Hematopoietic stem cells in the bone marrow give rise to lymphoid progenitors, which subsequently differentiate into B and T lymphocytes. Here we show that the proto-oncogene LRF plays an essential role in the B versus T lymphoid cell fate decision. We demonstrate that LRF is key for instructing early lymphoid progenitors to develop into B lineage cells by repressing T cell-instructive signals produced by the cell fate signal protein, Notch. We propose a new model for lymphoid lineage commitment, in which LRF acts as a master regulator of B versus T lineage fate decision.All hematopoietic cells are generated from a small subset of pluripotent stem cells (HSCs) via lineage-restricted progenitors. In adult mice, HSCs reside in the bone marrow (BM) and give rise to lymphoid restricted-progenitors (1), which subsequently develop into B and T lymphocytes in the BM and thymus, respectively. This developmental process is coordinated by the differentiation-stage specific expression of distinct sets of genes. Although some of the transcriptional regulators that play key roles in early stages of lymphocyte development are known (2,3), the precise molecular mechanisms by which lymphoid restricted-progenitors are instructed towards B or T cell fates are still largely undefined.

Immunoincompetence after allogeneic hematopoietic stem cell transplantation (HSCT) affects in particular the T-cell lineage and is associated with an increased risk for infections, graft failure and malignant relapse. To generate large numbers of T-cell precursors for adoptive therapy, we cultured mouse hematopoietic stem cells (HSCs) in vitro on OP9 mouse stromal cells expressing the Notch-1 ligand Delta-like-1 (OP9-DL1). We infused these cells, together with T-cell-depleted mouse bone marrow or purified HSCs, into lethally irradiated allogeneic recipients and determined their effect on T-cell reconstitution after transplantation. Recipients of OP9-DL1-derived T-cell precursors showed increased thymic cellularity and substantially improved donor T-cell chimerism (versus recipients of bone marrow or HSCs only). OP9-DL1-derived T-cell precursors gave rise to host-tolerant CD4+ and CD8+ populations with normal T-cell antigen receptor repertoires, cytokine secretion and proliferative responses to antigen. Administration of OP9-DL1-derived T-cell precursors increased resistance to infection with Listeria monocytogenes and mediated significant graft-versus-tumor (GVT) activity but not graft-versus-host disease (GVHD). We conclude that the adoptive transfer of OP9-DL1-derived T-cell precursors markedly enhances T-cell reconstitution after transplantation, resulting in GVT activity without GVHD.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative therapy for hematological malignancies. However, graft-versus-host disease (GVHD) and relapse after allo-HSCT remain major impediments. Chimeric antigen receptors (CARs) direct tumor cell recognition of adoptively transferred T cells.1–5 CD19 is an attractive CAR target, expressed in most B cell malignancies as well as normal B cells.6,7 Clinical trails using autologous CD19-targeted T cells have shown remarkable outcomes in various B cell malignancies8–15. The use of allogeneic CAR T cells poses a concern of increased GVHD, which however has not been reported in selected patients infused with donor-derived CD19-CAR T cells after allo-HSCT.16,17 To understand the mechanism whereby allogeneic CD19-CAR T cells may mediate anti-lymphoma activity without significant GVHD, we studied donor-derived CD19-CAR T cells in allo-HSCT and lymphoma models in mice. We demonstrate that alloreactive T cells expressing CD28-costimulated CD19-CARs experienced enhanced T cell stimulation, resulting in progressive loss of effector function and proliferative potential, clonal deletion, and significantly decreased GVHD. Concurrently, other CAR T cells present in bulk donor T cell populations retained their anti-lymphoma activity consistent with the requirement for engaging both the TCR and the CAR to accelerate T cell exhaustion. In contrast, first generation and 4-1BB-costimulated CARs increased GVHD. These findings could explain reduced risk of GVHD with cumulative TCR and CAR signaling.

Regenerative therapies that use allogeneic cells are likely to encounter immunological barriers similar to those that occur with transplantation of solid organs and allogeneic hematopoietic stem cells (HSCs). Decades of experience in clinical transplantation hold valuable lessons for regenerative medicine, offering approaches for developing tolerance-induction treatments relevant to cell therapies. Outside the field of solid-organ and allogeneic HSC transplantation, new strategies are emerging for controlling the immune response, such as methods based on biomaterials or mimicry of antigen-specific peripheral tolerance. Novel biomaterials can alter the behavior of cells in tissue-engineered constructs and can blunt host immune responses to cells and biomaterial scaffolds. Approaches to suppress autoreactive immune cells may also be useful in regenerative medicine. The most innovative solutions will be developed through closer collaboration among stem cell biologists, transplantation immunologists and materials scientists.

Velardi et al. show that sex steroids regulate thymopoiesis by directly modulating Notch signaling, and provide a novel clinical strategy to boost immune regeneration.

We present a strategy for adoptive immunotherapy using T-lineage committed lymphoid precursor cells generated by Notch1-based culture. We found that allogeneic T-cell precursors can be transferred to irradiated individuals irrespective of major histocompatibility complex (MHC) disparities and give rise to host-MHC restricted and host-tolerant functional allogeneic T cells, improving survival in irradiated recipients as well as enhancing anti-tumor responses. T-cell precursors transduced to express a chimeric receptor targeting hCD19 resulted in significant additional anti-tumor activity, demonstrating the feasibility of genetic engineering of these cells. We conclude that ex vivo generated MHC-disparate T-cell precursors from any donor can be used universally for 'off-the-shelf' immunotherapy, and can be further enhanced by genetic engineering for targeted immunotherapy.

Graft-versus-host disease (GVHD) is a major complication of allogeneic hematopoietic stem cell transplantation (HSCT).Migration of donor-derived T cells into GVHD target organs plays a critical role in the development of GVHD and chemokines and their receptors are important molecules involved in this process. Here, we demonstrate in murine bone marrow transplantation models that the expression of the inflammatory CC chemokine receptor 2 (CCR2) on donor-derived CD8 ؉ T cells is relevant for the control of CD8 ؉ T-cell migration and development of GVHD. Recipients of CCR2-deficient (CCR2 ؊/؊ ) CD8 ؉ T cells developed less damage of gut and liver than recipients of wild-type CD8 ؉ T cells, which correlated with a reduction in overall GVHD morbidity and mortality. Assessment of donor CD8 ؉ T-cell target organ infiltration revealed that CCR2 ؊/؊ CD8 ؉ T cells have an intrinsic migratory defect to the gut and liver. Other causes for the reduction in GVHD could be excluded, as alloreactive proliferation, activation, IFN-␥ production and cytotoxicity of CCR2 ؊/؊ CD8 ؉ T cells were intact. Interestingly, the graft-versustumor effect mediated by CCR2 ؊/؊ CD8 ؉ T cells was preserved, which suggests that interference with T-cell migration by blockade of CCR2 signaling can separate GVHD from GVT activity. ( IntroductionAllogeneic hematopoietic stem cell transplantation (HSCT) is a well-established therapy for a variety of malignant and nonmalignant disorders of the hematopoietic system and for certain solid tumors. 1 A major complication limiting the success and wider application of allogeneic HSCT is the occurrence of acute graft-versus-host disease (GVHD), which is a rapidly progressive illness with immunosuppression, cachexia and specific target organ damage of liver, intestines, skin, and lung. 2 Donor-derived alloreactive T cells play a major role in the pathogenesis of GVHD and depletion of T cells from the donor cell inoculum remains the most effective approach to prevent the development of disease. 3 However, alloreactive donor T cells also display graft-versus-tumor (GVT) activity, which is increasingly being recognized as an important component of the overall antitumor effect of an allogeneic HSCT. 4 Recent murine bone marrow transplantation studies suggest that specifically interfering with T-cell migration represents an attractive therapeutic approach toward the goal of amelioration of GVHD without reducing GVT activity. 5,6 It is known that 3 families of migration molecules (selectins, chemokines, integrins, and their respective ligands and receptors) control T-cell migration in homeostasis and inflammation 7 and members of all 3 families have been identified as important players during GVHD. 5 CC chemokine receptor 2 (CCR2) and its main ligand chemokine ligand 2 (CCL2) are among the chemokine receptor-ligand pairs that control leukocyte migration during inflammatory processes. 8,9 CCL2 (originally termed monocyte chemoattractant protein-1 [MCP-1]) belongs to the family of inflammatory CC chemokines and was one...

Graft-versus-host disease (GVHD) is a serious complication of allogeneic bone marrow transplantation, and donor T cells are indispensable for GVHD. Current therapies have limited efficacy, selectivity, and high toxicities. We used a novel flow cytometry technique for the analysis of intracellular phosphorylation events in single cells in murine BMT models to identify and validate novel GVHD drug targets. [1][2][3][4][5][6][7] This method circumvents the requirement for large numbers of purified cells, unlike western blots. We defined a signaling profile for alloactivated T cells in vivo and identified the phosphorylation of ERK1/2 and STAT-3 as important events during T-cell (allo)activation in GVHD. We establish that interference with STAT-3 phosphorylation can inhibit T-cell activation and proliferation in vitro and GVHD in vivo. This suggests that phosphospecific flow cytometry is useful for the identification of promising drug targets, and ERK1/2 and STAT-3 phosphorylation in alloactivated T cells may be important for GVHD. (Blood. 2008;112:5254-5258) IntroductionAnalysis of in vivo T-cell signaling with Western blots is limited by requirements for large numbers of cells and the inability to access subpopulations. We used a flow cytometric method for measuring phosphorylated epitopes in single cells [1][2][3][4][5][6][7] to establish a signaling profile for alloactivated T cells in vivo in murine graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation. We identify and validate molecular targets important for GVHD pathobiology and demonstrate the importance of ERK1/2 and STAT-3 phosphorylation for T-cell alloactivation in vivo. Finally, we confirm that small molecule inhibitors of STAT-3 phosphorylation can attenuate T-cell activation, proliferation, and GVHD. MethodsBone marrow transplantation, transfer of CFSE-labeled T cells, and GVHD assessment were described previously 8,9 and discussed in Document S1 (available on the Blood website; see the Supplemental Materials link at the top of the online article). In vitro stimulation of T cells with cytokines, anti-CD3, anti-CD3 ϩ CD28 antibody, or irradiated stimulator cells were previously described 1,9 and further discussed in Document S1. All protocols were approved by the Institutional Animal Care and Use Committee of Memorial Sloan-Kettering Cancer Center.Cell-surface staining, intracellular phospho-specific staining, flow cytometry, and data analysis were previously described 2,4-6 and further discussed in Document S1. The T(X) population comparison metric was described previously 10 and its use is further discussed in the Document S1.Finally, a list of phospho-specific antibodies and inhibitors used in this study can also be found in Document S1. Results and discussionWe first validated the antibodies for this study (Figure S1A-C and accompanying text), and then examined subsets of splenic T cells in the normal mouse. When we divided CD4 T cells into naive (CD44 lo CD62L hi ), effector memory (CD44 hi CD62L lo ), and central memory (C...