mAb Pretreatment Research Articles

Dog leukocyte antigen-haploidentical stem cell allografts after anti-CD44 therapy and reduced-intensity conditioning in a preclinical canine model.

We previously described a nonmyeloablative hematopoietic stem cell transplantation regimen in dog leukocyte antigen (DLA)-identical littermate recipients consisting of low-dose total body irradiation (TBI) before and mycophenolate mofetil (MMF)/cyclosporine (CSP) given after transplant to control both graft-vs-host and residual host-vs-graft reactions. In this study, we sought to develop a reduced-intensity regimen to achieve engraftment across major histocompatibility complex barriers in DLA-haploidentical littermate recipients. We tested a regimen of 450-cGy TBI with or without postgrafting MMF/CSP for 28 and 35 days, respectively, and with the administration of monoclonal antibody (mAb) S5 (anti-CD44), at a dose of 0.2 mg/kg/day from days -7 through -2, prior to receiving TBI. One of six dogs conditioned with 450-cGy TBI alone achieved engraftment of granulocyte colony-stimulating factor-mobilized peripheral blood stem cells. Three of six dogs achieved sustained donor cell engraftment using 450-cGy TBI and posttransplantation MMF/CSP. None of three dogs given mAb S5 followed by 450-cGy TBI showed signs of donor cell engraftment. However, when S5 mAb pretreatment was added to 450-cGy TBI and postgrafting MMF/CSP, 10 of 12 dogs achieved sustained engraftment (p = 0.008 or 0.007 vs 450-cGy alone or to S5 + 450-cGy TBI without MMF/CSP, respectively), with only three dogs developing severe graft-vs-host disease on this short regimen of immunosuppression. These results show that engraftment across a DLA haplotype-mismatched barrier can be achieved after reduced-intensity conditioning when mAb S5 directed at CD44 is added to this regimen.

Perturbation of naive TCR transgenic T cell functional responses and upstream activation events by anti-CD4 monoclonal antibodies

It is well established that non-depleting anti-CD4 monoclonal antibodies (mAb) have potent immunomodulatory properties in vivo and as such can induce a profound state of tolerance. Receptor blockade, CD4 modulation, or the transmission of a negative signal have all been proposed to explain their effects, however their precise mechanism of action, particularly at the level of cellular signaling, remains obscure. Experiments were thus carried out to examine the underlying mechanisms of action of two non-depleting anti-mouse CD4 mAb, YTS177 and KT6, which differ in their ability to modulate CD4 expression. The effects of the mAb were examined on CD4(+) T cells derived from D0.11.10 TCR transgenic mice. Functional studies revealed that both mAb could effectively block antigen-driven proliferation and IL-2 production but had only modest effects at higher peptide doses. Importantly, mAb pre-treatment of T cells stimulated by sub-optimal peptide seemed to induce an anergy-like state making them unresponsive to subsequent re-stimulation. Analysis of intracellular signaling demonstrated that certain key upstream events such as the phosphorylation of Zap-70 and LAT were also blocked by mAb pretreatment which may be due to interference with stable T cell-APC conjugate formation.

Acute Hepatotoxicity ofPseudomonas aeruginosaExotoxin A in Mice Depends on T Cells and TNF

AbstractThe most potent virulence factor of Pseudomonas aeruginosa, its exotoxin A (PEA), inhibits protein synthesis, especially in the liver, and is a weak T cell mitogen. This study was performed to correlate hepatotoxic and possible immunostimulatory features of PEA in vivo. Injection of PEA to mice caused hepatocyte apoptosis, an increase in plasma transaminase activities, and the release of TNF, IFN-γ, IL-2, and IL-6 into the circulation. Most strikingly, liver damage depended on T cells. Athymic nude mice or mice depleted of T cells by anti-Thy1.2 mAb pretreatment failed to develop acute hepatic failure, and survival was significantly prolonged following T cell depletion. Neutralization of TNF or lack of TNF receptors prevented liver injury. In the liver, TNF was produced by Kupffer cells before hepatocellular death occurred. After T cell depletion, Kupffer cells failed to produce TNF. Transaminase release was significantly reduced in perforin knockout mice, and it was even elevated in lpr/lpr mice. These results demonstrate that PEA induces liver damage not only by protein synthesis inhibition but also by TNF- and perforin-dependent, Fas-independent, apoptotic signals.

PRETREATMENT OF ISLETS WITH MITOMYCIN C (MMC) AND BLOCKAGE OF DIRECT ANTIGEN PRESENTATION INDUCE UNRESPONSIVENESS TO ISLET XENOGRAFT

124 Introduction: We have previously shown that rat ICAM-1 on APCs can interact with mouse LFA-1 on T cells across species barrier and blockage of this pathway using species specific mAbs produces prolongation of islet xenograft survival. We have also demonstrated that infusion of donor splenocytes pretreated with MMC, induced unresponsiveness to rat cardiac allografts. In this study we try to minimize immunosuppression and induce unresponsiveness to xenograft using blockage of costimulatory pathway with anti-donor ICAM-1 mAb and graft pretreatment with MMC. Materials and Methods: Collagenase-digested WS (RT1k) rat islets, purified by Ficoll density gradient, were incubated for 30 mins with MMC at a dose 10 µg/ml and subsequently cultured for 20 hrs. These crude 300-400 islets were transplanted into the renal subcapsular space of streptozotocin-induced diabetic C57BL/6 (H-2b) mice. Recipients were given anti-recipient (mouse) LFA-1 mAb or/and mouse anti-rat ICAM-1 mAb at a dose of 0.05 mg i.p. on days 0 and 1 after transplantation. Mice bearing xenografts over 100 days were challenged with freshly isolated crude WS islets. Results: Significant prolongation was obtained by transplantation of MMC-treated islets (group 2 vs. 1). When these islets were transplanted into the animals given anti-mouse LFA-1 mAb or combination of anti-mouse LFA-1 and anti-rat ICAM-1 mAbs, graft survival over 100 days was 44% and 75% respectively. Animals given anti-recipient LFA-1 and anti-donor ICAM-1 mAbs were more prone to accept the second graft than animals given anti-recipient LFA-1 mAb alone (group 5 vs. 4; p<0.001). The results were summarized in the table.TableConclusion: Unresponsiveness to crude xenogeneic islets can be induced by graft pretreatment with MMC and blockage of costimulatory pathway using species specific anti-donor ICAM-1 mAb under low-dose immunosupression with anti-recipient LFA-1 mAb.

Cytokine Gene Expression during the Elicitation Phase of Contact Sensitivity: Regulation By Endogenous IL-4

Recent studies have focused on characterizing the cytokine profile produced in the epidermis during the sensitization phase of contact sensitivity (CS). Some prior studies have also identified altered individual cytokine mRNA profiles in skin or draining lymph nodes (or several cytokine mRNA profiles in the epidermis) during the elicitation phase of CS. In this study we determined the dynamics of appearance of a battery of cytokine mRNA levels in both the epidermis and dermis during the elicitation phase of CS. We isolated mRNA from dispase-separated epidermis and dermis of TNCB-sensitized and naive BALB/c mice at various times after TNCB challenge. Changes in IFN-gamma and IL-4 mRNA levels (by semiquantitative RT-PCR) were more reproducible and dramatic than those of other cytokines studied (IL-1beta, IL-2, IL-10, and IL-12 p40). Compared to naive mice, sensitized mice had significantly elevated IL-4 mRNA signals 9 and 24 h (dermis), and 24 h (epidermis), after TNCB challenge. The increased IL-4 mRNA levels were mast-cell-independent, because sensitized mast-cell-deficient mice showed similar increases in IL-4 mRNA. To examine the role of endogenous IL-4 in CS elicitation, sensitized mice were treated with anti-IL-4 mAb 1 h before challenge. In accord with prior studies, anti-IL-4 mAb-pretreated mice showed increased ear swelling 24 h after challenge compared to mice pretreated with isotype control mAb. Anti-IL-4 mAb pretreatment also enhanced IFN-gamma, IL-2, IL-12 p40, and IL-1beta (but not IL-10) mRNA signals in the dermis of sensitized and challenged mice. These data indicate that IL-4 is produced in murine skin during the elicitation phase of CS and is an important down-modulator of inflammation. IL-4 may blunt CS by regulating local production of proinflammatory cytokines.

In vivo anti-CD3-driven cell activation. Cellular source of induced tumor necrosis factor, interleukin-1 beta, and interleukin-6.

In vivo anti-CD3-driven cell activation. Cellular source of induced tumor necrosis factor, interleukin-1 beta, and interleukin-6.

CD48 is a low affinity ligand for human CD2.

COS cells transiently transfected with human CD48 were found to bind human PBL, whereas mock or CD7-transfected COS cells failed to bind human lymphocytes. Binding of PBL to CD48 transfectants was almost totally inhibited by either CD48 mAb pretreatment of COS cells or CD2 mAb pretreatment of PBL, implying an interaction between CD2 and CD48. This conclusion was confirmed by the demonstration that a highly fluorescent, multimeric form of rCD2 bound to CD48 transfected COS cells in a CD48-dependent manner. Additional mAb blocking studies revealed that CD48 interacts with the T11(1) region of CD2, the same region of CD2 that binds LFA-3 (CD58). Thus, CD48 and CD58 represent alternative and possibly competing ligands for CD2, although based on blocking studies with soluble CD2, CD48 interacts with CD2 with approximately a 100-fold lower affinity that CD58.

Detection of a glycosylation-dependent ligand for the T lymphocyte cell adhesion molecule CD2 using a novel multimeric recombinant CD2-binding assay.

The CD2 molecule plays an important role in T cell adhesion by interacting with the ligands CD58 (LFA-3) and CD59. In order to detect additional ligands for CD2, potentially of low binding affinity, we have prepared a highly fluorescent, multimeric form of rCD2 whose binding to cells can be quantified by flow cytometry. Initial studies demonstrated that binding of multimeric rCD2 to cells was CD2-specific, concentration and time dependent, and saturable. The negative charge on cells was also found to play a critical role in the efficiency of multimeric rCD2 binding. Analysis of binding of multimeric rCD2 to 17 CD58+ cell types revealed that only 8 of the cells exhibited binding. Failure of multimeric rCD2 to interact with the other cells could not be explained by differences in CD58 expression, suggesting that, in terms of CD2 binding, there are qualitative differences in CD58 on different cell types. Binding of multimeric rCD2 to six of the seven reactive cells was virtually totally inhibited by CD58 mAb pretreatment, whereas binding to the erythroleukemic line K562 was only partially blocked, suggesting the existence of another CD2 ligand. Subsequent studies demonstrated that the putative new ligand is not CD59, and that it interacts with a different region of the CD2 molecule than CD58, probably a site located between the T11(1) and T11(2) epitopes. The binding affinity of CD2 for the new ligand is 10-fold lower than for CD58 and, based on studies with truncated rCD2, the binding site for the new ligand is located within the amino-terminal 105 amino acids of the CD2 polypeptide. Unlike CD58, the new ligand is tunicamycin sensitive suggesting that it contains a N-linked carbohydrate structure that is essential for functional activity.

HEART ALLOGRAFTS IN MURINE SYSTEMS

Activated CD4+ Th2 cells release cytokines (IL-4,-10) that block activation & cytokine (IL-2/IFN-gamma) release by proinflammatory T (CD4+,CD8+) effector cells. To test the hypothesis that peripheral tolerance to alloantigen is linked to differential activation of CD4+ Th2 cells we measured cytokine transcripts in heart grafts (C57BL/10----C3H/HeJ) and spleens of mice rendered "tolerant" by donor-specific blood transfusion, anti-CD4 mAb pretreatment, and cyclosporine administration. The expression of IL-2/IFN-gamma transcripts was reduced greater than 90% in grafts from tolerant recipients. IL-4/IL-10 transcripts were generally enhanced and persisted in graft and recipient spleen. Accordingly adoptive transfer studies were performed to determine whether Th2-like effectors, which express Fc receptors (FcR), mediate suppression in this model. Unfractionated mononuclear cells (MC) (5 x 10(6), isolated from spleens of heart graft recipients made tolerant by DST, prolonged the survival of test grafts greater than 90 days in irradiated (680 rads) recipients reconstituted with a sufficient number of MC from spleens of naive C3H to precipitate rejection of the test graft in 18.2 days (MST, n = 5). Conversely adoptive transfer of inocula depleted of FcR+ cells on immune complex columns or with anti-FcR mAb 24G2 caused test grafts to be rejected in 8-11 days. These results suggest that peripheral tolerance to alloantigen may be linked to differential activation of Th2 cells that induce anergy by suppression. The possibility that Th2-like effectors mediate peripheral tolerance to self is discussed.

Humoral tolerance in xenogeneic BMT recipients conditioned by a nonmyeloablative regimen.

We have recently demonstrated that mixed xenogeneic chimerism and donor-specific tolerance can be produced across a species barrier using a nonmyeloablative conditioning regimen (1). This regimen involves pretreatment of B10 mice with mAbs against CD4+, CD8+, Thy1+, and NK1+ cells, followed by a low dose (3 Gy) of whole-body irradiation and a higher dose (7 Gy) of local irradiation to the thymus and administration of T cell-depleted (TCD) F344 strain rat BMC. Although initial mixed chimerism and de novo maturation of donor rat T cells can be demonstrated in such animals, chimerism is gradually lost, and is no longer detectable by 6 months following BMT (1). When rat skin was grafted onto such animals 4 months following BMT, however, donor-specific skin graft survival was markedly prolonged, while non-donor type rat skin grafts were rapidly rejected (1). These results suggested that a state of donor-specific T cell tolerance existed, and that loss of chimerism was not due to a T cell-mediated immune mechanism. In order to evaluate the possibility that a humoral mechanism might mediate delayed loss of xenogeneic bone marrow grafts, we have now examined sera at various times for the presence of antibody against donor cells. Groups of animals not receiving the complete tolerizing mAb pretreatment regimen produced antidonor lymphocytotoxic antibody in response to BMT and skin grafting. Flow cytometric studies demonstrated high levels of IgM and of IgG of all subclasses against rat BMC and spleen cells in these control mice immunized by BMT. In contrast, such antibodies were not detectable in sera from animals receiving BMT following pretreatment with the tolerance-inducing mAb regimen. Furthermore, the tolerant animals did not develop cytotoxic antibodies or high levels of IgM or IgG against donor BMC after loss of hematopoietic chimerism. Donor-type skin grafts were eventually rejected, but rejection of these and repeat skin grafts did not lead to a cytotoxic antibody response. Low levels of rat BMC-binding IgM antibody were also detected in sera of tolerant mice, but the intensity of staining of rat BMC was lower than that of control animals receiving conditioning without BMT. These results suggest that a state of tolerance exists among cells responsible for T cell-dependent IgG antibody subclasses and natural IgM antibodies in animals receiving BMT following this nonmyeloablative conditioning regimen.

Studies on the participation of different T cell subsets in rat liver allograft rejection

In this study, we investigated which subsets of rat T cells (CD8 + vs. CD4 + ) are involved in the rejection of liver allografts by the in vivo administration of monoclonal antibody (OX-8 or OX-38, and W3/25 MAb) into thymectomized recipient Lewis (RTI1) rats prior to DA (RTIa) liver transplantation. We also compared the results of allograft survival of liver and heart transplants under the same experimental conditions. In order to deplete either CD8 + T cells or CD4 + T cells from recipient animals, 0.4 ml of OX-8 (ascitic form) or a 0.8 ml cocktail of MAb W3/25 and OX-38 (0.4 ml each) was injected into thymectomized recipient rats, respectively. Untreated Lewis rats consistently rejected donor DA liver grafts between 9 and 11 days (n = 7, 9.8 days ± 1.1 days). In contrast, anti-CD8 MAb pretreatment extended the survival times of DA liver grafts for up to 40 days (n = 5, 26.8 days ± 8.4 days). Furthermore, survival of DA liver grafts was significantly prolonged in Lewis rats that had been pretreated with anti-CD4 MAb (n = 7,35.6 days ± 17.9 days). Two out of seven recipient animals survived for more than 60 days. For heart transplantation, untreated Lewis rats rejected DA heart grafts between 6 and 8 days after operation (n = 6, 6.5 days ± 1.2 days). Anti-CD4 MAb treatment prolonged heart graft survival for more than 60 days in all cases (n = 3, > 60 days). However, there was virtually no effect of anti-CD8 MAb treatment on heart graft survival (n = 4, 7.0 days ± 0.9 days). These results suggested that when whole MHC disparity prevailed between donor and recipient, both subsets of T cells were required for the rejection of liver allografts and that class II reactive T cells predominantly mediated liver graft rejection. Furthermore, CD8 + T cells played a differential role in the rejection of rat liver and heart allograft.

Studies on the participation of different T cell subsets in rat liver allograft rejection. Comparison of liver with heart graft.

In this study, we investigated which subsets of rat T cells (CD8+ vs. CD4+) are involved in the rejection of liver allografts by the in vivo administration of monoclonal antibody (OX-8 or OX-38, and W3/25 MAb) into thymectomized recipient Lewis (RTI(l)) rats prior to DA (RTI(a)) liver transplantation. We also compared the results of allograft survival of liver and heart transplants under the same experimental conditions. In order to deplete either CD8+ T cells or CD4+ T cells from recipient animals, 0.4 ml of OX-8 (ascitic form) or a 0.8 ml cocktail of MAb W3/25 and OX-38 (0.4 ml each) was injected into thymectomized recipient rats, respectively. Untreated Lewis rats consistently rejected donor DA liver grafts between 9 and 11 days (n = 7, 9.8 days +/- 1.1 days). In contrast, anti-CD8 MAb pretreatment extended the survival times of DA liver grafts for up to 40 days (n = 5, 26.8 days +/- 8.4 days). Furthermore, survival of DA liver grafts was significantly prolonged in Lewis rats that had been pretreated with anti-CD4 MAb (n = 7, 35.6 days +/- 17.9 days). Two out of seven recipient animals survived for more than 60 days. For heart transplantation, untreated Lewis rats rejected DA heart grafts between 6 and 8 days after operation (n = 6, 6.5 days +/- 1.2 days). Anti-CD4 MAb treatment prolonged heart graft survival for more than 60 days in all cases (n = 3, > 60 days). However, there was virtually no effect of anti-CD8 MAb treatment on heart graft survival (n = 4, 7.0 days +/- 0.9 days). These results suggested that when whole MHC disparity prevailed between donor and recipient, both subsets of T cells were required for the rejection of liver allografts and that class II reactive T cells predominantly mediated liver graft rejection. Furthermore, CD8+ T cells played a differential role in the rejection of rat liver and heart allograft.

Anti-IL-6 monoclonal antibodies protect against lethal Escherichia coli infection and lethal tumor necrosis factor-alpha challenge in mice.

Potentially fatal physiologic and metabolic derangements can occur in response to bacterial infection in animals and man. Recently it has been shown that alterations in the levels of circulating cytokines such as IL-6 and TNF-alpha occur shortly after bacterial challenge. To understand better the role of IL-6 in inflammation, we investigated the effects of in vivo anti-mouse IL-6 antibody treatment in a mouse model of septic shock. Rat anti-mouse IL-6 neutralizing mAb was produced from splenocytes of an animal immunized with mouse rIL-6. This mAb, MP5-20F3, was a very potent and specific antagonist of mouse IL-6 in vitro bioactivity, demonstrated using the NFS60 myelomonocytic and KD83 plasmacytoma target cell lines, and also immunoprecipitated radiolabeled IL-6. Anti-IL-6 mAb pretreatment of mice subsequently challenged with lethal doses of i.p. Escherichia coli or i.v. TNF-alpha protected mice from death caused by these treatments. Pretreatment of E. coli-challenged mice with anti-IL-6 led to an increase in serum TNF bioactivity, in comparison to isotype control antibody, implicating IL-6 as a negative modulator of TNF in vivo. Anti-TNF-alpha treatment of mice challenged i.p. with live E. coli resulted in a 70% decrease in serum IL-6 levels, determined by immunoenzymetric assay, compared to control antibody, thereby supporting a role for TNF-alpha as a positive regulator of IL-6 levels. We conclude that IL-6 is a mediator in lethal E. coli infection, and suggest that antagonists of IL-6 may be beneficial therapeutically in life-threatening bacterial infection.

Involvement of CD45 in MHC Class II-allospecific cytotoxicity

Twenty-seven different CD45 monoclonal antibodies (mAb) were assessed for their ability to block cytotoxicity of alloreactive CD4 + MHC class II-specific or CD8 + class I-specific human T cell clones ( n = 3 and 5, respectively). Twelve of 27 blocked the former but only 1 27 the latter, although all 27 significantly inhibited MHC-unrestricted lysis of K562 cells by either CD4 + or CD8 + clones. MAb pretreatment of effector cells but not target cells resulted in retention of blocking. Crosslinking the CD45 with goat anti-mouse Ig serum did not result in blockade of lysis by class I-specific clones or reveal blocking of class II-specific clones not inhibited by mAb alone. These results suggest that CD45 molecules may be predominantly involved in MHC class II-specific but not class I-specific allocytotoxicity as well as MHC-unrestricted natural killer-like cytotoxicity.

Antibodies to cachectin/tumor necrosis factor reduce interleukin 1 beta and interleukin 6 appearance during lethal bacteremia.

Cytokines secreted in response to invading micro-organisms are important mediators of detrimental hemodynamic and metabolic changes in the host. To test whether cachectin/TNF plays a role in triggering release of other cytokines in the setting of infection, anesthetized baboons were passively immunized against systemic cachectin/TNF before infusion of a LD100 dose of live Escherichia coli. Bacteremia led to significant increases in circulating levels of cachectin/TNF, IL-1 beta, and IL-6. Although bacterial endotoxin/lipopolysaccharide is a potent stimulus for the synthesis and release of IL-1 and IL-6 in vitro, specific neutralization of cachectin/TNF in vivo with mAb pretreatment significantly attenuated both the IL-1 beta and the IL-6 responses despite fulminant overwhelming bacteremia. These data suggest that cachectin/TNF is essential for the initiation or amplification of IL-1 and IL-6 release during lethal gram-negative septic shock syndrome.