Remko Schotte

High-throughput epitope discovery reveals frequent recognition of neo-antigens by CD4 + T cells in human melanoma

Tumor-specific neo-antigens that arise as a consequence of mutations are thought to be important for the therapeutic efficacy of cancer immunotherapies. Accumulating evidence suggests that neo-antigens may be commonly recognized by intratumoral CD8+ T cells, but it is unclear whether neo-antigen–specific CD4+ T cells also frequently reside within human tumors. In view of the accepted role of tumor-specific CD4+ T-cell responses in tumor control, we addressed whether neo-antigen–specific CD4+ T-cell reactivity is a common property in human melanoma.

The ETS Transcription Factor Spi-B Is Required for Human Plasmacytoid Dendritic Cell Development

A number of transcription factors that act as molecular switches for hematopoietic lineage decisions have been identified. We recently described the ETS transcription factor Spi-B to be exclusively expressed in plasmacytoid dendritic cells (pDCs), but not in myeloid DCs. To assess whether Spi-B is required for pDC development we used an RNA interference knock down approach to specifically silence Spi-B protein synthesis in CD34+ precursor cells. We observed that a knock down of Spi-B mRNA strongly inhibited the ability of CD34+ precursor cells to develop into pDCs in both in vitro assays as well as in vivo upon injection into recombination activating gene 2−/− γ common−/− mice. The observed effects were restricted to the pDC lineage as the differentiation of pro–B cells and CD14+ myeloid cells was not inhibited but slightly elevated by Spi-B knock down. Knock down of the related ETS factor PU.1 also inhibited in vitro development of CD34+ cells into pDCs. However, in contrast to Spi-B, PU.1 knock down inhibited B cell and myeloid cell development as well. These results identify Spi-B as a key regulator of human pDC development.

Neoantigen landscape dynamics during human melanoma–T cell interactions

Recognition of neoantigens that are formed as a consequence of DNA damage is likely to form a major driving force behind the clinical activity of cancer immunotherapies such as T-cell checkpoint blockade and adoptive T-cell therapy. Therefore, strategies to selectively enhance T-cell reactivity against genetically defined neoantigens are currently under development. In mouse models, T-cell pressure can sculpt the antigenicity of tumours, resulting in the emergence of tumours that lack defined mutant antigens. However, whether the T-cell-recognized neoantigen repertoire in human cancers is constant over time is unclear. Here we analyse the stability of neoantigen-specific T-cell responses and the antigens they recognize in two patients with stage IV melanoma treated by adoptive T-cell transfer. The T-cell-recognized neoantigens can be selectively lost from the tumour cell population, either by overall reduced expression of the genes or loss of the mutant alleles. Notably, loss of expression of T-cell-recognized neoantigens was accompanied by development of neoantigen-specific T-cell reactivity in tumour-infiltrating lymphocytes. These data demonstrate the dynamic interactions between cancer cells and T cells, which suggest that T cells mediate neoantigen immunoediting, and indicate that the therapeutic induction of broad neoantigen-specific T-cell responses should be used to avoid tumour resistance.

Development of human plasmacytoid dendritic cells depends on the combined action of the basic helix‐loop‐helix factor E2‐2 and the Ets factor Spi‐B

Plasmacytoid dendritic cells (pDC) are central players in the innate and adaptive immune response against viral infections. The molecular mechanism that underlies pDC development from progenitor cells is only beginning to be elucidated. Previously, we reported that the Ets factor Spi‐B and the inhibitors of DNA binding protein 2 (Id2) or Id3, which antagonize E‐protein activity, are crucially involved in promoting or impairing pDC development, respectively. Here we show that the basic helix‐loop‐helix protein E2‐2 is predominantly expressed in pDC, but not in their progenitor cells or conventional DC. Forced expression of E2‐2 in progenitor cells stimulated pDC development. Conversely, inhibition of E2‐2 expression by RNA interference impaired the generation of pDC suggesting a key role of E2‐2 in development of these cells. Notably, Spi‐B was unable to overcome the Id2 enforced block in pDC development and moreover Spi‐B transduced pDC expressed reduced Id2 levels. This might indicate that Spi‐B contributes to pDC development by promoting E2‐2 activity. Consistent with notion, simultaneous overexpression of E2‐2 and Spi‐B in progenitor cells further stimulated pDC development. Together our results provide additional insight into the transcriptional network controlling pDC development as evidenced by the joint venture of E2‐2 and Spi‐B.

Spi-B inhibits human plasma cell differentiation by repressing BLIMP1 and XBP-1 expression.

The terminal differentiation of B cells into antibody-secreting plasma cells is tightly regulated by a complex network of transcription factors. Here we evaluated the role of the Ets factor Spi-B during terminal differentiation of human B cells. All mature tonsil and peripheral blood B-cell subsets expressed Spi-B, with the exception of plasma cells. Overexpression of Spi-B in CD19(+) B cells inhibited, similar to the known inhibitor BCL-6, the expression of plasma cell-associated surface markers and transcription factors as well as immunoglobulin production, ie, in vitro plasma cell differentiation. The arrest in B-cell differentiation enforced by Spi-B was independent of the transactivation domain, but dependent on the Ets-domain. By chromatin immunoprecipitation and assays using an inducible Spi-B construct BLIMP1 and XBP-1 were identified as direct target genes of Spi-B mediated repression. We propose a novel role for Spi-B in maintenance of germinal center and memory B cells by direct repression of major plasma cell factors and thereby plasma cell differentiation.

Synergy between IL-15 and Id2 Promotes the Expansion of Human NK Progenitor Cells, Which Can Be Counteracted by the E Protein HEB Required To Drive T Cell Development

The cytokine IL-15 and the inhibitor of DNA binding (Id)2, which negatively regulates the activity of basic helix-loop-helix transcription factors, have been shown to play key roles in NK cell development. Consistent with this, exogenous IL-15 added to human thymic progenitor cells stimulated their development into NK cells at the expense of T cells both in fetal thymic organ culture and in coculture with stromal cells expressing the Notch ligand Delta-like 1. Overexpression of Id2 in thymic progenitor cells stimulated NK cell development and blocked T cell development. This, in part, is attributed to inhibition of the transcriptional activity of the E protein HEB, which we show in this study is the only E protein that enhanced T cell development. Notably, Id2 increased a pool of lineage CD1a–CD5+ progenitor cells that in synergy with IL-15 furthered expansion and differentiation into NK cells. Taken together, our findings point to a dualistic function of Id2 in controlling T/NK cell lineage decisions; T cell development is impaired by Id2, most likely by sequestering HEB, whereas NK cell development is promoted by increasing a pool of CD1a–CD5+ NK cell progenitors, which together with IL-15 differentiate into mature NK cells.

Developmental origin of pre-DC2

It is generally accepted that dendritic cells can be generated from either myeloid or lymphoid derived progenitors. Ample information has been collected on the development and nature of myeloid DC type 1 (DC1). In contrast, our current understanding on the origin and function of the lymphoid derived DC type 2 (DC2) is still limited but is increasing rapidly. Here we will summarize recent findings on the developmental origin of the precursor of DC2 (pre-DC2). The presence of pre-DC2 has been revealed in bone marrow, fetal liver, and cord blood, where they develop from hematopoietic stem cells (HSC) most likely via an intermediate pro-DC2 stage. Both in human and mouse, development of pre-DC2 depends on the cytokine FLT3-ligand (FLT3-L). In addition, transcription factors such as Spi-B and members of the basic helix-loop helix (bHLH) family have been shown to be involved in the proper differentiation of HSC into pre-DC2. The human thymus contains a population of cells that closely resembles the peripheral pre-DC2, including interferon (INF)-a production after viral stimulation. Some phenotypic differences have been observed however. Furthermore, we have shown that the thymic microenvironment is able to support development of pre-DC2 from HSC in vivo. A thymus independent pathway of pre-DC2 development exists as well, although at present it is not clear where these extrathymic pre-DC2 are generated. In regard of the absence of a phenotypic defined pro-DC2 population in the thymus, we speculate that development of thymic pre-DC2 may differ from peripheral pre-DC2. The challenge of the near future will be to determine the role of pre-DC2 during thymic T cell development.

Critical assessment of human antibody generation in humanized mouse models

Immunodeficient mice reconstituted with human hematopoietic stem cells provide a small-animal model for the study of development and function of human hematopoietic cells in vivo. However, in the current models, the immune response, and especially the humoral response by the human immune cells is far from optimal. The B cells found in these mice exhibit an immature and abnormal phenotype correlating with a reduced capacity to produce antigen-specific affinity matured antibodies upon infection or immunization. Herein, we review the current state of knowledge of development, function and antibody production of human B cells and discuss the obstacles for the improvement of these models.

Isolation and in vitro generation of gene-manipulated human plasmacytoid and conventional dendritic cells.

Our understanding of human lymphocyte development has increased significantly over the past 20 years. In particular, our insight into human T- and B-cell development has improved (1, 2). Nonetheless, there are many gaps in our understanding, particularly regarding the early stages of development of hematopoietic progenitor cells (HPCs) into downstream lineage-biased and lineage-restricted precursors and the molecular mechanisms underlying these activities. The same holds true for our knowledge of human dendritic cell (DC) development. While the amount of data on the different subsets of conventional DCs (cDCs) and plasmacytoid DCs (pDCs) rapidly increases in mice (3, 4), the developmental stages of different DC subsets in humans remain poorly defined (2). The relatively easy access to patient material and therefore human precursor cells that can be isolated from these tissues combined with the availability of in vitro and in vivo differentiation assays allows studies in the field of human hematopoietic development, including that of DCs. In addition, the opportunities to manipulate gene expression, by stable overexpression of a gene of interest or RNA interference-mediated knockdown, generate valuable information about the mechanisms underlying lineage commitment and differentiation.

Erratum: Corrigendum: High-throughput epitope discovery reveals frequent recognition of neo-antigens by CD4+ T cells in human melanoma

Nat. Med. 21 81–85 (2015); published online 22 December 2014; corrected after print 18 August 2016 In the version of this article initially published, the article did not mention some restrictions on the availability of reagents. The following text has been added to the HTML and PDF versions of the paper: “The retroviral vectors containing BCL-6 and BCL-xL have been generated by a for-profit company, AIMM Therapeutics, which makes the plasmids available.