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Selective in vivo deletion of alloactivated TH1 cells by OKT3 monoclonal antibody in acute rejection
Immunology Letters 57 (1997) 151 – 153
Selective in vivo deletion of alloactivated TH1 cells by OKT3 monoclonal antibody in acute rejection P. Reinke a, H. Schwinzer b, C. Ho¨flich c, C. Ode-Hakim c, W.D. Do¨cke c, U. Frei a, H.D. Volk c,* a
Department of Nephrology and Intensi6e Care, Virchow Clinic, Humboldt-Uni6ersity, Berlin, Germany b Department of Surgery, Di6ision of Transplant Immunology, Medical School, Hanno6er, Germany c Institute of Medical Immunology, Uni6ersitatsklinikum Charite, Humboldt-Uni6ersitat Zu Berlin, Schumannstr.20 /21, 10098 Berlin, Germany
Abstract The OKT3 monoclonal antibody (mAb) recognizing the CD3 complex on human T-cells has been shown to be an effective immunosuppressive agent for the treatment and the prevention of acute rejection episodes in allograft recipients [1]. Following the initial doses of OKT3 mAb, activation of T lymphocytes and monocytes is observed. This is accompanied by a massive cytokine release, particularly following the first injection. The mAb opsonizes the circulating T-cells and the coated cells disappear quickly from circulation. OKT3 mAb is commonly administered for 5 – 10 days. The manifestation of side effects weeks (cytomegalovirus infection/disease, bacterial and fungal infections) or even months (Epstein-Barr-Virus related lymphoproliferative disease) after therapy as well as the good long-term effects on graft function suggest long-lasting immunosuppressive effects. Since peripheral T-cells reappear in the circulation already during therapy (with modulated CD3/T-cell receptor complex) and T-cell counts reach commonly pretreatment levels within 2–3 days after cessation of OKT3 mAb, the long-lasting immunosuppressive effects are not simply explainable by T-cell depletion. We wondered whether T-cells reappearing in the circulation after cessation of therapy, were functionally different from those before OKT3 mAb therapy. Our data suggest a selective depletion of activated T-cells particularly of type 1-like T-cells by OKT3 mAb resulting in long-lasting immune deviation that may explain the long-term effects of OKT3 mAb treatment. © 1997 Elsevier Science B.V. Keywords: OKT3 cells; T lymphocytes; Monoclonal antibody
1. Patients and methods Peripheral blood mononuclear cells (PBMC) from eight long-term renal allograft recipients (1 – 8 years after transplantation) were prepared by standard procedure before (day 0), during (day +1), and after (day + 8, day + 14) OKT3 mAb therapy (7 × 5 mg/d bolus injection) because of histologically proven steroid-resistant late acute rejection. To reduce OKT3 mAb induced cytokine release all patients received prophylactic dose of 1 mg/kg body weight (b.w.) of methylprednisolone i.v. on the first day. Four patients additionally received a 2 h pentoxiphylline infusion (4 mg/kg b.w./ * Corresponding author. Tel: +49 30 2802 5501; fax: + 49 2802 5461. 0165-2478/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 5 - 2 4 7 8 ( 9 7 ) 0 0 0 9 2 - 8
day i.v. starting 30 min prior to OKT3 mAb application) on the first 2 days. Maintenance immunosuppressive medication consisted of cyclosporin A (blood levels between 120 and 150 ng/ml), azathiprine (1 mg/kg b.w./day) and methylprednisolone (4–8 mg/day p.o.). For in vitro studies PBMC were collected from heparinized blood of healthy donors. Mixed lymphocyte reaction (MLR) was performed by co-culturing of 106 irradiated allogeneic stimulator PBMC and 106 responder PBMC in 1 ml RPMI-1640 (Biochrom, Berlin, Germany) cell culture medium supplemented with 20% heat-inactivated AB-serum, 2 mM glutamine and antibiotics. Apoptosis assay was performed cytofluorometrically by using the TUNEL-technique according to the sup-
P. Reinke et al. / Immunology Letters 57 (1997) 151–153
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pliers recommendation (Boehringer – Mannheim, Mu¨nchen, Germany). For cytokine gene expression analysis total RNA was extracted from purified T-cells by a matrix method as recommended by the manufacturer (Dianova, Hamburg, Germany) and reversely transcribed into cDNA. A cDNA equivalent of about 5 ng total RNA was taken for each competitive RT-PCR analysis. RT-PCR analysis was carried out as described in detail elsewhere [2]. Briefly, known amounts of the control fragment DNA were added in different dilutions to unknown constant amounts of sample cDNA for competitive coamplification with specific primers. The proportion of PCR products amplified from control fragment and target cDNA were estimated after separation on 1.5% agarose gel by measuring the intensity of ethidium bromide luminescence with an imaging system (Herolab, Wiesloch, Germany). The relative concentration of the pertinent target cDNA in each sample was estimated from the concentration of control fragment DNA that achieved equilibrium between its own amplification and the amplification of the target cDNA. It was expressed as arbitrary units (AU), defined as the lowest concentration of control fragment that yields a detectable amplification product given one particular primer pair and the PCR conditions used. To correct for variations across different RNA/cDNA preparations, all samples to be compared were adjusted to contain equal input cDNA concentrations on the basis of their GAPDH cDNA content, which was also determined by competitive RT-PCR.
2. Results and discussion T-cells from kidney transplant patients suffering from late acute rejection, expressed elevated level of interferon-g (IFNg), tumornecrosis factor-a (TNF), interleukin-4 (IL-4), and IL-10 mRNA level as compared with healthy donors or transplant patients without graft deterioration (Table 1). IL-2 mRNA was rarely detectable, probably because of cyclosporin A treatment.
Table 1 Expression of cytokine mRNA (AU) in PBMC from transplant patients Cytokine
With rejection (n= 8)
Without rejection (n = 9)
IFNg IL-2 TNF IL-4 IL-10
98 AU* 2 AUa 8000 AU* 45 AU* 65 AU*
0.5 AU* n.d. 220 AU 0.5 AU 0.4 AU
a
Only in 2/8 patients detectable * PB0.01.
cyclosporine A).
Table 2 Cytokine gene expression in PBMC beforea (100%), during (day 1) and after (day 14) OKT3 monoclonal antibody treatment Cytokine
Day +1
Day +14
IFNg IL-2 TNF IL-4 IL-10
7%* n.d. 21%* n.d.* 5%*
11%* n.d. 21%* 112%* 8%*
a
Day 0 = 100%. * PB0.01 versus day 0.
In vivo depletion of T-cells from circulation (day +1: B 50 T-cells/ml blood= B 5% of pretreatment level) by OKT3 mAb reduced dramatically the cytokine mRNA level of PBMC at day + 1 suggesting their T-cell origin (IFNg: 7%, TNF: 21%, IL-4: n.d., IL-10: 5% of pretreatment level, PB 0.01 for all four cytokines). Between day +3 and + 5 T-cells expressing CD4 or CD8 reappear in the circulation and reach 50–80% of pretreatment level before cessation of OKT3 Mab therapy. However, they show no or only very low level (mean fluorescence level B 10% of pretreatment level) expression of the CD3/TCR complex. This phenomenon is not a result of blocking by the therapeutic mAb because mAb recognizing epitopes distinct from the OKT3 epitope were used for flow cytometry analysis (data not shown). In order to analyze the cytokine pattern of these T-cells with modulated CD3 molecule mRNA/cDNA was prepared at day + 8, immediately after cessation of OKT3 mAb treatment. The cytokine mRNA expression in these day +8 T-cells showed very low level (B 5% of pretreatment level) for IFNg, TNF, and IL-10. By contrast, IL-4 expression reached higher level (25–50% of pretreatment level) in these cells. Similar data were found at day + 14 (Table 2). IL-4 expression was even higher and reached nearly pretreatment level (75–150% of pretreatment level) but no or only marginal expression of IL-10, IFNg, and TNF was detectable although the levels were slightly higher as compared with day +8. In order to study whether the cytokine pattern in cells reappearing in circulation after cessation of therapy is related to the therapeutic success, the patients were divided into responders (normalization of function—follow-up for 6 months) or non-responders (further deterioration of graft function). Although the low number of patients allows for a preliminary statement only, we found clear relationship between the stable downregulation of IFNg gene expression and therapeutic success (responders: B 2% of pretreatment level, n= 5; non-responders: 25–78% of pretreatment level, n= 3). In contrast to this, downregulation of other cytokines or persistence of IL-4 was not indicative of a favorable outcome of antirejection therapy (not shown).
P. Reinke et al. / Immunology Letters 57 (1997) 151–153
What may be the mechanism of long-lasting Thl/Th2 shift following OKT3 therapy? It is well established that activated T-cells are very sensitive to activation-induced apoptosis, particularly via the Fas/FasL pathway. In fact, in vitro alloantigenactivated (MLC) T-cells are more sensitive than resting memory or virgin T-cells to OKT3 mAb induced apoptosis. MLC primed T-cells were restimulated by crosslinked OKT3 mAb at day 8 of culture and apoptosis was measured 4 h later by TUNEL-assay. OKT3 mAb induced apoptosis in a subset of naive (LFA-1 +dim) and resting memory-type (LFA-1 bright) T-cells (B 20% and B 40%, respectively), whereas most of the alloantigen-primed T-cells (LFA-1 + bright, CD69 + / CD25 + ) showed signs of apoptosis (75 – 85 %). The primed T-cells showed a IFNg/IL-4 mRNA ratio of 2.2 which decreased following OKT3 stimulation to 0.8 (PB0.05) suggesting Thl cells seemed to be slightly more sensitive than Th2 cells to activation-induced apoptosis. T-cells reappearing in the peripheral blood after OKT3 cessation, showed low level expression of the TCR/CD3 complex for several days. We wondered whether stimulation of those cells may result in immune deviation. In fact, T-cells derived from three patients during OKT3 mAb therapy were cultured in presence of autologous serum (with therapeutically injected OKT3 mAb) or AB-serum. After 24 h culture, the CD3/TCR expression level recovered to nearly normal level in the AB cultures but
.
.
153
remained low in the autologous serum cultures. The cells were washed and stimulated by OKT3 in culture medium containing FCS. 24 h later, the mRNA/cDNA were prepared and analyzed by semiquantitative RTPCR. The IFNg/IL-4 ratio was 1.5 9 1.2 and 0.1 90.4 in the AB serum and autologous serum cultures, respectively. These results suggest, that TCR triggering of T-cells with modulated CD3/TCR complex induces an immune deviation resulting in an inversed IFNg/IL-4 ratio. That means for the in vivo situation that restimulation of T-cells reappearing in the periphery with low CD3/TCR density by alloantigen (but also by viral antigen) may prefer a Th2 response. The preferential killing of activated T-cells by OKT3 mAb, particularly of Thl cells, and the immune deviation following restimulation of T-cells reappearing after therapy, may explain the long-lasting immunosuppressive effects. Because there is no difference in the fate of alloantigen or viral antigen-triggered T-cells, it would also explain the problems with viral infections following OKT3 treatment.
References [1] L. Chatenoud, Anti-CD3 and anti-TCR monoclonal antibodies, in: J.F. Bach (Ed.), T-cell Directed Immunointervention, Blackwell, Oxford, 1993, pp. 157 – 169. [2] S. Ode-Hakim, W.D. Do¨cke, F. Kern, F. Emmrich, H.D. Volk, P. Reinke, Delayed-type hypersensitivitylike mechanisms dominate late acute rejection episodes in renal allograft recipients, 61 (1996) 1233 – 1240.
Selective in vivo deletion of alloactivated TH1 cells by OKT3 monoclonal antibody in acute rejection P. Reinke a, H. Schwinzer b, C. Ho¨flich c, C. Ode-Hakim c, W.D. Do¨cke c, U. Frei a, H.D. Volk c,* a
Department of Nephrology and Intensi6e Care, Virchow Clinic, Humboldt-Uni6ersity, Berlin, Germany b Department of Surgery, Di6ision of Transplant Immunology, Medical School, Hanno6er, Germany c Institute of Medical Immunology, Uni6ersitatsklinikum Charite, Humboldt-Uni6ersitat Zu Berlin, Schumannstr.20 /21, 10098 Berlin, Germany
Abstract The OKT3 monoclonal antibody (mAb) recognizing the CD3 complex on human T-cells has been shown to be an effective immunosuppressive agent for the treatment and the prevention of acute rejection episodes in allograft recipients [1]. Following the initial doses of OKT3 mAb, activation of T lymphocytes and monocytes is observed. This is accompanied by a massive cytokine release, particularly following the first injection. The mAb opsonizes the circulating T-cells and the coated cells disappear quickly from circulation. OKT3 mAb is commonly administered for 5 – 10 days. The manifestation of side effects weeks (cytomegalovirus infection/disease, bacterial and fungal infections) or even months (Epstein-Barr-Virus related lymphoproliferative disease) after therapy as well as the good long-term effects on graft function suggest long-lasting immunosuppressive effects. Since peripheral T-cells reappear in the circulation already during therapy (with modulated CD3/T-cell receptor complex) and T-cell counts reach commonly pretreatment levels within 2–3 days after cessation of OKT3 mAb, the long-lasting immunosuppressive effects are not simply explainable by T-cell depletion. We wondered whether T-cells reappearing in the circulation after cessation of therapy, were functionally different from those before OKT3 mAb therapy. Our data suggest a selective depletion of activated T-cells particularly of type 1-like T-cells by OKT3 mAb resulting in long-lasting immune deviation that may explain the long-term effects of OKT3 mAb treatment. © 1997 Elsevier Science B.V. Keywords: OKT3 cells; T lymphocytes; Monoclonal antibody
1. Patients and methods Peripheral blood mononuclear cells (PBMC) from eight long-term renal allograft recipients (1 – 8 years after transplantation) were prepared by standard procedure before (day 0), during (day +1), and after (day + 8, day + 14) OKT3 mAb therapy (7 × 5 mg/d bolus injection) because of histologically proven steroid-resistant late acute rejection. To reduce OKT3 mAb induced cytokine release all patients received prophylactic dose of 1 mg/kg body weight (b.w.) of methylprednisolone i.v. on the first day. Four patients additionally received a 2 h pentoxiphylline infusion (4 mg/kg b.w./ * Corresponding author. Tel: +49 30 2802 5501; fax: + 49 2802 5461. 0165-2478/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 5 - 2 4 7 8 ( 9 7 ) 0 0 0 9 2 - 8
day i.v. starting 30 min prior to OKT3 mAb application) on the first 2 days. Maintenance immunosuppressive medication consisted of cyclosporin A (blood levels between 120 and 150 ng/ml), azathiprine (1 mg/kg b.w./day) and methylprednisolone (4–8 mg/day p.o.). For in vitro studies PBMC were collected from heparinized blood of healthy donors. Mixed lymphocyte reaction (MLR) was performed by co-culturing of 106 irradiated allogeneic stimulator PBMC and 106 responder PBMC in 1 ml RPMI-1640 (Biochrom, Berlin, Germany) cell culture medium supplemented with 20% heat-inactivated AB-serum, 2 mM glutamine and antibiotics. Apoptosis assay was performed cytofluorometrically by using the TUNEL-technique according to the sup-
P. Reinke et al. / Immunology Letters 57 (1997) 151–153
152
pliers recommendation (Boehringer – Mannheim, Mu¨nchen, Germany). For cytokine gene expression analysis total RNA was extracted from purified T-cells by a matrix method as recommended by the manufacturer (Dianova, Hamburg, Germany) and reversely transcribed into cDNA. A cDNA equivalent of about 5 ng total RNA was taken for each competitive RT-PCR analysis. RT-PCR analysis was carried out as described in detail elsewhere [2]. Briefly, known amounts of the control fragment DNA were added in different dilutions to unknown constant amounts of sample cDNA for competitive coamplification with specific primers. The proportion of PCR products amplified from control fragment and target cDNA were estimated after separation on 1.5% agarose gel by measuring the intensity of ethidium bromide luminescence with an imaging system (Herolab, Wiesloch, Germany). The relative concentration of the pertinent target cDNA in each sample was estimated from the concentration of control fragment DNA that achieved equilibrium between its own amplification and the amplification of the target cDNA. It was expressed as arbitrary units (AU), defined as the lowest concentration of control fragment that yields a detectable amplification product given one particular primer pair and the PCR conditions used. To correct for variations across different RNA/cDNA preparations, all samples to be compared were adjusted to contain equal input cDNA concentrations on the basis of their GAPDH cDNA content, which was also determined by competitive RT-PCR.
2. Results and discussion T-cells from kidney transplant patients suffering from late acute rejection, expressed elevated level of interferon-g (IFNg), tumornecrosis factor-a (TNF), interleukin-4 (IL-4), and IL-10 mRNA level as compared with healthy donors or transplant patients without graft deterioration (Table 1). IL-2 mRNA was rarely detectable, probably because of cyclosporin A treatment.
Table 1 Expression of cytokine mRNA (AU) in PBMC from transplant patients Cytokine
With rejection (n= 8)
Without rejection (n = 9)
IFNg IL-2 TNF IL-4 IL-10
98 AU* 2 AUa 8000 AU* 45 AU* 65 AU*
0.5 AU* n.d. 220 AU 0.5 AU 0.4 AU
a
Only in 2/8 patients detectable * PB0.01.
cyclosporine A).
Table 2 Cytokine gene expression in PBMC beforea (100%), during (day 1) and after (day 14) OKT3 monoclonal antibody treatment Cytokine
Day +1
Day +14
IFNg IL-2 TNF IL-4 IL-10
7%* n.d. 21%* n.d.* 5%*
11%* n.d. 21%* 112%* 8%*
a
Day 0 = 100%. * PB0.01 versus day 0.
In vivo depletion of T-cells from circulation (day +1: B 50 T-cells/ml blood= B 5% of pretreatment level) by OKT3 mAb reduced dramatically the cytokine mRNA level of PBMC at day + 1 suggesting their T-cell origin (IFNg: 7%, TNF: 21%, IL-4: n.d., IL-10: 5% of pretreatment level, PB 0.01 for all four cytokines). Between day +3 and + 5 T-cells expressing CD4 or CD8 reappear in the circulation and reach 50–80% of pretreatment level before cessation of OKT3 Mab therapy. However, they show no or only very low level (mean fluorescence level B 10% of pretreatment level) expression of the CD3/TCR complex. This phenomenon is not a result of blocking by the therapeutic mAb because mAb recognizing epitopes distinct from the OKT3 epitope were used for flow cytometry analysis (data not shown). In order to analyze the cytokine pattern of these T-cells with modulated CD3 molecule mRNA/cDNA was prepared at day + 8, immediately after cessation of OKT3 mAb treatment. The cytokine mRNA expression in these day +8 T-cells showed very low level (B 5% of pretreatment level) for IFNg, TNF, and IL-10. By contrast, IL-4 expression reached higher level (25–50% of pretreatment level) in these cells. Similar data were found at day + 14 (Table 2). IL-4 expression was even higher and reached nearly pretreatment level (75–150% of pretreatment level) but no or only marginal expression of IL-10, IFNg, and TNF was detectable although the levels were slightly higher as compared with day +8. In order to study whether the cytokine pattern in cells reappearing in circulation after cessation of therapy is related to the therapeutic success, the patients were divided into responders (normalization of function—follow-up for 6 months) or non-responders (further deterioration of graft function). Although the low number of patients allows for a preliminary statement only, we found clear relationship between the stable downregulation of IFNg gene expression and therapeutic success (responders: B 2% of pretreatment level, n= 5; non-responders: 25–78% of pretreatment level, n= 3). In contrast to this, downregulation of other cytokines or persistence of IL-4 was not indicative of a favorable outcome of antirejection therapy (not shown).
P. Reinke et al. / Immunology Letters 57 (1997) 151–153
What may be the mechanism of long-lasting Thl/Th2 shift following OKT3 therapy? It is well established that activated T-cells are very sensitive to activation-induced apoptosis, particularly via the Fas/FasL pathway. In fact, in vitro alloantigenactivated (MLC) T-cells are more sensitive than resting memory or virgin T-cells to OKT3 mAb induced apoptosis. MLC primed T-cells were restimulated by crosslinked OKT3 mAb at day 8 of culture and apoptosis was measured 4 h later by TUNEL-assay. OKT3 mAb induced apoptosis in a subset of naive (LFA-1 +dim) and resting memory-type (LFA-1 bright) T-cells (B 20% and B 40%, respectively), whereas most of the alloantigen-primed T-cells (LFA-1 + bright, CD69 + / CD25 + ) showed signs of apoptosis (75 – 85 %). The primed T-cells showed a IFNg/IL-4 mRNA ratio of 2.2 which decreased following OKT3 stimulation to 0.8 (PB0.05) suggesting Thl cells seemed to be slightly more sensitive than Th2 cells to activation-induced apoptosis. T-cells reappearing in the peripheral blood after OKT3 cessation, showed low level expression of the TCR/CD3 complex for several days. We wondered whether stimulation of those cells may result in immune deviation. In fact, T-cells derived from three patients during OKT3 mAb therapy were cultured in presence of autologous serum (with therapeutically injected OKT3 mAb) or AB-serum. After 24 h culture, the CD3/TCR expression level recovered to nearly normal level in the AB cultures but
.
.
153
remained low in the autologous serum cultures. The cells were washed and stimulated by OKT3 in culture medium containing FCS. 24 h later, the mRNA/cDNA were prepared and analyzed by semiquantitative RTPCR. The IFNg/IL-4 ratio was 1.5 9 1.2 and 0.1 90.4 in the AB serum and autologous serum cultures, respectively. These results suggest, that TCR triggering of T-cells with modulated CD3/TCR complex induces an immune deviation resulting in an inversed IFNg/IL-4 ratio. That means for the in vivo situation that restimulation of T-cells reappearing in the periphery with low CD3/TCR density by alloantigen (but also by viral antigen) may prefer a Th2 response. The preferential killing of activated T-cells by OKT3 mAb, particularly of Thl cells, and the immune deviation following restimulation of T-cells reappearing after therapy, may explain the long-lasting immunosuppressive effects. Because there is no difference in the fate of alloantigen or viral antigen-triggered T-cells, it would also explain the problems with viral infections following OKT3 treatment.
References [1] L. Chatenoud, Anti-CD3 and anti-TCR monoclonal antibodies, in: J.F. Bach (Ed.), T-cell Directed Immunointervention, Blackwell, Oxford, 1993, pp. 157 – 169. [2] S. Ode-Hakim, W.D. Do¨cke, F. Kern, F. Emmrich, H.D. Volk, P. Reinke, Delayed-type hypersensitivitylike mechanisms dominate late acute rejection episodes in renal allograft recipients, 61 (1996) 1233 – 1240.