Monoclonal OKT3 Research Articles

Massive elevation of procalcitonin plasma levels in the absence of infection in kidney transplant patients treated with pan-T-cell antibodies.

To determine the value of procalcitonin (PCT) monitoring in transplant patients receiving pan-T-cell antibody therapy. Retrospective clinical study. A collaborative study between the Institute of Medical Immunology, the Department of Nephrology and Internal Intensive Care, both Charite, Humboldt University Berlin, and the Department of Laboratory Medicine, Friedrichshain Hospital, Berlin, Germany. Thirty-one patients were included in the study: 8 kidney transplant patients with acute rejection episodes, 5 receiving OKT3 monoclonal antibody therapy, 3 receiving steroid bolus therapy; 21 patients undergoing renal transplantation, 11 receiving ATG perioperatively, 10 without ATG administration; 2 patients undergoing renal transplantation and receiving anti-IL-2R mAb. Procalcitonin (PCT) and tumor necrosis factor (TNF) alpha plasma levels were measured in infection-free transplant patients treated with the pan-T-cell antibodies ATG or OKT3. We found PCT plasma concentrations up to 600 ng/ml (reference < 0.5 ng/ ml), which are comparable to those seen in severe sepsis. Increases in TNF-alpha plasma levels preceded the rises in PCT. After peaking on day 1 of therapy the PCT plasma concentrations returned to normal values independently of further antibody administration. In contrast, steroid bolus therapy or anti-interleukin 2 receptor mAb administration did not increase plasma PCT or TNF-alpha levels. PCT monitoring for evaluating infectious complications in kidney transplant patients must be very careful during pan-T-cell antibody therapy.

Factors affecting neutrophil and platelet reconstitution following T cell-depleted bone marrow transplantation: differential effects of growth factor type and role of CD34+ cell dose

We have performed univariate and multivariate analysis to determine the factors that affect the kinetics of neutrophil and platelet recovery in 546 recipients of T cell-depleted (TCD) marrow allografts. All patients received marrow depleted of mature CD3(+) T cells by complement-mediated lysis using T(10)B(9)-1A3 (n = 489) or Muromonab-Orthoclone OKT3 (n = 57) monoclonal antibodies. Neutrophil engraftment to 0.5 x 10(9)/1 and platelet engraftment to 20 x 10(9)/l were assessed as endpoints. Factors significantly affecting neutrophil or platelet engraftment in the univariate analysis included patient age, T cell dose, anti-thymocyte globulin use, gender, diagnosis at transplant, CMV serostatus, HLA mismatch, CD34 cell dose (n = 249), and growth factor use and type. These variables were included in the multivariate Cox proportional hazards regression model. The results showed that a faster rate of neutrophil engraftment was independently associated with CD34(+) cell dose > or = 5 x 10(6)/kg and most strongly with growth factor administration. Faster platelet engraftment was associated with transplantation for chronic leukemia, CD34(+) cell dose > or = 2 x 10(6)/kg, an HLA matched related donor, and the absence of growth factor use. G-CSF had a higher relative risk (RR) of enhancing neutrophil engraftment than GM-CSF and significantly delayed platelet engraftment. The combined use of G-CSF + GM-CSF was similar to G-CSF alone. The enhancing effect of G-CSF for neutrophil recovery was most striking for patients who engrafted to 0.5 x 10(9)/1 at or before day 12 (RR = 9.5, P < 0.0001) compared to patients who received no growth factor. Conversely, the delaying effect of G-CSF on platelet engraftment was strongest for patients engrafting on or before day 25 (RR = 0.4, P = 0.0004). Of the independent variables affecting engraftment kinetics in recipients of TCD marrow allografts only growth factor, and to a limited extent, CD34(+) cell dose can be controlled by the clinician. A higher CD34(+) cell dose enhances the rate of both neutrophil and platelet engraftment whereas for G-CSF the benefits of myeloid growth factor use in enhancing neutrophil recovery may be partly offset by a delay in platelet engraftment.

Use of OKT3 for acute myocarditis in infants and children

Acute viral myocarditis triggers an autoimmune phenomenon that aggressive immunosuppressive therapy with monoclonal OKT3 may suppress. We treated 5 patients, aged 15 months to 16.5 years, who had acute viral myocarditis and left ventricular ejection fraction (LVEF) of 5% to 20%, with a combination immunosuppressive regimen that included OKT3, intravenous immunoglobulin, methylprednisone, cyclosporine, and azathioprine. Within 2 weeks of therapy, all patients demonstrated normalization of LVEF to 50% to 74%, and on mid-term follow-up, we have found no recurrence of heart failure or progression to dilated cardiomyopathy. In patients with severe acute myocarditis, aggressive immunosuppressive regimen based on OKT3 is safe and may inhibit or reverse the immune response, resulting in dramatic improvement in myocardial function.

Reduction of the HIV-1-infected T-cell reservoir by immune activation treatment is dose-dependent and restricted by the potency of antiretroviral drugs.

Treatments combining T-cell activating agents and potent antiretroviral drugs have been proposed as a possible means of reducing the reservoir of long-lived HIV-1 infected quiescent CD4 T-cells. To analyse the effect of such therapies on HIV-1 dynamics and T-cell homeostasis. A mathematical framework describing HIV-1 dynamics and T-cell homeostasis was developed. Three patients who were kept on a particularly potent course of highly active antiretroviral therapy (HAART) were treated with the anti-CD3 monoclonal antibody OKT3 and interleukin (IL)-2. Plasma HIV-RNA, and HIV-RNA and DNA in peripheral blood mononuclear cells and lymph node mononuclear cells were measured. These results and other published studies on the use of IL-2 alone were assessed using our mathematical framework. We show that outcome of treatment is determined by the relative rates of depletion of the infected quiescent T-cell population by activation and of its replenishment through new infection. Which of these two processes dominates is critically dependent on both the potency of HAART and also the degree of T-cell activation induced. We demonstrate that high-level T-cell stimulation is likely to produce negative outcomes, both by failing to reduce viral reservoirs and by depleting the CD4 T-cell pool and disrupting CD4/CD8 T-cell homeostasis. In contrast, repeated low-level stimulation may both aid CD4 T-cell pool expansion and achieve a substantial reduction in the long-lived HIV-1 reservoir. Our analysis suggests that although treatment that activates T-cells can reduce the long-lived HIV-1 reservoir, caution should be used as high-level stimulation may result in a negative outcome.

BIOENGINEERED MONOCLONALS-EASIER ON PATIENTS, BUT STILL HARD ON REJECTION?

BIOENGINEERED MONOCLONALS-EASIER ON PATIENTS, BUT STILL HARD ON REJECTION?

The fate of renal allografts treated with OKT3 for steroid-resistant rejection

WITH CURRENT immunosuppressive protocols, acute rejection occurs in 30 to 70% of kidney transplant recipients, and there are data suggesting that one or more acute rejection episodes may have detrimental effects in graft survival, both in the short and long term. It is also accepted that steroid-resistant rejection may carry a worse prognosis for reversal, and that even those grafts that are rescued with antilymphocyte therapies might be fated to a short survival. The monoclonal antibody OKT3 is used for the treatment of steroid-resistant rejection in 20 to 40% of kidney transplant recipients and the short-term reversal rates are around 80 to 90%. However, the fate of OKT3rescued kidneys is less well established. We analysed graft function and survival and patient survival, in the short and long term, in patients that never had an acute rejection episode, in those with acute rejection(s) treated with highdose steroids, and in patients in whom the corticosteroids were followed by OKT3 as rescue therapy for rejection.

Age-related impairments in TCR/CD3 activation of ZAP-70 are associated with reduced tyrosine phosphorylations of ζ-chains and p59 fyn/p56 lck in human T cells

The expression and catalytic activity of the protein tyrosine kinase (PTK) ZAP-70 are needed for normal intracellular signaling through the T-cell receptor (TCR)/CD3 complex. However, the possible effect of aging on the catalytic activity of ZAP-70 in human peripheral blood T cells stimulated via the TCR/CD3 complex is unknown. The current studies show that T cells from a substantial proportion of elderly humans (12) exhibit significant reductions in the catalytic activity, but not expression of ZAP-70 when stimulated by ligation of the TCR/CD3 with cross-linked anti-CD3ε monoclonal antibody OKT3. In addition, the reduced catalytic activity of ZAP-70 in T cells from elderly subjects was not restored to the normal levels in response to ligation of CD4 receptors, suggesting defects in PTKs linked to both CD3 and CD4 receptors. Other experiments demonstrated that the age-related impairments of ZAP-70 activation in anti-CD3-stimulated T cells were accompanied by decreased tyrosine phosphorylations of ζ-chains and autophosphorylations of the PTKs p56 lck/p59 fyn. Moreover, the age-related defects in these early TCR/CD3-mediated phosphorylation events were readily detectable in both CD45RO + memory and CD45RA + naive T cells. Thus, these results suggest that defects in early TCR/CD3-mediated phosphorylation events among CD45RO + memory and CD45RA + naive T cells from certain elderly humans may contribute to impaired induction of ZAP-70 catalytic activity.

Tyrosine phosphorylation in peripheral T cells of kidney transplant recipients: analyses of baseline levels and response to T cell receptor stimulation.

Impaired immunosurveillance in recipients of organ transplants has been attributed to alleviation of T cell functions. We analyzed the phosphorylation of tyrosine residues in T cells of peripheral blood, and after T cell receptor (TCR) stimulation. The TCR was stimulated by OKT3 monoclonal antibody (mAb) in non-separated heparinized blood specimens of patients (n=64) and healthy controls (n=25). After fixation and red cell lysis, lymphocytes were permeabilized by saponin. Subsequently, intracellular phosphotyrosine residues and surface CD3 antigen were stained simultaneously with specific mAbs. We analyzed transplant recipients and healthy donors for baseline levels of total cellular tyrosine phosphorylation and for increase in phosphotyrosine content following stimulation by OKT3. Phosphotyrosine levels were significantly lower in non-stimulated T cells of kidney transplant recipients compared to controls (p=0.004). There was a marked variability in the levels of tyrosine phosphorylation among transplanted patients (p=0.02). T cell receptor stimulation by OKT3 mAb in vitro led to a strong increase of tyrosine phosphorylation in all specimens of patients and healthy controls. In conclusion, we demonstrated decreased phosphotyrosine levels in T cells of kidney transplant recipients compared to healthy donors. However, increase in tyrosine phosphorylation was not impaired in all patients as a result of TCR stimulation.

Age-related defects in Th1 and Th2 cytokine production by human T cells can be dissociated from altered frequencies of CD45RA + and CD45RO + T cell subsets

The present study investigated whether age-related changes in the production of Th1 and Th2 cytokines by human T cells might be linked to altered frequencies of naive (CD45RA +) and memory (CD45RO +) T cell subsets. T cells from healthy elderly humans ( n=32) stimulated with anti-CD3ε monoclonal antibody OKT3 plus PMA produced significantly lower levels of IL-2 and IFNγ (Th1 type) and of IL-4 (Th2 type) cytokines compared with T cells from young subjects. Although considerable heterogeneity was observed in the levels of cytokines produced by activated T cells from elderly individuals, linear regression analysis failed to demonstrate any significant shift in Th1 to Th2 type cytokine profiles of human T cells during aging. Sufficient T cells were available from eighteen elderly subjects to quantitate the levels of cytokine production in parallel with flow cytometry analysis of the frequencies of CD45RA + naive and CD45RO + memory T cells. Compared with the group of young subjects, the elderly group exhibited significant decreases in the frequencies of naive T cells with reciprocal increases in memory T cells. However, defects in Th1 and Th2 cytokine production were not significantly correlated with altered frequencies of naive/memory T cells among elderly individuals. In addition, those elderly individuals with normal frequencies of naive/memory T cells exhibited decreases in cytokine production comparable to the reductions observed for elderly donors with alterations in the frequencies of naive/memory T cells. These findings suggest that age-related defects in Th1 and Th2 cytokine production cannot be attributed entirely to alterations in the frequencies of naive/memory T cell subsets and point toward intrinsic aberrancies within human T cell cytokine networks during aging.

Single Center Experience of En Bloc Kidney Transplantation and Review of Literature

Cadaveric renal transplant was started at King FaisalSpecialist Hospital and Research Centre in January 1987. Asthe number of end-stage renal disease patients requiringtransplant was steadily increasing, and the shortage of organswas becoming more severe, we began using cadavericpediatric kidneys in 1989. When the age of the donor was lessthan three years, we used both kidneys together (en bloc) forone recipient.Materials and MethodsFrom 1989 to 1997, we performed seven en bloctransplants from cadaveric donors aged between seven monthsand three years. Five of the seven patients were adults, and twowere pediatric recipients. It was the second transplant in thefirst three recipients. The donor aorta and vena cava weresewn at their proximal ends. The distal end of the aorta wasanastomosed to the external iliac artery and the vena cava tothe external iliac vein. The ureters were sutured to make asingle opening and anastomosed to the bladder as extravesicalureteroneocystostomy (Figure 1).All the patients except Case numbers 3 and 7 receivedquadruple sequential therapy ALG or ATG, prednisone,cyclosporin or FK506 and Imuran or M ycophenolate Mofetil(MMF). The monoclonal antibody OKT3 was used for steroid-resistant rejection. FK506 was used as first-line treatment, oras rescue therapy in resistant rejection. Of late, MMF is beingused as an initial immunosuppression.ResultsCase number 1 received an en bloc kidney transplant in1989, and was given anti-thymocyte globulin (ATG) asinduction. The patient developed two episodes of biopsy-proven rejection at 21 days and at two months, and was givenOKT3 for 10 days. The kidneys never functioned andtransplant nephrectomy was performed on the 68th day. The

ANALYSIS OF IL-2 TRANSCRIPTIONAL EVENTS IN ANERGIZED HUMAN T-CELLS

263 Introduction: The mechanisms responsible for the induction and maintenance of clonal anergy are not well understood. We have established a human in vitro model of T-cell anergy to explore the perturbations in cell signaling at the level of IL-2 gene transcription, and to define the contribution of other cytokines to this effect. Methods: An in vitro model of clonal anergy was established using peripheral T-lymphocytes from healthy human donors. CD4+-T-cells were anergized by pre-stimulation with the anti-CD3 monoclonal antibody OKT3 followed by restimulation 72 hours later with OKT3 with or without anti-CD28. Proliferation was measured by [3H]- thymidine incorporation and IL-2 production using ELISA. Results: CD4+-T-cells anergized with OKT3 displayed a marked reduction in proliferation (4,824 ± 461 cpm vs 134,134 ± 36505 cpm, p=0.0036).) and IL-2 production (<4 pg/ml vs 512 ± 49 pg/ml, p<0.0001) compared with controls. Simultaneous treatment with anti-CD28 prevented induction of anergy measured either by proliferation (80,639 ± 10652 cpm, p=0.0002) or IL-2 production (471 ± 22 pg/ml, p<0.0001). Co-incubation with IL-10 reduced cellular proliferation in OKT3/CD28 pretreated cells by 19% (p = n.s.) and reduced IL-2 production by 40% (p=0.0024), suggesting an additional effect of IL-10 in anergizing T-cells. Binding of the transcription factor AP-1 was measured by gel-shift using a non-radioactive method. Anergized T-cells demonstrated a clearly reduced binding activity of the AP-1 protein complex, as did fully stimulated (anti-CD3+anti-CD28) T-cells treated with anti-CD40L MoAb, suggesting that members of the Fos- and Jun-family play a crucial role in the signaling events leading to clonal anergy. Supershift experiments performed using specific antibodies against c-Fos, FosB, JunB and JunD confirmed that the binding of c-Fos, JunB and JunD, but not of FosB was reduced in anergized cells by comparison with controls. Conclusion: T-cell anergy induced by OKT3 is characterized by reduced proliferation and a profound decrease in IL-2 production. Anergy can be prevented by co-incubation with anti-CD28 and re-established by IL-10 or anti-CD40L. Anergy is accompanied by a defect in AP-1 binding to the IL-2 gene promoter, with selective reduction in binding of c-Fos, JunB and JunD.

In vivo generation of C4d, Bb, iC3b, and SC5b-9 after OKT3 administration in kidney and lung transplant recipients.

OKT3 monoclonal antibody therapy results in an acute clinical syndrome (ACS) associated with the release of tumor necrosis factor and sequestration of neutrophils in the lungs. We have previously shown that inhibition of tumor necrosis factor does not completely eliminate OKT3-ACS, suggesting that other factors also contribute to the ACS. The current studies analyzed complement activation in vivo during the first hour after OKT3 administration. Renal (n=4) and lung (n=4) transplant recipients received OKT3 as treatment for rejection and induction therapy, respectively. Complement activation products C4d, Bb, iC3b, and SC5b-9 were measured by ELISA. Hemodynamic parameters were also monitored in the lung transplant recipients. Neutrophil expression of CD11a, CD11b, and CD18 was monitored by flow cytometry. Controls included patients receiving methylprednisolone for rejection (n=4), two adults with adult respiratory distress syndrome who received extracorporeal membrane oxygenation, and normal volunteers (n=5). P values less than 0.05 (*) were considered significant. Increases in the plasma levels of C4d, Bb, iC3b, and SC5b-9 were observed in seven of eight patients after OKT3 administration. Mean values (n=8) at 0, 15, and 60 min (in microg/ml) were as follows-C4d: 1.865, 2.644*, and 2.607*; Bb: 0.245, 0.411, and 0.385; iC3b: 10.881, 17.242*, and 15.145*; and SC5b-9: 0.232, 0.269, and 0.302*. An increase in CD11b and CD18 and a decrease of CD11a on neutrophils in parallel with complement activation was observed. In lung transplant recipients, C3 activation correlated with increases in mean pulmonary and central venous pressures (P<0.05). As compared with extracorporeal membrane oxygenation, which activated classical and alternative pathways, OKT3 predominantly activated complement by the classical pathway. Methylprednisolone pulses did not activate complement. Complement activation is an early event after OKT3 administration and is associated with the increased expression of adhesion molecules on neutrophils and with pulmonary hemodynamic changes. Effective therapeutic approaches to the control of early monoclonal antibody side effects may require measures that limit complement activation in addition to reducing cytokine activity.

Expansion of Philadelphia Chromosome–Negative CD3+CD56+ Cytotoxic Cells From Chronic Myeloid Leukemia Patients: In Vitro and In Vivo Efficacy in Severe Combined Immunodeficiency Disease Mice

We have developed culture conditions for the efficient expansion of cytotoxic effector cells from peripheral blood mononuclear cells (PBMNCs) by the timed addition of interferon-γ (IFN-γ), interleukin-2 (IL-2), and the monoclonal antibody (MoAb) OKT3. These cells, termed cytokine-induced killer (CIK) cells, are composed primarily of T cells, and the population of cells with the greatest cytotoxic activity is an otherwise rare population of CD3+CD56+ cells that expand dramatically under these culture conditions. CIK cells were expanded from PBMNCs from 13 patients with chronic myeloid leukemia (CML). These cultures contained a variable number of T cells at the start of the culture (median 44%, range 1% to 64%), yet after 21 to 28 days of culture, virtually all of the cells were CD3+ T cells (median 97%, range 90% to 99%). The CD3+CD56+subset of cells expanded significantly (median 25-fold, range 2.2- to 525-fold). CIK cells from all patients showed cytotoxicity against the tumor cell lines OCI-LY8 and K562. In four patients the expanded CIK cells suppressed colony growth of autologous CML blast cells and myeloid progenitor cells. Allogeneic CIK cells from normal donors also suppressed CML colony growth but did not inhibit growth of normal hematopoietic colonies. Twelve of the 13 cultures were exclusively composed of Philadelphia (Ph)-negative cells and one culture had 1 out of 20 Ph-positive metaphases after 4 weeks in culture. Intracellular cytokine production was assayed by fluorescence-activated cell sorter (FACS), and the expanded T-cell cultures produced IL-2, IFN-γ, and tumor necrosis factor- (TNF-), but not IL-4. Both the CD4+ and CD8+ subsets secreted this cytokine profile. To test the in vivo activity of the expanded CIK cells, CML was engrafted into severe combined immunodeficiency disease (SCID) mice using matrigel. After 4 weeks, 4 × 107autologous CIK cells were injected intravenously by tail vein injection into groups of mice, and the animals were sacrificed after a total of 18 weeks. Bcr-abl was detected in the bone marrow or spleen of 5 out of 6 control mice and only 2 out of 13 mice who received the autologous CIK cells (P = .02). In an additional series of animals, the mice did not engraft with CML but instead developed large human Epstein-Barr virus–associated lymphomas by 12 weeks. The mice who received autologous CIK cells at 4 weeks had either no tumor (5) or small tumors (5), whereas all 10 mice that received CIK cells at week 8 developed lymphomas; however, these were not as large as in the 10 control mice who did not receive CIK cells (P = .03). This study shows that CIK cells, which are Ph chromosome–negative, can be expanded from patients with CML and have potent in vitro and in vivo efficacy against autologous tumor cells. © 1998 by The American Society of Hematology.

Expansion of Philadelphia Chromosome–Negative CD3+CD56+ Cytotoxic Cells From Chronic Myeloid Leukemia Patients: In Vitro and In Vivo Efficacy in Severe Combined Immunodeficiency Disease Mice

AbstractWe have developed culture conditions for the efficient expansion of cytotoxic effector cells from peripheral blood mononuclear cells (PBMNCs) by the timed addition of interferon-γ (IFN-γ), interleukin-2 (IL-2), and the monoclonal antibody (MoAb) OKT3. These cells, termed cytokine-induced killer (CIK) cells, are composed primarily of T cells, and the population of cells with the greatest cytotoxic activity is an otherwise rare population of CD3+CD56+ cells that expand dramatically under these culture conditions. CIK cells were expanded from PBMNCs from 13 patients with chronic myeloid leukemia (CML). These cultures contained a variable number of T cells at the start of the culture (median 44%, range 1% to 64%), yet after 21 to 28 days of culture, virtually all of the cells were CD3+ T cells (median 97%, range 90% to 99%). The CD3+CD56+subset of cells expanded significantly (median 25-fold, range 2.2- to 525-fold). CIK cells from all patients showed cytotoxicity against the tumor cell lines OCI-LY8 and K562. In four patients the expanded CIK cells suppressed colony growth of autologous CML blast cells and myeloid progenitor cells. Allogeneic CIK cells from normal donors also suppressed CML colony growth but did not inhibit growth of normal hematopoietic colonies. Twelve of the 13 cultures were exclusively composed of Philadelphia (Ph)-negative cells and one culture had 1 out of 20 Ph-positive metaphases after 4 weeks in culture. Intracellular cytokine production was assayed by fluorescence-activated cell sorter (FACS), and the expanded T-cell cultures produced IL-2, IFN-γ, and tumor necrosis factor-α (TNF-α), but not IL-4. Both the CD4+ and CD8+ subsets secreted this cytokine profile. To test the in vivo activity of the expanded CIK cells, CML was engrafted into severe combined immunodeficiency disease (SCID) mice using matrigel. After 4 weeks, 4 × 107autologous CIK cells were injected intravenously by tail vein injection into groups of mice, and the animals were sacrificed after a total of 18 weeks. Bcr-abl was detected in the bone marrow or spleen of 5 out of 6 control mice and only 2 out of 13 mice who received the autologous CIK cells (P = .02). In an additional series of animals, the mice did not engraft with CML but instead developed large human Epstein-Barr virus–associated lymphomas by 12 weeks. The mice who received autologous CIK cells at 4 weeks had either no tumor (5) or small tumors (5), whereas all 10 mice that received CIK cells at week 8 developed lymphomas; however, these were not as large as in the 10 control mice who did not receive CIK cells (P = .03). This study shows that CIK cells, which are Ph chromosome–negative, can be expanded from patients with CML and have potent in vitro and in vivo efficacy against autologous tumor cells.© 1998 by The American Society of Hematology.

Phosphorylation and coupling of ζ-chains to activated T-cell receptor (TCR)/CD3 complexes from peripheral blood T-cells of elderly humans

Aging is often accompanied by altered T-cell signaling and functions. Signals mediated through the T-cell receptor (TCR)/CD3 complex are associated with tyrosine phosphorylations of ζ-chains by the regulated activities of protein tyrosine kinases p56 lck and p59 fyn as well as protein tyrosine phosphatases. In the present investigation, the coupling and phosphorylation of ζ-chains to TCR/CD3 immunocomplexes were examined in peripheral blood T-cells from 13 elderly and young humans stimulated by ligation of the TCR/CD3 with cross-linked anti-CD3 ϵ monoclonal antibody OKT3. Western blots analyzing the non-covalent coupling of ζ-chains to TCR/CD3 immunocomplexes from Brij-96 detergent lysates of anti-CD3 ligated T-cells showed that the levels of ζ-chains within TCR/CD3 immunocomplexes from T-cells of elderly and young subjects did not significantly differ. By contrast, the levels of phosphorylated ζ-chains generated during in vitro phosphorylations of TCR/CD3 immunocomplexes from elderly subjects were significantly reduced and averaged 44% of those observed for anti-CD3 ϵ ligated T-cells from young subjects. Analyses of the levels of ζ-chain coupling and phosphorylations in T-cells from each of the 13 elderly individuals also showed that the reductions in ζ-chain phosphorylations were heterogeneous and unrelated to modest reductions in coupling. Furthermore, the age-related decreases in ζ-chain phosphorylations were not due to diminished frequencies of CD3 ϵ + cells or densities of CD3 ϵ surface receptors and could be observed without reductions in ϵ-chain phosphorylations. These results suggest that aberrancies of ζ-chain phosphorylations can occur in T-cells of elderly humans independent from any uncoupling of ζ-chains to activated TCR/CD3 complexes.

Specific targeting of cytokine-secreting cells: a bispecific diabody recognizing human interleukin-6 and CD3 induces T cell-mediated killing.

Cytokines have been implicated in the pathophysiology of many diseases. Although there have been many attempts to neutralize the activity of cytokines in vivo and in vitro, no strategies have been developed to specifically eliminate cells that overexpress cytokines. Considering the fact that cytokines in part remain cell associated on secretion, we have constructed a bispecific diabody consisting of a nonneutralizing scFv antibody recognizing human interleukin-6 (IL-6) and an scFv corresponding to the monoclonal antibody (mAb) OKT3, which recognizes and activates the human T cell receptor. Here we show that the diabody recognized both human IL-6 and human CD3. In the presence of human T cells, the diabody induced killing of human hepatoma cells that had been transfected with a human IL-6 cDNA. The extent of killing was dependent on the ratio of effector/target cells and increased with increasing concentrations of the diabody. Untransfected control cells or human hepatoma cells that secrete the IL-6-related cytokine leukemia inhibitory factor (LIF) remained unaffected. We conclude that diabodies recognizing cytokines can be used to specifically target cytokine-secreting cells.

Mycophenolate mofetil, together with cyclosporin A, prevents anti-OKT3 antibody response in kidney transplant recipients.

OKT3 monoclonal antibody, a murine IgG2a monoclonal antibody targeting the T cell CD3 antigen, elicits a neutralizing humoral response in 20 to 50% of kidney transplant recipients when the concomitant immunosuppression consists of CsA-Sandimmun (SAND) and azathioprine (AZA). In the present study, we investigated the impact of the newer agents, CsA-Neoral (NEO) and mycophenolate mofetil (MMF) on OKT3 sensitization. Sixty-two consecutive kidney transplant recipients received prophylactic OKT3 (5 mg/d) from days 0 to 13, together with steroids. Concomitant immunosuppression consisted of either AZA + SAND (n=20), AZA + NEO (n=31), or MMF + NEO (n=11). The following doses were used: AZA, 2 mg/kg per d from days 0 to 13, then 1 mg/kg per d; MMF, 2 g/d starting on day 1; and CsA, either SAND or NEO, 6 mg/kg per d from day 6. At least two serum samples per month were available during the initial 3 mo for each patient. IgG anti-OKT3 antibodies were first evaluated by enzyme-linked immunosorbent assay. Patients were considered sensitized if their serum scored positive at a dilution > or = 1/1000. Peak titers of IgG anti-OKT3 antibodies and the incidence of patients harboring neutralizing anti-idiotypic antibodies were also determined. A first reduction in OKT3 sensitization was seen in patients receiving Neoral instead of Sandimmun (AZA + SAND: 10 of 20 [50%] patients sensitized versus 6 of 31 [19%] in the AZA + NEO group; P=0.03). This was probably related to the achievement of higher mean CsA trough blood levels in the NEO group during the first month (253+/-44 versus 186+/-49 ng/ml in SAND patients). Peak antibody titers and the proportion of patients with anti-idiotypic antibodies were similar in the AZA + SAND and AZA + NEO groups. A further reduction in the sensitization rate was observed with the replacement of AZA by MMF (MMF + NEO: 0% sensitized patients; P=0.0013). It is concluded that the combination of CsA-Neoral and MMF efficiently prevents sensitization against OKT3.

Tacrolimus for steroiD-resistant liver rejection in children

Abstract Eighteen pediatric liver transplant recipients were converted from cyclosporine-based immunosuppression to tacrolimus for refractory rejection episodes affecting 21 grafts. Before conversion, steroid boluses were applied to all episodes followed by OKT3 monoclonal antibodies in 3 of them. Baseline biopsy showed cellular rejection in 18 patients and ductopenia in 3 cases. Thirteen episodes initiated within the first 2 postoperative weeks, and 8 occurred beyond the 21st day. A previous steroiD-responsive episode of rejection was noted in 4 patients. Tacrolimus was administered by the oral route to obtain trough blood levels in the range 6–15 ng/ml. Reversal of rejection was obtained in 15 patients (71.4%). Complete normalization of liver function tests was achieved in 10 out of 12 patients who were followed for more than 6 months. A refractory evolution affected 6 patients (28.5 %). Significant factors predictive for tacrolimus-resistant rejection were identified as ductopenia on baseline biopsy, previous episodes of acute rejection, late onset rejection (beyond 21st posttransplant (day), and a longer time of evolution of rejection prior to conversion.

TRENDS IN IMMUNOSUPPRESSION AFTER PANCREAS TRANSPLANTATION: EVOLUTION OR REVOLUTION?

694 According to Registry data, nearly 10,000 pancreas transplants (PTXs) have been performed through 1997, with 88% being simultaneous kidney-PTXs (SKPT). Current 1 year patient survival exceeds 90% and pancreas graft survival exceeds 80% for SKPT, 70% for sequential PTX after kidney transplant (PAKT), and 60% for PTX alone (PTA). Rejection accounts for 32% of graft failures in the first year after PTX. However, improving outcomes are expected to continue with the evolution of treatment protocols. Most PTX centers employ quadruple immunosuppression with anti-lymphocyte induction (ALI) with either the monoclonal antibody OKT3 or polyclonal agents such as ATG. From 1987-1997, the use of polyclonal agents decreased from 61% to 29%, while the use of OKT3 for induction increased from 28% to 50%. The proportion of patients receiving ALI therapy was higher in PTA recipients (94%) than in SKPT (88%) or PAKT (86%) recipients. In recent years, there has been an overall decline in the use of ALI therapy from 90% during 1987-1993 to 83% of pancreas recipients transplanted during 1994-1997. Maintenance immunosuppression has been triple therapy consisting of a calcineurin inhibitor [cyclosporine (CSA) or tacrolimus (TAC)], corticosteroids, and an anti-metabolite [azathioprine (AZA) or mycophenolate mofetil (MMF)]. Prior to 1995, nearly all PTX recipients were managed with CSA-Sandimmune. In the Sandimmune era, graft survival following SKPT or PTA was higher in patients receiving ALI. However, PAKT recipients receiving OKT3 induction had reduced graft survival compared to either polyclonal ATGs or no induction. In the last 3 years, TAC-based therapy has been used in approximately 20% of cases and the microemulsion formulation of CSA (Neoral) has replaced Sandimmune. From 1994-1997, the results of TAC versus CSA-based therapy have been similar for SKPT and PTA recipients. However, one year graft survival for PAKTs receiving TAC is significantly higher. In addition, MMF is replacing AZA in contemporary PTX immunosuppression. At present, a number of centers are conducting various trials with new drug combinations including either CSA-Neoral or TAC in combination with steroids and MMF with or without ALI therapy. From 1994-1997, the 1 year rates of immunologic graft loss have decreased to 2% after SKPT, 9% after PAKT, and 16% after PTA. Immunosuppressive strategies will continue to evolve to achieve effective control of rejection while minimizing injury to the allograft(s) and risks to the patient. New regimens must not only address the issue of specific drug toxicities but also long-term economic, metabolic, and quality of life outcomes.

RECOMBINANT HUMAN TUMOR NECROSIS FACTOR RECEPTOR Fc FUSION PROTEIN(TNFR:Fc) THERAPY IN KIDNEY TRANSPLANT RECIPIENTS RECEIVING OKT3 INDUCTION THERAPY

294 The monoclonal antibody OKT3 is widely used for induction and treatment of rejection in organ transplant recipients. However, initial doses of the drug are often associated with a cytokine-induced acute clinical syndrome (ACS), which appears to be primarily mediated by TNFα. Several approaches have been used to minimize OKT3-ACS symptoms including pretreatment with corticosteroids, IL-10, and anti-TNF monoclonal antibodies. This study examined the safety of a recombinant human TNF receptor Fc fusion protein(TNFR:Fc) administered post-operatively to kidney transplant recipients, and the ability of the drug to reduce OKT3-ACS symptoms. Methods: Sixteen renal allograft recipients undergoing OKT3 induction therapy were randomized into 4 groups of 4 patients each. All patients received intraoperatively IV azathioprine (Imuran) 3 mg/kg and methylprednisolone 3 mg/kg 1 hour prior to 2.5 mg IV OKT3. Daily IV dosing of 2.5 mg OKT3 was continued for 12 days and CsA was initiated when serum creatinine ≤3.0. The 3 treatment groups received TNFR:Fc (4, 8, or 16 mg/m2) 1 hour prior to OKT3 administration on days 0 and 3. ACS symptoms were assessed on post-transplant days 1 to 3 using a scoring system. Serum cytokine levels(IL-2, IL-4, IL-6, IFNγ, GM-CSF, endothelin-1, NO) were determined using ELISA, RIA, and colorimetry. Serum creatinine and C-reactive protein (CRP) levels were also followed. Results: For most parameters tested there were no differences between groups. Noteworthy findings are summarized:TableThe etiology of the higher incidence of infection in treated patients was unclear. The infections were minor, resolved, and typical of the patient population. The small size of the study and confounding factors such as gender and transplant type (cadaveric vs. living) likely contributed to the observed difference. There were no other differences in adverse event profiles between the two groups. Conclusion: This study suggests that TNFR:Fc is well tolerated by renal transplant patients receiving OKT3 induction therapy. TNFR:Fc therapy may offer some improvement in symptoms associated with OKT3-ACS. Considering the evidence implicating TNFα in the pathogenesis of acute rejection. TNFR:Fc may be a useful adjuvant immunomodulator in the subacute period (1 to 6 months) following transplant, allowing a reduction or earlier withdrawal of steroids.