Treatment of acute kidney allograft rejection with a non-mitogenic CD3 antibody.

T3/4.A is a non-mitogenic murine IgA mAb to human CD3 that was selected for clinical studies to provide an alternative for the mitogenic, T cell-activating, therapeutic mAb OKT3. Previously, we reported that T3/4.A is better tolerated in humans than the IgG2a-CD3 mAb T3/4.2a. Here we report the results of a phase II clinical trial to assess the immunosuppressive potential of T3/4.A. Eighteen first kidney transplant recipients with a first rejection episode were included. Baseline immunosuppression consisted of cyclosporin and prednisolone. Rejection treatment consisted of 5 mg mAb per day during 10 days. Fourteen patients responded, of whom four experienced a second rejection within 2 weeks, one experienced chronic rejection after 2.5 years, whereas the others remained rejection-free after treatment (median duration of follow-up 42 months). Four patients did not respond and eventually lost their graft. These results are similar to treatment results with OKT3, as reported in the literature. Following the first dose of T3/4.A, side effects were limited, and reduced compared to OKT3-treated controls. On the second day, 15 patients developed transient vomiting and/or diarrhoea, which coincided with elevated serum levels of proinflammatory cytokines. Minimal or even no side effects occurred during the remaining days, which is in sharp contrast to that seen generally during OKT3 treatment. Both T cell numbers and TCR expression were reduced during the therapy. We conclude that T3/4.A is a good alternative for OKT3 to treat rejection episodes in renal transplant recipients.

Pharmacokinetics of murine anti-human CD3 antibodies in man are determined by the disappearance of target antigen.

Therapy with monoclonal antibodies (mAbs) is characterized by a molar ratio of receptor to drug that is higher than usual in pharmacotherapy. As a consequence, changes in the amount of receptors induced by the therapy may have important consequences for pharmacokinetics. We therefore analyzed the pharmacokinetics and pharmacodynamics of an experimental therapeutic CD3 antibody, CLB-T3/4.A (murine IgA), which was given as a rejection treatment to renal transplant patients. Patients were treated with 5 mg of the mAb, as a daily bolus injection, during 10 days. Mean trough levels of mAbs increased during the 1st week, and decreased thereafter. However, about one-third of the patients had continuously rising trough levels and about one-third displayed a steady state, that was reached only after 4 days. On the first day of treatment, mAb concentrations showed a biphasic plasma disappearance curve. On subsequent days, monophasic plasma disappearance curves were observed with mean half-lives of 6 to 8 h. Administration of the mAb induced disappearance of target antigen from the peripheral blood, which could explain the difference in kinetics between day 1 and subsequent days shown by a simulation of the multidose curve of plasma concentrations, based on target antigen depletion. We conclude that at this dose the pharmacokinetics of CLB-T3/4.A were to a great extent determined by antibody-induced changes in antigen in peripheral blood. Moreover, determinations of pharmacokinetic and pharmacodynamic parameters based on single-dose data and traditional compartment models were inadequate for the purpose of prediction and extrapolation.

The effect of two different dosages of intravenous immunoglobulin on the incidence of recurrent infections in patients with primary hypogammaglobulinemia. A randomized, double-blind, multicenter crossover trial.

BACKGROUND: In patients with hypogammaglobulinemia, substitution with immunoglobulin is the treatment of choice to reduce both frequency and severity of bacterial infections. Even with treatment, however, infections still occur in these patients. OBJECTIVE: To determine whether doubling the standard dose of intravenous immunoglobulin would affect the incidence and duration of infections. DESIGN: Multicenter, double-blind, randomized, crossover study. SETTING: 15 outpatient clinics in the Netherlands. PATIENTS: 43 patients with primary hypogammaglobulinemia, 41 of whom completed the protocol. INTERVENTION: Patients received standard-dose immunoglobulin therapy for 9 months, followed by a 3-month washout period, and high-dose intravenous immunoglobulin therapy for 9 months, or vice versa. MEASUREMENTS: The primary outcome measures were total number and duration of infections. Other measures were periods of fever, hospital admissions, use of antibiotics, absence from school or work, and trough levels of serum immunoglobulin. Side effects from the study medication were also recorded. RESULTS: Compared with the standard dose of intravenous immunoglobulin (adults, 300 mg/kg of body weight every 4 weeks; children, 400 mg/kg every 4 weeks), high-dose therapy (adults, 600 mg/kg every 4 weeks; children, 800 mg/kg every 4 weeks) significantly reduced the number (3.5 vs. 2.5 per patient; P = 0.004) and duration (median, 33 days vs. 21 days; P = 0.015) of infections. Trough levels of IgG increased significantly during high-dose therapy. The incidence and type of side effects did not differ significantly for the two dosages. CONCLUSION: In patients with hypogammaglobulinemia, doubling the standard dose of intravenous immunoglobulin significantly reduced the number and duration of infections.

OKT3 and IL-2 treatment for purging of the latent HIV-1 reservoir in vivo results in selective long-lasting CD4+ T cell depletion.

Activation of resting T cells has been proposed to purge the reservoir of HIV-1-infected resting CD4+ T cells. We therefore treated three HIV-1-infected patients on antiretroviral therapy with OKT3, a CD3 monoclonal antibody, and recombinant human IL-2. Here we report the profound and partially long-lasting host responses induced by the OKT3 and IL-2 treatment. OKT3/IL-2 induced a strong but transient release of plasma cytokines and chemokines. The percentage CD4+ and CD8+ cells in the blood expressing the activation marker CD38 transiently increased to almost 100%, and in lymph nodes we "observed" a 10-fold increase in the number of dividing Ki67+ cells and increased numbers of apoptotic cells. Following OKT3/IL-2 treatment, a long-lasting depletion of CD4+ cells in the peripheral blood and lymph nodes occurred, suggesting the physical deletion of these cells. Increases in CD4+T cell numbers during the two year followup period were due mainly to increased memory cell numbers. CD8+ cells were also depleted in the blood, but less severely in lymph nodes, and returned to baseline levels within several weeks.

Non FcR-binding murine antihuman CD3 monoclonal antibody is capable of productive TCR signalling and induces proliferation in the presence of costimulation.

CLB T3/4.A is a non FcR-binding CD3 mAb of the murine IgA isotype, which may be used as an alternative for the mitogenic OKT3 mAb in the treatment of acute cellular rejection after organ transplantation. We studied TCR signalling and T cell activation in response to T3/4.A in normal human PBMC in vitro. T3/4.A induced a rapid rise in free cytoplasmic Ca(2+), not different from the response to mitogenic CD3 mAb. However, protein tyrosine phosphorylation and, particularly, MAPK activation, were reduced as compared to mitogenic CD3 mAb. T3/4.A enhanced expression of both CD69 and CD25, but proliferation and detectable cytokine production did not occur. Addition of either CD28 mAb or IL-2 induced a strong proliferative response, which was accompanied by cytokine production. At higher mAb concentrations, T cell activation decreased, which correlated with TCR downmodulation. To exclude the possibility that activation by T3/4.A depends on interaction of murine IgA Fc with as yet unknown FcR, we showed that also with CD3 mAb F(ab')2 fragments upregulation of activation molecules occurred, as well as proliferation in the presence of costimulation. We conclude that the non FcR-binding murine IgA mAb T3/4.A acts as a partial agonist and leads to proliferation and cytokine production only in the presence of appropriate costimuli. These findings may explain the mitigated cytokine release syndrome observed in vivo with some nonmitogenic CD3 mAbs.

Changes in the composition of circulating CD8+ T cell subsets during acute epstein-barr and human immunodeficiency virus infections in humans.

In response to viral infection, unprimed naive CD8(+), major histocompatibility complex class I-restricted, virus-specific T cells clonally expand and differentiate into memory- and effector-type cells. Changes in CD8(+) subset distribution were studied in 17 subjects with acute human immunodeficiency virus type 1 infection and in 14 subjects with acute Epstein-Barr virus (EBV) infection, with combined CD45RO, CD27, and CD28 monoclonal antibodies. A vast expansion of memory-type CD45RO(+)CD27(+)CD8(+) T cells, with high expression of the cell-cycle marker Ki-67, was observed in both infections. Strikingly, CD45RO(+)CD27(+)CD28(-) cells increased >10-fold in acute viral infection and had high Ki-67 expression. In acute EBV infection, a substantial portion of the expanded T cells were EBV-peptide specific. These cells resided mainly in the CD45RO(+)CD27(+) subpopulation, with most in the CD27(+)CD28(-) subpopulation. Content of perforin expression, as a measure of cytotoxic capacity, was relatively low in the CD27(+)CD28(+) T cells and highest in the CD27(-)CD28(-) subpopulation.

Nonradioactive techniques for measurement of in vitro T-cell proliferation: alternatives to the [(3)H]thymidine incorporation assay.

T-cell proliferation is an important in vitro parameter of in vivo immune function and has been used as a prognostic marker of human immunodeficiency virus type 1 (HIV-1) disease progression. The proliferative capacity of T cells in response to various stimuli is commonly determined by a radioactive assay based on incorporation of [(3)H]thymidine ([(3)H]TdR) into newly generated DNA. In order to assess techniques for application in laboratories where radioactive facilities are not present, two alternative methods were tested and compared to the [(3)H]TdR assay as a "gold standard." As an alternative, T-cell proliferation was measured by flow cytometric assessment of CD38 expression on T cells and by an enzyme-linked immunosorbent assay (ELISA) based on bromo-2'-deoxyuridine (BrdU) incorporation. Peripheral blood mononuclear cells (PBMCs), either in whole blood or Ficoll-Isopaque separated, from a total of 26 HIV-1-positive and 18 HIV-1-negative Dutch individuals were stimulated with CD3 monoclonal antibody (MAb) alone, a combination of CD3 and CD28 MAbs, or phytohemagglutinin. BrdU incorporation after 3 days of stimulation with a combination of CD3 and CD28 MAbs correlated excellently with the [(3)H]TdR incorporation in both study groups (HIV-1 positives, r = 0.96; HIV-1 negatives, r = 0.83). A significant correlation of absolute numbers of T cells expressing CD38 with [(3)H]TdR incorporation, both in HIV-1-positive (r = 0.96) and HIV-1-negative (r = 0.84) individuals, was also observed under these conditions. The results of this study indicate that determination of both the number of CD38-positive T cells and BrdU incorporation can be used as alternative techniques to measure the in vitro T-cell proliferative capacity. The measurement of CD38 expression on T cells provides the additional possibility to further characterize the proliferating T-cell subsets for expression of other surface markers.

Immuno-activation with anti-CD3 and recombinant human IL-2 in HIV-1-infected patients on potent antiretroviral therapy.

BACKGROUND: A stable reservoir of latently infected, resting CD4 T cells has been demonstrated in HIV-1-infected patients despite prolonged antiretroviral treatment. This is a major barrier for the eradication of HIV by antiretroviral agents alone. Activation of these cells in the presence of antiretroviral therapy might be a strategy to increase the turnover rate of this reservoir. METHODS: Three HIV-1-positive patients on potent antiretroviral therapy, in whom plasma viremia had been suppressed to below 5 copies/ml for at least 26 weeks, were treated with a combination of OKT3 (days 1-5) and recombinant human IL-2 (days 2 6). RESULTS: The side-effects were fever, headache, nausea, diarrhea, and in one of the patients transient renal failure and seizures. The regimen resulted in profound T cell activation. In one patient plasma HIV-1 RNA transiently increased with a peak at 1500 copies/ml. In the other two patients plasma HIV-1 RNA levels remained below the detection limit, but HIV-1 RNA levels in the lymph nodes increased two- to threefold. All patients developed antibodies against OKT3. CONCLUSION: OKT3/IL-2 resulted in T cell activation and proliferation, and could stimulate HIV replication in patients having achieved prolonged suppression of plasma viremia. OKT3/IL-2 therapy was toxic and rapidly induced antibodies against OKT3.

Treatment of patients with systemic sclerosis with extracorporeal photochemotherapy (photopheresis).

BACKGROUND: Effective treatment modalities for systemic sclerosis, a life-threatening and disabling disease, are still lacking. Possible efficacy of photopheresis has been reported in several studies. Because of the complexity of the treatment, placebo-controlled trials are difficult to perform. OBJECTIVE: We investigated the effect of photopheresis on clinical parameters (skin score and internal organ functions), immunologic parameters, and quality of life. METHODS: Nineteen patients with progressive systemic sclerosis of less than 5 years' duration were randomized into 2 groups. One group (group A) received photopheresis for 1 year, the other group (group B) received no treatment at all. After 1 year the groups switched (crossover design). Photopheresis was performed on 2 consecutive days every 4 weeks; the psoralens were administered parenterally. The main outcome parameter was the skin score after 1 year of treatment compared with that of the control group. RESULTS: The average skin score improved with 5.4% (standard error [SE], 20. 8%) in group A and deteriorated with 4.5% (SE, 13.8%) in group B (not significant; P =.71). Before crossover, the average increase in skin score was 5.3% (means of entire group). No change was observed in other clinical parameters. Approximately 1 year after crossover, the skin score reversed to what would have been expected with an average increase of 5.3% per year. There was also no effect on immunologic parameters. Quality of life did not change during treatment. CONCLUSION: We were not able to show that photopheresis, performed as described above, is an effective treatment in systemic sclerosis. The difference in average skin score was statistically and clinically insignificant. Despite the small sample size, we concluded that the magnitude of the observed changes is too small to justify photopheresis as a regular treatment.

Immune reconstitution after 2 years of successful potent antiretroviral therapy in previously untreated human immunodeficiency virus type 1-infected adults.

Today's antiretroviral combination regimens can induce significant and sustained decreases in human immunodeficiency virus (HIV)-RNA levels, allowing the immune system to recover. To what extent immune reconstitution is possible and what factors determine the outcome have thus far not been resolved. We studied 19 subjects, treated for 2 years with protease inhibitor-containing triple therapy, who had a strong suppression of HIV-RNA levels. CD4+ T-cell numbers increased from medians of 170 to 420x106 cells/L, but in a number of subjects T-cell numbers did not further increase after week 72, without having reached normal values. Long-term CD4+ T-cell change was mainly caused by a slow but continuous increase in naive CD4+ T cells (CD45RA+CD62L+) and was predicted by the baseline number of these cells. Our data indicate that long-term immunological recovery is gradual, even during strong suppression of viral replication, not always complete, and dependent on the preexisting level of naive CD4+ T cells.

CD4dullCD8bright double-positive T-lymphocytes have a phenotype of granzyme Bpos CD8pos memory T-lymphocytes.

BACKGROUND: T-lymphocytes that co-express CD4 and CD8 antigens may be found in small percentages in the peripheral blood of healthy individuals, and have a CD4brightCD8dull phenotype. CD4dullCD8bright T-lymphocytes have been found only in temporal association with some viral infections. METHODS: Four-colour flow cytometric analysis of peripheral blood mononuclear cells from a renal transplant recipient with cytomegalovirus infection was performed. RESULTS: A small but clearly distinguishable subpopulation of CD4dullCD8bright double-positive T-lymphocytes was detected, that exhibited phenotypic characteristics of cytotoxic T-lymphocytes and were granzyme B positive. Furthermore, no naive cells appeared to be present within this subpopulation. CONCLUSIONS: CD4dullCD8bright double-positive T-lymphocytes are enriched for memory and effector cytotoxic T cells.

Immune restoration does not invariably occur following long-term HIV-1 suppression during antiretroviral therapy. INCAS Study Group.

BACKGROUND: Current antiretroviral treatment can induce significant and sustained virological and immunological responses in HIV-1-infected persons over at least the short- to mid-term. OBJECTIVES: In this study, long-term immune reconstitution was investigated during highly active antiretroviral therapy. METHODS: Patients enrolled in the INCAS study in The Netherlands were treated for 102 weeks (range 52-144 weeks) with nevirapine (NVP) + zidovudine (ZDV) (n = 9), didanosine (ddl) + ZDV (n = 10), or NVP + ddl + ZDV (n = 10). Memory and naïve CD4+ and CD8+ T cells were measured using CD45RA and CD27 monoclonal antibodies (mAb), T-cell function was assayed by CD3 + CD28 mAb stimulation, and plasma HIV-1 RNA load was measured by ultra-direct assay (cut-off < 20 copies/ml). RESULTS: Compared to both double combination regimens the triple combination regimen resulted in the most sustained increase in CD4+ T cells (change in CD4+, + 253 x 10(6) cells/l; standard error, 79 x 10(6) cells/l) and reduction of plasma HIV-1 RNA. In nine patients (31%) (ddl + ZDV, n = 2; NVP + ddl + ZDV, n = 7) plasma HIV-1 RNA levels remained below cut-off for at least 2 years. On average, these long-term virological responders demonstrated a significantly higher increase of naïve and memory CD4+ T cells (P = 0.01 and 0.02, respectively) as compared with patients with a virological failure, and showed improved T-cell function and normalization of the naïve; memory CD8+ T-cell ratio. However, individual virological success or failure did not predict the degree of immunological response. T-cell patterns were independent of baseline CD4+ T-cell count, T-cell function, HIV-1 RNA load or age. Low numbers of naïve CD4+ T cells at baseline resulted in modest long-term naïve T-cell recovery. CONCLUSIONS: Patients with prolonged undetectable plasma HIV-1 RNA levels during antiretroviral therapy do not invariably show immune restoration. Naïve T-cell recovery in the setting of complete viral suppression is a gradual process, similar to that reported for immune recovery in adults after chemotherapy and bone marrow transplantation.

Reduced naive and increased activated CD4 and CD8 cells in healthy adult Ethiopians compared with their Dutch counterparts.

To assess possible differences in immune status, proportions and absolute numbers of subsets of CD4+ and CD8+ T cells were compared between HIV- healthy Ethiopians (n = 52) and HIV- Dutch (n = 60). Both proportions and absolute numbers of naive CD4+ and CD8+ T cells were found to be significantly reduced in HIV Ethiopians compared with HIV- Dutch subjects. Also, both proportions and absolute numbers of the effector CD8+ T cell population as well as the CD4+CD45RA-CD27- and CD8+CD45RA-CD27- T cell populations were increased in Ethiopians. Finally, both proportions and absolute numbers of CD4+ and CD8+ T cells expressing CD28 were significantly reduced in Ethiopians versus Dutch. In addition, the possible association between the described subsets and HIV status was studied by comparing the above 52 HIV- individuals with 32 HIV+ Ethiopians with CD4 counts > 200/microliter and/or no AIDS-defining conditions and 39 HIV+ Ethiopians with CD4 counts < 200/microliter or with AIDS-defining conditions. There was a gradual increase of activated CD4+ and CD8+ T cells, a decrease of CD8+ T cells expressing CD28 and a decrease of effector CD8+ T cells when moving from HIV- to AIDS. Furthermore, a decrease of naive CD8+ T cells and an increase of memory CD8+ T cells in AIDS patients were observed. These results suggest a generally and persistently activated immune system in HIV- Ethiopians. The potential consequences of this are discussed, in relation to HIV infection.

Expression of granzyme B during primary cytomegalovirus infection after renal transplantation.

CD8+ T cells employ granzyme B (GrB) to induce apoptosis in target cells. Increased expression of GrB has been put forward as a diagnostic marker in transplant rejection and viral infection. Three-color flow cytometric analysis revealed that peripheral blood CD8+ T lymphocytosis during primary cytomegalovirus infection after renal transplantation resulted from expansion of a CD8+GrB+CD62L+ T cell subset that was almost absent during stable transplant function or acute rejection. This expansion coincided with a temporary increase in systemic soluble GrB (sGrB) levels. No such increase was observed during stable transplant function or acute rejection. Thus, the primary immune response to cytomegalovirus infection is accompanied by appearance of CD8+GrB+CD62L+ T cells and increased sGrB levels in the peripheral blood compartment. Determination of the latter may provide a novel approach for monitoring viral infections.

Guidelines for the optimal use of muromonab CD3 in transplantation.

Muromonab CD3 (OKT3), the murine anti-CD3 monoclonal antibody (mAb) of the IgG-2a class, directed against the CD3 molecule on the surface of human T cells, has proven to be a powerful immunosuppressive agent in solid organ transplantation and has been shown to be superior to high-dose steroids as first-line treatment of acute allograft rejection. It is comparable to antithymocyte globulin (ATG) in treating steroid-resistant rejection and it is also effective as rescue treatment in ATG-resistant rejection. However, OKT3 treatment is followed by a substantial percentage of re-rejections, most of which respond well to steroids. In the early post-transplant period, a prophylactic course of OKT3 can delay the onset of acute rejection, in particular in immunologically high-risk patients. First-dose-related adverse effects (pyrexia, dyspnoea, headache, nausea, vomiting and diarrhoea) of OKT3 are severe, but usually transient and seldom life-threatening, provided overhydration has been corrected and steroids have been given before the first administration. These adverse effects are partly attributed to the release of cytokines as a result of mononuclear cell activation. In addition, complement activation and subsequent activation of neutrophils may play a role in their pathogenesis. After exposure of patients to OKT3 an increased incidence of infections and malignancies has been reported. However, most likely this merely reflects the total burden of immunosuppression. Xenosensitisation represents an important limitation to OKT3 treatment, although a second or third course can still be effective in patients with low antibody titres. In practice, the initiation of OKT3 treatment should be preceded by correction of overhydration, if necessary by means of dialysis or ultrafiltration. According to the instructions from the manufacturer the first dose of OKT3 is preceded by paracetamol (acetaminophen), an antihistamine and intravenous (IV) corticosteroids, in an attempt to mitigate the first dose-related symptoms. A regimen consisting of administration of 2 IV doses of 250mg methylprednisolone, given 6 hours and 1 hour before the first OKT3 injection, followed by a 2-hour infusion of OKT3, is most effective in diminishing OKT3-induced adverse effects.