The B7/CD28 family has profound modulatory effects in immune responses and constitutes important targets for the development of novel therapeutic drugs against human diseases. Here we describe a new CD28 homolog (CD28H) that has unique functions in the regulation of the human immune response and is absent in mice. CD28H is constitutively expressed on all naive T cells. Repetitive antigenic exposure, however, induces a complete loss of CD28H on many T cells, and CD28H-negative T cells have a phenotype of terminal differentiation and senescence. After extensive screening in a receptor array, a B7-like molecule, B7 homolog 5 (B7-H5), was identified as a specific ligand for CD28H. B7-H5 is constitutively found in macrophages and could be induced on dendritic cells. The B7-H5/CD28H interaction co-stimulates human T cell growth and cytokine production, selectively via an AKT-dependent signaling cascade. Our study identifies a novel co-stimulatory pathway regulating human T cell responses.

Therapy-resistant microenvironments represent a major barrier towards effective elimination of disseminated malignancies. Here, we show that select microenvironments can underlie resistance to antibody-based therapy. Using a humanized model of treatment-refractory B-cell leukemia, we find that infiltration of leukemia cells into the bone marrow rewires the tumor microenvironment to inhibit engulfment of antibody-targeted tumor cells. Resistance to macrophage-mediated killing can be overcome by combination regimens involving therapeutic antibodies and chemotherapy. Specifically, the nitrogen mustard cyclophosphamide induces an acute secretory activating phenotype (ASAP), releasing CCL4, IL8, VEGF and TNFα from treated tumor cells. These factors induce macrophage infiltration and phagocytic activity in the bone marrow. Thus, the acute induction of stress-related cytokines can effectively target cancer cells for removal by the innate immune system. This synergistic chemo-immunotherapeutic regimen represents a potent strategy for using conventional anti-cancer agents to alter the tumor microenvironment and promote the efficacy of targeted therapeutics.

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Clinical and preclinical applications of human hematopoietic stem cells (HSCs) are often limited by scarcity of cells. Expanding human HSCs to increase their numbers while maintaining their stem cell properties has therefore become an important area of research. Here, we report a robust HSC coculture system wherein cord blood CD34 þ CD133þ cells were cocultured with mesenchymal stem cells engineered to express angiopoietin-like-5 in a defined medium. After 11 days of culture, SCID repopulating cells were expanded *60-fold by limiting dilution assay in NOD-scid Il2rg À=À (NSG) mice. The cultured CD34 þ CD133 þ cells had similar engraftment potential to uncultured CD34 þ CD133 þ cells in competitive repopulation assays and were capable of efficient secondary reconstitution. Further, the expanded cells supported a robust multilineage reconstitution of human blood cells in NSG recipient mice, including a more efficient T-cell reconstitution. These results demonstrate that the expanded CD34 þ CD133 þ cells maintain both short-term and long-term HSC activities. To our knowledge, this *60-fold expansion of SCID repopulating cells is the best expansion of human HSCs reported to date. Further development of this coculture method for expanding human HSCs for clinical and preclinical applications is therefore warranted.

Increasing demand for human hematopoietic stem cells (HSCs) in clinical and research applications necessitates expansion of HSCs in vitro. Before these cells can be used they must be carefully evaluated to assess their stem cell activity. Here, we expanded cord blood CD34+ CD133+ cells in a defined medium containing angiopoietin like 5 and insulin-like growth factor binding protein 2 and evaluated the cells for stem cell activity in NOD-SCID Il2rg−/− (NSG) mice by multi-lineage engraftment, long term reconstitution, limiting dilution and serial reconstitution. The phenotype of expanded cells was characterized by flow cytometry during the course of expansion and following engraftment in mice. We show that the SCID repopulating activity resides in the CD34+ CD133+ fraction of expanded cells and that CD34+ CD133+ cell number correlates with SCID repopulating activity before and after culture. The expanded cells mediate long-term hematopoiesis and serial reconstitution in NSG mice. Furthermore, they efficiently reconstitute not only neonate but also adult NSG recipients, generating human blood cell populations similar to those reported in mice reconstituted with uncultured human HSCs. These findings suggest an expansion of long term HSCs in our culture and show that expression of CD34 and CD133 serves as a marker for HSC activity in human cord blood cell cultures. The ability to expand human HSCs in vitro should facilitate clinical use of HSCs and large-scale construction of humanized mice from the same donor for research applications.