific transcription factors. The expression of CD2 in several of these mutants was rescued by gene transfer using a genomic 28.5-kb CD2 fragment, suggesting that the enhancer function of this gene may be dependent on the enhancer site. These observations suggest that the coordinate expressions of CD2 and TCR-beta genes share common regulatory mechanisms involving factors regulating chromatin structure and accessibility.

Differential sensitivity of human naive and memory CD4+ T cells for dexamethasone.

Human CD45RA+ ('naive') and CD45RO+ ('memory') CD4+ T cells were compared with respect to their sensitivity to dexamethasone (DEX). In three different activation pathways, i.e. (i) immobilized anti-CD3, (ii) immobilized anti-CD3 plus soluble anti-CD28 and (iii) soluble anti-CD2 plus soluble anti-CD28, naive CD4+ T cells appeared more sensitive to DEX than memory CD4+ T cells. In the anti-CD3 system this difference in sensitivity was apparent at a suboptimal DEX concentration. Addition of anti-CD28 rendered the cells largely insensitive to DEX, indicating that the CD28 pathway is less dependent of the DEX-sensitive transcription factor AP-1. However, the alternative pathway of T cell activation through CD2/CD28 triggering was highly sensitive to DEX when naive cells were studied; in the case of memory cells, at least a 10-fold higher DEX concentration was needed to achieve a comparable inhibition. The strong inhibitory effect of DEX on naive CD4+ T cells stimulated via the alternative pathway was completely abrogated by activation of protein kinase C (PKC) with phorbol myristate acetate. Our data suggest that at least two different mechanisms contribute to DEX resistance, i.e. CD28 triggering and PKC activation, which may occur more effectively in memory cells making them less sensitive to DEX.

The presence and cytotoxicity of CD16+ CD2- subset from PBL and NK cells in long-term IL-2 cultures enhanced by Prothymosin-alpha.

The aim of this study was to characterize the LAK cell subpopulation on which Prothymosin-alpha (ProT alpha) exerts its enhancing effect on cytotoxicity. We investigated the role of ProT alpha on LAK induction from peripheral blood lymphocytes (PBL) and NK-enriched cells, both cultured for 5 weeks with IL-2 and ProT alpha. The different cultures were separated into several subsets throughout the culture time using two color fluorescence activated cell sorting (FACS) and CD56, CD16, CD2 and CD8 monoclonal antibodies. Each cell subset was then tested for cytotoxicity against K562 and Daudi cells. The CD16+ CD2- subset from both, PBL and NK-enriched cells, was the only subset on which ProT alpha had an effect, significantly enhancing this subpopulation. Within the CD16 population, the cells CD16+ CD2+ were the most cytotoxic, although CD16+ CD2- cells were also cytotoxic. ProT alpha only potentiated the cytotoxicity of CD16+ CD2- subset significantly, with 29% and 41% for K562 and Daudi cells, respectively.