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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.     

Long-term survival of segmental pancreas isografts in NOD/Lt mice treated with anti-CD4 and anti-CD8 monoclonal antibodies

Spontaneously diabetic nonobese diabetic (NOD/Lt) mice were treated with anti-T-cell monoclonal antibodies (mAbs) at the time of grafting with vascularized segmental pancreas isografts. Recipients were either untreated or given anti-CD4 and/or anti-CD8 mAbs (0.5 mg/20-g mouse on each of 4 consecutive days), which reduced target cell levels to <5% of normal. Graft function was monitored by measuring blood glucose (BG) levels. Transplants were removed for histological examination when BG returned to >20 mmol/l for two consecutive readings. Isografts from 3- to 4-week-old prediabetic mice placed in untreated diabetic NOD mice ceased functioning in 9-13 days with a mean survival time (MST) +/- SD of 10 +/- 2. Treatment with anti-CD4 prolonged survival significantly (MST = 61 +/- 35 days, P < 0.05 compared with untreated control mice). Anti-CD8 treatment was less effective, but it still significantly improved graft survival (MST = 24 +/- 9 days, P < 0.05 compared with untreated control mice). Anti-CD8 plus anti-CD4 treatment was highly effective in inhibiting autoimmune destruction of the grafts (MST = 97 +/- 8 days). This clearly demonstrates that transient inactivation of most T-cells with anti-CD4 plus anti-CD8 mAbs effectively controls autoimmune disease in the isograft, despite recovery of CD4 and CD8 T-cells to normal levels. Although insulitis developed in the long-term grafts, insulitis scores did not increase between 33 and 100 days, and none of the mice progressed to IDDM in 100 days. Histology showed a predominantly peri-islet T-cell and macrophage infiltrate with ductal expression of the cytokines interleukin (IL)-4, IL-2, and interferon-gamma. There was little infiltrate or expression of cytokines within the islets. Thus, mAb treatment at the time of grafting allowed isograft survival and prevented progression from insulitis to beta-cell destruction.     

Induction of tolerance with nondepleting anti-CD4 monoclonal antibodies is associated with down-regulation of TH2 cytokines

BACKGROUND: Induction of tolerance with anti-CD4 has mainly focused on monoclonal antibodies (mAbs) that deplete CD4+ T cells. In this study, the mechanisms by which nondepleting anti-CD4 mAbs induce tolerance in the Dark Agouti to PVG rat heart graft model were examined. METHODS: Five anti-CD4 mAbs were tested. Immunohistology and cytokine mRNA profiles were analyzed within grafts. Effects of combining anti-CD4 therapy with alloantibody (alloAb), interleukin (IL)-4, and anti-IL-4 mAb were also examined. RESULTS: All mAbs tested induced indefinite graft survival (>150 days), with blocking of alloAb production. Exogenous alloAb did not restore rejection. Similar T cell receptor alphabeta+, CD8+, IL-2 receptor+ T cell, macrophage, and natural killer cell infiltration and comparable MHC II and intercellular adhesion molecule-1 levels were seen in rejecting and tolerant grafts. mRNA for IL-2, interferon-gamma, lymphotoxin, tumor necrosis factor-alpha, transforming growth factor-beta, cytolysin, and granzyme-A/B was comparable, although inducible nitric oxide synthase was slightly reduced in tolerant grafts. IL-4 and IL-5 were significantly reduced in tolerant grafts, although IL-6, IL-10, and IL-13 levels were similar; this was consistent with partial T helper (Th)2 response inhibition, which was also manifested by inhibited alloAb. The combination of alloAb, IL-4, or anti-IL-4 mAb with anti-CD4 did not prevent tolerance induction. CONCLUSIONS: This study demonstrated that anti-CD4 mAb therapy did not inhibit activation and infiltration of Th1 and CD8+ effector T cells. Preferential induction of Th2 responses, especially IL-4, was not essential for the induction of tolerance. Our studies also found no evidence to support induction of anergy or transforming growth factor-beta as mechanisms of tolerance induction. These results question whether IL-4 is required for induction of transplantation tolerance.     

Treatment of a newly established transgenic model of chronic arthritis with nondepleting anti-CD4 monoclonal antibody

We established a novel animal model for rheumatoid arthritis (RA) by following backcrossing to DBA/1 of (SWR/J x DBA/1)F1 TCR-beta Tg mice, previously reported to be highly susceptible to collagen-induced arthritis. These mice evolved, upon collagen type II immunization, into a chronic arthritis that histopathologically resembles RA. The availability of such a model prompted us to study the role of CD4+ T cells throughout the evolution of disease. Here, we show that administration of nondepleting anti-CD4 not only prevented the evolution of disease but also treated established arthritis. Moreover, functional analyses of T cells isolated from anti-CD4-treated mice demonstrated that the mechanism of protection is not achieved by suppression of the Th1 population but is mediated by induction of collagen type II-specific T cell anergy. Our study suggests that: 1) CD4+ T cells have a fundamental role both in the induction and in the perpetuation of disease; 2) targeting T cells may be an appropriate therapeutic option; and 3) a suitable and well-balanced anti-CD4 treatment may be a valid approach to the control of RA.     

The development of anti-CD79 monoclonal antibodies for treatment of B-cell neoplastic disease

The B-cell antigen receptor (BCR) consists of cell surface IgM associated with the CD79 alpha/beta heterodimer. In this paper we describe a panel of monoclonal antibodies (mAbs) recognising the extracellular regions of human CD79 alpha and beta. FACS analysis demonstrated that the mAbs bind to a range of Burkitt's lymphoma lines, a mouse B-cell line (JO-72) transfected with human CD79 alpha and beta, and tumour biopsies from NHL patients. The specificity of the mAbs was confirmed by immunoprecipitation. The Ka for the binding of IgG from the anti-CD79 alpha mAbs to cell surface CD79 alpha on Ramos cells was 3 x 10(8) M-1, and their maximum level of binding, 1.7-2 x 10(5) molecules/cell, matched that obtained with anti-Fc mu and anti-Fd mu mAbs. All four anti-CD79 beta mAbs were of lower affinity. Interestingly, in growth arrest studies, we found that while all anti-Fc mu mAbs caused profound inhibition of proliferation of Ramos cells, a range of other anti-BCR mAbs, which included the anti-CD79, anti-Fab mu, anti-gamma and anti-idiotype reagents, all performed poorly giving a maximum of 25% inhibition. These differences in performance are believed to relate to the ability of anti-BCR mAbs to cross-link neighbouring surface BCR and suggest that, unlike anti-Fc mu which favours cross-linking, most of these mAbs are binding in a monogamous, non-cross-linking, union with the BCR.     

Effects of administration of monoclonal antibodies (anti-CD4 or anti-CD8) on the development of autoimmune diseases in (NZW x BXSB)F1 mice

(NZW x BXSB)F1 (W/BF1) mice spontaneously develop autoimmune diseases, characterized by lymphadenopathy, lupus nephritis, and immune thrombocytopenia associated with various autoantibodies such as anti-DNA, anti-platelet and anti-cardiolipin antibodies (Abs). In the present study, we investigate the effects of administration of monoclonal Abs (anti-CD4 or anti-CD8 mAb) on the development of autoimmune diseases in W/BF1 mice. MAb was administered from the age of 7 weeks. Prolongation of survival rate and reduction of severity of autoimmune diseases were observed after treatment with anti-CD4 mAb. However, anti-CD8 mAb treatment accelerated the diseases. Serum levels of IFN-gamma and IL-10 in old W/BF1 mice were significantly high, whereas IL-4 levels were low in comparison with those of young W/BF1 mice; the expression of mRNA of IFN-gamma, IL-4 or IL-10 in CD4+ T cells of old W/BF1 mice was parallel to the serum levels of each cytokine. These observations suggest that CD4+ cells are involved in the development of autoimmune diseases in W/BF1 mice, and that CD8+ cells have a suppressive effect on the development of autoimmune diseases in W/BF1 mice.     

Perspectives of application of anti-CD4 antibodies in the treatment of autoimmune diseases

Escherichia coli JM83 [F- ara delta(lac-proAB) rpsL [phi 80d delta (lacZ)M15]] in midlog growth phase at 30 degrees C were exposed to 60 Hz sinusoidal magnetic field of 3 mT of nonuniform diverging flux, inducing a nonuniform electric field with a maximum intensity of 32 microV/cm using an inductor coil. Exposed and unexposed control cells were maintained at 30.8 +/- 0.1 degrees C and 30.5 +/- 0.1 degrees C, respectively. Quadruplicate samples of exposed and unexposed E. coli cells were simultaneously radiolabeled with 35S-L-methionine at 10 min intervals over 2 hr. Radiochemical incorporation into proteins was analyzed via liquid scintillation counting and by denaturing 12.5% polyacrylamide gel electrophoresis. The results showed that E. coli exposed to a 60 Hz magnetic field of 3 mT exhibited no qualitative or quantitative changes in protein synthesis compared to unexposed cells. Thus small prokaryotic cells (less than 2 microns x 0.5 micron) under constant-temperature conditions do not alter their protein synthesis following exposure to 60 Hz magnetic fields at levels at 3 mT.     

Biohybrid artificial pancreas. Long-term function of discordant islet xenografts in streptozotocin diabetic rats

DNA sequences are presented for two members of the wheat Em gene family. The sequences correspond to the two linked genes at the Xem-1AL locus. Comparisons of these sequences with that of another wheat Em gene and two Em cDNA clones reveals substantial homology within the protein-coding regions, and the presence in the 5'-flanking regions of the genomic sequences of motifs characteristic of ABA-responsive cis-acting elements.     

Long-term survival of skin allografts induced by donor splenocytes and anti-CD154 antibody in thymectomized mice requires CD4(+) T cells, interferon-gamma, and CTLA4

Treatment of C57BL/6 mice with one transfusion of BALB/c spleen cells and anti-CD154 (anti-CD40-ligand) antibody permits BALB/c islet grafts to survive indefinitely and BALB/c skin grafts to survive for approximately 50 d without further intervention. The protocol induces long-term allograft survival, but the mechanism is unknown. We now report: (a) addition of thymectomy to the protocol permitted skin allografts to survive for > 100 d, suggesting that graft rejection in euthymic mice results from thymic export of alloreactive T cells. (b) Clonal deletion is not the mechanism of underlying long-term graft survival, as recipient thymectomized mice were immunocompetent and harbor alloreactive T cells. (c) Induction of skin allograft acceptance initially depended on the presence of IFN-gamma, CTLA4, and CD4(+) T cells. Addition of anti-CTLA4 or anti-IFN-gamma mAb to the protocol was associated with prompt graft rejection, whereas anti-IL-4 mAb had no effect. The role of IFN-gamma was confirmed using knockout mice. (d) Graft survival was associated with the absence of IFN-gamma in the graft. (e) Long-term graft maintenance required the continued presence of CD4(+) T cells. The results suggest that, with modification, our short-term protocol may yield a procedure for the induction of long-term graft survival without prolonged immunosuppression.     

Prolongation of porcine islet xenograft survival in mice after therapy with immunosuppressive peptides

BACKGROUND: Recently, peptides derived from the heavy chain of HLA-B2702 have been shown to modulate immune responses. In this study, we examined the use of these peptides for immunosuppression in a pig to mouse islet xenograft model. METHODS: Purified porcine islets were transplanted in autoimmune (non-obese diabetic) and non-autoimmune (streptozotocin-injected CBA or C57/Bl6) diabetic mice. Various dosing regimens of HLA-derived peptides with and without antilymphocyte therapy were administered to recipient mice. Graft rejection was determined by daily serum glucose determinations, and, at selected time points, grafts were removed to demonstrate function and provide immunohistochemical examination. RESULTS: HLA-derived peptides were demonstrated to prolong graft survival in both pretransplant and posttransplant treatment regimens. This effect was increased with concomitant antilymphocyte therapy. CONCLUSIONS: Further elucidation of the mechanism of action of these immunomodulatory peptides may help in the development of novel immunosuppressive protocols.     

Gallium-68 chelate imaging of human colon carcinoma xenografts pretargeted with bispecific anti-CD44V6/anti-gallium chelate antibodies

Recently, we demonstrated the feasibility of combining improved tumor-to-tissue contrasts and PET imaging for immunoscintigraphic tumor localization using a multistep targeting technique that consists of the administration of an antitumor/antihapten bispecific monoclonal antibody (BS-MAb), a blocker to saturate the antihapten binding sites of the BS-MAb that are still present in the circulation, and a low molecular weight Ga chelate, labeled with positron emitter 68Ga, serving as the hapten. Due to this technique, the biodistribution of the radiolabeled hapten is governed mainly by the binding characteristics of both the antitumor and the antihapten part of the BS-MAb. For a future clinical implementation of the method, we investigated MAb VFF18, which is reactive with the adhesion molecule CD44V6, a tumor-associated antigen, and up-regulated in colon, squamous cell and pancreas carcinoma, and two anti-Ga chelate MAbs, which are highly selective for only one of the two enantiomers (optical isomers) of the inherently racemic Ga chelate. METHODS: From the VFF18 MAb and the anti-Ga chelate MAbs, two BS-MAbs containing the same antitumor parts, but different antihapten parts, were prepared and tested for multistep targeting in human colon carcinoma-bearing nude mice. RESULTS: Despite identical biodistributions of both BS-MAbs and their very similar affinities for the corresponding Ga chelate enantiomers, tumor uptake of the two enantiomers 1 hr postinjection was significantly different [8.7 +/- 1.9% versus 5.8% +/- 1.6% of the injected dose/g (%i.d./g)], with tumor-to-blood ratios being higher for the BS-MAb showing the lower tumor uptake (7.6 +/- 1.6 versus 4.7 +/- 0.6). From data obtained with each BS-MAb, a similar initial tumor binding of approximately 15.5%i.d./g, but different in vivo half-lives of the corresponding BS-MAb-enantiomer immune complexes, could be estimated. Pretargeting with a mixture of both BS-MAbs followed by the administration of the racemic Ga chelate resulted in the lowest tumor uptake (3.9% +/- 1.5%i.d./g). PET imaging of nude mice with the enantiomeric, as well as with the racemic, 68Ga chelate demonstrated a clear delineation of tumors against blood pool background. CONCLUSION: Multistep immunoscintigraphy with BS-MAbs markedly increases tumor-to-tissue ratios in nude mice and enables PET imaging. Using a BS-MAb containing MAb VFF18, a more sensitive localization of CD44V6-positive tumors in patients should also be obtained.     

Anti-CD45 and anti-CD52 (Campath) monoclonal antibodies effectively eliminate systematically disseminated human non-Hodgkin's lymphoma B cells in Scid mice

Severe combined immunodeficient (Scid) mice inoculated with the human (t(14;18)-positive B cell lines DoHH2 and BEVA develop lethal systemically disseminated lymphoma (de Kroon et al., Leukemia 8:1385, and Blood 80 [suppl 1]:436). These models were used to study the therapeutic effect of rat-anti-human CD52 (Campath-1G) or CD45 monoclonal antibodies (mAbs) on systemically disseminated tumor cells and on tumor cells present in solid tumor masses. Both mAbs were effective in inhibiting growth of systemically disseminated malignant cells. When treatment with anti-CD52 or anti-CD45 mAbs at a dose of 30 micrograms/mouse/d for 4 days was started 24 hours after intravenous inoculation of human DoHH2 or BEVA cells, a 3-log kill of tumor cells was observed as measured by prolonged survival. After treatment, surviving animals injected with high numbers of BEVA cells showed tumor masses in liver, kidney, and mesenteric lymph nodes. In contrast to nontreated animals, however, only low numbers of malignant cells were found in peripheral blood, and bone marrow was free of tumor cells. Similarly, after mAb treatment of mice inoculated subcutaneously (sc) with DoHH2 cells, no tumor cells could be found in the bone marrow, and few DoHH2 cells could be detected in the peripheral blood, spleen, liver, kidney, or lung. In contrast, tumor cells present in subcutaneous tumors and axillary lymph nodes were relatively unaffected by mAb therapy. The presence of rat immunoglobulin (Ig) could be demonstrated on surviving tumor cells. The presence of murine macrophages in areas in these tumors that were depleted of DoHH2 cells suggested that the mAb-mediated antitumor effect observed in the Scid mouse model is mediated by cellular mechanisms. Apparently these mechanisms were not sufficient to eliminate the fast-growing tumor cells present in the protected sites. Our results indicate that treatment with anti-CD52 or anti-CD45 mAbs potentially may be useful as adjuvant immunotherapy for systemically disseminated B cell lymphoma.     

Prolongation of survival of skin allograft with anti-Thy-1 monoclonal antibody in mice

Skin allograft rejection is mainly mediated by T lymphocytes. We investigated the cytotoxicity of anti-Thy-1 McAb to murine thymocytes in vitro, and evaluated the prolonging effect on the survival of murine skin allografts (BALB/c--C57BL/6) in vivo with the McAb. The results showed murine thymocytes were destroyed in vitro by the McAb with complement; the survival of skin allograft was prolonged in vivo with the McAb. Lymphocyte infiltration in skin allografts was inhibited. These results provide a valuable reference for the clinical usefulness of anti-CD3 McAb in prolonging survival of human skin allografts in burn patients.     

Successful induction of adjuvant arthritis in mice by treatment with a monoclonal antibody against IL-4

Adjuvant arthritis (AA) is an experimental model of autoimmune disease in rats induced by immunization with Mycobacterium tuberculosis (MT). Induction of AA in other species, including mice, has been shown to be difficult. In the present study, we found that AA could be induced in mice if the animals were treated with a mAb (11B11 mAb) against IL-4. Histologically, the joints exhibited synovial edema with infiltration of many neutrophils in the early phase of inflammation. In its late phase, there were proliferation of synovium, cell infiltrate in which mononuclear cells predominated, and destruction of cartilage and subchondral bone. The joint inflammation was passively transferred to normal syngeneic recipient mice with lymphoid cells but not with sera from mice immunized with MT followed by treatment with the anti-IL-4 Ab. Delayed-type hypersensitivity (DTH) and proliferative responses of lymphoid cells to purified protein derivative were markedly augmented in 11B11 mAb-treated mice. Furthermore, the induction of arthritis was associated with a marked decrease in IL-4 secretion but a significant increase in IFN-gamma and IL-2 production. Thus, the neutralization of IL-4 by an anti-IL-4 Ab appears to be required for the induction of AA in mice.     

Soluble tumour necrosis factor receptor release after anti-CD3 monoclonal antibody treatment in mice is independent of tumour necrosis factor-alpha release

The involvement of TNF-alpha in the release of soluble TNF receptors was assessed in mice, treated with anti-CD3 monoclonal antibodies. After treatment with three different anti-CD3 mAb, we simultaneously studied serum levels of TNF-alpha, soluble TNF receptor P55 and P75. All three anti-CD3 mAb triggered the release of both of the soluble TNF receptors, whereas only one of the anti-CD3 mAb triggered TNF-alpha release. These data demonstrate that in our model soluble TNF receptor release is independent of TNF-alpha release.     

Antibody response to islet antigens in anti-CD4/prednisolone immune intervention of type 1 diabetes

The O intermediate of the photocycle of bacteriorhodopsin (BR) was studied by absorption kinetic measurements at different actinic light densities. With increasing exciting flash intensity, the relative yield of O slightly increases, while that of Mf strongly decreases at the expense of Ms. Kinetic calculations and the optical anisotropy of O show that O can be formed only from Mf although Mf and O have different light intensity dependences. In order to resolve the apparent contradiction, a phenomenologically new cooperative regulatory mechanism seems to be necessary.     

Clinical and immunological effects of treatment with murine anti-CD3 monoclonal antibody along with interleukin 2 in patients with cancer

Anti-CD3 mAb and interleukin 2 (IL-2) were used in a Phase I study to treat 29 patients with cancer. The anti-CD3 was given as an i.v. bolus infusion over 10 min followed by two i.v. 96-h continuous infusions of IL-2 at 3 x 10(6) units/m2/day with a 3-day rest between the IL-2 infusions. Four patients were treated with 6, 18, 60, and 300 microgram/m2 anti-CD3. One patient received 3000 microgram/m2 anti-CD3. This patient developed profound hypotension and the IL-2 infusions were delayed for 2 weeks. Two patients were treated at an intermediate dose of 600 microgram/m2. These patients developed dose-limiting toxicities including hypotension, dyspnea and increased blood urea nitrogen, creatinine, and bilirubin. They were unable to complete their first course of therapy. In an effort to achieve a dose of anti-CD3 which would activate T cells in vivo, pentoxifylline was given to blunt the toxicities seen with anti-CD3 thought to be due predominantly to the cytokine syndrome and tumor necrosis factor release. Four patients received p.o. pentoxifylline to cover an anti-CD3 dose of 600 microgram/m2. The IL-2 infusion was initiated 1 week after the mAb. While there was an anti-CD3 dose-dependent increase in serum tumor necrosis factor level 1 h after mAb infusion, pentoxifylline did not reduce the serum tumor necrosis factor level. There was also an anti-CD3 dose-dependent increase in the serum soluble IL-2 receptor levels. Other immune parameters monitored, including in vitro cytotoxic and proliferative responses and lymphocyte count, were similar to treatment courses with IL-2 alone. Fourteen of 26 patients examined developed human anti-murine antibodies following a single dose of anti-CD3. There were no objective antitumor responses. We conclude that in vivo treatment with anti-CD3 did not enhance T cell activity or expansion with subsequent IL-2 infusion and that the combination of anti-CD3 followed by IL-2 did not improve upon the antitumor activity previously seen with IL-2 alone.     

Comparison of workshop CD45R monoclonal antibodies with OvCD45R monoclonal antibodies in sheep

The reactivity of the antibovine monoclonal antibodies (mAbs) comprising temporary cluster TC1 was compared with that of two OvCD45R mAbs on sheep cells. Three of the mAbs--CC31, CC99 and CC103--did not cross-react with sheep cells. All the workshop mAbs precipitated two molecules of apparent molecular weight (MW) 200 kDa and 220 kDa while the antisheep CD45R mAb 20-96 precipitated a single band of 220 kDa. Cell surface expression was examined by single colour FACS (fluorescence activated cell sorting) analysis of efferent and afferent lymph cells and peripheral blood lymphocytes and the distribution of the antigens on CD4+, CD8+ and T19+ (WC1) and B cells was determined by two colour fluorescence staining. By cellular distribution and immunohistology the TC1 mAbs could be divided into four distinct groups which differed from a fifth group comprising the two OvCD45R antibodies.