anti-CD45RB mAb Research Articles

Synergistic tolerance induced by LF15-0195 and anti-CD45RB monoclonal antibody through suppressive dendritic cells.

LF 15-0195 (LF), a novel analogue of 15-deoxyspergualin (DSG), inhibits maturation of dendritic cells (DC). Anti-CD45RB is a monoclonal antibody (mAb) that blocks activation of T-helper (Th) 1 cells and generates T-regulatory cells. This study addressed whether these two reagents act synergistically to inducing tolerance, and investigated associated cellular mechanisms. BALB/c recipients were treated by a short course of mAb alone, LF alone, or the combination of both agents. Mice that accepted a C57BL/6 cardiac allograft for more than 100 days were considered tolerant. Splenic DC were purified using positive selection for CD11c. Bone marrow DC were generated by culture with interleukin-4 and granulocyte-macrophage colony-stimulating factor. Surface marker expression was determined by fluorescence-activated cell sorter analysis. DC function was assessed by the ability to stimulate or inhibit T cells in vitro. Although monotherapy with LF or mAb failed to induce tolerance, combination therapy resulted in long-lasting acceptance of allogeneic hearts (>200 days) and secondary donor skin grafts (>100 days). DC from tolerant recipients possessed lower major histocompatibility complex class II and CD40 expression, and were poorer co-stimulators for T-cell proliferation than control DC. Furthermore, DC from tolerant mice induced Th2 differentiation, suppressed overall T-cell proliferation, and were poor presenters of T cells specific for antigen to pigeon cytochrome c 81-104. The combination of LF and anti-CD45RB mAb induced stable tolerance. The synergy of these two approaches appears to be mediated through formation of tolerogenic DC and T-regulatory cells.

Inhibitory feedback loop between tolerogenic dendritic cells and regulatory T cells in transplant tolerance.

An active role of T regulatory cells (Treg) and tolerogenic dendritic cells (Tol-DC) is believed important for the induction and maintenance of transplantation tolerance. However, interactions between these cells remain unclear. We induced donor-specific tolerance in a fully MHC-mismatched murine model of cardiac transplantation by simultaneously targeting T cell and DC function using anti-CD45RB mAb and LF 15-0195, a novel analog of the antirejection drug 15-deoxyspergualin, respectively. Increases in splenic Treg and Tol-DC were observed in tolerant recipients as assessed by an increase in CD4(+)CD25(+) T cells and DC with immature phenotype. Both these cell types exerted suppressive effects in MLR. Tol-DC purified from tolerant recipients incubated with naive T cells induced the generation/expansion of CD4(+)CD25(+) Treg. Furthermore, incubation of Treg isolated from tolerant recipients with DC progenitors resulted in the generation of DC with Tol-DC phenotype. Treg and Tol-DC generated in vitro were functional based on their suppressive activity in vitro. These results are consistent with the notion that tolerance induction is associated with a self-maintaining regulatory loop in which Tol-DC induce the generation of Treg from naive T cells and Treg programs the generation of Tol-DC from DC progenitors.

INDUCTION OF TOLERANCE BY SIGNAL ONE BLOCKADE WITH ANTI-CD45RB REQUIRES NEGATIVE SIGNALING THROUGH CTLA-4.

450 We have shown that blockade of Signal One with 3 doses of anti-CD45RB induces tolerance in 50-60% of murine islet allograft recipients (PNAS. 1998. 95:3821). Engagement of CTLA-4 is required for induction of tolerance to nominal antigen (Immunity. 1997. 6:411). Here, we evaluate the role of negative signaling through CTLA-4 in tolerance induction by anti-CD45RB in a murine islet allograft model through use of a blocking anti-CTLA-4 mAb. Chemically diabetic C57BL/6 mice were transplanted with 350-400 BALB/c islets under the left kidney capsule. Recipients were untreated, or treated with: anti-CD45RB (MB23G2), 100 ug IV on days −1,0, and 5; anti-CTLA-4 (UC10-4F10), 200 ug IP days −1, 0, 1; or the combination of anti-CTLA-4 plus anti-CD45RB. (Table)TableTreatment with anti-CTLA-4 alone had no effect on graft survival compared to untreated controls. Preliminary data indicate that anti-CTLA-4 does not affect CD45 isoform expression. In contrast, anti-CD45RB treatment induces a shift from high to low Mr (CD45RBLo) CD45 isoforms. Importantly, CD45RBLo T cells have been reported to exhibit increased CTLA-4 expression (J. Immunol. 1998. 161:5855). We hypothesize that the shift towards CD45RBLo isoforms induced by anti-CD45RB mAb augments CTLA-4 expression and that this contributes towards the generation of tolerance. Concordantly, blockade of CTLA-4-mediated negative signals with anti-CTLA-4 abrogates tolerance induction by anti-CD45RB. Understanding the role and mechanisms of negative signaling pathways in induction and maintenance of tolerance is critical for development of novel tolerance strategies in solid organ and cell transplantation.

DISCORDANT ISLET XENOTRANSPLANTATION; LONG-TERM SURVIVAL FOLLOWING SHORT-TERM TREATMENT WITH ANTI-CD45RB AND CYCLOPHOSPHAMIDE

125 We have recently reported that a short treatment course of anti-CD45RB mAb (MB23G2) is able to induce tolerance to islet allografts in mice. (Auersvald et al, Transplantation 63: 1355 ). Furthermore, when combined with cyclophosphamide (CTX), anti-CD45RB was able to prolong survival of concordant islet xenografts. In this study, we examine the immunosuppressive effect of anti-CD45RB mAb and CTX in a discordant model of porcine islet transplantation into mice. C57BL/6 (H-2b) streptozotocin-induced diabetic mice were used as recipients. Adult porcine islets (3500 islet equivalents) were transplanted under the left kidney capsule. Recipient mice were either left untreated (n=2) or received a combination of anti-CD45RB mAb (100 mg IV on days -1 to 10, 20, 21) plus CTX (20 mg/kg IV on days -1, 1, 0, 3, 5, 19, 20) (n=11). Results: Untreated mice show rapid onset of hyperglycemia (Median day 4). Treated mice had a median graft survival of 37 days. Significantly, in 4/11 mice (36%), indefinite graft survival (>120 days) was achieved. After nephrectomy, long-term survivors reverted to hyperglycemia. Analysis of splenocytes on day 6 post-transplant, reveals that CTX causes a decrease in B cells and a relative increase in CD4 and CD8 cells. Anti-CD45RB induced a marked decrease in the intensity of CD45RB expression after transplantation. This suggests that this mAb induces a shift from CD45RBHi to CD45RBLo isoforms in xenograft recipients, as previously demonstrated in untransplanted animals. Histologic examination of long-term surviving grafts shows very well preserved islet tissue surrounded by a lymphocytic infiltrate within the renal capsule and in the renal parenchyma immediately underlying the xenograft. The infiltrating lymphocytes stained strongly positive for IL-4 and to a much lesser extent, IL-2, by immunohistochemistry. Within in the islets themselves, there were only rare lymphocytes, staining positive for IL-4, but not IL-2. In conclusion, this study shows that a short course of anti-CD45RB combined with CTX can induce significant prolongation of discordant islet xenografts with indefinite survival in over one third of the recipients.

Antibody-mediated targeting of CD45 isoforms: a novel immunotherapeutic strategy.

CD45 is a family of transmembrane protein tyrosine phosphatases exclusively expressed by hematopoietic cells and critically involved in the regulation of T cell activation signals. We now demonstrate that three 100-microg doses of anti-CD45RB mAb MB23G2 can induce long-term engraftment of islets into major histcompatibility complex-disparate chemically diabetic mice. Long-term graft survivors (>120 days) were tolerant to new islet allografts from the original donor strain. MB23G2 induced a temporary decrease in number circulating leukocytes but had no effect on leukocyte number in other lymphoid compartments. Histologic examination of allografts from treated and untreated recipients revealed a similar peri-islet infiltration on day 6. Eleven days after transplant, the peri-islet infiltrate in treated animals persisted, but in marked contrast to untreated control animals, there was no insulitis and islet integrity was preserved. The peri-islet infiltrate from treated animals showed a mild increase in CD4 cells, a decrease in CD8 cells, and decreased intensity of CD45RB expression. Treatment of naive animals with anti-CD45RB (MB23G2) resulted in a shift in CD45 isoform expression on T cells with a loss of higher molecular weight isoforms and increased expression of lower molecular weight (CD45R0) isoform. This shift in CD45 isoform expression from CD45RBHi to CD45RBLo was associated with an increase in the intragraft expression of transcripts for interleukin (IL) 4 and IL-10, consistent with the expected activity of this distinct immunoregulatory T cell subset. Antibody-mediated targeting of CD45 may induce tolerance through novel mechanisms and have direct applicability to clinical transplantation in humans.

Indefinite islet allograft survival in mice after a short course of treatment with anti-CD45 monoclonal antibodies.

Although islet cell transplantation is considered an ideal form of endocrine replacement for type I diabetes, clinical application in humans is still not feasible. New immunosuppressive strategies are clearly needed to control inexorable rejection. CD45 is a family of transmembrane protein tyrosine phosphatases critically involved in the regulation of lymphocyte activation signals. Anti-CD45RB monoclonal antibody can prevent rejection of murine renal allografts. Here, we examine the consequences of targeting CD45 in murine islet cell transplantation. Diabetic mice recipients received islet allografts under the kidney capsule and were divided into seven groups. Recipients received no treatment (controls) or anti-CD45RB monoclonal antibody (mAb; MB23G2 or C363.16A) at different dosages and treatment intervals. All untreated control animals lost islet function, becoming hyperglycemic within 10-17 days after transplantation. Animals treated with either anti-CD45RB mAb showed a significant prolongation of islet allograft survival when compared with controls. Anti-CD45RB MB23G2 at 100 microg/day, given on days -1, 0, and 5 was particularly effective, inducing indefinite islet allograft survival in 60% of recipients. These results indicate that anti-CD45 mAbs are potent immunomodulatory agents, able to sustain indefinite islet allograft function after a short treatment course in the highly immunogenic model of islet transplantation.

Microbial induction of co-stimulatory activity for CD4 T-cell growth

The activation of naive CD4 T cells by antigen is a critical step in the initiation of an immune response; it requires both ligation of the T-cell receptor (TCR) and the delivery of co-stimulatory factors by accessory cells. We have examined the role of syngeneic accessory cells in the response of purified normal CD4 T cells to anti-CD3 antibody as ligand. We show that the ability to deliver co-stimulatory signals is inducible in B cells by microbial products such as bacterial lipopolysaccharide (LPS), mitogenic influenza viruses, and synthetic polyinosinic-polycytidylic acid (poly-I:C) as a mimic of viral infection. LPS stimulation for 16 h allows the co-stimulatory activity of B cells to become resistant to paraformaldehyde fixation. LPS induction of fixation-resistant co-stimulator activity requires new protein synthesis, as it is inhibited by cycloheximide. Using the anti-CD45RB mAb 16A as marker for naive and memory CD4 T cells, we show that B cells activated by LPS and by poly-I:C can provide co-stimulatory signal to both naive and memory CD4 T cells. By contrast, zymosan particles, which are known to activate macrophages in a variety of assays, do not activate B cells to become co-stimulatory, but do induce this activity in macrophages. These data demonstrate that a variety of infectious agents or their constituents can induce accessory cells to become co-stimulatory for CD4 T cells. They are interpreted in light of a proposed role for two classes of recognition in the induction of the immune responses, specific recognition of antigens and non-specific recognition of infectious agents. These data support the contention that the immune system uses this mechanism to discriminate infectious non-self from non-infectious self.

Glycosylation of CD45: carbohydrate composition and its role in acquisition of CD45RO and CD45RB T cell maturation‐related antigen specificities during biosynthesis

The contribution of oligosaccharides to the biochemical composition and antigen heterogeneity of the phosphotyrosine phosphatase CD45 glycoproteins has been studied on the K-562 erythroleukemic cell line. Treatment of immunoprecipitated CD45 glycoproteins with distinct exo- and endoglycosidases revealed the presence of highly sialylated O- and N-linked complex carbohydrates in the composition of mature CD45 glycoproteins. Incubation of K-562 cells with the N-glycosylation inhibitor tunicamycin blocked carbohydrate processing during biosynthesis of CD45 proteins, generating unglycosylated polypeptides similar in size to those resulting from digestion of CD45 proteins with a mixture of both N- and O-glycanases. Epitopes defining the T cell maturation related CD45R0 and CD45RB antigen specificities were present on the mature 180- and 190-kDa K-562 CD45 proteins, respectively. However, the CD45R0 and CD45RB epitopes were not detected on the high mannose biosynthetic CD45 precursors. Furthermore, treatment of CD45 proteins with O-glycanase or neuraminidase resulted in the loss of both CD45R0 and CD45RB epitopes, although reactivity of the anti-CD45R0 and anti-CD45RB mAb was not affected by mAb preincubation with either sialic acids or sialyllactose in solution. From these results we conclude that the blockade of early steps of N-glycosylation during carbohydrate processing resulted in the inhibition of subsequent incorporation of O-linked sugars on CD45 polypeptides, thus preventing the late acquisition of the CD45R0 and CD45RB determinants on the 180- and 190-kDa CD45 polypeptides.