Mesenchymal stem cells protect NOD mice from diabetes by inducing regulatory T cells.

AIMS/HYPOTHESIS: Displaying immunomodulatory capacities, mesenchymal stem cells (MSCs) are considered as beneficial agents for autoimmune diseases. The aim of this study was to examine the ability of MSCs to prevent autoimmune diabetes in the NOD mouse model. METHODS: Prevention of spontaneous insulitis or of diabetes was evaluated after a single i.v. injection of MSCs in 4-week-old female NOD mice, or following the co-injection of MSCs and diabetogenic T cells in irradiated male NOD recipients, respectively. The frequency of CD4(+)FOXP3(+) cells and Foxp3 mRNA levels in the spleen of male NOD recipients were also quantified. In vivo cell homing was assessed by monitoring 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE)-labelled T cells or MSCs. In vitro, cell proliferation and cytokine production were assessed by adding graded doses of irradiated MSCs to insulin B9-23 peptide-specific T cell lines in the presence of irradiated splenocytes pulsed with the peptide. RESULTS: MSCs reduced the capacity of diabetogenic T cells to infiltrate pancreatic islets and to transfer diabetes. This protective effect was not associated with the modification of diabetogenic T cell homing, but correlated with a preferential migration of MSCs to pancreatic lymph nodes. While injection of diabetogenic T cells resulted in a decrease in levels of FOXP3(+) regulatory T cells, this decrease was inhibited by MSC co-transfer. Moreover, MSCs were able to suppress both allogeneic and insulin-specific proliferative responses in vitro. This suppressive effect was associated with the induction of IL10-secreting FOXP3(+) T cells. CONCLUSIONS/INTERPRETATION: MSCs prevent autoimmune beta cell destruction and subsequent diabetes by inducing regulatory T cells. MSCs may thus offer a novel cell-based approach for the prevention of autoimmune diabetes and for islet cell transplantation.

[Hand allografts: experience from Lyon team]. / Allogreffe de mains: expérience lyonnaise.

Hand allograft is a method in the stage of clinical experimentation, which is reserved in France for the treatment of bilateral traumatic amputees. This study reports the Lyon team experience, which is pioneer in this domain. Four patients (3 males and 1 female) underwent seven (one unilateral and three bilateral) hand transplantations from September 1998 to February 2007. The level of amputation was at the wrist or at the mid-forearm. Delay since hand loss ranged from 2.5 to 9 years. The surgical protocol was elaborated and planned case by case. All recipients received the same immunosuppressive treatment. Episodes of acute rejection were observed in the first 3 months after transplantation, which were easily managed after a few days increasing oral prednisone doses and applying topical immunosuppressants. Currently the patients receive the doses of immunosuppressants comparable to those in kidney-grafted patients. We have not registered any severe complication of immunosuppressive treatment up till now (7 years follow-up for the earliest graft). We performed analytical and functional clinical, as well as questionnaire evaluation of patients. The first case (unilateral graft) resulted in graft failure at 2 years due to non-compliance of the patient. The three bilateral graftees demonstrate a favorable evolution despite some immunological (hyperglycemia, serum sickness) and surgical (thrombosis, osteomyelitis, skin loss) complications, which could be managed. The middle and long-term follow-up evaluation revealed good to excellent sensorimotor recovery of 4 hands in both male recipients (4 and 7 years) with satisfactory social adaptation, higher or equal to those expected after post-traumatic replantations at the equivalent level and higher to those obtained with currently available myoelectric prosthesis. The last patient, a young female who has been grafted in February 2007, receives ongoing reeducation course and shows normal progress of functional restoration of both hands. The encouraging results of this clinical experimentation make us currently consider hand allografting as reasonable and useful both for the patients and for evolution of research in composite tissues allotransplantation (CTA). Further long-term careful research and worldwide monitoring of all patients with hand allografts is required to, on the one part, state on the authorization of this surgery, and, on the other part, to better elucidate the mechanisms of successful CTA.

Alloreaction increases or restores CD40, CD54, and/or HLA molecule expression in acute myelogenous leukemia blasts, through secretion of inflammatory cytokines: Dominant role for TNFbeta, in concert with IFNgamma.

We have previously reported that alloreaction can lead to activation of dendritic cells through secretion of inflammatory cytokines. Here, we addressed whether alloreaction-derived cytokines may also lead to acute myelogenous leukemia (AML) blast differentiation. With this aim, supernatant (sn) harvested from major or minor histocompatibility antigen-mismatched mixed lymphocyte reaction (MLR) were used to culture French American Bristish (FAB) type M4 or M5 AML blasts. Our results showed that the secreted factors induced upregulation of CD40, CD54, and/or HLA molecules in AML blasts. Protein fractionation, blockade experiments and exogenous cytokine reconstitution demonstrated the involvement of TNF in the upregulation of CD54, CD40 and HLA-class II molecules, and of IFNgamma in the increase of HLA-class I and class II molecule expression. But, in line of its much higher levels of secretion, TNFbeta, rather than TNFalpha, was likely to play a preponderant role in AML blast differentiation. Moreover TNFbeta and IFNgamma were also likely to be involved in the AML blast differentiation-mediated by HLA-identical donor T-cell alloresponse against recipient AML blasts. In conclusion, we show herein that upon allogeneic reaction, TNFbeta secretion contributes, in concert with IFNgamma, to increase or restore surface molecules involved in AML blast interaction with T cells.

Human umbilical vein endothelial cells increase ex vivo expansion of human CD34(+) PBPC through IL-6 secretion.

BACKGROUND: Ex vivo expansion of hematopoietic stem cells (HSC) can help reduce cytopenia following transplantation, especially in NHL patients whose BM is deficient because of extensive chemotherapy. We have previously reported that human umbilical vein endothelial cells (HUVEC) can contribute to improved PBPC expansion when used in co-culture with CD34(+) cells. METHODS: We evaluated the roles of direct HUVEC CD34(+) contact and HUVEC-produced soluble factors. We cultured CD34(+) PBPC harvested from NHL patients in four different conditions: (1) liquid culture without HUVEC; (2) co-culture in contact with HUVEC; (3) co-culture with HUVEC but without direct contact; (4) liquid culture with HUVEC-conditioned medium (CM). Thrombopoietin (Tpo), Flk2Flt3 ligand (FL) and c-kit ligand (KL) with or without rhIL-6 were added to these four culture conditions. RESULTS AND DISCUSSION: Our results showed that HUVEC co-culture or addition of HUVEC-CM to Tpo, FL and KL (TFK) improved CD34(+) PBPC expansion compared with liquid culture, as determined by total viable nucleated cells (TNC), colony-forming cell assay (CFC) and week-6 cobblestone area-forming cells (Wk-6 CAFC) expansions. Non-contact culture led to similar PBPC expansion as contact co-culture; moreover, HUVEC-CM improved PBPC expansion. However, when rhIL-6 was added to HUVEC-CM with TFK, no significant difference was observed. Finally, high quantities of IL-6 were detected in HUVEC-CM and addition of anti-IL-6 Ab inhibited the positive effect of HUVEC on PBPC expansion. Our results thus suggest that HUVEC may improve PBPC expansion, at least through IL-6 secretion.

Human mesenchymal stem cells suppress induction of cytotoxic response to alloantigens.

Mesenchymal stem cells (MSC) fail to induce allogeneic responses in mixed lymphocyte reaction assays. Because MSC express HLA class I molecules, here we investigated whether they could be recognized as allogeneic targets by cytolytic T lymphocytes (CTL). With this aim, CTL precursor (CTLp) frequencies were measured following stimulation of T cells with either allogeneic mononuclear cells (MNC) or MSC originated from the same human bone marrow donor. Lysis of MSC was measured at day 10 of culture in standard chromium release assays. In addition, allogeneic PHA blast T cells or B-EBV lymphoblastoid cell lines (LCLs) generated from the same donor were used as positive controls of lysis. Our results showed that when allogeneic MNC were used to stimulate T cells, a high CTLp frequency was detected towards MSC targets. However, when MSC were used as stimulators, CTLp frequencies were markedly altered whatever the targets used, i.e.: MSC, PHA blast T cells or EBV-B LCLs. Moreover, when graded concentrations of MSC were added together with MNC upon stimulation of alloreactive T cells, we observed a dose-dependent decrease in CTLp frequencies towards MSC targets. This inhibition of MSC lysis was partially overcome by adding exogenous rh-IL-2 from the beginning of cultures. In addition, this suppressive effect was totally reproduced when, instead of MSC, supernatant harvested from MSC cultures was added to allogeneic MNC, upon stimulation of alloreactive T cells. In conclusion, our results demonstrate that MSC which can be recognized as targets by pre-activated alloreactive CTLs, may be able to suppress differentiation of CTL precursors into CTL effectors through secretion of suppressive factors.

From hemorheology to vascular mechanobiology: An overview.

Almost all of the cells of the human body are subjected to mechanical stresses. In endothelial cells, mechanical stresses can vary from some milli-Pascal (shear stress) to one ore more Pascal (hydrostatic pressure). Now it is know that mechanical stresses have a decisive part cellular physiology. However, if the main biological effects of mechanical stress are well related, the mechanisms allowed the relation between mechanical stress to physiological phenomenon remain nearly unknown (mechanotransduction phenomenon). In this work, through personal results and published works, the authors considers all the effects of mechanical stresses and the possible hypothesis.

Cyclosporin A inhibits dendritic cell maturation promoted by TNF-alpha or LPS but not by double-stranded RNA or CD40L.

Here, we investigated the influence of cyclosporin A (CsA) on dendritic cell (DC) generation. With this aim, human DC were propagated from monocytes in serum-free medium with granulocyte macrophage-colony stimulating factor and interleukin-4. DC were then exposed to tumor necrosis factor alpha (TNF-alpha) for maturation. Our results show that CsA does not impair commitment of monocytes into DC, as assessed by loss of CD14 and increase of CD40 and CD1a. However, TNF-alpha-induced DC maturation was affected, as CsA-treated DC expressed lower levels of human leukocyte antigen and costimulatory molecules but sustained levels of CD1a, and less DC expressed DC-lysosomal-associated-membrane-protein (LAMP) and CD83. Accordingly, CsA inhibited the allostimulatory and accessory cell functions of DC. Surprisingly, when other maturation stimuli were used, we observed that CsA significantly inhibited maturation induced by lipopolysaccharides but not by polyribocytidylic acid or CD40 ligand, as assessed by DC phenotype and functions. Therefore, our results indicate that CsA may differentially affect DC maturation.

Generation of helper and cytotoxic CD4+T cell clones specific for the minor histocompatibility antigen H-Y, after in vitro priming of human T cells by HLA-identical monocyte-derived dendritic cells.

BACKGROUND: There is now convincing evidence that minor histocompatibility antigens (mHag) may play a significant role in the pathogenesis of graft-versus-host disease after HLA-identical bone marrow transplantation. Indeed, in this clinical situation, T cells specific for mHag have been isolated. Here, we addressed whether one can generate mHag-specific T cells in vitro, without any in vivo immunization, among healthy blood donors. METHODS: We used monocyte-derived dendritic cells (Mo-DCs) as antigen presenting cells to induce primary responses between healthy HLA-identical siblings, in mixed lymphocyte dendritic cell reactions (MLDCRs). RESULTS: We show that CD4+ T-cell clones, specific for the mHag H-Y, can be generated in vitro. These clones were derived from a gender-mismatched positive MLDCR pair of HLA-identical siblings and were restricted by the HLA DQB1*0502 molecule. In addition, these CD4+ T clones were also able to lyse allogeneic targets with the same pattern of restriction and specificity than helper function. Finally, acute myeloid leukemia (AML) blast cells were susceptible to lysis by these clones. CONCLUSIONS: Altogether, these results predict that Mo-DCs could help to generate class II-associated, mHag-specific, T-cell lines or clones in vitro, between healthy blood donors, without any need of transplantation-mediated immunization.

Outcome of unrelated bone marrow donor searches in 174 children resulting in 45 patients transplanted in the HLA-matched and -mismatched situation.

The aim of the study was to evaluate the outcome of unrelated bone marrow donor (UBMD) searches initiated for 174 children between 1986 and 1997. Seven patients were registered twice so that a total of 181 UBMD searches took place. At the time of registration, patients suffered from hematological malignancies (n = 121), non-malignant hemopathies (n = 26) and inborn errors (n = 34). Forty-five of the patients (26%) were given transplants from unrelated donors of whom 26 (58%) were HLA-mismatched transplants. Our strategy accepted HLA mismatches at the time of donor selection, using Thymoglobuline as part of the conditioning regimen. Of the 45 patients given unrelated donor transplants, overall survival was 60% at 3 years and concerned 27 patients of whom 14 were from HLA-mismatched donors. Disease-free survival for hematological malignancies was 65% in HLA-matched transplants and 50% in HLA-mismatched transplants. For some patients (16%) urgency led us to use alternative options: non-identical related donor (n = 14), autograft (n = 10), related cord blood transplant (n = 4). For others, UBMD searches were stopped because of favorable evolution (n = 29), death (n = 24), disease progression (n = 22) or other reasons (n = 21). By the end of the follow-up period, 88 patients had died (50%), 75 (43%) are currently alive with or without being transplanted of whom eight are still having active searches and 11 are no longer contactable. In conclusion, in severe disease in children, an immediate transplant from a partially matched donor might be preferable to a prolonged search for a full match. Consequently, this strategy increases the number of patients for whom a suitable donor can be found. We have chosen this option in order not to delay BMT; in so doing we have obtained encouraging results which include high overall survival, low incidence of acute GVHD grade III-IV and low percentage of relapse even in mismatched pairs.

Distinct subsets of dendritic cells resembling dermal DCs can be generated in vitro from monocytes, in the presence of different serum supplements.

We recently demonstrated that dendritic cells (DCs) can be generated from monocytes in the presence of high concentrations of human serum (HS), provided the extra-cellular pH is maintained at plasma values. Because monocyte-derived DCs (Mo-DCs) can also be generated in the presence of fetal calf serum (FCS) or serum-free medium, we have investigated whether these different culture supplements influence DC generation. With this aim, purified monocytes were cultured with GM-CSF plus IL-4 for 6 days and were further exposed to TNF-alpha for 2 additional days, in the presence of HS, autologous plasma (AP), FCS, or X-VIVO 20, a serum-free medium. Our results show that good yields of functionally mature DCs can reproducibly be obtained in the presence of HS or AP, as assessed by CD83 and CD86 up-regulation, dextran-FITC uptake, allogeneic MLR assays and the induction of an autologous response. Interestingly, the effect of serum on DC generation was probably not only quantitative, but also qualitative, since (i) the majority of HS- or AP-cultured DCs expressed CD83 with very weak levels of CD1a, whereas CD83+ DCs cultured in FCS or X-VIVO were mostly CD1a++; (ii) HS- and AP-cultured DCs were much more granular and heterogeneous than FCS- or X-VIVO-cultured DCs, and (iii) the presence of Birbeck-like granules was preferentially observed in HS- or AP-cultured DCs, as assessed by electron microscopy. That these different cells resemble dermal DCs (DDCs) was further supported by the observations that most of the cells displayed intracytoplasmic FXIIIa in the absence of Lag antigen, and expressed E-cadherin at very low levels. Altogether, our results indicate that starting from the same monocytic population, different subsets of DCs can be generated, depending on the culture conditions. Thus, HS or AP favors the generation of fully mature DCs that resemble activated dermal DCs, whereas FCS, or X-VIVO preferentially leads to the generation of less mature CD1a++ dermal-like DCs.

Generation of stable monocyte-derived dendritic cells in the presence of high concentrations of homologous or autologous serum: influence of extra-cellular pH.

Recent studies have highlighted the high degree of differentiation of monocytes. Indeed, dendritic cells (DC) can be generated from monocytes, in the presence of appropriate cytokines. However, human serum is usually avoid in such cultures. Here, we report that human serum does not inhibit generation of mature DC from blood monocytes, but rather that extra-cellular pH may play an important role in the regulation of monocyte differentiation. Indeed, monocytes cultured at pH 7.4 in the presence of high concentrations of human serum developed efficiently into mature DC, as opposed with monocytes cultured at pH 7. These pH 7.4 cultured DC presented features characteristic of mature DC, at the phenotypical, functional and morphological levels. In addition, these DC were stable, with respect to their sustained expression of CD83 and CD86, upon withdrawal of cytokines. Finally, when autologous plasma was used instead of homologous serum, differentiation of monocytes into mature DC was efficient, as well. Thus, altogether, our data show the importance of extra-cellular pH on differentiation of monocyte-derived DC in the presence of human serum, which should be maintained at plasma levels.

Defective cytokine expression but adult-type T-cell receptor, CD8, and p56lck modulation in CD3- or CD2-activated T cells from neonates.

Expression of IL-2, interferon-gamma, and IL-3 mRNA and proteins was investigated in peripheral blood mononuclear cells from cord blood after activation with phytohemagglutinin, CD2, or CD3 MAb. The results showed that interferon-gamma and IL-3 expression was decreased in cord peripheral blood mononuclear cells when compared with expression observed in adult peripheral blood mononuclear cells, irrespective of the stimulation used. In addition, in newborn cells a defect in IL-2 secretion and mRNA expression was observed in response to CD2 or CD3 MAb but not in response to phytohemagglutinin-mediated activation. We further analyzed the modulation of nonlymphokine genes under the same protocol of stimulations. The results indicate that in newborn cells, despite a reduced lymphokine expression observed after CD2 or CD3 MAb activation, the up-regulation of the T-cell receptor, CD8, and p56lck was similar to that found in adult cells, as was also found after phytohemagglutinin activation of both types of cells. These data are in favor of a deficient T-cell responsiveness to CD2 or CD3 MAb in newborn cells. This impairment of the T-cell response appears to selectively affect lymphokine gene expression because the modulation of other genes also implicated in T cell activation is not altered.

Contribution of p56lck to the upregulation of cytokine production and T cell proliferation by IL-2 in human CD3-stimulated T cell clones.

Interaction of the interleukin 2 receptor (IL-2R) beta chain with the lymphocyte-specific protein tyrosine kinase (PTK), p56lck, has led to the speculation that p56lck participates in growth signal transduction. Although activation of T cells with interleukin 2 (IL-2) results in the activation of p56lck, accumulating data support the notion that Lck does not play an essential role in mitogenic signal delivery from the IL-2R. Since this src-related PTK has been shown to enhance TCR/CD3-mediated T cell responsiveness, here we investigated whether activation of Lck by IL-2 could contribute to enhance TCR/CD3-mediated T cell functions. This was achieved by using human CD4(+)-cloned T cells and comparing the effects of IL-2 on p56lck kinase activation and cytokine production. Results show that p56lck kinase activity increased as early as 1 min, reached a maximum within 5 min and decreased within 60 min after IL-2 stimulation. Such treatment with IL-2 also resulted in enhancing T cell responsiveness to CD3+PMA stimulation, as assessed by IL-2 and IFN-gamma secretion and by T cell proliferation. This increase of T cell functions was correlated with IL-2-induced p56lck activation in both dose-response and time-course experiments. Taken together these results strongly suggest that activation of Lck by IL-2 may play a role in regulating CD3-mediated T cell functions.

Contribution of tyrosine kinases to the selective orientation of human CD4+ bifunctional cloned T cells toward proliferation or cytolytic function.

We show that T cell activation of human CD4+ cloned T cells through the CD2 molecule can induce either autocrine proliferation or cytolysis, depending on the pair of anti-CD2 mAbs used for stimulation, that is, D66/T11(1) or GT2/T11(1), respectively. As the earliest biochemical event after CD2 stimulation is likely the induction of tyrosine phosphorylation of various proteins, we investigated whether differential activation of protein tyrosine kinases (PTKs) could contribute to the selective induction of each function. Results show that herbimycin A, a potent PTK inhibitor, markedly decreased the induction of both proliferation and cytolysis. This implies a regulatory role for tyrosine phosphorylation in the induction of each function by CD2. However, that PTKs are differentially activated upon induction of proliferation by D66/T11(1) or cytotoxic function by GT2/T11(1) emanated from two different approaches. First, immunoblotting total cellular extracts with an anti-phosphotyrosine mAb showed different patterns of tyrosine phosphorylation depending on the pair of CD2 mAbs used for stimulation. Second, a differential activation of p56lck, a src-related PTK, was observed after stimulation with D66/T11, and GT2/T11(1). Although induction of proliferation by D66/T11(1) was correlated with increased Lck activity, this was not observed when cells were triggered to lyse by GT2/T11(1). Thus, by providing striking correlative evidences linking differences in PTK activation with induction of different functions in bifunctional cloned T cells, our results strongly suggest that PTKs may contribute to the selective orientation of T cell functions at a single-cell level.

Requirements for lysis of activated T cells by class-II-restricted cytolytic T-lymphocytes.

In the present study, we explored the specific requirements for lysis of human activated T cells by CD4+ CTLs. This was achieved by using human CD4+ T cell lines or clones specific for a peptidic fragment of influenza virus as both CTL effectors and target T cells (TTCs). Our results further establish that human activated T cells expressing HLA-DR molecules can present Ag to and be lysed by CD4+ HLA-DR restricted CTLs. This killing is Ag specific and HLA-DR restricted. It can be observed whether TTCs are heterologous or autologous, CD4+ or CD8+. However, we find that in our model: (a) TTCs are able to present artificially processed peptidic fragments of Ag, but not the corresponding natural Ag in the context of class II determinants, even if they can process whole virus in the context of class I determinants; (b) TTCs must express high density of HLA-DR molecules on their membrane; (c) preincubation of TTCs with high concentrations of peptide is required; and (d) interestingly enough, addition of free peptide at similar concentration during the cytolytic assay to replace TTC preincubation inhibits TTC lysis by at least two different mechanisms, i.e., cold-target inhibition in which CTLs serve as their own cold targets and inhibition at the effector cell level. From these results, one can conclude that stringent conditions are required for lysis of activated T cells by class-II-restricted CTLs.

Comparison of T cell functional changes during childhood with the ontogeny of CDw29 and CD45RA expression on CD4+ T cells.

The ontogeny of the peripheral blood mononuclear cells' responsiveness to various activators during childhood was studied and compared to the expression of CDw29 and CD45RA molecules at the surface of CD4+ T cells. The results show that newborn peripheral blood mononuclear cells are characterized by a responsiveness to mitogens that is higher than that observed in adults, at least shortly after stimulation. This contrasts with a clear decreased response to CD2 and CD3 MAb at any time after stimulation. These functional characteristics correlate with a low density of CDw29 antigen on virtually all CD4+ T cells and a high density of CD45RA antigen on most CD4+ T cells at birth. These patterns of reactivity and phenotype are similar to those found among naive adult T cells. When ageing, the response to mitogens becomes rapidly similar to the adult's values, whereas the responses to CD2 or CD3 MAb are more gradually acquired. This slow rate of functional changes grossly parallels the increase of CDw29+ CD4+ and the decrease of CD45RA+ CD4+ T cell subsets. These changes finally lead to the immunophenotypic and functional characteristics that are typical of adult memory T cells. These results suggest that iterative antigenic stimulations both induce memory T cells and create the conditions to improve the overall immune competence.

Helper or cytolytic functions can be selectively induced in bifunctional T cell clones.

By using bifunctional T cell populations, we have shown in this report that elicitation of helper versus cytolytic function depends on the stimulatory signal at the membrane. Interestingly enough, the transduction of these signals is likely to be achieved via different metabolic pathways. Thus, helper function is associated with intracellular Ca2+ mobilization and PLC activation, while cytolysis can occur even in the absence of detectable levels of these second messengers. These results indicate that selective activation through the same membrane-transducing molecule may orientate T cell function through qualitatively or quantitatively different second messengers. This would be an important part of immune regulation.

Activation signals via CD2 molecule and interleukin 2 receptor act in synergy for helper function induction.

The membrane CD2 molecule appears to play an important role in T cell activation. Indeed, T cell stimulation by some combinations of anti-CD2 monoclonal antibodies (mAb) can result in antigen-independent expression of helper function as assessed by proliferation and lymphokine secretion. We report here that T cell stimulation by a combination of two anti-CD2 mAb recognizing GT2 and T11(1) epitopes, respectively, cannot alone induce T helper clones to proliferate when preincubated in culture medium devoid of exogenous interleukin 2 (IL2). The concerted action of both anti-GT2 + T11(1) mAb and exogenous recombinant IL2 is required to induce cloned helper T cells to produce IL 2 and interferon-gamma to significantly increase IL2 receptors (IL2R) and finally to divide by an autocrine mechanism, whereas each signal alone has no effect. This therefore suggests that, under some conditions of CD2 stimulation, two minimal signals may be delivered through CD2 and IL2R and act synergistically to achieve a complete expression of T helper cell functions. Moreover, analysis of phosphatidylinositol and phosphatidic acid metabolic changes mediated by each signal separately or together suggests that, in this model, IL2 increases the phosphoinositide turnover induced by anti-CD2 antibodies up to a level required for helper function acquisition.