IL-10-Producing Potential Treg Precursor in Placenta.

The maternal immune system needs to be tolerant of allogeneic fetal tissue for reproductive success. The regulatory immune cell network plays an essential role in maintaining maternal tolerance to the fetus. We herein demonstrate in a green fluorescent protein (GFP)/IL-10 reporter mouse system that unique IL-10-expressing cells exist presumably in chorionic villi within the placenta. Flow cytometric analysis revealed that these IL-10- expressing cells exhibit a unique CD19 negative, CD3 negative, and B220 positive phenotype. Interestingly, these cells were enriched during in vitro culture, but well-known stimuli for T cells and B cells failed to enhance their growth, suggesting that the CD19- CD3- B220+ cells were self renewing. Unexpectedly, in an adoptive cell trans fer experiment, IL-10 production was detected in Sca-1+ CD4+ CD25+ regulatory T cells (Treg). To our knowledge, this is the first report to identify IL-10-producing CD19- CD3- B220+ cells in the fetus. These cells may rep resent a potential progenitor of Sca-1+ Treg or pluripotent precursor cells for immune tolerance.

Lymphoid regeneration from gene-corrected SCID-X1 subject-derived iPSCs.

X-linked Severe Combined Immunodeficiency (SCID-X1) is a genetic disease that leaves newborns at high risk of serious infection and a predicted life span of less than 1 year in the absence of a matched bone marrow donor. The disease pathogenesis is due to mutations in the gene encoding the Interleukin-2 receptor gamma chain (IL-2Rγ), leading to a lack of functional lymphocytes. With the leukemogenic concerns of viral gene therapy there is a need to explore alternative therapeutic options. We have utilized induced pluripotent stem cell (iPSC) technology and genome editing mediated by TALENs to generate isogenic subject-specific mutant and gene-corrected iPSC lines. While the subject-derived mutant iPSCs have the capacity to generate hematopoietic precursors and myeloid cells, only wild-type and gene-corrected iPSCs can additionally generate mature NK cells and T cell precursors expressing the correctly spliced IL-2Rγ. This study highlights the potential for the development of autologous cell therapy for SCID-X1 subjects.

IL-7 and IL-15 instruct the generation of human memory stem T cells from naive precursors.

Long-living memory stem T cells (T(SCM)) with the ability to self-renew and the plasticity to differentiate into potent effectors could be valuable weapons in adoptive T-cell therapy against cancer. Nonetheless, procedures to specifically target this T-cell population remain elusive. Here, we show that it is possible to differentiate in vitro, expand, and gene modify in clinically compliant conditions CD8(+) T(SCM) lymphocytes starting from naive precursors. Requirements for the generation of this T-cell subset, described as CD62L(+)CCR7(+)CD45RA(+)CD45R0(+)IL-7Rα(+)CD95(+), are CD3/CD28 engagement and culture with IL-7 and IL-15. Accordingly, T(SCM) accumulates early after hematopoietic stem cell transplantation. The gene expression signature and functional phenotype define this population as a distinct memory T-lymphocyte subset, intermediate between naive and central memory cells. When transplanted in immunodeficient mice, gene-modified naive-derived T(SCM) prove superior to other memory lymphocytes for the ability to expand and differentiate into effectors able to mediate a potent xenogeneic GVHD. Furthermore, gene-modified T(SCM) are the only T-cell subset able to expand and mediate GVHD on serial transplantation, suggesting self-renewal capacity in a clinically relevant setting. These findings provide novel insights into the origin and requirements for T(SCM) generation and pave the way for their clinical rapid exploitation in adoptive cell therapy.

Systemic human T cell developmental processes in humanized mice cotransplanted with human fetal thymus/liver tissue and hematopoietic stem cells.

BACKGROUND: In many humanized mouse models, there are few T cells in the engrafted human cell, whereas the number of B cells is high. We attempted to overcome this limitation and investigate whether the entire process of human T cell development arose similarly to the process in humans, as previously reported. METHODS: To produce an advanced humanized mice model, we transplanted human fetal liver/thymus tissue subrenally and injected human CD34(+) stem cells intravenously into NOD/SCID/IL2Rgamma null (NSG) mice. RESULTS: Humanized mice transplanted with fetal thymus/liver tissues and fetal liver-derived CD34(+) stem cells (FLT+FLCD34) showed higher levels of human cells and T cells than mice transplanted with fetal liver-derived CD34(+) stem cells only (FLCD34). In the transplanted thymus tissue of FLT+FLCD34 mice, thymus seeding progenitors (TSPs), early thymic progenitors (ETPs), pre-T cells, and all the other human T cell populations were identified. In the periphery, FLT+FLCD34 mice have high levels of CD45RA(+) T cells; conversely, FLCD34 mice have higher levels of CD45RO(+) T cells. The CD45RO(+) T cells of FLCD34 mice proliferated rapidly after stimulation and exhibited innate T cells properties, expressing PLZF (promyelocytic leukemia zinc finger protein). CONCLUSION: Human T cells educated by mouse MHC II in mice without a human thymus differ from normal human T cells. On the basis of these findings, numerous T cell-tropic human diseases could be explored in our humanized mice and molecular aspects of human T cell development could be also studied extensively.

Greater than the sum of their parts: combination strategies for immune regeneration following allogeneic hematopoietic stem cell transplantation.

Cytoreductive conditioning regimes designed to allow for successful allogeneic hematopoietic stem cell transplantation (allo-HSCT) paradoxically are also detrimental to recovery of the immune system in general but lymphopoiesis in particular. Post-transplant immune depletion is particularly striking within the T cell compartment which is exquisitely sensitive to negative regulation, evidenced by the profound decline in thymic function with age. As a consequence, regeneration of the immune system remains a significant unmet clinical need. Over the past decade studies have revealed several promising therapeutic strategies to address ineffective lymphopoiesis and post-transplant immune deficiency. These include the use of cytokines such as IL-7, IL-12 and IL-15; growth factors and hormones like keratinocyte growth factor (KGF), insulin-like growth factor (IGF)-1 and growth hormone (GH); adoptive transfer of ex vivo-generated precursor T cells (pre-T) and sex steroid ablation (SSA). Moreover, recently several novel approaches have been proposed to generate whole thymii ex vivo using stem cell technologies and bioscaffolds. Increasingly, however, when transferred to the clinic, these strategies alone are not sufficient to restore thymopoiesis in all patients leading to the potential of combination strategies as a way to reign in non-responders. Synergistic enhancement in combination may be due to differential targets may therefore be effective in improving clinical outcomes in the transplant settings as well as in other lymphopenic states induced by high dose chemotherapy/radiation therapy or HIV, and may also be useful in improving responses to vaccination and augmenting anti-tumor immunotherapy.

Concurrent visualization of trafficking, expansion, and activation of T lymphocytes and T-cell precursors in vivo.

We have developed a dual bioluminescent reporter system allowing noninvasive, concomitant imaging of T-cell trafficking, expansion, and activation of nuclear factor of activated T cells (NFAT) in vivo. NFAT activation plays an important role in T-cell activation and T-cell development. Therefore we used this system to determine spatial-temporal activation patterns of (1) proliferating T lymphocytes during graft-versus-host disease (GVHD) and (2) T-cell precursors during T-cell development after allogeneic hematopoietic stem cell transplantation (HSCT). In the first days after HSCT, donor T cells migrated to the peripheral lymph nodes and the intestines, whereas the NFAT activation was dominant in the intestines, suggesting an important role for the intestines in the early stages of alloactivation during development of GVHD. After adoptive transfer of in vitro-derived T-cell receptor (TCR) H-Y transgenic T-cell precursors into B6 (H-2(b)) hosts of both sexes, NFAT signaling and development into CD4(+) or CD8(+) single-positive cells could only be detected in the thymus of female recipients indicating either absence of positive selection or prompt depletion of double-positive thymocytes in the male recipients. Because NFAT plays an important role in a wide range of cell types, our system could provide new insights into a variety of biologic processes.

The Lmo2 oncogene initiates leukemia in mice by inducing thymocyte self-renewal.

The LMO2 oncogene causes a subset of human T cell acute lymphoblastic leukemias (T-ALL), including four cases that arose as adverse events in gene therapy trials. To investigate the cellular origin of LMO2-induced leukemia, we used cell fate mapping to study mice in which the Lmo2 gene was constitutively expressed in the thymus. Lmo2 induced self-renewal of committed T cells in the mice more than 8 months before the development of overt T-ALL. These self-renewing cells retained the capacity for T cell differentiation but expressed several genes typical of hematopoietic stem cells (HSCs), suggesting that Lmo2 might reactivate an HSC-specific transcriptional program. Forced expression of one such gene, Hhex, was sufficient to initiate self-renewal of thymocytes in vivo. Thus, Lmo2 promotes the self-renewal of preleukemic thymocytes, providing a mechanism by which committed T cells can then accumulate additional genetic mutations required for leukemic transformation.