Diverse and heritable lineage imprinting of early haematopoietic progenitors.

Haematopoietic stem cells (HSCs) and their subsequent progenitors produce blood cells, but the precise nature and kinetics of this production is a contentious issue. In one model, lymphoid and myeloid production branch after the lymphoid-primed multipotent progenitor (LMPP), with both branches subsequently producing dendritic cells. However, this model is based mainly on in vitro clonal assays and population-based tracking in vivo, which could miss in vivo single-cell complexity. Here we avoid these issues by using a new quantitative version of 'cellular barcoding' to trace the in vivo fate of hundreds of LMPPs and HSCs at the single-cell level. These data demonstrate that LMPPs are highly heterogeneous in the cell types that they produce, separating into combinations of lymphoid-, myeloid- and dendritic-cell-biased producers. Conversely, although we observe a known lineage bias of some HSCs, most cellular output is derived from a small number of HSCs that each generates all cell types. Crucially, in vivo analysis of the output of sibling cells derived from single LMPPs shows that they often share a similar fate, suggesting that the fate of these progenitors was imprinted. Furthermore, as this imprinting is also observed for dendritic-cell-biased LMPPs, dendritic cells may be considered a distinct lineage on the basis of separate ancestry. These data suggest a 'graded commitment' model of haematopoiesis, in which heritable and diverse lineage imprinting occurs earlier than previously thought.

Alpha beta T cell receptor transfer to gamma delta T cells generates functional effector cells without mixed TCR dimers in vivo.

The successful application of T cell-based immunotherapeutic applications depends on the availability of large numbers of T cells with the desired Ag specificity and phenotypic characteristics. Engineering of TCR-transferred T lymphocytes is an attractive strategy to obtain sufficient T cells with an Ag specificity of choice. However, the introduction of additional TCR chains into T cells leads to the generation of T cells with unknown specificity, due to the formation of mixed dimers between the endogenous and introduced TCR chains. The formation of such potentially autoaggressive T cells may be prevented by using gammadelta T cells as recipient cells, but the in vivo activity of such TCR-engineered gammadelta T cells has not been established. In the present study, we have investigated the in vivo functionality of TCR-transduced gammadelta T cells, in particular their Ag specific proliferative capacity, Ag specific reactivity, in vivo persistence, and their capacity to mount recall responses. The results demonstrate that alphabeta TCR engineering of gammadelta T cells forms a feasible strategy to generate Ag-specific effector T cells that do not express mixed TCR dimers. In view of increasing concerns on the potential autoimmune consequences of mixed TCR dimer formation, the testing of alphabeta TCR engineered gammadelta T cells in clinical trials seems warranted.

One naive T cell, multiple fates in CD8+ T cell differentiation.

The mechanism by which the immune system produces effector and memory T cells is largely unclear. To allow a large-scale assessment of the development of single naive T cells into different subsets, we have developed a technology that introduces unique genetic tags (barcodes) into naive T cells. By comparing the barcodes present in antigen-specific effector and memory T cell populations in systemic and local infection models, at different anatomical sites, and for TCR-pMHC interactions of different avidities, we demonstrate that under all conditions tested, individual naive T cells yield both effector and memory CD8+ T cell progeny. This indicates that effector and memory fate decisions are not determined by the nature of the priming antigen-presenting cell or the time of T cell priming. Instead, for both low and high avidity T cells, individual naive T cells have multiple fates and can differentiate into effector and memory T cell subsets.

Recruitment of antigen-specific CD8+ T cells in response to infection is markedly efficient.

The magnitude of antigen-specific CD8+ T cell responses is not fixed but correlates with the severity of infection. Although by definition T cell response size is the product of both the capacity to recruit naïve T cells (clonal selection) and their subsequent proliferation (clonal expansion), it remains undefined how these two factors regulate antigen-specific T cell responses. We determined the relative contribution of recruitment and expansion by labeling naïve T cells with unique genetic tags and transferring them into mice. Under disparate infection conditions with different pathogens and doses, recruitment of antigen-specific T cells was near constant and close to complete. Thus, naïve T cell recruitment is highly efficient, and the magnitude of antigen-specific CD8+ T cell responses is primarily controlled by clonal expansion.

Dissecting T cell lineage relationships by cellular barcoding.

T cells, as well as other cell types, are composed of phenotypically and functionally distinct subsets. However, for many of these populations it is unclear whether they develop from common or separate progenitors. To address such issues, we developed a novel approach, termed cellular barcoding, that allows the dissection of lineage relationships. We demonstrate that the labeling of cells with unique identifiers coupled to a microarray-based detection system can be used to analyze family relationships between the progeny of such cells. To exemplify the potential of this technique, we studied migration patterns of families of antigen-specific CD8(+) T cells in vivo. We demonstrate that progeny of individual T cells rapidly seed independent lymph nodes and that antigen-specific CD8(+) T cells present at different effector sites are largely derived from a common pool of precursors. These data show how locally primed T cells disperse and provide a technology for kinship analysis with wider utility.

Long-term functionality of TCR-transduced T cells in vivo.

To broaden the applicability of adoptive T cell therapy to cancer types for which tumor-specific T cells cannot routinely be isolated, an effort has been made to develop the transfer of tumor-specific TCR genes into autologous T cells as a novel immunotherapeutic approach. Although such TCR-modified T cells have been shown to react to Ag encounter and can be used to break tolerance to defined self-Ags, the persistence and capacity for renewed expansion of TCR-modified T cells has not been analyzed. To establish whether TCR-transduced T cells can provide recipients with long-term Ag-specific immune protection, we analyzed long-term function of TCR transduced T cells in mouse model systems. We demonstrate that polyclonal populations of T cells transduced with a class I restricted OVA-specific TCR are able to persist in vivo and respond upon re-encounter of cognate Ag as assessed by both proliferation and cytolytic capacity. These experiments indicate that TCR gene transfer can be used to generate long-term Ag-specific T cell responses and provide a useful model system to assess the factors that can promote high-level persistence of TCR-modified T cells.

An inducible caspase 9 safety switch can halt cell therapy-induced autoimmune disease.

Transfer of either allogeneic or genetically modified T cells as a therapy for malignancies can be accompanied by T cell-mediated tissue destruction. The introduction of an efficient "safety switch" can potentially be used to control the survival of adoptively transferred cell populations and as such reduce the risk of severe graft-vs-host disease. In this study, we have tested the value of an inducible caspase 9-based safety switch to halt an ongoing immune attack in a murine model for cell therapy-induced type I diabetes. The data obtained in this model indicate that self-reactive T cells expressing this conditional safety switch show unimpaired lymphopenia- and vaccine-induced proliferation and effector function in vivo, but can be specifically and rapidly eliminated upon triggering. These data provide strong support for the evaluation of this conditional safety switch in clinical trials of adoptive cell therapy.