Effects Of Alemtuzumab On Lymphocytes In Cynomolgus Model

Alemtuzumab(Campath-1H)is a humanized CD52-specific monoclonal antibody that causes profound depletion of most immunocompetent cells,including T-and B-lymphocytes,monocytes,and NK cells.It has been used therapeutically in chronic lymphocytic leukemia,multiple sclerosis,and it is increasingly used as an induction agent in solid organ transplantation,including kidney,liver and small intestinal transplantation.Although alemtuzumab has been studied off-label for use in organ transplantation over 10 years,the development of this antibody was challenging because of few animal models to access drug safety and efficacy.The antibody is not equivalently recognized by murine or rat effector cells,nonhuman primates are the most informative animal models,because the species are phylogenetically proximate to human.In clinical transplantation,lymphocyte depletion of>99%can be seen after a single dose with varying rates of cellular recovery depending upon the subpopulation of interest.There are far more lymphocytes in the intestine than in the peripheral blood,making the intestinal mucosa the largest immunologic organ in the body.Intestinal lymphocytes play a major role in immunologic homeostasis.Alemtuzumab has been used as an induction agent in organ transplantation for many years,but the effect of alemtuzumab on lymphocytes in small intestine was not clear.In our study,we chose cynomolgus monkey as animal model.Selected monkeys were received alemtuzumab.The changes of lymphocytes in peripheral blood,spleen,inguinal lymph nodes,IEL,LPL,and interaction of mucosal addressin cell adhesion molecule-1(MAdCAM-1)and lymphocyte homing receptor were examined before and after treatment,in order to know whether the use of alemtuzumab in preclinical transplantation models is feasible,and further studies can be investigated using this model.Part ? Establishment of alemtuzumab used for induction therapy in cynomolgus model and effects of alemtuzumab on peripheral blood,spleen and lymph node lymphocytesObjective:Although alemtuzumab has been studied off-label for use in organ transplantation over 10 years,the development of this antibody was challenging because of few animal models to access drug safety and efficacy.We aim to establish the model of alemtuzumab used for induction therapy in cynomolgus monkeys,and to observe effects of alemtuzumab on peripheral blood,spleen and lymph node lymphocytes.Methods:Male cynomolgus,3 to 5 years old,3 to 5.5 kg,without CD52 antigen on erythrocytes were selected by flow cytometry.Monkeys without CD52 antigen on erythrocytes were randomly assigned to treatment or control groups.The treatment groups received alemtuzumab with a single dose of 3.0 mg/kg intravenously,whereas the control group received the same volume of normal saline.Blood samples were obtained from three alemtuzumab-treated monkeys prior to treatment,on d 3,6,9,14,21,35,56 for complete blood count(CBC)and flow cytometric analysis(FACS),and these monkeys were euthanized on day 56.Three monkeys were euthanized as control and other groups of three alemtuzumab-treated monkeys were euthanized on day 9,14,35.Sections of spleen and inguinal lymph nodes were obtained for histopathology.Data were analyzed via commercial software package SPSS 13.0 and by using one-way ANOVA and t test.P values<0.05 were considered statistically significant.Results:Fifteen of 40 cynomolgus monkeys without CD52 antigen on erythrocytes were selected and there was no difference between selected and unselected monkeys on blood physiological and biochemistry parameters.Powerful depletion of lymphocytes and monocytes from blood followed by gradual repopulation was observed.Monocytes returned to pretreatment levels by day 14,while CD20+ B cells,CD8+ T cells,CD4+ T cells returned to pretreatment levels by day 21,35,56.Lymphocytes depletion and repopulation of spleen and inguinal lymph nodes following alemtuzumab treatment were confirmed by histopathology.Conclusions:We successfully established the model of alemtuzumab used for induction therapy in cynomolgus monkeys.Depletion of lymphocytes in peripheral blood was powerful and repopulation occurred gradually.Lymphocyte depletion and repopulation occurred in peripheral blood,spleen and lymph node.This model can be used in preclinical transplantation.Part ? Effect of alemtuzumab on intestinal lymphocytes in cynomolgus modelObjective:To observe the alternation of intraepithelial lymphocytes and lamina propria lymphocytes and the alternation of mucosal addressin cell adhesion molecule-1(MAdCAM-1)and lymphocyte homing receptor following alemtuzumab treatment in cynomolgus model.Methods:Fifteen male cynomolgus,3 to 5 years old,3 to 5.5 kg,without CD52 antigen on erythrocytes were randomly assigned to treatment or control groups.The treatment groups received alemtuzumab with a single dose of 3.0 mg/kg intravenously,whereas the control group received the same volume of normal saline.Three monkeys were euthanized as control and other groups of three alemtuzumab-treated monkeys were euthanized on day 9,14,35,56.Sections of ileum were obtained for isolation of IEL and LPL,for a hemocytometer and flow cytometry.Ileum adjoining ileocecal junction was obtained for histopathology and immunofluorescence examination.Total protein of intestinal mucosa was extracted with RIPA solution and the expressing change of MAdCAM-1 protein was analyzed by Western Blot.Blood samples were obtained for flow cytometric analysis of integrin ?7 on T cells.Data were analyzed via commercial software package SPSS 13.0 and by using one-way ANOVA.P values<0.05 were considered statistically significant.Results:IEL and LPL decreased significantly 9 days after alemtuzumab treatment,remained in low levels on day 14,increased gradually on day 35 and returned to pretreatment levels by day 56.Depletion and repopulation of IEL and LPL were confirmed by immunofluorescence.The phenotypes of IEL and LPL were changed 14 days after alemtuzumab treatment,the percentage of CD4-CD8+,TCR??+ T cells of IEL,CD3-CD20+ B cells,TCR??+ T cells of LPL,decreased significantly while CD4+CD8+ T cells of IEL,CD4+CD8-?CD4-CD8+?CD4+CD8+ T cells of LPL,increased significantly compared with control group.There was no difference of phenotypes of IEL and LPL between 56 day group and control group.Histopathology of ileum revealed that villi were atrophied,shortened and epithelium of ileum arranged disorderly at 9 days after administration,while villi were integrated and epithelium were compact-arrayed at 56 days after administration.Western Blot showed the expression of MAdCAM-1 protein decreased significantly 9 days after alemtuzumab treatment,remained in low level on day 14,returned to pretreatment levels by day 35.Results were confirmed by immunofluorescence.Flow cytometric analysis showed the expression of integrin ?7 on T cells increased significantly 9 days after alemtuzumab treatment,remained in high level on day 14,returned to pretreatment levels by day 35.Conclusions:The lymphocyte depletion and repopulation occurred in IEL and LPL after alemtuzumab treatment in cynomolgus model,and the phenotypes of IEL and LPL were affected.Histopathological changes of ileum could also be found after alemtuzumab treatment.Change in the expression of MAdCAM-1 protein and change in the expression of integrin ?4?7 on T cells is related.The specific binding of MAdCAM-1 and integrin ?4?7,may be the possible mechanism of lymphocyte homing to gut in cynomolgus model.