Cytokines and cytotoxic pathways in engraftment resistance to purified allogeneic hematopoietic stem cells.

The way that allogeneic hematopoietic cells are rejected is not completely understood. Regimen-resistant populations, including natural killer (NK) cells and lymphocytes, are thought to mediate the allograft barrier. In this report, the mechanism by which recipient cell populations resist engraftment of purified allogeneic hematopoietic stem cells (HSCs) was examined in mice. To define the immunoregulatory pathways involved in allogeneic hematopoietic cell resistance, HSC transplantations were performed in immune-defective recipients. Recipients were wild-type mice treated with alpha-NK cell antibodies or knockout strain mice lacking expression of CD8, perforin, Fas ligand, or 1 of the following cytokines: tumor necrosis factor alpha, transforming growth factor beta, interferon gamma, interleukin 4, or interleukin 10. Elimination of a single cytotoxic pathway was ineffective in reducing engraftment resistance, although mice treated with a polyclonal antibody that recognizes NK-cell determinants or CD8 expression showed a profound reduction in the engraftment barrier. Posttransplantation chimerism analysis revealed regeneration of host hematopoiesis in some experimental groups. These studies show, for the first time, that elimination of selected cytokines does not alter allogeneic hematopoietic resistance. Furthermore, the chimerism data reinforce the importance of competition for HSC niches in conjunction with immune mechanisms in resistance to long-term HSC engraftment.

Purified allogeneic hematopoietic stem cell transplantation blocks diabetes pathogenesis in NOD mice.

Purified hematopoietic stem cells (HSCs) were transplanted into NOD mice to test whether development of hyperglycemia could be prevented. Engraftment of major histocompatibility complex-mismatched HSCs was compared with bone marrow (BM) grafts. HSCs differed from BM because HSCs were more strongly resisted and HSC recipients retained significant levels of NOD T-cells, whereas BM recipients were full donor chimeras. Despite persistent NOD T-cells, all HSC chimeras were protected from hyperglycemia, and attenuation of islet lesions was observed. T-cell selection was altered in allogeneic HSC recipients as demonstrated by deletion of both donor and host superantigen-specific T-cells. Syngeneic and congenic hematopoietic cell transplants were also performed to differentiate the influence of the preparative regimen(s) versus the allografts. Unlike the allogeneic HSC transplantations, syngeneic or congenic grafts did not retard diabetes development. In a pilot study, overtly diabetic NOD mice were cured by co-transplantation of allogeneic HSCs and donor-matched islets. We conclude that allogeneic HSC transplants block allo- and autoimmunity, despite residual host T-cell presence. These data demonstrate for the first time that purified HSC grafts block development of autoimmune diabetes and illuminate how HSC grafts alter thymic and peripheral T-cell responses against auto- and alloantigens.

Visualization of effective tumor targeting by CD8+ natural killer T cells redirected with bispecific antibody F(ab')(2)HER2xCD3.

HER2 is an attractive immunotherapeutic target for neoplastic disease because this cell surface molecule is overexpressed on a large fraction of malignant tumor cells. To directly assess therapeutic responses to targeted therapy by noninvasive in vivo imaging in small animals, human HER2-expressing ovarian carcinoma cells were genetically modified with a firefly luciferase gene, and light emission was used for visualization of tumor growth and response to therapy. This imaging approach was able to demonstrate in real-time tumor regression in a HER2 xenograft mouse model by adoptive transfer of in vitro induced and expanded cytotoxic CD8+ natural killer T (NKT) cells retargeted with a humanized bispecific antibody F(ab')(2)HER2xCD3. Immunotherapy with effector cells alone or a humanized monoclonal antibody anti-p185(HER2) (4D5-8) resulted in significant but slower reduction in tumor burden. Long-term survival of tumor xenografts correlated inversely with visible residual tumor burden. In vitro, F(ab')(2)HER2xCD3 substantially augmented cytotoxic activity of CD8+ NKT cells. By flow-sorting, CD8+ NKT cells coexpressing CD56 were found to have the highest redirected killing ability. Treatment with concanamycin A or EGTA abrogated CD8+ NKT cytotoxicity indicating that perforin is a major pathway of tumor cell lysis. In contrast, when CD8+ NKT cell were cross-linked with F(ab')(2)HER2xCD3 neither the immunosuppressants cyclosporine A and FK506, nor the increase of intracellular cyclic AMP by dibutyryl cyclic AMP were able to inhibit cytotoxicity demonstrating that signaling via the CD3 antigen changes the biological activity of non-MHC-restricted effector cells. These studies have demonstrated that CD8+ NKT cells can be successfully redirected to tumor cells using bispecific antibodies and offer a promising strategy for adoptive immunotherapy of neoplastic diseases.