Characterization Of The In Vivo Effects And Potentionial Risks Of Antithymocyte Globulin Therapy In Humanized Mice

Background:Allogeneic hematopoietic cell and organ transplantation is widely used to treat patients with hematological malignancies, inherited hematological diseases or end stage of organ failure. However, HLA-matched donors are not available for most patients and transplantation across HLA barriers inevitably causes alloimmune responses, such as the graft-vs.-host disease(Gv HD) following allogeneic hematopoietic cell transplantation, and host-vs.-graft(HVG) reactions or immune rejection, leading to transplant failure or even life-threatening complications. Because T cells play a critical role in this process, elimination or functional inhibition of these cells is effective in ameliorating these complications. Thus, the development of effective and less toxic drugs or protocols for inhibiting T cell alloresponses remains a goal in the filed of transplantation.Antithymocyte globulin(ATG) is used for human T cell depletion in the clinic. Because ATG is a polyclonal antibody produced by immunization of animals(such as rabbits or horses) with human thymocytes or human T cell line cells, it has a wide antigen recognition spectrum. ATG not only recognizes the X cell surface antigens expressed by T cells(e.g., CD2, CD3, CD4, and CD8), but also binds to antigens expressed by other lymphohematopoietic cells including B cells(CD19 and CD20), NK cells(CD16 and CD56) and dendritic cells(CD11b, CD80 and CD86), as well as CD45 and MHC-I that are ubiquitiously expressed on almost all lymphohematopoietic cells. There is a concern that such a wide antigen recognition spectrum may make it difficult to predict the cytodepletion effects of ATG in patients, and also potentially increase the risk associated with its off-target effects.Because ATG specifically recognizes human antigens, but not the counterperts of other species(such as mice or rats), conventional animal models cannot be used to evaluate the in vivo effects of ATG. Although analysis of peripheral blood cells from ATG-treated patients provided important information help understand the in vivo cytodepletion effects of ATG, the susceptibilities of human lymphohematopoietic cells in different tissues including the spleen, lymph nodes, thymus and bone marrow, to depletion by ATG remains relatively unknown.We have previously shown that humanized mice constructed by transplantation of human fetal thymus and fetal liver-derived hematopoietic stem cells into immunodeficient mice(also known as BLT mice) have systemic repopulation by human lymphohematopoietic cells. These humanized mice are considered one of the best humanized mouse models for assessing human immune function in vivo. Thus, in my doctoral thesis, I used this humanized mouse model to investigate the in vivo effects of ATG. Objective:The study is aimed to comprehensively characterize the in vivo cytodepletion effects of ATG in humanized mice, evaluate the possible risk of ATG as a part of pre-conditioning regimens during hematopoietic stem cell transplantation; and determine the efficacy of ATG in depleting human thymocytes and explore the potential use of ATG in constructing humanized mice with a personalized human immune system. Methods:Humanized mice with the human immune system were made by transplantation of fetal thymic tissue fragments(under the recipient kidney capsule) and human fetal liver-derived CD34+ cells(i.v.) into NOD/SCID or NSG mice which received 2 Gy total body irradiation couple hours ahead; Determine different subsets of immune cells by flow cytometric analysis; Confirm the depletion effect of ATG to lymphoid organs by H/E staining of tissue sections; To mimic personalized mouse models, HLA-A*0201+fetal thymus and HLA-A*0201- fetal CD34+ hematopoietic stem cell were transplanted into immunodeficiency mice; ATG was given through the tail vein injection. Results:We found that ATG was capable to bind to almost all lineages of human peripheral blood lymphocytes as well as human HSCs purified either from XII human fetal liver cells or human bone marrow cells.This result suggests that ATG might not only deplete human T cells but also might eliminate human lymphoid progenitor cells and HSCs in vivo.Our results showed that ATG-treated humanized mice had a different extent of decrease in human CD3+ T cells, CD19+ B cells and CD14+ monocyte in peripheral blood and lymphoid organ, like spleen;We also firstly discovered that ATG administration a very high dose could deplete human thymocyte including mature T cells and T cell precursor in human thymic grafts.Furthermore, we found that although in vivo regimen by ATG was capable to deplete almost all human lymho-hematopietic cells in blood, spleen and human thymic grafts, the level of human cells with different lineages in mouse bone marrows almost did not change. Together, these results suggest that the bone marrow microenvironment is likely to lead human immune cells to “escape” from the ATG-mediated cell depletion.ATG as part of a preparative conditioning regimen for HSC transplantation is proven to inhibit Gv HD caused by allogeneic hematopoietic stem cells in sets of clinical trials. We used humanized mice to mimic ATG as part of a pre-conditioning for HSC transplantation. To our surprise, unlike the cases happen in clinic, we found that human lympho-hematopoiesis was completely inhibited as a result of human HSC engraftment failure. To the contrary, whenever ATG regimens were postponded to 3 weeks after HSC transplantation, the infused human HSCs already settled in bone marrows, human lympho-hematopoiesis recovered to normal level gradually even with high ATG dosage. Taken together, our results show that ATG regimens at the time of human HSC transplantation may increase potential risk of hematopoietic stem cell engraftment failure.The personalized mouse model made by co-transplantation of patient bone marrow-derived hematopoietic stem cell and human HLA partial-matched fetal thymic tissue fragment could “replicate” the patient-specific immune system, which is considered a powerful model for personalized medicine. However, the bottleneck of this model is that the allogeneic T cells derived from existenting human T cell progenitors resident in the human fetal thymic graft can reject human hematopoietic stem cells collected from allogeneic patients. Taking advantage of ATG’s ability to deplete human thymocytes, we proposed a new strategy to establish personalized mouse model, in which the rejection of patient-derived hematopoietic cells by T cells derived from allogeneic the thymic graft can be avoided. Conclusions:(1) ATG not only depletes human immune cells in peripheral blood, but also eliminates these cells in lymphoid organs in vivo. However, human T cells seemed to be more sensitive than B cells to depletion by ATG, as ATG-treated mice showed a significant reduction in peripheral blood human CD3+T cells, but not CD19+ B cells.(2) ATG treatment could efficiently deplete human T cell progenitors in engrafted human thymic tissues and destroy thymic structure which may explain the reason why some ATG treated patients have sustained low level of human T cells for a long time.(3)Although intravenous injection of ATG almost completely deplete human lymphoid cells in peripheral blood, spleen and human thymic tissues, the human lympho-hematopoietic cells in the bone marrow were almost unaffected which imply that the immune suppressive bone marrow microenvironment might protective human immune cells against ATG-mediated depletion. Also, this outcome suggests that the human T cells residing in patient bone marrow(mostly memory T cells) might resist from ATG-mediated depletion in the clinic trials and cause Gv HD or Hv G syndrome relapse after withdraw of ATG regimens.(4) Our data show that ATG may inhibit human lymphohematopoiesis by depleting hematopoietic stem cells when treated at the time of human HSC transplantation while engrafted human HSCs were unaffected by ATG even at a very high dose, suggesting that inclusion of ATG as pre-condition regimen for the patients received HSC transplantation may rise the potential risk of human HSC engraftment failure.(5) The capability of ATG to deplete human thymocytes efficiently provides a mean to optimize the protocol to establish personalized mouse model.In conclusion, our results provide significant insights that help understand the in vivo cytodepletion effects of ATG, and refine the clinical use of ATG as a part of the conditioning regimen in allogeneic transplantation.