Nondepleting anti-CD4 Antibody Research Articles

CTLA4-Ig Effectively Controls Clinical Deterioration and Immune Condition in a Murine Model of Foxp3 Deficiency

PurposeFOXP3 deficiency results in severe multisystem autoimmunity in both mice and humans, driven by the absence of functional regulatory T cells. Patients typically present with early and severe autoimmune polyendocrinopathy, dermatitis, and severe inflammation of the gut, leading to villous atrophy and ultimately malabsorption, wasting, and failure to thrive. In the absence of successful treatment, FOXP3-deficient patients usually die within the first 2 years of life. Hematopoietic stem cell transplantation provides a curative option but first requires adequate control over the inflammatory condition. Due to the rarity of the condition, no clinical trials have been conducted, with widely unstandardized therapeutic approaches. We sought to compare the efficacy of lead therapeutic candidates rapamycin, anti-CD4 antibody, and CTLA4-Ig in controlling the physiological and immunological manifestations of Foxp3 deficiency in mice.MethodWe generated Foxp3-deficient mice and an appropriate clinical scoring system to enable direct comparison of lead therapeutic candidates rapamycin, nondepleting anti-CD4 antibody, and CTLA4-Ig.ResultsWe found distinct immunosuppressive profiles induced by each treatment, leading to unique protective combinations over distinct clinical manifestations. CTLA4-Ig provided superior breadth of protective outcomes, including highly efficient protection during the transplantation process.ConclusionThese results highlight the mechanistic diversity of pathogenic pathways initiated by regulatory T cell loss and suggest CTLA4-Ig as a potentially superior therapeutic option for FOXP3-deficient patients.

Editorial: Current Concepts of Cellular and Biological Drugs to Modulate Regulatory T Cell Activity in the Clinic.

The Editorial on the Research Topic Current Concepts of Cellular and Biological Drugs to Modulate Regulatory T Cell Activity in the Clinic Regulatory T (Treg) cells are essential for the maintenance of peripheral tolerance and prevent the development of autoimmunity and allergy. While on the one hand being indispensable for the perpetuation of tolerance to harmless antigens or self-antigens, Treg cells contribute to cancer pathogenesis and progression (1). Hence, the potential to treat a multitude of different human diseases by pharmacological modulation of Treg cells is enormous. Consequently, this T cell population is in the focus of biomedical research and development. Currently, isolated and in vitro expanded Treg cells as cellular therapeutics are intensively tested in clinical trials for their ability to prevent and ameliorate graft-versus-host disease (GvHD) and first biologicals to modulate Treg cell activity directly in man are being developed and tested in preclinical and clinical trials. This Research Topic comprised seven review articles; the authors discuss selected developments on the way from basic research on manipulation of peripheral tolerance mechanisms toward clinical application. Mahnke et al. present an updated view on the critical role of dendritic cells (DC) in control and maintenance of peripheral tolerance under non-inflammatory conditions based on findings obtained in preclinical mouse models showing that factors of local microenvironment and extrinsic inflammatory signals dictate the function of these “sentinels of immunity” (2): inducers of tolerance versus immunity. Analogous to this overview of basic DC research in the mouse, Raker et al. provide an overview of translational research on human tolerogenic DC, the important role of IL-10 for stability of their phenotype and their potential as cellular therapeutics for reestablishment of peripheral tolerance in patients with autoimmune or allergic diseases (Raker et al.). Hahn et al. discuss current developments in establishment of humanized mouse models for preclinical testing of novel biologicals to manipulate Treg cell suppressive function in vivo. Additionally, the risk and benefit of cellular therapies using in vitro expanded Treg cells or pharmacological modulators of Treg cell function like rapamycin or IL-2 is comparatively discussed by Perdigoto et al. Complementary, Pham et al. provide an updated view on the use of low dose IL-2 in combination with tissue-specific antigens as a therapeutic approach to expand and activate antigen-specific Treg cells in patients with type I diabetes (Pham et al.), an approach that will be currently used by different groups for treatment of T cell-mediated autoimmunity. A well-known strategy for tolerance induction first described by Hermann Waldman’s group in 1990 is the application of non-depleting anti-CD4 antibodies that induces a long lasting antigen-specific tolerance in mouse models of solid organ transplantation (3). In this Research Topic, Konig et al. report on their clinical experiences by using a humanized anti-CD4 antibody (BT-061, Tregalizumab) for treatment of rheumatoid arthritis in patients. This anti-CD4 mAb, first tested in patients with psoriasis, induces suppressive function of human Treg without blocking T helper cell activities per se. Additionally, Hui et al. discuss recent advances of management of advanced non-small cell lung cancer by PD-1-blocking antibodies and their impact on cytokine-induced killer cell activity. In summary, this Research Topic of Frontiers in Immunology provides a snapshot of our recent understanding of Treg cell biology and function with a special focus on current concepts of therapeutic strategies to modulate peripheral tolerance mechanisms and Treg cell activity in humans. Given the recent advances in our understanding of the molecular mechanisms underlying cell type- and ­tissue-specific Treg cell function, it seems promising that Treg cell-based cellular therapeutics as well as novel biologicals to directly and cell-specifically modulate Treg cell function are entering the clinic, and first results of their potential as tolerance modifiers will be available in near future.

Anti-CD4 treatment inhibits autoimmunity in scurfy mice through the attenuation of co-stimulatory signals

A major concept in autoimmunity is that disruption of Foxp3+ regulatory T cells (Tregs) predisposes to breach of tolerance. This is exemplified by the Foxp3-linked disorder termed IPEX (immunodysregulation, polyendocrinopathy, enteropathy, X-linked) which affects newborn children. There has been considerable clinical interest in the role of non-depleting anti-CD4 antibodies as a means of upregulating the function of Foxp3+ Tregs in order to control detrimental inflammatory responses such as transplant rejection. However, according to the paradigm of a Treg-dependent mechanism of action, the effectiveness of anti-CD4 antibodies as a therapy for human autoimmune diseases is unclear considering that Treg function might be intrinsically impaired. Specifically, anti-CD4 therapy is expected to fail in patients suffering from the IPEX syndrome due to the lack of functional Foxp3+ Tregs. Taking advantage of natural Foxp3 mutant scurfy (sf) mice closely resembling the IPEX syndrome, and genetically engineered mice depleted of Foxp3+ Tregs, we report here that anti-CD4 treatment induces tolerance independent of Foxp3+ Tregs. This so far undefined mechanism is dependent on the recessive non-infectious tolerization of autoreactive T cells. Treg-independent tolerance alone is powerful enough to suppress both the onset and severity of autoimmunity and reduces clinically relevant autoantibody levels and liver fibrosis. Mechanistically, tolerance induction requires the concomitant activation of autoreactive T cells and is associated with the down-regulation of the co-stimulatory TNF-receptor superfamily members OX40 and CD30 sustaining CD4+ T cell survival. In the light of ongoing clinical trials, our results highlight an unexpected potency of anti-CD4 antibodies for the treatment of autoimmune diseases. Particularly, CD4 blockade might represent a novel therapeutic option for the human IPEX syndrome.

Generation of highly effective and stable murine alloreactive Treg cells by combined anti‐CD4 mAb, TGF‐β, and RA treatment

The transfer of alloreactive regulatory T (aTreg) cells into transplant recipients represents an attractive treatment option to improve long-term graft acceptance. We recently described a protocol for the generation of aTreg cells in mice using a nondepleting anti-CD4 antibody (aCD4). Here, we investigated whether adding TGF-β and retinoic acid (RA) or rapamycin (Rapa) can further improve aTreg-cell generation and function. Murine CD4(+) Tcells were cultured with allogeneic B cells in the presence of aCD4 alone, aCD4+TGF-β+RA or aCD4+Rapa. Addition of TGF-β+RA or Rapa resulted in an increase of CD25(+)Foxp3(+)-expressing Tcells. Expression of CD40L and production of IFN-γ and IL-17 was abolished in aCD4+TGF-β+RA aTreg cells. Additionally, aCD4+TGF-β+RA aTreg cells showed the highest level of Helios and Neuropilin-1 co-expression. Although CD25(+)Foxp3(+) cells from all culture conditions displayed complete demethylation of the Treg-specific demethylated region, aCD4+TGF-β+RA Treg cells showed the most stable Foxp3 expression upon restimulation. Consequently, aCD4+TGF-β+RA aTreg cells suppressed effector T-cell differentiation more effectively in comparison to aTreg cells harvested from all other cultures, and furthermore inhibited acute graft versus host disease and especially skin transplant rejection. Thus, addition of TGF-β+RA seems to be superior over Rapa in stabilising the phenotype and functional capacity of aTreg cells.

Alpha-1,2-Mannosidase and Hence N-Glycosylation Are Required for Regulatory T Cell Migration and Allograft Tolerance in Mice

BackgroundSpecific immunological unresponsiveness to alloantigens can be induced in vivo by treating mice with a donor alloantigen in combination with a non-depleting anti-CD4 antibody. This tolerance induction protocol enriches for alloantigen reactive regulatory T cells (Treg). We previously demonstrated that alpha-1,2-mannosidase, an enzyme involved in the synthesis and processing of N-linked glycoproteins, is highly expressed in tolerant mice, in both graft infiltrating leukocytes and peripheral blood lymphocytes.Principal FindingsIn this study we have identified that alpha-1,2-mannosidase expression increases in CD25+CD4+ Treg when they encounter alloantigen in vivo. When alpha-1,2-mannosidase enzyme activity was blocked, Treg retained their capacity to suppress T cell proliferation in vitro but were unable to bind to physiologically relevant ligands in vitro. Further in vivo analysis demonstrated that blocking alpha-1,2-mannosidase in Treg resulted in the migration of significantly lower numbers to the peripheral lymph nodes in skin grafted mice following adoptive transfer, where they were less able to inhibit the proliferation of naïve T cells responding to donor alloantigen and hence unable prevent allograft rejection in vivo.SignificanceTaken together, our results suggest that activation of alloantigen reactive Treg results in increased alpha-1,2-mannosidase expression and altered N-glycosylation of cell surface proteins. In our experimental system, altered N-glycosylation is not essential for intrinsic Treg suppressive capacity, but is essential in vivo as it facilitates Treg migration to sites where they can regulate immune priming. Migration of Treg is central to their role in regulating in vivo immune responses and may require specific changes in N-glycosylation upon antigen encounter.

Non-depleting anti-CD4 antibody alleviates ovalbumin-induced airway inflammation and T-helper type 2 responses in mice (140.13)

Abstract Bronchial asthma is an emerged chronic inflammatory disorder of the airways. It is becoming urgent to identify target molecules for the preventive and therapeutic purposes. Non-depleting anti-CD4 monoclonal antibody, YTS177, has been used to induce immunotolerance in allograft transplantation through antigen-specific Treg cells. This study aims for determining whether YTS177 is able to inhibit the ovalbumin (OVA)-induced allergic inflammation in an asthmatic murine model. The injection of YTS177 before and after OVA-sensitization was able to suppress airway hyperresponsiveness, pulmonary eosinophilia, OVA-specific immunoglobulin (Ig) E synthesis, and the production of T helper (Th) 2 cytokines from OVA-activated splenocytes. Furthermore, YTS177 had the therapeutic capacity to relieve the attack of asthmatic symptoms when it was administrated after the mice were sensitized and challenged with OVA. However, the depletion of CD25+ cells did not reverse YTS177-induced hyporesponsive status. The state might be resulted from the angery of CD4+ T cells with the increased level of anergy factor-Cbl-b in activated CD4+ cells cultured with YTS177. Moreover, the high level of cell cycle inhibitor p27Kip1 and increased number of G1 phase arrested-T cells were detected. Therefore, p27Kip1 might be involved in the preventive and therapeutic efficacy of YTS177 for allergic diseases.

Regulatory T cells in HIV infection: pathogenic or protective participants in the immune response?

Regulatory T cells in HIV infection: pathogenic or protective participants in the immune response?

Non-depleting Anti-CD4 Antibody not only Prevents Onset but Resolves Sialadenitis in NOD Mice

The non-obese diabetic (NOD) mouse spontaneously develops lymphocytic infiltrates in the salivary glands (sialadenitis) and provides an useful rodent model of human Sjögren's syndrome (SS). Non-depleting anti-CD4 antibodies have been shown to ameliorate Type 1 diabetes in NOD mice and also vasculitis in MRL/lpr mice. This study shows that a short course of treatment with the non-depleting anti-CD4 monoclonal antibody, YTS 177, completely prevents salivary infiltration and reverses ongoing pathology in the salivary gland.

CD25+CD4+ REGULATORY T CELLS GENERATED IN VIVO PREVENT ISLET ALLOGRAFT REJECTION BY BLOCKING THE RECRUITMENT OF EFFECTOR CELLS INTO THE GRAFT

O178* The concept of CD25+CD4+ regulatory T cells (T-reg) is well established in various autoimmune and transplantation models and a growing body of evidence suggests that operational transplantation tolerance depends on active T cell regulation mediated by these T-regs. Previously, we have demonstrated that T-reg generated in vivo prior to transplant by pre-treatment with donor-specific blood transfusion (DST) plus anti-CD4 monoclonal antibody can regulate donor-specific allograft rejection in a CTLA-4 and IL-10 dependent manner. Aim: The aim of this study was to ask whether T-reg generated by the same protocol are capable of preventing the rejection of a life-sustaining islet allograft, and if so, explore the mechanisms involved. Methods: T cell depleted CBA (H-2k) recipients were rendered diabetic by streptozotocin (blood glucose >20 mmol/L). These mice were then reconstituted with 1x105 CD45RBhigh CD4+ effector cells from naïve mice with or without co-transfer of 4x105 CD25+CD4+ from cell donors tolerized with nondepleting anti-CD4 antibody on days –28 and –27 plus a C57BL/6 (H-2b) DST on day –27. The naïve CD45RBhigh CD4+ cells used as effectors in this model co-express human CD52 as a transgene which allows them to be distinguished phenotypically from putative T-regs following adoptive transfer. On day 0, reconstituted, diabetic animals were transplanted with 400 C57BL/6 islets under the kidney capsule. Graft function was assessed by daily blood glucose measurements and rejection was defined as a blood glucose level ≥14.5 mmol/L on two consecutive days. Cellular infiltration was quantified by immunofluorescence using anti-hCD52-FITC. Four separate planes were analysed with an average of 4 high power fields (hpf) examined per plane. Results and Conclusion: Mice reconstituted with CD45RBhigh CD4+ effector cells alone rejected their grafts acutely (MST=18, n=7). This was completely prevented by co-transfer of CD25+CD4+ T cells isolated from tolerized animals (MST>70, n=10). These animals remained normoglycemic (8-13 mmol/L, normal range 8-12 mmol/L). In the absence of CD25+CD4+ T cells, CD45RBhigh CD4+ effector cells rapidly infiltrated the graft site and accumulated until day 15. However, in the presence of CD25+CD4+ T cells, at day 5 there was a 7-fold decrease in the number of infiltrating effector cells (CD45RBhigh only 3.66 ± 0.33; CD45RBhighplus CD25+ 0.47 ± 0.15 cells/hpf, p<0.0001; syngeneic control 0.57 ± 0.2). To our knowledge, this is the first demonstration that CD25+CD4+ T cells generated in vivo by immunomodulation can protect a life-sustaining, functional primary islet allograft. The effect of regulation on the recruitment of effector cells into the graft provides an insight into one of the mechanisms in which regulatory T cells can prevent allograft rejection and confirms that the generation of CD25+CD4+ regulatory cells should be considered as an important strategy for providing operational tolerance in clinical transplantation.

Nondepleting anti-CD4 and soluble interleukin-1 receptor prevent autoimmune destruction of syngeneic islet grafts in diabetic NOD mice.

Successful islet transplantation in type 1 diabetes requires tolerance induction of both allo- and autoreactive T-cell responses. Monoclonal antibodies targeting the CD4 coreceptor on T-helper cells have been shown to be effective in this regard. In type 1 diabetes, there is some evidence to suggest that cytokines such as interleukin (IL)-1 may be involved in beta-cell destruction. The high glucose levels associated with type 1 diabetes are also known to be toxic to beta cells. The tempo of T-cell and macrophage infiltration into syngeneic islets transplanted into diabetic nonobese diabetic (NOD) mice was examined by immunohistochemistry. We investigated the ability of a nondepleting anti-CD4 monoclonal antibody (YTS177) to induce tolerance to syngeneic islet grafts in female spontaneous diabetic NOD mice and in an adoptive transfer model of diabetes in NOD mice. The spontaneous model was used to test the effect on graft function of perioperative insulin therapy in mice treated with YTS177. The ability of soluble interleukin (sIL)-1 receptor (R) type II (sIL-1RII) to inhibit IL-1 effects in syngeneic islet transplants was also assessed. Cellular infiltration of CD3 cells and macrophages into the islet graft coincided with loss of graft function in untreated mice. Self-tolerance to beta cells was restored with YTS177, allowing long-term graft survival in a proportion of animals. The use of perioperative insulin therapy increased the number of successful grafts in spontaneously diabetic NOD mice treated with YTS177. The combination of YTS177 with sIL-1RII significantly improved the rates of graft survival compared with graft survival in YTS177-treated spontaneously diabetic NOD mice. Nondepleting anti-CD4 antibodies restore self tolerance to syngeneic islet transplants in diabetic NOD mice. Insulin therapy improves graft survival in mice treated with YTS177. Preventing the action of IL-1 greatly improves graft survival induced with YTS177.

Use of a non-depleting anti-CD4 antibody to modulate the immune response to coagulation factors VIII and IX.

The generation of antibodies to therapeutic factors VIII or IX is a major problem in the management of haemophilia and places potential limitations on the application of gene therapy. We have investigated the administration of a non-depleting anti-CD4 antibody for modulation of the immune response to human recombinant coagulation factors VIII and IX. In mice given these clotting factors, co-administration of anti-CD4 antibody significantly reduced the appearance of factor-specific antibodies. These data provide evidence that the neutralizing antibody response to exogenous coagulation factors may be controllable if non-depleting anti-CD4 antibody is co-administered at the time of initial replacement therapy.

A humanized, nondepleting anti-CD4 antibody that blocks virus entry inhibits virus replication in rhesus monkeys chronically infected with simian immunodeficiency virus.

Therapeutic approaches that interfere with viral entry hold promise in preventing or treating HIV infection. Hu5A8, a humanized monoclonal antibody against CD4, was previously shown to inhibit HIV and SIV replication in vitro and was safely administered to rhesus monkeys without depleting CD4(+) T cells. This antibody completely suppressed replication of six different SIVmac 251 primary isolates in vitro. Twice weekly administration of 3-mg/kg doses of hu5A8 for 2 to 4 weeks to SIV-infected rhesus monkeys resulted in sustained plasma antibody levels of > or =20 microg/ml during treatment and 5- to 50-fold decreases in plasma viremia, although suppression of viral replication was transient. Two of three treated monkeys developed antibody responses against the administered monoclonal antibody. Loss of antiviral effect was not temporally associated with anti-hu5A8 antibody responses or due to activation of CD4(+) T cells by hu5A8. However, SIV isolated after hu5A8 treatment was approximately 5-fold more resistant to suppression by hu5A8 than SIV isolates obtained from the same monkeys before treatment. The rapid development of resistance may have resulted from SIV variants that infect cells by a CD4-independent mechanism. These results support the overall concept of anti-CD4 monoclonal antibody treatment to suppress AIDS virus replication in vivo while demonstrating important issues as to its clinical feasibility.

Ability of a Nondepleting Anti-CD4 Antibody to Inhibit Th2 Responses and Allergic Lung Inflammation Is Independent of Coreceptor Function

Nondepleting anti-CD4 Abs have been used in vivo to induce Ag-specific immunological tolerance in Th1 responses, including tissue allograft rejection and autoimmune diabetes. To examine whether this Ab (YTS177.9) acts by provoking a Th2 shift, we tested the effect in a mouse model of allergic lung inflammation. Interestingly, nondepleting anti-CD4 treatment induces tolerance to allergens as well, especially when given during initial priming. In vitro studies indicate that the effect of the Ab is independent of CD4 coreceptor function, as Ab treatment also inhibits proliferation and induces a persistent anergy in naive CD4 T cells stimulated by anti-CD3/CD28. Moreover, the Ab stimulated a distinct pattern of tyrosine phosphorylation in T cells even in the absence of TCR triggering, suggesting that signaling through CD4 alone induces significant physiological changes in T cell function. These results show that tolerance induced by anti-CD4 triggering is not a simple shift in Th1/Th2 effector function or depletion of Ag-specific cells, but may instead induce a persistent clonal anergy capable of blocking subsequent immunity.

Anti-CD4 therapy in treatment of rheumatoid arthritis--have the die been cast?

The hypothesis of rheumatoid arthritis (RA) as a T cell mediated disease has led to the development of numerous therapeutic approaches that target the function of T cells. The development of monoclonal antibodies against the T cell surface molecule CD4 has raised hopes to achieve a major progress in the treatment of RA. However, after encouraging results in early open studies, double blind trials were unable to demonstrate the efficacy of anti-CD4-therapy in RA. There are numerous reasons to explain the failure of this treatment approach. Besides the fact that the T cell hypothesis of RA has repeatedly been challenged, pharmacological problems or an inappropriate selection of outcome criteria have to be considered. The final evaluation of anti-CD4 therapy in RA will be possible only after the testing of newly developed non-depleting anti-CD4 antibodies.

ATTENUATION OF IL-2 RECEPTOR SIGNALING BY CD4-LIGATION - MOLECULAR MECHANISMS

666 Administration of non-depleting anti-CD4 antibody can lead to long-term allograft survival and possibly transplantation tolerance in rodents, but mechanisms underlying in vivo immunomodulation by CD4 ligands are incompletely understood. Using peptomer, a CD4-binding peptide multimer, we have previously identified attenuation of IL-2-induced nuclear translocation of STAT5 as a downstream effect of CD4 ligation (CD4L). To further characterize this interference of CD4L with IL-2 receptor (IL-2R) signaling, we examined proximal elements of IL-2-activated signal transduction cascades as potential molecular targets of such ligation. Purified primed human T cells were stimulated with IL-2 (100U/ml, 10-60 min.) alone or after pretreatment with peptomer (1 hr.). By flow cytometry, IL-2R α-chain expression, which is regulated by IL-2 via STAT5, was unaffected in the time frame studied. In anti-phosphotyrosine immunoblots, IL-2-induced tyrosine phosphorylation of IL-2R β-chain immunoprecipitates was also unaltered by preceding CD4L with peptomer. However, anti-phosphotyrosine immunoblots of whole cell lysates revealed that peptomer attenuates IL-2-induced tyrosine phosphorylation of 130 kD protein(s) indistinguishable from JAKs 1 and 3 on reprobing with antibodies against these kinases. As IL-2R associated JAK3 is essential for STAT5 activation, we examined JAK3 immunoprecipitates from the cells and found that peptomer pretreatment indeed inhibits JAK3 tyrosine phosphorylation by IL-2. Cytoskeletal sequestration of another kinase, p56lck, which associates with CD4 and the IL-2R, also correlates with CD4L-induced immune modulation and is preventable by pre-incubation with cytochalasin D (CyD) to inhibit cytoskeletal actin polymerization. We thus asked if CyD could block the attenuation of IL-2R signaling by antecedent CD4L, and if p56lck was required for this effect. Nuclear extracts of human T cells treated as above and with or without CyD-pre-incubation(0.5µM, 2-3 hrs.) were analyzed in gel shift assays. CyD was found to protect IL-2-induced nuclear translocation of STAT5 from attenuation by CD4L. Identical results were obtained with a p56lck-deficient human lymphoid cell line. These studies identify JAK3 as a novel target of CD4 ligands and indicate that CD4L can interfere with IL-2R signaling via attenuation of IL-2-induced JAK3 tyrosine phosphorylation. Polymerized cytoskeletal actin, but not p56lck, appears to be required for this interference. Our observations delineate new molecular mechanisms underlying CD4L-induced immune modulation and thus provide fresh insights into the possible genesis of transplantation tolerance induced by therapies targeting CD4.

ATTENUATION OF IL-2 RECEPTOR SIGNALING BY CD4-LIGATION-MOLECULAR MECHANISMS.

666 Administration of non-depleting anti-CD4 antibody can lead to long-term allograft survival and possibly transplantation tolerance in rodents, but mechanisms underlying in vivo immunomodulation by CD4 ligands are incompletely understood. Using peptomer, a CD4-binding peptide multimer, we have previously identified attenuation of IL-2-induced nuclear translocation of STAT5 as a downstream effect of CD4 ligation (CD4L). To further characterize this interference of CD4L with IL-2 receptor (IL-2R) signaling, we examined proximal elements of IL-2-activated signal transduction cascades as potential molecular targets of such ligation. Purified primed human T cells were stimulated with IL-2 (100U/ml, 10-60 min.) alone or after pretreatment with peptomer (1 hr.). By flow cytometry, IL-2R α-chain expression, which is regulated by IL-2 via STAT5, was unaffected in the time frame studied. In anti-phosphotyrosine immunoblots, IL-2-induced tyrosine phosphorylation of IL-2R β-chain immunoprecipitates was also unaltered by preceding CD4L with peptomer. However, anti-phosphotyrosine immunoblots of whole cell lysates revealed that peptomer attenuates IL-2-induced tyrosine phosphorylation of 130 kD protein(s) indistinguishable from JAKs 1 and 3 on reprobing with antibodies against these kinases. As IL-2R-associated JAK3 is essential for STAT5 activation, we examined JAK3 immunoprecipitates from the cells and found that peptomer pretreatment indeed inhibits JAK3 tyrosine phosphorylation by IL-2. Cytoskeletal sequestration of another kinase, p56lck, which associates with CD4 and the IL-2R, also correlates with CD4L-induced immune modulation and is preventable by pre-incubation with cytochalasin D (CyD) to inhibit cytoskeletal actin polymerization. We thus asked if CyD could block the attenuation of IL-2R signaling by antecedent CD4L, and if p56lck was required for this effect. Nuclear extracts of human T cells treated as above and with or without CyD-pre-incubation (0.5μM, 2-3 hrs.) were analyzed in gel shift assays. CyD was found to protect IL-2-induced nuclear translocation of STAT5 from attenuation by CD4L. Identical results were obtained with a p56lck-deficient human lymphoid cell line. These studies identify JAK3 as a novel target of CD4 ligands and indicate that CD4L can interfere with IL-2R signaling via attenuation of IL-2-induced JAK3 tyrosine phosphorylation. Polymerized cytoskeletal actin, but not p56lck, appears to be required for this interference. Our observations delineate new molecular mechanisms underlying CD4L-induced immune modulation and thus provide fresh insights into the possible genesis of transplantation tolerance induced by therapies targeting CD4.

Nondepleting anti-CD4 antibody treatment prolongs lung-directed E1-deleted adenovirus-mediated gene expression in rats.

E1-deleted adenoviral vectors are efficient vectors for somatic cell gene therapy, but transgene expression is limited in part by a cytotoxic T cell response directed against virally transduced cells. Moreover, the development of a neutralizing antibody response limits secondary gene transfer with these vectors. Therapy with a depleting anti-CD4 antibody permits prolonged transgene expression in the lung and liver of mice. Furthermore, transient depletion of CD4+ lymphocytes blocks neutralizing antibody production and therefore allows repeat administration and expression of E1-deleted recombinant adenovirus. In this study, we investigated the efficacy of a novel nondepleting anti-CD4 antibody (RIB 5/2) in a model of lung-directed gene therapy in outbred rats. Treatment with RIB 5/2 permitted prolonged reporter gene expression and reduced adenovirus-induced peribronchial and alveolar inflammation in the lung. Moreover administration of RIB 5/2 blocked the development of an anti-adenoviral neutralizing antibody response in the lung and permitted secondary administration and expression of a recombinant adenovirus. These data support the role of immunomodulation in prolonging in vivo transgene expression by recombinant adenovirus.

Transplantation tolerance induced by antigen pretreatment and depleting anti-CD4 antibody depends on CD4+ T cell regulation during the induction phase of the response.

Adult mice pretreated with donor-specific transfusion and depleting anti-CD4 antibody 28 days before transplant accept fully allogeneic heart grafts and become specifically tolerant without further treatment. The induction of tolerance in this model is not simply a function of CD4+ T cell ablation, but appears to depend on residual CD4+ T cells which escape depletion and engage donor alloantigen during a transient period of antibody blockade. To test the hypothesis that these CD4+ T cells might be responsible for regulating immune responses toward the graft, mice were reconstituted with naive recipient leukocytes at various times after pretreatment. Reconstitution either shortly after pretreatment or shortly after transplant had little effect on graft survival. However, when pretreated mice were given an additional dose of depleting anti-CD4 antibody at the time of transplant to target putative regulatory cells, naive leukocytes were able to cause acute graft rejection. These data suggest that in clinical transplantation specific T cell regulation might develop following pretreatment with antigen and non-depleting anti-CD4 antibodies. Such an approach could provide donor-specific unresponsiveness prior to transplant without the risks associated with sustained CD4+ T cell depletion.

NONDEPLETING ANTI-CD4 ANTIBODIES IN TRANSPLANTATION

CD4+ T cells are obvious targets for immunotherapy since they appear to be pivotal in rejection responses. Current interest in clinical transplantation is focused on the potential use of nondepleting anti-CD4 antibodies largely because depletion of CD4+ T cells may lead to prolonged periods of nonspecific immunosuppression. We have examined the efficacy of two nondepleting antibodies, KT6 and YTS 177.9, in a mouse cardiac allograft model. KT6 causes abrupt and prolonged occupation of CD4 molecules in vivo, while YTS 177.9 results in rapid and almost complete modulation of CD4 from the cell surface. In identical perioperative two-dose protocols, equivalent doses of KT6 were more effective than YTS 177.9 (MST 58 days and 24 days, respectively). When the treatment protocols were redesigned to include one or two additional doses of antibody, given as CD4 molecules were either released from blockade (KT6) or re-expressed following modulation (YTS177.9), the YTS 177.9 protocol led only to a slight improvement in graft survival (MST 51 days), whereas the modified KT6 regimen gave indefinite survival in 100% of the recipients. These data clearly show that modulating anti-CD4 antibodies may be far less effective than antibodies that effect prolonged CD4 occupation, a significant observation especially in relation to the proposed clinical use of the antibody OKT4A, which appears to cause modulation rather than CD4 blockade.

Induction of tolerance in peripheral T cells with monoclonal antibodies

Our goal has been to develop ways to tolerize the mature immune system to any defined antigen. In this report we show that peripheral (post-thymic) T cells of mice can become tolerant to a range of antigens (human and rat immunoglobulins, and bone marrow and skin grafts that differ at multiple minor transplantation antigens). In the case of human gamma globulin (HGG), this required that the antigen be given under the cover of a short course of non-depleting anti-CD4 antibody, while for tolerance to skin and marrow grafts anti-CD8 antibody was also required. Tolerance to HGG could be reinforced by repeated injections of HGG, but was lost in the absence of any further exposure to antigen. This reversal of tolerance with time was due to new T cells being exported from the thymus, as it was not observed in tolerized, adult thymectomized mice. In contrast, tolerance to marrow and skin grafts was permanent, presumably because the established grafts acted as a continuous source of antigen to reinforce the tolerant state. Tolerance could not be broken by the infusion of unprimed spleen cells and in one example (tolerance to Mls-1a) there was clear evidence that specific peripheral T cells were anergic. We propose that anergic cells may themselves participate in reinforcing the tolerant state by competing at sites of antigen presentation.