Greet Verstichel

Functionally Mature CD4 and CD8 TCRαβ Cells Are Generated in OP9-DL1 Cultures from Human CD34+ Hematopoietic Cells

Human CD34+ hematopoietic precursor cells cultured on delta-like ligand 1 expressing OP9 (OP9-DL1) stromal cells differentiate to T lineage cells. The nature of the T cells generated in these cultures has not been studied in detail. Since these cultures do not contain thymic epithelial cells which are the main cell type mediating positive selection in vivo, generation of conventional helper CD4+ and cytotoxic CD8+ TCRαβ cells is not expected. Phenotypically mature CD27+CD1− TCRγδ as well as TCRαβ cells were generated in OP9-DL1 cultures. CD8 and few mature CD4 single-positive TCRαβ cells were observed. Mature CD8 single-positive cells consisted of two subpopulations: one expressing mainly CD8αβ and one expressing CD8αα dimers. TCRαβ CD8αα and TCRγδ cells both expressed the IL2Rβ receptor constitutively and proliferated on IL-15, a characteristic of unconventional T cells. CD8αβ+ and CD4+ TCRαβ cells were unresponsive to IL-15, but could be expanded upon TCR stimulation as mature CD8αβ+ and CD4+ T cells. These T cells had the characteristics of conventional T cells: CD4+ cells expressed ThPOK, CD40L, and high levels of IL-2 and IL-4; CD8+ cells expressed Eomes, Runx3, and high levels of granzyme, perforin, and IFN-γ. Induction of murine or human MHC class I expression on OP9-DL1 cells had no influence on the differentiation of mature CD8+ cells. Similarly, the presence of dendritic cells was not required for the generation of mature CD4+ or CD8+ T cells. These data suggest that positive selection of these cells is induced by interaction between T precursor cells.

In vitro human embryonic stem cell hematopoiesis mimics MYB independent yolk sac hematopoiesis

Although hematopoietic precursor activity can be generated in vitro from human embryonic stem cells, there is no solid evidence for the appearance of multipotent, self-renewing and transplantable hematopoietic stem cells. This could be due to short half-life of hematopoietic stem cells in culture or, alternatively, human embryonic stem cell-initiated hematopoiesis may be hematopoietic stem cell-independent, similar to yolk sac hematopoiesis, generating multipotent progenitors with limited expansion capacity. Since a MYB was reported to be an excellent marker for hematopoietic stem cell-dependent hematopoiesis, we generated a MYB-eGFP reporter human embryonic stem cell line to study formation of hematopoietic progenitor cells in vitro. We found CD34+ hemogenic endothelial cells rounding up and developing into CD43+ hematopoietic cells without expression of MYB-eGFP. MYB-eGFP+ cells appeared relatively late in embryoid body cultures as CD34+CD43+CD45−/lo cells. These MYB-eGFP+ cells were CD33 positive, proliferated in IL-3 containing media and hematopoietic differentiation was restricted to the granulocytic lineage. In agreement with data obtained on murine Myb−/− embryonic stem cells, bright eGFP expression was observed in a subpopulation of cells, during directed myeloid differentiation, which again belonged to the granulocytic lineage. In contrast, CD14+ macrophage cells were consistently eGFP− and were derived from eGFP-precursors only. In summary, no evidence was obtained for in vitro generation of MYB+ hematopoietic stem cells during embryoid body cultures. The observed MYB expression appeared late in culture and was confined to the granulocytic lineage.

RHAMM/HMMR (CD168) is not an ideal target antigen for immunotherapy of acute myeloid leukemia

Background Criteria for good candidate antigens for immunotherapy of acute myeloid leukemia are high expression on leukemic stem cells in the majority of patients with acute myeloid leukemia and low or no expression in vital tissues. It was shown in vaccination trials that Receptor for Hyaluronic Acid Mediated Motility (RHAMM/HMMR) generates cellular immune responses in patients with acute myeloid leukemia and that these responses correlate with clinical benefit. It is not clear however whether this response actually targets the leukemic stem cell, especially since it was reported that RHAMM is expressed maximally during the G2/M phase of the cell cycle. In addition, tumor specificity of RHAMM expression remains relatively unexplored. Design and Methods Blood, leukapheresis and bone marrow samples were collected from both acute myeloid leukemia patients and healthy controls. RHAMM expression was assessed at protein and mRNA levels on various sorted populations, either fresh or after manipulation. Results High levels of RHAMM were expressed by CD34+CD38+ and CD34- acute myeloid leukemia blasts. However, only baseline expression of RHAMM was measured in CD34+CD38- leukemic stem cells, and was not different from that in CD34+CD38- hematopoietic stem cells from healthy controls. RHAMM was significantly up-regulated in CD34+ cells from healthy donors during in vitro expansion and during in vivo engraftment. Finally, we demonstrated an explicit increase in the expression level of RHAMM after in vitro activation of T cells. Conclusions RHAMM does not fulfill the criteria of an ideal target antigen for immunotherapy of acute myeloid leukemia. RHAMM expression in leukemic stem cells does not differ significantly from the expression in hematopoietic stem cells from healthy controls. RHAMM expression in proliferating CD34+ cells of healthy donors and activated T cells further compromises RHAMM-specific T-cell-mediated immunotherapy.

In vitro generation of mature, naive antigen-specific CD8⁺ T cells with a single T-cell receptor by agonist selection

Peripheral blood T cells transduced with a tumor-specific T-cell receptor (TCR) face problems of auto-reactivity and lack of efficacy caused by cross-pairing of exogenous and endogenous TCR chains, as well as short term in vivo survival due to activation and growth factor-induced differentiation. We here studied an alternative strategy for the efficient generation of naive CD8+ T cells with a single TCR. TCR-transduced human postnatal thymus-derived and adult mobilized blood-derived hematopoietic progenitor cells (HPCs) were differentiated to CD4+CD8+ double-positive T cells using OP9-Delta-like 1 (OP9-DL1) cultures. Addition of the agonist peptide induced double positive cells to cross-present the peptide, leading, in the absence of co-stimulation, to cell cycle arrest and differentiation into mature CD8+ T cells. Comprehensive phenotypic, molecular and functional analysis revealed the generation of naive and resting CD8+ T cells through a process similar to thymic positive selection. These mature T cells show a near complete inhibition of endogenous TCRA and TCRB rearrangements and express high levels of the introduced multimer-reactive TCR. Upon activation, specific cytokine production and efficient killing of tumor cells were induced. Using this strategy, large numbers of high-avidity tumor-specific naive T cells can be generated from readily available HPCs without TCR chain cross-pairing.

Notch induces human T-cell receptor γδ+ thymocytes to differentiate along a parallel, highly proliferative and bipotent CD4 CD8 double-positive pathway

In wild-type mice, T-cell receptor (TCR) γδ+ cells differentiate along a CD4 CD8 double-negative (DN) pathway whereas TCRαβ+ cells differentiate along the double-positive (DP) pathway. In the human postnatal thymus (PNT), DN, DP and single-positive (SP) TCRγδ+ populations are present. Here, the precursor–progeny relationship of the various PNT TCRγδ+ populations was studied and the role of the DP TCRγδ+ population during T-cell differentiation was elucidated. We demonstrate that human TCRγδ+ cells differentiate along two pathways downstream from an immature CD1+ DN TCRγδ+ precursor: a Notch-independent DN pathway generating mature DN and CD8αα SP TCRγδ+ cells, and a Notch-dependent, highly proliferative DP pathway generating immature CD4 SP and subsequently DP TCRγδ+ populations. DP TCRγδ+ cells are actively rearranging the TCRα locus, and differentiate to TCR− DP cells, to CD8αβ SP TCRγδ+ cells and to TCRαβ+ cells. Finally, we show that the γδ subset of T-cell acute lymphoblastic leukemias (T-ALL) consists mainly of CD4 SP or DP phenotypes carrying significantly more activating Notch mutations than DN T-ALL. The latter suggests that activating Notch mutations in TCRγδ+ thymocytes induce proliferation and differentiation along the DP pathway in vivo.

Antigen receptor-redirected T cells derived from hematopoietic precursor cells lack expression of the endogenous TCR/CD3 receptor and exhibit specific antitumor capacities

ABSTRACT Recent clinical studies indicate that adoptive T-cell therapy and especially chimeric antigen receptor (CAR) T-cell therapy is a very potent and potentially curative treatment for B-lineage hematologic malignancies. Currently, autologous peripheral blood T cells are used for adoptive T-cell therapy. Adoptive T cells derived from healthy allogeneic donors may have several advantages; however, the expected occurrence of graft versus host disease (GvHD) as a consequence of the diverse allogeneic T-cell receptor (TCR) repertoire expressed by these cells compromises this approach. Here, we generated T cells from cord blood hematopoietic progenitor cells (HPCs) that were transduced to express an antigen receptor (AR): either a CAR or a TCR with or without built-in CD28 co-stimulatory domains. These AR-transgenic HPCs were culture-expanded on an OP9-DL1 feeder layer and subsequently differentiated to CD5+CD7+ T-lineage precursors, to CD4+ CD8+ double positive cells and finally to mature AR+ T cells. The AR+ T cells were largely naive CD45RA+CD62L+ T cells. These T cells had mostly germline TCRα and TCRβ loci and therefore lacked surface-expressed CD3/TCRαβ complexes. The CD3− AR-transgenic cells were mono-specific, functional T cells as they displayed specific cytotoxic activity. Cytokine production, including IL-2, was prominent in those cells bearing ARs with built-in CD28 domains. Data sustain the concept that cord blood HPC derived, in vitro generated allogeneic CD3− AR+ T cells can be used to more effectively eliminate malignant cells, while at the same time limiting the occurrence of GvHD.