anti-CD45RB mAb Research Articles

Analysis of CD45 Isoforms Displayed By Human Peripheral Blood Mononuclear Cells

CD45 is the most prevalent glycoprotein on the surface of mature leukocytes and hematopoietic stem/progenitor cells (HSPCs). Accordingly, this glycoprotein has been intensively interrogated since its discovery, and antibody-drug-conjugates (ADCs) and radioimmunoconjugates targeting the extracellular domain of CD45 have been explored in pre-clinical and clinical trials as a potential replacement of, or supplement to, existing myeloablative regimens for hematopoietic stem cell transplant (HSCT) preparation. Importantly, human CD45 consists of five distinct isoforms, resulting from assortment of three alternatively spliced exons (known as "A", "B", and "C"). Though monoclonal antibodies (mAbs) directed to each of these exons, and to the CD45 isoform bearing no such exons (i.e., the "CD45RO" isoform) are available, our understanding of the display of these distinct exons is grossly incomplete. To date, the most well-characterized leukocyte subset differentially expressing CD45 isoforms is the human T cell. By contrast, the human myeloid compartment's expression of specific CD45 isoforms has been largely unstudied. Given this, we sought to comprehensively interrogate CD45 isoform expression on human peripheral blood mononuclear cells following defined in vitro manipulations of the cells. CD4+ and CD8+ human T cells were isolated from peripheral blood of healthy donors and activated with anti-CD3 and anti-CD28 antibodies for 5 days. Before and after activation, we performed flow cytometry combined with western blot analysis to assess shifts to CD45RA, CD45RB, CD45RC, and CD45RO expression. We isolated baseline human CD14+ monocytes and utilized the same techniques to ascribe CD45 isoforms to resting human monocytes. Upon activation of human T cells, our analyses recapitulated the expected transition of these cells from CD45RA+ to CD45RO+. However, strikingly, all native and in vitro-activated human T cells universally react with anti-CD45RB mAb as determined by both flow cytometry and western blotting. Similarly, 100% of human CD14+ monocytes display CD45RB and CD45RO, as determined by flow cytometry and western blotting. The universal reactivity of human T cells with anti-CD45RB illustrates that canonical CD45RA-/CD45RO+ memory and activated T cells co-express the CD45RB isoform. In a similar manner, baseline human CD14+ monocytes co-express CD45RO and CD45RB isoforms. The capability to unveil the ubiquity of CD45RB isoform expression was enabled by concurrent use of exon-specific mAbs. These data indicate a previously unappreciated complexity in human CD45 isoform display, draw caution on simply stratifying CD45 expression on the basis of CD45RA or CD45RO phenotypes, and highlight a broader significance of the CD45RB isoform among human T cells and monocytes, and within the greater human hematologic system.

Selective FcγR Co-engagement on APCs Modulates the Activity of Therapeutic Antibodies Targeting T Cell Antigens

The co-engagement of fragment crystallizable (Fc) gamma receptors (FcγRs) with the Fc region of recombinant immunoglobulin monoclonal antibodies (mAbs) and its contribution to therapeutic activity has been extensively studied. For example, Fc-FcγR interactions have been shown to be important for mAb-directed effector cell activities, as well as mAb-dependent forward signaling into target cells via receptor clustering. Here we identify a function of mAbs targeting Tcell-expressed antigens that involves FcγR co-engagement on antigen-presenting cells (APCs). In the case of mAbs targeting CTLA-4 and TIGIT, the interaction with FcγR on APCs enhanced antigen-specific Tcell responses and tumoricidal activity. This mechanism extended to an anti-CD45RB mAb, which led to FcγR-dependent regulatory Tcell expansion in mice.

CD45 ligation reduces Tregs’ motility and enhances recognition of activating signals in vitro

Abstract Regulatory T cells (Tregs) play a pivotal role in modulating immune response hence supporting immune tolerance. Recently, CD45 ligation with anti-CD45RB mAb MB23G2 (anti-CD45RB) was reported to enhance graft-survival; evidence shows it was by homeostatic Treg proliferation in vivo. While many in vivo data support the notion that anti-CD45RB supports Tregs expansion, it is still unclear if the ligation ex vivo is sufficient to enhance Tregs’ avidity for activation and further promote their expansion and function. Here, we were to see if anti-CD45RB ligation can enhance Treg’s sensitivity to perceive activating signals in varying strength in vitro. To test this hypothesis, mouse CD4+ T cells (Foxp3-GFP linked B6 mouse, both Foxp3+ and Foxp3−, or Tregs and Tconvs, respectively) were seeded onto surfaces micropatterned with an antibody targeting the CD3 on T cell (anti-CD3) with and without the presence of anti-CD45RB. These surfaces were created by first microcontact printing of 2-μm diameter features of anti-CD3 at various concentrations (i.e., 1, 5, and 20 μg/mL) on glass coverslips and back-filled with ICAM-1. The effect of anti-CD45RB on Treg and Tconv activation was compared by the ratios of phosphorylation of Lck at Tyr 394 to 505 sites, cell registration on anti-CD3 dot array, and motility of each cell type on varying concentration of activation signals. Overall results show that anti-CD45RB specifically slow-down Treg motility and, therefore, enhance the probability to contact to activating signals (cell registration) and improve T cell activation indicated by ratios of phospho-Lck at active over inactive sites. This study assures the importance of further investigation on the role of anti-CD45RB in Treg activation mechanism.

Sample sparing platform for microscopy shows that CD45RB ligation increases Treg sensitivity to activation signals in vitro

Abstract Small subpopulations of cells, e.g. regulatory T cells, often play key roles in the function of the entire immune system. While their smaller number makes them difficult to work with in conventional immunocytochemistry assays or flow cytometry, microscopy can provide functional and spatial information on a single cell basis. Combined with surface modification technology such as synthetic lipid bilayers and micropatterned antibodies, we are in a unique position to be able to study the spatial mobility of intracellular signals (e.g. show that mobility of Lck differs between mouse and human T cells). Even still, conventional microscopy platforms such as 96-well plates or flow cell systems require a minimum of 10k to 1 million cells and does not allow for an interchangeable surface. Conical wells with a 1mm bottom and 5mm top diameter (microwells), laid out in a 96-well configuration were mass manufactured out of polypropylene from Protolabs, and adhered to surface modified glass coverslips. 1k cells seeded and centrifuged at 100g for 10 mins resulted in 81% of the cells landing on an image-able area. Based on the report that anti-CD45RB mAb MB23G2 enhances graft-survival, we tested the hypothesis that anti-CD45RB ligation in vitro increases Treg sensitivity to activation signals. 3 different activation signal levels (1, 5, and 20 μg/mL), 2 cell types, and 2 treatment conditions conventionally required at least 150k cells per sample, or 1.8M cells total. On the microwell, we performed the experiment with 8k cells per sample, with quadruplicates for a total of 384k cells, to show that the ratio of phosphorylation of Lck at Tyr 394 to 505 sites, and alignment to micropatterned anti-CD3 dots are increased in Tregs treated with anti-CD45RB.

CD45 ligation expands Tregs by promoting interactions with DCs.

Regulatory T cells (Tregs), which express CD4 and FOXP3, are critical for modulating the immune response and promoting immune tolerance. Consequently, methods to expand Tregs for therapeutic use are of great interest. While transfer of Tregs after massive ex vivo expansion can be achieved, in vivo expansion of Tregs would be more practical. Here, we demonstrate that targeting the CD45 tyrosine phosphatase with a tolerogenic anti-CD45RB mAb acutely increases Treg numbers in WT mice, even in absence of exogenous antigen. Treg expansion occurred through substantial augmentation of homeostatic proliferation in the preexisting Treg population. Moreover, anti-CD45RB specifically increased Treg proliferation in response to cognate antigen. Compared with conventional T cells, Tregs differentially regulate their conjugation with DCs. Therefore, we determined whether CD45 ligation could alter interactions between Tregs and DCs. Live imaging showed that CD45 ligation specifically reduced Treg motility in an integrin-dependent manner, resulting in enhanced interactions between Tregs and DCs in vivo. Increased conjugate formation, in turn, augmented nuclear translocation of nuclear factor of activated T cells (NFAT) and Treg proliferation. Together, these results demonstrate that Treg peripheral homeostasis can be specifically modulated in vivo to promote Treg expansion and tolerance by increasing conjugation between Tregs and DCs.

Protection of Porcine Islet Xenografts in Mice Using Sertoli Cells and Monoclonal Antibodies

To remedy the shortage of human donor islets, xenotransplantation of neonatal porcine islets (NPI) provides an attractive alternative source of donor tissue so long as graft rejection can be circumvented. Thus, in this study, we sought to determine whether cotransplantation of NPI with Sertoli cells (SC) combined with a short-course treatment of monoclonal antibody (mAb) could provide long-term islet xenograft survival. NPI alone or NPI cotransplanted with neonatal porcine SC were transplanted into diabetic C57BL/6 mice. These mice were left untreated or were treated with a short course of antileukocyte function associated antigen-1 (LFA-1), anti-CD154, or anti-CD45RB mAb. Blood glucose levels were monitored twice a week to assess graft function. At more than 100 days posttransplantation or on the day of rejection, graft-bearing kidneys were collected for characterization using immunohistochemistry. None of the untreated control mice transplanted with NPI alone (0/5) or NPI cotransplanted with SC (0/8) achieved normoglycemia. However, of the mice receiving NPI alone, 3 of 7 treated with anti-LFA-1 mAb, 2 of 7 treated with anti-CD154 mAb, and 1 of 7 treated with anti-CD45RB mAb achieved long-term graft survival (>100 days). These proportions improved considerably when NPI were cotransplanted with SC, as 15 of 15 mice treated with anti-LFA-1 mAb, 7 of 8 mice treated with anti-CD154 mAb, and 4 of 9 mice treated with anti-CD45RB mAb achieved long-term graft survival. These results show that transient administration of anti-LFA-1 mAb or anti-CD154 mAb is efficacious in prolonging NPI xenograft survival when islets are cotransplanted with SC. Interleukin-4 and Serpina3n may be important mediators of protection observed in this model.

Rapamycin Combined with Anti-CD45RB mAb and IL-10 or with G-CSF Induces Tolerance in a Stringent Mouse Model of Islet Transplantation

BackgroundA large pool of preexisting alloreactive effector T cells can cause allogeneic graft rejection following transplantation. However, it is possible to induce transplant tolerance by altering the balance between effector and regulatory T (Treg) cells. Among the various Treg-cell types, Foxp3+Treg and IL-10–producing T regulatory type 1 (Tr1) cells have frequently been associated with tolerance following transplantation in both mice and humans. Previously, we demonstrated that rapamycin+IL-10 promotes Tr1-cell–associated tolerance in Balb/c mice transplanted with C57BL/6 pancreatic islets. However, this same treatment was unsuccessful in C57BL/6 mice transplanted with Balb/c islets (classified as a stringent transplant model). We accordingly designed a protocol that would be effective in the latter transplant model by simultaneously depleting effector T cells and fostering production of Treg cells. We additionally developed and tested a clinically translatable protocol that used no depleting agent.Methodology/Principal FindingsDiabetic C57BL/6 mice were transplanted with Balb/c pancreatic islets. Recipient mice transiently treated with anti-CD45RB mAb+rapamycin+IL-10 developed antigen-specific tolerance. During treatment, Foxp3+Treg cells were momentarily enriched in the blood, followed by accumulation in the graft and draining lymph node, whereas CD4+IL-10+IL-4− T (i.e., Tr1) cells localized in the spleen. In long-term tolerant mice, only CD4+IL-10+IL-4− T cells remained enriched in the spleen and IL-10 was key in the maintenance of tolerance. Alternatively, recipient mice were treated with two compounds routinely used in the clinic (namely, rapamycin and G-CSF); this drug combination promoted tolerance associated with CD4+IL-10+IL-4− T cells.Conclusions/SignificanceThe anti-CD45RB mAb+rapamycin+IL-10 combined protocol promotes a state of tolerance that is IL-10 dependent. Moreover, the combination of rapamycin+G-CSF induces tolerance and such treatment could be readily translatable into the clinic.

Role of T and Dendritic Cells in Mouse Islet Allografts Treated With Anti-CD45RB Monoclonal Antibodies

Currently lifelong immunosuppression is required for organ transplant recipients. Anti-CD45RB monoclonal antibody (mAb) prolongs graft survival by mechanisms that are not yet clear. Therefore, we investigated the role of T and dendritic cells (DC) in islet allografts treated with anti-CD45RB mAb after transplantation of 200 allogeneic islets (BALB/c mouse) under the kidney capsules of diabetic C57BL/6 mice treated with intraperitoneal injections of 100 μg of anti-CD45RB mAb on days 0, 1, 3, 5, and 7. We observed a tilt of the ratios of Th1/Th2 and Tc1/Tc2 to Th2 and Tc2. The numbers of naïve and memory T cells were down-regulated in peripheral blood after transplantation. In addition, the maturation, endocytosis, and interleukin-12 secreted by DC derived from bone marrow cells was suppressed in recipient mice. Therefore, anti-CD45RB mAb alleviated, rejection by suppressive effects on T-lymphocyte subsets and DC.

Subcutaneous immunization with heat shock protein-65 reduces atherosclerosis in Apoe−/− mice

ObjectiveTo modulate atherosclerosis by combining subcutaneous immunization with heat shock protein 65 (hsp65) in alum adjuvant and anti-CD45RB monoclonal antibodies (mAb). Methods8 week old Apoe−/− mice on normal chow were treated for 12 weeks: group A received hsp65-alum immunization combined with anti-CD45RB mAb, group B hsp65-alum immunization combined with isotype control antibody, and group C mock vaccine combined with isotype control antibody. ResultsUnexpectedly, atherosclerotic lesions in the aortic root were significantly reduced in both hsp65-alum immunization groups (A and B) compared with the control group (C). Significantly elevated antibody titers against hsp65 were detected in both groups along with a significant increase in MHC class II expression on B cells. Body weight, total cholesterol and triglyceride levels were not different between groups. Treatment with anti-CD45RB antibody mediated a shift on CD4+ T cells from the CD45RBhigh to CD45RBlow isoform with a relative increase in CD4+Foxp3+ regulatory T cells (Treg) in an overall reduced T cell pool. Furthermore, anti-CD45RB treatment mediated a transient reduction of peripheral leukocytes and increased IFN-γ and IL-17A plasma levels. ConclusionsSubcutaneous immunization with hsp65-alum protects Apoe−/− mice against progression of early atherosclerosis. Administration of anti-CD45RB antibody provided no incremental benefit to the athero-protective effects of hsp65-alum treatment alone.

A New Combined Therapy (anti-CD45RB mAb + rapamycin + IL-10) Able to Induce Active Long-term Tolerance in a Stringent Pre-clinical Model of Allogeneic Islet Transplantation

A New Combined Therapy (anti-CD45RB mAb + rapamycin + IL-10) Able to Induce Active Long-term Tolerance in a Stringent Pre-clinical Model of Allogeneic Islet Transplantation

Long-Term Limb Allograft Survival Using a Short Course of Anti-CD45RB Monoclonal Antibody, LF 15-0195, and Rapamycin in a Mouse Model

The purpose of this study was to determine if a short course of monoclonal antibody (mAb) against CD45RB, LF 15-0195, and rapamycin would achieve long-term survival by inducing tolerance in a mouse limb transplant model. Group 1 (n=9) consisted of nine isogenic (C57BL/6) transplants. Group 2 (n=3) included C57BL/6-to-BALB/c transplants receiving no drug therapy. Group 3 mice (n=4) were treated with mAb (3 mg/kg) and LF (2 mg/kg), and Group 4 (n=13) was treated with mAb, LF, and rapamycin (2 mg/kg). Both treatment groups received drug treatment for only 14 days posttransplantation. Animals were sacrificed if they displayed evidence of rejection or when deemed to be tolerant (defined as >day 100). All isografts had normal histology and graft function on day 100. Untreated C57BL/6-to-BALB/c allografts developed acute rejection within 10 days. The combination of mAb and LF prolonged allograft survival to a mean of 39+/-7 days. In Group 4, two animals had to be sacrificed at days 28 and 76 due to acute urinary retention. Transplant tolerance was achieved in 8 of the remaining 11 animals with a mean survival time of 100+/-12 days. Donor specific tolerance was demonstrated through permanent acceptance of skin grafts from the donor strain and rejection of skin grafts from C3H mice. Three Group 4 animals showed clinical and histological signs of mild, chronic rejection. Dendritic cells isolated from tolerant recipients exerted a suppressive effect in mixed lymphocyte reaction. A short course of anti-CD45RB mAb and LF 15-0195 prolonged limb allograft survival. The addition of rapamycin induced limb allograft tolerance which is associated with the generation of tolerogenic dendritic cells that suppressed T-cell proliferation.

CD45RB Ligation Inhibits Allergic Pulmonary Inflammation by Inducing CTLA4 Transcription

CD45, a type I transmembrane protein tyrosine phosphatase expressed on nucleated hemopoietic cells, is prominently involved in T cell activation. Ligation of CD45RB isoforms has been associated with transplant tolerance. A recent genotyping analysis of asthma indicates a correlation with CD45 splicing. In this study, we administered an anti-CD45RB mAb (aCD45) in a murine model of allergic asthma and found that CD45RB ligation decreases allergic responses. aCD45 decreases allergen-induced pulmonary eosinophilia, bronchoalveolar lavage IL-13, IgE, and airway responses. Also, aCD45 increases the expression of CTLA4, a negative regulator of T cell activation. Furthermore, CD45RB signals no longer decrease allergic inflammation when CTLA4 is inhibited. These data support a role for CTLA4 in CD45RB-mediated inhibition of allergic inflammation. T cells and splenocytes stimulated with aCD45 exhibited increased CTLA4 levels, and analysis of CTLA4 promoter gene constructs identified a CD45RB-inducible regulatory region localized from -335 to -62 bp relative to the transcription start site. Together, these findings suggest that CD45RB signals mediate a novel role in the modulation of allergic inflammation, orchestrated by T cells through induction of CTLA4 transcription.

Anti-CD45RB Monoclonal Antibody Prolongs Renal Allograft Survival in Cynomolgus Monkeys

Previously, an anti-CD45RB monoclonal antibody (mAb) has been shown to induce murine allograft tolerance. The present study was performed to assess the ability of an anti-human CD45RB mAb to prevent rejection in a monkey MHC-mismatched kidney transplant model. The recipients were allocated into the following treatment groups: (1) isotype control IgG; (2) mouse anti-human CD45RB IgG1 (6G3); (3) human-mouse chimeric anti-CD45RB-IgG1 (C6G3-IgG1); (4) human-mouse chimeric anti-CD45RB-IgG2 (C6G3-IgG2); (5) tacrolimus at a subtherapeutic dose and (6) tacrolimus and C6G3-IgG1 in combination. Monotherapy with anti-CD45RB mAb significantly prolonged renal allograft survival to a median survival of 21 days. Adding a subtherapeutic dose of tacrolimus improved the efficacy of the anti-CD45RB mAb, achieving a median survival of 85 days, whereas a subtherapeutic dose of tacrolimus alone only moderately prolonged survival to 27 days. Treatment with anti-CD45RB mAb resulted in an alteration of the CD45RB(hi) : CD45RB(lo) cell ratio in the peripheral blood. We have, for the first time, demonstrated that an anti-human CD45RB mAb (6G3) can prolong graft survival. Induction with an anti-CD45RB mAb improves the efficacy of tacrolimus in the prevention of rejection. These encouraging results indicate that an anti-CD45RB mAb may be valuable in future clinical transplantation.

Tolerance Induction Through Megadose Bone Marrow Transplantation with Two-Signal Blockade 1

Induction of mixed chimerism is currently the most promising concept for clinical tolerance induction; however, the toxicity of the required host conditioning for allogeneic bone marrow transplantation (BMT) should be overcome. Therefore, we explored tolerogenic effectiveness of megadose BMT with anti-CD45RB and anti-CD154 mAb (two-signal blockade) in murine recipients without conditioning. Recipient B6 mice of BALB/c skin allograft received conditioning and an optimal dose (2x10(7) cells) of BMT. For a megadose BMT model, the conditioning was not performed; instead, megadose (2x10(8) cells) of BM was transplanted. The recipients were then treated with anti-CD45RB mAb and anti-CD154 mAb alone or their combination. Flow cytometry was performed to analyze the degree and distribution of donor-derived cells, peripheral deletion of Vbeta5 or Vbeta11 T cells and intrathymic presence of donor MHC class II+ cells. Induction of chimerism-based tolerance to skin allograft was further determined. High levels ( approximately 23.7%) of mixed and multi-lineage chimerism-based tolerance to skin allograft were induced in the recipients (91%) treated with the optimal-dose BMT and the two-signal blockade. The megadose BMT could replace the recipient conditioning and establish low (approximately 10%) and stable multilineage chimerism. Donor-specific tolerance to skin allograft was induced in these chimeras through clonal deletion of donor-reactive cells. The megadose BMT with the two-signal blockade could effectively establish mixed and multi-lineage chimerism and induce donor-specific tolerance, suggesting its potential for clinical application.

4-1BB promotes long-term survival in skin allografts treated with anti-CD45RB and anti-CD40L monoclonal antibodies

4-1BB (CD137) is a T-cell co-stimulatory molecule that promotes T cell activation. Using a skin transplantation model, we observed that simultaneous administration of monoclonal antibodies (mAb) targeting CD45RB and CD40L prolonged skin allograft in co-stimulation blockade (CTLA4-Ig and anti-CD40L mAb)–resistant mice, because of reducing CD8 + T cells and CD4 + CD45RB high T cells. Anti-CD45RB mAb (45RB) blocks the activation of T helper 1 (Th1) cells and generates regulatory T cells (T reg). The experimental design included five groups: group 1, control; group 2, 45RB-MR1; group 3, 45B-MR1 + 4-IBBL; group 4, anti-CD4 mAb plus group 3 treatment; group 5, anti-CD8 mAb plus group 3 treatment. In this study we highlight the involvement of 4-1BB/4-1BBL in the development of T-cell responses. C57BL/6 recipients of BALB/c skin grafts were treated with 45RB, anti-CD40L mAb (MR1), and antagonistic anti-4-1BBL mAb (4-1BBL) on days 0, 2, 4, 6, and 8 posttransplantation. Additional 4-1BBL further prolonged skin graft survival, although the percentage of splenocyte-derived CD8 + T cells was reduced similarly in both groups. Use of 4-1BBL seems to have additive effects on T reg cells, which play a major role in the maintenance of tolerance. Even after immunosuppressive therapy in combination with CD4 + T-cell depletion, we did not achieve prolonged graft survival, possibly because of the absense of T reg cells, which require CD4-independent CD8 + T cells, based on the observation of increasing proportion of CD8 + T cells in similar degree as the control group.

Changes in expression of T-cell activation-related molecules and cytokines during tolerance induction in an allogeneic skin transplantation murine model

Bone marrow transplantation after treatment with busulfan and costimulatory blockade with monoclonal antibodies (mAb) cytotoxic T lymphocyte antigen 4 (CTLA4)-Ig and anti-CD154 mAb or two-signal blockade using anti-CD45RB and anti-CD154 mAb are nonmyeloablative treatment regimens for allogeneic transplantation. There may be differences in the mechanisms of donor cell engraftment and reactive cell deletion by which these regimens induce donor-specific tolerance. Therefore, this study was performed to investigate changes in T cells and cytokines during tolerance induction toward allogeneic skin grafts. BALB/c and C57BL/6 mice were used as donors and recipients, respectively. Skin and bone marrow transplantations were performed and busulfan was administered. Three groups were treated with mAb as follows: group 1, anti-CD154 mAb; group 2, anti-CD154 plus anti-CD45RB mAb; and group 3, anti-CD154 mAb plus CTLA4-Ig. The proportions of CD4 + or CD8 + T cells and the expression of CD45RB isoforms on splenocytes were measured using flow cytometry and the production of cytokines by CD4 + T cells using enzyme-linked immunosorbent assay. Group 2 showed a significant reduction in the proportions of CD8 + T cells and CD45RB high isoforms compared with groups 1 and 3. The levels of interleukin (IL)-2 and IL-4 in group 2 were lower and higher than those of groups 1 and 3, respectively. In conclusion, the combined use of anti-CD154 and anti-CD45RB mAb decreases the CD8 + T-cell population and the expression of CD45RB, resulting in a Th2 cytokine profile, which may be a characteristic mechanism leading to donor cell engraftment and reactive cell deletion for donor-specific tolerance.

Inhibition of ICAM-1/LFA-1 interaction prevents allograft tolerance induced by anti-CD45RB

Abstract Introduction: Long-term allograft tolerance may be established by elimination of peripheral graft-reactive cell, by the induction of regulatory cells, or by induced ignorance through alteration of lymphocyte trafficking. We have investigated the contribution of the intracellular adhesion molecules ICAM-1/LFA-1 to tolerance induced by anti-CD45RB. Methods: Cardiac allografts from C3H mice were transplanted to either wild-type or ICAM-deficient C57BL/6 mice. Recipients were untreated, treated with anti-CD45RB mAb (100ug ip days 0, 1, 3, 5, and 7), or treated with a combination of anti-CD45RB and anti ICAM-1 or anti-LFA1 (50ug ip x5). Immunophenotyping of B cells by flow cytometry was performed after exposure of mice to alloantigen and anti-CD45RB. Results: Exposure of lymphocytes in vivo to anti-CD45RB and alloantigen induces upregulation of ICAM-1 on B cells. Although treatment with anti-CD45RB uniformly establishes permanent acceptance of C3H allografts (MST > 100days, n = 14), coadministration of either anti-ICAM-1 or anti-LFA-1 prevents tolerance induction (MST = 28.25 ± 2.96 and 14 ± 3.11 days, respectively). Neither anti-ICAM-1 or anti-LFA-1 therapy alone induced permanent allograft survival despite modest prolongation of survival. Treatment of genetically ICAM deficient B6 animals with anti-CD45RB also failed to induce tolerance (MST = 23.66 ± 3.57 days). Conclusions: These data suggest the involvement of ICAM-1/LFA-1 interactions during tolerance induction mediated by anti-CD45RB. Whether this effect is mediated by inter- or intracellular signaling through these molecules or by alterations of lymphocyte trafficking patterns is under investigation.

BLOCKING OF 4-1BBL PROMOTES LONG-TERM SURVIVAL IN SKIN ALLOGRAFT TREATED WITH ANTI-CD45RB AND ANTI-CD40L MONOCLONAL ANTIBODIES.

P943 Aims: 4-1BB (CD137) is a T cell costimulatory molecule that promotes T cell activation. Using a skin transplantation model, we have observed that simultaneous administration of monoclonal antibodies (mAb) targeting CD45RB and CD40L prolonged skin allograft in costimulation blockade (CTLA4-Ig and anti-CD40L mAb)-resistant mice because of reducing CD8+ T cells and CD4+CD45RBhigh T cells. Anti-CD45RB mAb (45RB) block the activation of Th1 cells and generates regulatory T cells (Treg). In this study, we highlight the involvement of 4-1BB/4-1BBL in the development of T cell responses. Methods: C57BL/6 recipients of BALB/c skin grafts were treated with 45RB, anti-CD40L mAb (MR1) and antagonistic anti-4-1BBL mAb (4-1BBL) on days 0, 2, 4, 6 and 8 posttransplant as the following. Group 1: control (No treatment), Group 2: 45RB+MR1, Group 3: 45RB+MR1+4-1BBL, Group 4: CD4 depletion+45RB+MR1+4-1BBL, Group 5: CD8 depletion+45RB+MR1+4-1BBL. The surface molecules of T cells were observed by using flow cytometry and cytokines were measured by ELISA. Results: Additional 4-1BBL (Group 3) more prolonged skin graft survival than Group 2. Even though the percentage of splenocyte-derived CD8+ T cells was reduced similarly in both groups (Group 2 and 3), the Group 3 augmented the proportion of Treg cells such as CD4+CD45RBlow and CD4+CD25+ T cells in comparison to the Group 2. Interestingly, Group 4, but not Group 5, prevented prolongation in allograft survival with high proportion of CD8+ T cells and CD25 expression on activated CD8+ T cells. The Group 5 enjoyed long-term graft survival with higher level of IL-4 than other groups. Conclusions: Use of 4-1BBL might have additive effects on Treg cells which play a major role in the maintenance of tolerance. Even after triple immunosuppressive therapy in combination with the depletion of CD4+ T cells, we could not achieve prolonged graft survival. This rejection might result from absent Treg cells and require CD4-independent CD8+ T cells based on the observation of increasing proportion of CD8+ T cells in similar degree as the control group.

Anti-CD45RB treatment induces t cell regulation by increasing CD4+CD25+CD45RB low cells

<b>Introduction:</b> We have shown that 3 doses of anti-CD45RB mAb induce longterm graft survival and same donor strain tolerance in ∼50% of C57BL/6 recipients of Balb/c islets. Anti-CD45RB treatment promotes tolerance by inducing a shift in CD45 isoform expression from CD45RB^High to CD45RB^Low and up-regulating CTLA-4 expression. Here, we examine whether anti-CD45RB treatment induces expression of regulatory cells. <b>Methods:</b> Lymphocytes were isolated from Balb/c mice (n = 3) and phenotyped by immunostaining and flow cytometry analysis following anti-CD45RB mAb treatment (3 doses: 100 μg/iv/dose). <b>Results:</b> Flow cytometry analysis showed a ∼55% increase of CD25 expression on CD4+ lymphocytes after treatment with 3 doses of anti-CD45RB. In addition, the CD25+CD45RB^LowCD4+ subset showed a threefold increase after anti-CD45RB treatment. Control untreated allogenic lymphocytes from C57BL/6 mice (n = 2) were then used as stimulator cells in mixed lymphocyte reactions (MLR) to demonstrate whether anti-CD45RB treated lymphocytes (unseparated population) could regulate proliferation. Results show that anti-CD45RB treated T cells do not proliferate when compared to untreated lymphocytes. In addition, when anti-CD45RB treated lymphocytes were cocultured with increased amounts of untreated T cells (1:1, 1:2, and 1:4 ratios), proliferation was increased in a dose-dependent manner (∼50, ∼75, and ∼90%, respectively) when compared to untreated cells. <b>Conclusions:</b> A short course of treatment with anti-CD45RB mAb induces a significant increase (300%) of CD25+CD45RB^LowCD4+ cells. This T cell subset has been shown to have regulatory properties in both mice and humans. In addition, anti-CD45RB treated lymphocytes inhibit thymidine incorporation and proliferation of allogeneic lymphocytes. Finally, when cocultured with untreated T cells, anti-CD45RB treated lymphocytes regulate the proliferation of allogeneic responder lymphocytes in a dose-dependent manner.

CD45RB-targeting strategies for promoting long-term allograft survival and preventingchronic allograft vasculopathy.

CD45RB is a potent immunomodulatory target to achieve long-term allograft survival. We evaluated the in vivo effect of anti-CD45RB monoclonal antibody (mAb) treatment in combination with conventional immunosuppression or costimulatory blockade strategies as a therapeutic modality for future clinical application. A fully MHC-mismatched vascularized mouse cardiac allograft model was used to test the interactions between anti-CD45RB mAb and conventional immunosuppressive drugs or costimulatory blockade of the CD40/CD154 or B7/CD28 pathway. Chronic rejection was examined histologically for development of chronic allograft vasculopathy. Cyclosporine significantly abrogated the effect of anti-CD45RB therapy. In contrast, rapamycin acted synergistically with anti-CD45RB mAb in promoting long-term allograft survival. CD154 blockade further enhanced the tolerogenic efficacy of anti-CD45RB mAb. These synergistic effects of combination treatments also prevented the development of chronic allograft vasculopathy. CD45RB-targeting strategy in combination with the use of rapamycin or costimulatory blockade promotes allograft tolerance and prevents chronic rejection.