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Chimeric antigen receptor (CAR) T-cells have shown great promise in the treatment of B-cell malignancies. For acute myeloid leukemia (AML), however, the optimal target surface antigen has yet to be discovered. Alternatively, T-cell receptor (TCR)-redirected T-cells target intracellular antigens, marking a broader territory of available target antigens. Currently, adoptive TCR T-cell therapy uses peripheral blood lymphocytes for the introduction of a transgenic TCR. However, this can cause graft-versus-host disease, due to mispairing of introduced and endogenous TCR chains. Therefore, we started from hematopoietic stem and progenitor cells (HSPC), that do not express a TCR yet, isolated from healthy donors, patients in remission after chemotherapy and AML patients at diagnosis. Using the OP9-DL1 in vitro co-culture system and agonist selection, TCR-transduced HSPC develop into mature tumor antigen-specific T-cells with only one TCR. We show here that this approach is feasible with adult HSPC from clinically relevant sources, albeit with slower maturation and lower cell yield compared to cord blood HSPC. Moreover, cryopreservation of HSPC does not have an effect on cell numbers or functionality of the generated T-cells. In conclusion, we show here that it is feasible to generate TA-specific T-cells from HSPC from adult healthy donors and patients and we believe these T-cells could be of use as a very valuable form of patient-tailored T-cell immunotherapy.

Human thymic CD8αα+ CD10+ PD-1+ αβ T cells selected through early agonist selection have been proposed as the putative thymic precursors of the human CD8αα+ intestinal intraepithelial lymphocytes (IELs). However, the progeny of these thymic precursor cells in human blood or tissues has not yet been characterized. Here, we studied the phenotypical and transcriptional differentiation of the thymic IEL precursor (IELp) lineage upon in vitro exposure to cytokines prominent in the peripheral tissues such as interleukin-15 (IL-15) and the inflammatory cytokines interleukin-12 (IL-12) and interleukin-18 (IL-18). We showed that only the CD1a− fraction of the CD10+ PD-1+ IELp population was able to proliferate with IL-15, suggesting that this subset had acquired functionality. These cells downregulated PD-1 expression and completely lost CD10 expression, whereas other surface markers such as CD95 and CXCR3 remained highly expressed. RNA-seq analysis of the IL-15-cultured cells clearly showed induction of innate-like and effector genes. Induction of the cytotoxic machinery by the CD10+ PD-1+ population was acquired in the presence of IL-15 and was further augmented by inflammatory cytokines. Our data suggest that only the CD1a− CD10+ PD-1+ population exits the thymus and survives in the periphery. Furthermore, PD-1 and CD10 expression is not an intrinsic property of this lineage, but rather characterizes a transient stage in differentiation. CD95 and CXCR3 expression combined with the absence of CD28, CCR7, and CD6 expression might be more powerful markers to define this lineage in the periphery.

We formerly demonstrated that vaccination with Wilms tumor 1 (WT1)-loaded autologous monocyte-derived dendritic cells (mo-DCs) can be a well-tolerated effective treatment in acute myeloid leukemia (AML) patients. Here, we investigated whether we could introduce the receptor for hyaluronic acid-mediated motility (RHAMM/HMMR/CD168), another clinically relevant tumor-associated antigen, into these mo-DCs through mRNA electroporation and elicit RHAMM-specific immune responses. While RHAMM mRNA electroporation significantly increased RHAMM protein expression by mo-DCs, our data indicate that classical mo-DCs already express and present RHAMM at sufficient levels to activate RHAMM-specific T cells, regardless of electroporation. Moreover, we found that RHAMM-specific T cells are present at vaccination sites in AML patients. Our findings implicate that we and others who are using classical mo-DCs for cancer immunotherapy are already vaccinating against RHAMM.

Recent clinical trials have shown that adoptive chimeric antigen receptor (CAR) T cell therapy is a very potent and possibly curative option in the treatment of B cell leukemias and lymphomas. However, targeting a single antigen may not be sufficient, and relapse due to the emergence of antigen negative leukemic cells may occur. A potential strategy to counter the outgrowth of antigen escape variants is to broaden the specificity of the CAR by incorporation of multiple antigen recognition domains in tandem. As a proof of concept, we here describe a bispecific CAR in which the single chain variable fragment (scFv) is replaced by a tandem of two single-antibody domains or nanobodies (nanoCAR). High membrane nanoCAR expression levels are observed in retrovirally transduced T cells. NanoCARs specific for CD20 and HER2 induce T cell activation, cytokine production and tumor lysis upon incubation with transgenic Jurkat cells expressing either antigen or both antigens simultaneously. The use of nanobody technology allows for the production of compact CARs with dual specificity and predefined affinity.

The thymus plays a central role in self-tolerance, partly by eliminating precursors with a T cell receptor (TCR) that binds strongly to self-antigens. However, the generation of self-agonist-selected lineages also relies on strong TCR signaling. How thymocytes discriminate between these opposite outcomes remains elusive. Here, we identified a human agonist-selected PD-1+ CD8αα+ subset of mature CD8αβ+ T cells that displays an effector phenotype associated with agonist selection. TCR stimulation of immature post-β-selection thymocyte blasts specifically gives rise to this innate subset and fixes early T cell receptor alpha variable (TRAV) and T cell receptor alpha joining (TRAJ) rearrangements in the TCR repertoire. These findings suggest that the checkpoint for agonist selection precedes conventional selection in the human thymus.

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 TCR alpha and TCR beta loci and therefore lacked surface-expressed CD3/TCR alpha beta 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.

While in vitro models exist to study human T cell development, they still lack the precise environmental stimuli, such as the exact combination and levels of cytokines and chemokines, that are present in vivo. Moreover, studying the homing of hematopoietic stem (HSC) and progenitor (HPC) cells to the thymus can only be done using in vivo models. Although species-specific differences exist, "humanized" models are generated to circumvent these issues. In this chapter, we focus on the humanized mouse models that can be used to study early T cell development. Models that study solely mature T cells, such as the SCID-PBL (Tary-Lehmann et al., Immunol Today 16:529-533) are therefore not discussed here, but have recently been reviewed (Shultz et al., Nat Rev Immunol 12:786-798).