Immunopathology of ATG Prior to Bone Marrow Transplantation in Mouse Model

Introduction: Polyclonal anti-thymocyte globulin (ATG) is mainly used in hematopoietic stem cell transplantation, as a prevention for graft rejection as well as for GVHD. Immunosuppressive properties of ATG have been considered primarily from the depletion of peripheral lymphocytes. However direct or indirect effects of ATG on other immune components still is controversial. In the present study we evaluated the immunopathologic effects of ATG on immune system in a mouse model.

Methods Ten to fourteen weeks old BALB/c and C57BL/6 mice, were injected intra- peritoneally or intravenously with different doses of ATG (0.2mg/kg -25mg/kg) at three dosage schedules. Considering bone marrow transplantation as day 0, ATG were given at days -10, -7 and -5 or -7, -5 and -1 or -5, -3 and -1. At day 0, mice were killed; spleen (SP), bone marrow (BM), lymph nodes (LN) and thymus were removed and analyzed for T-, B-, DC, NK-, T-reg and myeloid cells.

To evaluate the efficacy of immunosuppressive effect of ATG, a group of BALB/c mice were conditioned using busulfan (Bu) and ATG and compared to a control group of BALB/c conditioned with Bu (80mg/kg) followed by cyclophosphamide (200mg/kg). Both groups transplanted with BM and spleen cells from C57BL/6 and followed for engraftment and/or GVHD.

Results The administration of ATG (0.2- 4.5mg/kg) has resulted in an increase in spleen cellularity while in lymph node and thymus a decreased cellularity was observed. We have found that injecting 4.5mg/kg of ATG at day -7, -5 and -3 significantly decreased T-cell population in spleen and LN compare to Bu-Cy conditioning. The ratio CD4/CD8 increased after ATG treatment showing that CD8 cells are six-fold more sensitive to ATG treatment compared to CD4 lymphocyte. Interestingly T-reg cell population increase after ATG at day 0, however, the increment was negatively correlated with the administration time and positively correlated with the dose.

ATG treatment has resulted in an increase in B-cell population by two- and three- fold in spleen and lymph nodes, respectively. Moreover, 1.2- to 3.5-fold increase in DCs, NK and myeloid cells was observed in SP and LN. Thymus cellularity and cell phenotype was less affected while BM cellularity was not affected by ATG treatment. No differences in the ATG effect on cellularity or cell phenotype between IP and IV route was observed.

Despite the high immunosuppressive effect observed in T-cell population compared to that seen in Bu-Cy conditioning, no chimerism was observed when Cy was substituted with ATG (4.5 – 10 mg/kg).

Conclusion No donor chimerism could be obtained using ATG as a single agent up to 25mg/kg. ATG dose and the administration time are important factors affecting the repopulation of residual T-cells in spleen and lymph node that have to be considered in bone marrow transplantation setting.