Selective in vivo deletion of alloactivated Th1 cells by OKT 3 mAb in acute rejection

Novel imnunosuppressives

25 June 1997 - Oral presentations

T helper 2 (THP)-type cells decreased in the anti-CD4 treated group, numbers of interferon (IFN)-y producing T helper 1 (THl)-type cells were not affected, resulting in a significant increase in the THl/THP ratio (.Ol < p < 65). Conclueion: our data show that depleting CD4 mAb are ineffective in eliminating primed, IFN-y producing THl-&pe &Is and may give an explanation for the lack of beneffcial effects of depleting CD4 mAb on the ctinical course of human chronic autoimmune disease. This study was supported by grant 92-112 from the Dutch Society for Support of Research on Multiple Sclerosis.

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0.5.23.1

Novel immunosuppressives and xenotransplantation 6eledve

in vlvo deletion of alloactivated Thl cells

’ by OKT 3 mAb in acute rejection P. Reinke, H. Schwinzer, C. Hbflich, S. Ode-Hakim, W.D. D6cke, U. Frei, H.-D. Volk. Dept. Nephrol., Vinzhowklinikum & Dept. Med. Immunolosv; Charit Humboldt-University, Berlin, Germany: Dept. of Surgery; Div of Transplantimmunol~ Medical School Hannover. Germany

Introductbn: OKT 3 mAb is very effective for the treatment of severe acute rejection episodes in allogmft recipients. The manifestation of side effects weeks (CMV disease, bacterial and fungal infections) or even months (EBV related Ivmphoma) after theraov as well as the oood lona-term results on transolant fun&ion suggest long-lasting immunosu~pressive”effects. Since periphekl T cells reappear in the circulation already during the therapy and T cell counts usually reach pretreatment levels within 2-3 days after cessation of OKT 3 mAb, the long-lasting immunosuppressive effects is not simply explainable by T cell depletion. Reeultr: We wondered whether T cells reappearing in the circulation after cessation of therapy, were functionally different from those before therapy. T cells from kidney transplant patients suffenng from acute Rx, expressed elevated level of IL4, IL-lo, TNF and IFN-y mRNA as shown by quantitative RT-PCR. In vivo depletion of T cells by OKT 3 rnAb reduced dramatically the cytokine mRNA level at day 1 suggesting their T cell origin. Six days after cessation of therapy circulating T cells expressed IL4 mRNA level as high as before therapy whereas the expression of the other cytokine genes was still significantly reduced by a factor of 10 to >loOO. Remarkably, the degree of IFN-y downregulation correlated with the therapeutic success..What may be the mechanism of this THl/THP shift? It is well established that antigen-primed T cells are sensitive to Fas/FasL induced apoptosis. Indeed alloanttgen-triggered LFA-1 +++ memory-type T cells expressed high level of Fas (CD 95) and were more sensitive than resting memory or virgin T-cellsto OKT 3&duced(Fas/FasL mediated) apoptceis. Thl cells seemed to be slightly mom sensitive than Th2 cells. Moreover, T cells reappearing in the circulation after OKT 3 treatment, showed low level expression of the TCWCD 3 complex for several days. TCR crosslinking of these T cells resulted in immune deviation with high IL4 and low IFNy expression. Conclusion: OKT 3 selectively kill activated T cells in acute rejection resulting in loss of freshly actfvated effector T cells. Resting T cells (and some activated Th2 cells) survive with modulated TCR and express IL4 but not IFN-y following (allahriml) antigen contact. The immune deviation would explain the long-lasting antigen-specific immunosuppression.

10.5.23.2

1 Quantlflcation of TCWCW complex expression on human T-lymphocytes after in vivo tretment with murine IgA monoclonal antibody to human CD3

R.T. Meijer, S.L. Yong, R.J.M. ten Berge, P.T.A. Schellekens. Renal Transpkmt Unit and Clinical and Laboratorv lmmunolwv__ Unit, Academic Medical Centre. Amsterdam,

The Netherlands

.

Introduction:OKT3 is a murine IgGPa mAb to human CD3 that is succesfully used in the treatment of acute rejection in solid organ transplant recipients. Its immunosuppressive action is probably achieved primarily by direct interference with antigen recognition, by blocking and down modulation of the TCWCDB complex. 0KT3 mav however cause severe side effects. Previouslv. we reoorted that .a munne IgA mAb to CD3 (CLB T3/4.A) is better tolemted by humans than a corresponding murine IgGPa rnAb to CD3 (CLB T3/4.2a). Currently, we are investigating the immunosuppressive properties of this IgA mAb in renal

and xenotransplantation

373

transplant recipients suffering from an acute cellular rejection episode. We studied TCWCD3 complex expression and blocking in a quantitative manner in five patients who received an intravenous bolus of 5 rng of mAb daily for 10 davs. vMaterlaloand Methods: We used a three colour flowcytometer to analyse PBMNC taken from the patients immediateiv before the first mAb bofus and one to three times daily thereafter during the treatment. After the treatment had been completed, PBMNC were analysed in decreasing frequency for 3 to 6 months. Two rnAb to CD3 were used that bind in a mutually exclusive way to the e-chain: FITC labeled Leu4 and the aforementioned IgA mAb. Binding of the latter was detected with a FITC labeled rat mAb to mudne kappa light chain. In addiion, a PE labeled mAb to TCR@ and PECy5 labeled mAbs to CD4 and CD6 were used. Free CD3 and IgA rnAb bound in vivo were detected using Leu4 FITC and the rat mAb respectively. Total CD3 expression was detected using the rat mAb after prior in vitro saturation with the IgA rnAb to CD3. Cells of interest were oated on FSC and SSC and on exoression of CD4 and CD6 resoectivefy. Mean-Fluorescence Intensities (MFI) were calculated and related to’baseline MFls. resulting in relative MFls. Leu4 and anti-TCR@ gave almost identical relative MFls at each point of time. The sums of the relative MFls from Leu4 and the rat mAb without prior in vitro saturation with IgA to CD3 were almost identical to the relative MFls from the rat mAb after prior in vitro saturation with IgA mAb to CD3 at each point of time. Baseline TCWCDI expression as assessed by this method appeared to be fairfy constant dunng the follow up period. Finally, these flowcytometric data fitin with the corresponding _ MA _ rnAb serum levels in the oatients. as measured by ELISA. Conclusion: We conclude that with this method we can easily quantify TCWCDI expression as well as in vivo binding of IgA mAb to the CD3 antigen during a treatment course with this IgA rnAb.

Results:

, IO.5.23.3 1 The effect Thalidomide analogue CC-3052 on mltogen-induced TNF-dcytoklne production ex V/V0

J.B. Marriott, S. Cookson, M. Westby, M. Guckian, A.G. Dalgleish. Division of Onculog~ St George’s Hospital Medical School, Cmnmer Terrace, London, UK

Introduction: Thalidomide is a TNFa inhibitor and immunosuppressant which has been shown to inhibit activation of HIV replication in a monocyte cell line and has been used to treat patients with erythema nodosum leprosum, HIV associated aphthous ulceration, chronic GvHD and chronic tuberculosis. We have evaluated the properties of thalidomide analogues in terms of ability to down-regulate mitogen induced production of TNFa ex viva Also, we have assessed one of these analogues for cytokine modulating properties in vitro. Materials and Mods: Blood was collected for stimulation in an ax t&o culture system. Whole blood was diluted (1:5) in RPM and cultured (+/- analogue) with either LPS (1 &ml), PHA (2 &ml) or left unstlmulated. After 24 hours at 3pC/5%COs cell-free supematants were collected and stored in aliquots at -70°C unfl analysis bv ELISA. Whole blood was atso diluted (1:2) in RPhnl and cultured (+/- analogue) in the presence of LPS or PHA for 4 hours and 6 hours respectively prior to collection and storage of PBMC at -70°C for mRNA analvsis bv comoetitive PCR. Reeuttsr Oneof the’analogues (CC-3652) was found to be soluble in water, whereas thalidomide and other analogues are not. CC-3052 showed potent inhibition of TNFa protein and message (I&-l FM) in both PHA and LPS stimulated cultures derived from HIV positive and normal individuals. IFN-y and RANTES were also reduced in a similar manner to TNFa. an effect that could be mimicked using anti TNFa mAb. Furthermore, CC-3652 has greater stablfky in human plasma (half-life of 17.5 hours compared to 1.5 hours for thalidomide) and has no apparent toxicity, assessed using the trypan Mue exclusion test. At higher concentrations (100 tiM) general inhibition of cvtokine oroduction was observed, in line with reduced p&femtfve response and NK activity. Conclusion: CC-3652 is a potent TNFa down-reoulator which is also water soluble and highly stable. Apparent inhibition of pro-inflammatory and chernotactic molecules such as IFN-)I and RANTES is also associated with TNFa down regulation by CC-3052. This analogue would therefore seem to be a good candidate for use in inflammatory disease as well as other conditions that are associated with elevated levels of TNFa. However, further studies are needed to fully assess any toxicity prior to clinical evaluation.

1

Selective killing of CDW CD28- CDfi2L’” cells by the GalNAc-specific lectin from V/scum album

Amdt Bussing ‘, Uwe Pfiiller 2. ’ Krebstbrschung

He&&e, Dept. of App/ied lmmunolo~ Communal Hospital Herdecke, He&eke, Germany 2 Institute of Phyiochemistry; University WflewHerde&e, Witten, Germany

Introduction: The lectin domains of typ 2 and typ 4 plant tibosome-inactivating proteins, such as the lectins from Viscum album L. (mistletoe), can bind to any appropriate carbohydrate domain on cell surface receptors, enabeling the