Allograft rejection in CD4+ T cell-reconstituted athymic nude rats--the nonessential role of host-derived CD8+ cells.

PVG-rnu/rnu nude rats reject fully allogenic renal (DA) and skin (BN, AO) allografts after the adoptive transfer of naive CD4+ T cells alone, but rejection is accompanied by the accumulation of many nude-derived CD8+ leukocytes within the graft. In addition, mononuclear cells infiltrating the rejecting renal grafts in these animals display cytotoxic activity in vitro against specific and third-party alloantigens. In this investigation we have treated CD4+ T cell-restored nude rats bearing renal or skin allografts with the mAb MRC OX8 to deplete the host of CD8+ cells. In vivo treatment with OX8 completely eliminated CD8+ cells from rejecting grafts of both kidney and skin, but it did not prevent graft rejection, nor did OX8 treatment abolish the cytotoxic effector cells found in nude rat spleen or in graft-infiltrating cells (GIC) of rejecting renal allografts. The nature of the cytotoxic activity was examined with anti-CD3 mAb 1F4, which was shown to block conventional CD8+ Tc killing in vitro but did not inhibit allogeneic target cell lysis by spleen cells from nude rats. The cytotoxic activity found in GIC of rejecting allografts was not inhibited by anti-CD3 mAb, suggesting that these cytotoxic effector cells were CD3-CD8- and were of extrathymic origin. We conclude that non-thymus-derived CD8+ GIC are not essential for allograft rejection in CD4+ T cell-restored nude rats.     

The mechanism of anti-CD3 monoclonal antibodies. Mediation of cytolysis by inter-T cell bridging

OKT3, an anti-CD3 MAB, depletes T cells in vivo and is among the most potent inhibitors of acute allograft rejection. The mechanism of this inhibition is unknown. The present studies investigate whether anti-CD3 antibodies have the ability to crosslink CD3 on two different cells and induce TCR-dependent antibody-bridged cell-mediated cytolysis (TCR-ABCMC) between T cells. Two different anti-CD3 antibodies (OKT3 and CD3,3) and OKT3 F(ab')2 were all highly effective in inducing cytolysis of CD8+ and CD4+ T cells by CD8+ T cells, and CD8+ T cells by CD4+ T cells. Monovalent OKT3 Fab was 25-125-fold less potent than OKT3 F(ab')2. Monovalent CD3,X was totally ineffective. The necessity for intercellular bridging was evidenced by the observation that an anti-CD3:anti-CD4 (CD3,4) bispecific MAB (BSMAB) was effective in mediating lysis of CD4+ but not CD8+ T cells by CD8+ T cells. These studies indicate that neither FcR-mediated ADCC nor complement fixation is necessary for bivalent anti-CD3 MAB to lyse T cells. Inter-T cell TCR-ABCMC may be particularly effective in inflammatory tissues, such as rejecting allografts and autoimmune diseases, in which numerous cytolytic T lymphocytes are present in close association with other T cells.     

Evolving use of OKT3 monoclonal antibody for treatment of renal allograft rejection

OKT3, a monoclonal antibody reactive with a surface glycoprotein present on all postthymic T cells, was used to treat the initial acute episode of rejection in 30 recipients of cadaveric donor renal allografts. The first 16 patients received 1-5 mg daily for a period of 10-21 days during which the azathioprine and prednisone dosages were sharply reduced. Circulating T cells were eliminated within minutes after the first OKT3 infusion. T cells reactive with OKT3 remained depressed throughout the period of treatment, although a significant number of cells reactive with other T cells subset reagents became detectable after several days of OKT3 treatment. In all instances, the established rejection episode was reversed in 2-8 days without the addition of other immunosuppressive measures. Recurrent rejection occurred in 12 of 16 patients, but with further conventional immunosuppression, 50% of the renal allografts remain functional 20-44 months after transplantation. Fever, chills, and, in some instances, dyspnea following the first dose of OKT3 were the only side-effects observed. Most patients developed antiidiotypic or antimouse immunoglobulin antibodies without apparent clinical sequelae. In the subsequent 14 patients, modifications in the protocol included a steroid bolus prior to the first OKT3 infusion, limitation of therapy to 10 days, resumption of maintenance levels of azathioprine and prednisone prior to discontinuing OKT3, and addition of 3 i.v. doses of cyclophosphamide at the termination of treatment. Respiratory symptoms after the first infusion of the reagent have been eliminated. Antibody responses to OKT3 have been reduced, occurring in 38% as compared with 73% of patients treated previously. Recurrent rejection episodes observed in 8 of 14 patients have been reversible in all but one case. Allograft survival is 86% at 6-17 months posttransplantation. In the entire series of 30 OKT3-treated patients, only 4 grafts (13%) have been lost because of recurrent episodes of rejection.     

Composition of interstitial cellular infiltrate identified by monoclonal antibodies in renal biopsies of rejecting human renal allografts

Monoclonal antibodies and a four-layer immunoperoxidase technique were used to analyze and quantitate the various infiltrating cell types found in percutaneous renal biopsies of 25 patients undergoing acute renal allograft rejection. Cell markers included monoclonal antibodies to the human leukocyte-common antigen (PHM 1), mononuclear phagocytes (PHM 2, FMC 32), T cells and T cell subsets (OKT 3, OKT 4, and OKT 8), B cells (7.2), polymorphs (FMC 10, FMC 13), and plasma cells (OKT 10). The proportion of labelled interstitial cells was expressed as a percentage of the total number of infiltrating leukocytes identified with PHM 1, and correlated with the histologically graded intensity of rejection. In mild rejection 32% of the infiltrating cells were T lymphocytes, of which 90% were OKT-8-positive cytotoxic-suppressor cells, and 52% were macrophages. Similarly, in moderate rejection T cells composed 42% of the infiltrate (with 67% of T cells expressing OKT 8 antigen), and macrophages formed 38% of the total cells. By contrast, in severe rejection, the T cell component was decreased to 15% of the cells, of which 78% were OKT-8-positive; these were preponderantly macrophages (60%) and polymorphs (22%). These studies demonstrate that cytotoxic-suppressor T cells and macrophages are the major cells mediating acute interstitial graft rejection.     

Immunopathology of renal allograft rejection analyzed with monoclonal antibodies to mononuclear cell markers

The composition of the mononuclear cell infiltrate in rejecting renal allografts was determined on 96 renal biopsies and 22 nephrectomy specimens by the use of monoclonal antibodies to mononuclear cell surface markers and an indirect immunoperoxidase staining technique. During rejection the composition of the infiltrate was heterogeneous, with T cells (T11), monocytes (OKM1) and HLA-DR expressing mononuclear cells the most frequent sub-populations. B cells (B1) and activated T cells, identified by OKT10, were always in the minority. The T cells infiltrate usually included the helper/inducer (T4) and cytotoxic (T8) subclasses, which suggests that both may contribute to the mediation of rejection. Whether T4 or T8 predominated in the graft did not relate to the ratio of T4:T8 in blood, the HLA A, B or DR incompatibilities of the graft, or the immunosuppressive used. The frequency of T11, T4, T8, HLA-DR positive cells and monocytes, but not B cells, increased with the severity of rejection and was similar in biopsies from patients immunosuppressed with Cyclosporine (CSA) to those given a combination of azathioprine, prednisone and antilymphocyte globulin (AZA). Severe rejection episodes which did not respond to treatment with corticosteroids were more often characterized by a predominance of T8 over T4 cells and T cells infiltrating the glomeruli. In grafts with evidence of cellular rejection, renal tubular cells were shown to have a marked increase in their expression of HLA-DR antigens compared to normal kidneys or grafts with minimal rejection. The expression of HLA-DR antigens on graft tubular cells correlated with the presence of T cells in the interstitium and the severity of rejection, except for moderate rejection in CSA treated biopsies, in which HLA-DR expression was lower than in AZA biopsies. These immunopathological studies have demonstrated that a variety of potential effector cells exist within the graft, and several features have been identified which may assist in assessing the prognosis of the rejection episode.     

Alterations in T lymphocyte subpopulations associated with renal allograft rejection

The peripheral blood OKT3 (total T), OKT4 (T helper/inducer), and OKT8 (T suppressor/cytotoxic) cells were determined by flow cytometry on twenty consecutive recipients of HLA-nonidentical cadaveric renal allografts. The absolute number of cells in all three populations decreased significantly posttransplantation, but no differences were found between patients experiencing rejection and those in quiescence. An OKT4/OKT8 ratio of greater than or equal to 1.7, either pretransplant or posttransplant, uniformly identified patients who subsequently experienced rejection. However, an OKT4/OKT8 ratio of less than 1.7 did not identify patients with a low risk of rejection. Pretransplant splenectomy was performed in 6 of 7 patients who rejected despite a low ratio. Serial monitoring of the OKT4/OKT8 ratio posttransplantation determined that an increase in the ratio of greater than or equal to 0.5 was a sensitive (81%) and specific (91%) indicator of a rejection episode. Graft survival was improved in patients with a high posttransplant OKT4/OKT8 ratio. These results indicate that the balance of helper and suppressor cell function may be of critical importance to the fate of an allograft, and that the alterations in this balance can be used to assist in the clinical management of allograft recipients.     

HuM291, a humanized anti-CD3 antibody, is immunosuppressive to T cells while exhibiting reduced mitogenicity in vitro.

BACKGROUND: OKT3, a mouse monoclonal antibody (Ab) specific for the human CD3 complex on T cells, is a potent immunosuppressive agent used for the treatment of acute allograft rejection. The utility of the drug has been limited by a neutralizing anti-mouse Ab response and adverse side effects resulting from T cell activation and systemic cytokine release. T cell activation is caused by OKT3-mediated cross-linking of T cells and Fc receptor-bearing cells. Studies in the mouse model have shown that global T cell activation is not necessary for immunosuppression, as Fc receptor-nonbinding anti-CD3 Abs can suppress graft rejection in the absence of the activation effects seen with Fc receptor-binding Abs. Thus, a humanized anti-CD3 antibody with a low affinity for Fc receptors might improve immunosuppressive therapy by reducing the side effects associated with OKT3. METHODS: We developed a mouse monoclonal Ab, M291, which competes with OKT3 for binding to T cells. Humanized, complementary-determining region-grafted versions of M291 featuring various Fc were engineered, including a previously described IgG2 mutant deficient in Fc receptor binding (HuM291). RESULTS: Compared with OKT3 and HuM291-IgG1, HuM291 was significantly less mitogenic to T cells in vitro and induced the release of much lower levels of the cytokines tumor necrosis factor-alpha, interferon-gamma, and interleukin-10. Despite this reduction in T cell activation, HuM291 retained the ability to modulate the CD3 complex and inhibit the mixed lymphocyte reaction. CONCLUSIONS: When evaluated in vivo, HuM291 may be an immunosuppressive agent associated with less of the acute toxicity and immunogenicity seen with OKT3 therapy.     

Monoclonal antibodies in renal transplantation: a review

OKT3 is the first anti-CD3 monoclonal antibody available for treatment in humans. Over the last few years it has proven to be a very powerful immunosuppressive agent in renal transplantation. Clinical studies have shown that OKT3 is superior to high-dose steroids as first-line treatment for acute renal allograft rejection. Furthermore, it is comparable to antithymocyte globulin (ATG) in treating steroid-resistant rejection and is also effective as rescue treatment in ATG-and antilymphocyte globulin-(ALG-) resistant rejection. Despite its excellent rejection-reversal rate, OKT3 treatment is followed by a substantital percentage of re-rejections, most of which respond well to steroids. In the early post-transplantation period, a prophylactic course of OKT3 is very effective in preventing acute rejections, and in this respect it is probably equivalent to ATG. Indirect evidence exists that a prophylatic course of OKT3 may be beneficial in immunologically high-risk patients and in patients with delayed graft function. However, more clinical studies are required to answer the question whether OKT3 should be given as induction treatment, as first-line treatment, or as rescue treatment. To answer this question, the side effects of OKT3 should also be taken into account. First-dose-related side effects, although frequent and disturbing, are usually transient and seldom life-threatening, provided overhydration has been corrected and steroids have been given before the first administration. These side effects are attributed to the release of cytokines as a result of T-cell activation or lysis. After exposure of patients to OKT3 an increased incidence of infections and malignanies has been reported. However, it is not yet clear whether this is due to OKT3 as such, or whether it merely reflects the total burden of immunosuppression. Xenosensitization represents an important limitation to OKT3 treatment, although a second or third course can still be effective in patients with low antibody titers. The precise immunosuppressive mechanism of anti-CD3 monoclonal antibodies is yet unknown. Monitoring of patients treated with OKT3 revealed CD3 and/or T-cell antigen receptor depletion and immunological incompetence of remaining T cells. More clinical data are required to establish the correct dose and duration of OKT3 treatment. In conclusion, OKT3 is a powerful immunosuppressive agent but its real value in renal transplantation remains to be determined. A practical approach may be to reserve it for the treatment of steroid-resistant rejections. Research strategies for the development of new monoclonal antibodies for future use in renal transplant patients should be aimed at evading the major hazards of OKT3 treatment, namely side effects and xenoimmunization. Many promising monoclonal antibodies are currently under study: theoretically, anti-CD3/TCR monoclonal antibodies that are not mitogenic (such as CLB-T3/4.A, BMA 031, T10.B9.1A-31A, and OKT3 F[ab']2 fragments) may be expected to cause less immediate toxicity than OKT3. However, their immunosuppressive properties still have to be established. Monoclonal antibodies that are reactive with more restricted T-cell markers such as the interleukin-2 receptor (33B3.1 and anti-Tac), CD4 (BL4), and CD8 (Leu 2a) are generally better tolerated than OKT3. On theoretical grounds they may also be expected to cause fewer long-term complications. However, their immunosuppressive properties also remain to be established. In patients with high titers of anti-idiotypic antibodies to OKT3, WT 32 and perhaps RIV 9 and T10B9.1A–31A may be good alternatives.     

Phenotypic composition and in vitro functional capacities of unmodified fresh cells infiltrating acutely rejected human kidney allografts

Cell surface markers of isolated graft-infiltrating cells (GIC) were studied, and functional in vitro assays performed in 8 cases of acute irreversible rejection of human renal allografts. The GIC were mostly activated T cells (OKT11+, OKT3+, Ia+), with predominance of the cytotoxic/suppressor T cell phenotype (OKT8+). A small proportion of B cells and monocytes/macrophages were also present among these GIC. The GIC were able to proliferate with lectin of allogeneic stimulation and were strongly cytotoxic toward specific donor target cells. Within the T cell subset, OKT8+ cells displayed most of the specific cytotoxicity. Despite allograft morphology typical of cellular rejection, anti-HLA complement-dependent antibodies and antibody-dependent cell cytotoxicity were found in the eluted material from rejecting kidneys. The results of our phenotypic and functional testing of unmodified GIC (no enzyme treatment, no additional culture with or without interleukin 2), show that T cells, especially OKT8+ cells, are of paramount importance in the mechanism of this type of acute irreversible rejection of human renal allografts (i.e., to the point of allograft rupture), but other potential effector mechanisms are also present in situ.     

Effectiveness of a second course of OKT3 monoclonal anti-T cell antibody for treatment of renal allograft rejection

A second course of OKT3 monoclonal anti-T cell antibody was given to 21 recipients of kidney transplants. Rejections reversed in 43% of patients in whom 95% of rejections had reversed with their initial OKT3 course. Reversal was highly dependent upon the timing of rejection, anti-OKT3 antibody production, and T cell CD3 modulation. Rejections treated greater than 90 days after transplantation were resistant to OKT3 reversal. High-titer anti-OKT3 antibodies prevented OKT3 reversal of rejection, and effective CD3 (the cell surface target of OKT3) modulation was necessary for successful OKT3 reversal of rejection. Reexposure to OKT3 further stimulated anti-OKT3 antibody production and broadened the specificity of the antibodies produced. OKT3 can effectively and safely be used a second time for treatment of early T cell-mediated renal allograft rejections if high-titer anti-OKT3 antibodies have not been made.     

Function and surface phenotype of T lymphocytes infiltrating renal allografts in nonhuman primates treated with monoclonal antibodies

The phenotype and function of T lymphocyte cell lines established in vitro from kidney biopsies at the time of acute cellular rejection were studied using a nonhuman primate renal allograft model. Our objectives were to investigate the function and surface phenotype of cells that infiltrate renal allografts in animals that were untreated, that were given subtherapeutic cyclosporin, or that developed rejection after treatment with monoclonal antibodies to IL-2R B chain (CD25), immune cell adhesion molecule-1 (ICAM-1), or CD8. Lines from allograft biopsies and peripheral blood were expanded in vitro using solely human recombinant IL-2 and analyzed after 6-20 days in culture. We found that the large majority of cells cultured from cynomolgus allografts at the time of acute rejection or, when possible, assayed directly without culture, were CD3+4-8+ T lymphoblasts that possessed donor-specific cytolytic function and an NK-line, cytotoxic activity. In contrast, it was rarely possible to establish T cell lines exhibiting donor-specific cytotoxic activity from the blood except in the absence of immunosuppression or during CsA taper. A stable number of graft-derived CD4+8- cells was only observed in an unsuppressed animal 2 days after transplantation in the absence of manifest signs of rejection. Taken together, the above data indicate that similar T lymphocyte populations associated with allograft rejection are present in acutely rejecting allografts after the various types of immunosuppressive therapy. Since the infiltrating cells were similar to those obtained prior to therapy, recurrent rejection most likely represents cells that have escaped elimination. The T cells derived from monkey grafts differ from those from human renal allografts by the decreased frequency of CD4+ cells. Whether this difference is species-related or therapy-related is not known.     

CD103 mRNA levels in urinary cells predict acute rejection of renal allografts

BACKGROUND: CD103 is displayed on the cell surface of alloreactive CD8 cytotoxic T lymphocytes (CTLs) and is a critical component for the intraepithelial homing of T cells. Because intratubular localization of mononuclear cells is a feature of acute cellular rejection of renal allografts, we explored the hypothesis that CD103 messenger (m)RNA levels in urinary cells predict acute rejection. METHODS: We collected 89 urine specimens from 79 recipients of renal allografts. RNA was isolated from the urinary cells, and we measured CD103 mRNA levels and a constitutively expressed 18S ribosomal (r)RNA with the use of real-time quantitative polymerase chain reaction assay. RESULTS: CD103 mRNA levels, but not 18S rRNA levels, were higher in urinary cells from 30 patients with an episode of acute rejection (32 biopsies and 32 urine samples) compared with the levels in 12 patients with other findings on allograft biopsy (12 biopsies and 12 urine samples), 12 patients with biopsy evidence of chronic allograft nephropathy (12 biopsies and 12 urine samples), and 25 patients with stable graft function after renal transplantation (0 biopsies and 33 urine samples) (P = 0.001; one-way analysis of variance). Acute rejection was predicted with a sensitivity of 59% and a specificity of 75% using natural log-transformed value 8.16 CD103 copies per microgram as the cutoff value (P = 0.001). CONCLUSION: CD103 mRNA levels in urinary cells are diagnostic of acute rejection of renal allografts. Because CD103 is a cell surface marker of intratubular CD8 CTLs, a noninvasive assessment of cellular traffic into the allograft may be feasible by the measurement of CD103 mRNA levels in urinary cells.     

Impact of both donor and recipient strains on cardiac allograft survival after blockade of the CD40-CD154 costimulatory pathway

BACKGROUND: The effectiveness of anti-CD154 monoclonal antibodies in prolonging the survival of mouse allografts is dependent on the strain combination. In this report, we examined the impact of the donor and the recipient strains on the success of CD40-CD154 blockade. MATERIALS AND METHODS: Cardiac allograft survival was monitored in different donor/recipient strain combinations. Morphometric analyses on the allograft coronary arteries allowed quantification of vessel intimal thickening. RESULTS: Prolonged cardiac allograft survival after the administration of an anti-CD154 monoclonal antibody was found to be dependent on the donor and the recipient strains. The influence of the donor and the recipient strains lay in the ability of CD8 T cells to cause graft rejection despite CD40-CD154 blockade. Elimination of CD8 T cells before transplantation resulted in similar graft prolongation irrespective of the genotype of the donor or the recipient strain. CONCLUSION: These data show that both donor and recipient strains contribute to CD40-CD154-independent CD8 T-cell-mediated rejection.     

Interest in and limitations of monoclonal anti-T-cell antibodies for the follow-up of renal transplant patients

The OKT series of anti-T-cell monoclonals has been used on 442 occasions in 41 renal allograft recipients in a 6-12 month follow-up study. Standard immunosuppressive therapy (including antithymocyte globulin in 26 patients) tended to decrease the helper-inducer/suppressor-cytotoxic cell ratio (OKT4/OKT8). Conversely, 71% of 35 renal failure episodes were associated with increased OKT4/OKT8 ratios. Twenty-three percent of renal failure episodes were associated with dramatically decreased OKT4/OKT8 ratios. At least half of these cases could be explained by a cytomegalovirus infection. In fact, similar infections were found in 6 out of 17 patients with low OKT4/OKT8 values in the absence of renal failure. These results prompt us to use anti-T cell monoclonals for the diagnosis of rejection because only nine episodes of transient increase in the OKT4/OKT8 ratio were observed in the absence of rejection. The interest of this new method for the immunological follow-up of transplanted patients is, however, limited by the difficulty in interpreting a significant percentage of tests because of (1) the presence of doubly labeled cells (OKT4+OKT8+) or the significant discrepancy between the number of OKT3+ cells and total cells labeled with OKT4 and/or OKT8 antibodies; (2) gross lymphocytopenia--most often observed in patients receiving antithymocyte globulins plus steroids; and (3) the clinically unexplained shifts in the T cell subset ratios mentioned above.     

Levels of HBV-DNA and HBsAg after acute liver allograft rejection treatment by corticoids and OKT3

The aim of this work was to analyze whether the treatment of acute rejection of orthotopic liver transplants (OLT), either with corticoids or OKT3, has any effect on the levels of hepatitis B virus (HBV)-DNA and HBsAg in individuals which were originally affected by cirrhosis or fulminant hepatic failure as a result of B virus. We have found that HBV-DNA is present in macrophages, B cells and both CD4+ and CD8+ T cells after OLT in all cases studied. Interestingly, the levels of HBV-DNA and HBsAg in the serum analyzed were increased extremely rapidly in the patients treated with OKT3 in an acute rejection episode. However, the serum levels of HBV-DNA and HBsAg found were lower when the patients were treated with steroids, and were not found in non-treated patients. As the serum levels of HBV-DNA increase, the process of liver reinfection could be accelerated; therefore, these results may help to understand how OKT3 and corticoids immunosuppressive therapy may accelerate the reinfection of OLT by HBV. In conclusion, our results suggest that special care must be taken in the use of OKT3 in the treatment of acute liver rejection episodes in chronic or fulminant HBV transplanted patients.     

CD40 expression on graft infiltrates and parenchymal CD154 (CD40L) induction in human chronic renal allograft rejection

BACKGROUND: CD40-CD154 (CD40L) costimulatory signaling plays a pivotal role in the effector mechanisms of transplant graft rejection. In animal models, CD40-CD154 blockade induces long-term graft acceptance concurrent with an absence of chronic rejection (CR) lesions. Given the critical importance of CD40-CD154 interactions in the development of chronic transplant allograft rejection, the relevance of in situ CD40 and CD154 expression was assessed in human chronic renal allograft rejection. METHODS: The expression of CD40, CD154, CD68, and T-cell receptor (TCR)alpha/beta was analyzed by immunohistochemistry. Serial cryostat sections of snap-frozen core renal allograft biopsies were obtained from 30 renal transplant patients. Biopsy specimens received diagnoses of CR (N = 23) according to the Banff classification and were compared with controls (N = 7) consisting of stable allografts and normal kidney tissue. RESULTS: Striking CD40 staining of graft cellular infiltrates (P = 0.016) was observed in renal allografts with CR compared with controls. The CD40+ cellular infiltrates in CR were predominantly TCR alpha/beta + T cells and some CD68+ macrophages. These findings were contrasted by the low-level CD40 expression detected in glomeruli and tubules of CR and controls. However, glomerular induction of CD154 was observed in CR allografts (P = 0.028) as compared with controls. CD154 immunoreactivity was demonstrated on glomerular endothelial, epithelial, and mesangial cells. Moderate CD154 expression was detected on tubular epithelial cells, and only weak CD154 immunoreactivity was observed on the infiltrates in isolated CR cases. CONCLUSION: In human chronic renal allograft rejection, CD40 is expressed on graft-infiltrating cells of the T cell and macrophage compartments. CD154 expression is induced on glomerular and tubular epithelial cells during CR, demonstrating another novel source of CD154 expression. The data substantiate the potential contributory role of an interaction between CD40+ graft-destructive effector T cells and macrophages with CD154+ renal allograft parenchymal cells in the development of chronic renal allograft rejection.     

Treatment of acute graft-versus-host disease with a nonmitogenic anti-CD3 monoclonal antibody.

Treatment with the monoclonal antibody OKT3 specific for the CD3 complex associated with the T cell antigen receptor can reverse acute rejection of human renal allografts. However, efficacy of anti-CD3 antibodies for treatment of patients with acute graft-versus-host disease after marrow transplantation has not been established. The dose-limiting side effects resulting from T cell activation induced by some anti-CD3 antibodies in vivo have discouraged their use for this application. We now report a phase I-II study of GVHD treatment with the anti-CD3 antibody BC3, a monoclonal murine IgG2b that, unlike OKT3, does not activate T cells. Fourteen patients were treated with BC3 after progression of acute GVHD despite treatment with cyclosporine and corticosteroids, and three patients received BC3 as primary treatment for GVHD. BC3 was administered at a dose of 0.1 or 0.2 mg/kg/day for seven or eight days. Five patients achieved complete resolution of GVHD, eight patients had partial improvement, two patients had no change, and two patients had progression of GVHD on therapy. Responses were sustained in 8 of 13 patients. Mild chills, fever, hypertension, and chest discomfort occurred in various combinations following 6 of 17 (35%) initial infusions of BC3 and following 4 of 99 (4%) subsequent infusions. In each instance it was possible to continue BC3 therapy without adjusting the dose or treatment schedule. In each patient treated, the absolute count of peripheral blood lymphocytes decreased transiently but returned to baseline within 22 hr after the first infusion. Circulating T cells had surface CD3 molecules saturated by the infused antibody in all but one patient. Four patients survived longer than one year after treatment with antibody BC3, and 13 patients died of infection or organ failure. Administration of the nonmitogenic anti-CD3 antibody BC3 was associated with improvement in the clinical manifestations of GVHD with minimal acute toxicity. Efficacy of antibody treatment did not depend on depletion of circulating T cells. Therefore, antibody BC3 may be achieving therapeutic immunosuppression by modulating T cell function. Controlled studies in patients treated earlier in the course of GVHD should determine whether antibody BC3 can improve survival.     

Donor-Reactive CD8 Memory T Cells Infiltrate Cardiac Allografts Within 24-h Posttransplant in Naive Recipients

Normal immune responses stimulated by pathogenic and environmental antigens generate memory T cells that react with donor antigens and no currently used immunosuppressive drug completely inhibits memory T-cell function. While donor-reactive memory T cells clearly compromise graft outcomes, mechanisms utilized by memory T cells to promote rejection are largely unknown. In this study, we investigated how early endogenous memory cells infiltrate and express effector function in cardiac allografts. Endogenous CD8 memory T cells in nonsensitized recipients distinguish syngeneic versus allogeneic cardiac allografts within 24 h of reperfusion. CD8-dependent production of IFN-γ and CXCL9/Mig was observed 24 to 72 h posttransplant in allografts but not isografts. CXCL9 was produced by donor cells in response to IFN-γ made by recipient CD8 T cells reactive to donor class I major histocompatibility complex (MHC) molecules. Activated CD8 T cells were detected in allografts at least 3 days before donor-specific effector T cells producing IFN-γ were detected in the recipient spleen. Early inflammation mediated by donor-reactive CD8 memory T cells greatly enhanced primed effector T-cell infiltration into allografts. These results suggest that strategies for optimal inhibition of alloimmunity should include neutralization of infiltrating CD8 memory T cells within a very narrow window after transplantation.     

Contribution of CD40-CD154-mediated costimulation to an alloresponse in vivo.

BACKGROUND: Costimulation through CD40-CD154 plays an important role in T-cell activation. Although systemic administration of anti-CD154 antibody prevents or delays rejection of organ allografts in animal models, the molecular mechanisms responsible for this effect are not well defined. METHODS: We have previously demonstrated that priming of mice (H2d) with CD40-/- but not with wildtype naive B cells (H2b) leads to alloantigen-specific T-cell hyporesponsiveness in vitro. In the present study, we investigated whether such priming modifies allograft rejection in a major histocompatibility complex-mismatched murine cardiac transplantation model. RESULTS: Priming of hosts with donor-specific CD40-/- B cells delayed rejection of subsequently transplanted wild-type cardiac allografts by 8.0 days (P<0.001). The lack of CD40 on the cardiac graft delayed rejection in unprimed or primed hosts by 3-5 days. Prolongation of graft survival correlated with the failure of infused CD40-/- B cells to express B7.2 and ICAM-1 in vivo. CONCLUSIONS: Our data suggest that CD40-CD154 costimulation contributes to T cell priming to alloantigens in vivo and to a second set rejection phase in which donor antigens are presented to primed T cells.     

Cellular infiltrate in renal graft rejection: T lymphocyte subsets detected by monoclonal antibodies

We have examined the interstitial cellular infiltrate using monoclonal antibodies against T cells (Cris 1), helper/inducer T cells (OKT4) and suppressor/cytotoxic T cells (OKT8) by indirect immunofluorescence in renal biopsies taken from 14 transplanted patients during clinical episodes suggestive of acute (n = 9), chronic (n = 2) and no rejection (n = 3). Infiltrating T cells and T cell subsets were found to be significantly increased during all types of rejection (n = 11) as compared to no rejection (n = 3). Two types of biopsies could be distinguished according to the predominance of T cell subsets. In some biopsies (n = 6), OKT8+ cells were significantly more numerous that OKT4+ cells. In the remaining biopsies (n = 5), OKT4+ cells were more common that OKT8+ cells, the OKT4/OKT8 ratio being significantly higher. No association was observed between HLA mismatch and predominating T cell subset, neither for type nor outcome of graft rejection. Our results suggest that the OKT4+ cells may play a more important role than previously reported in renal graft rejection.