Severe combined immunodeficiency (SCID) is characterized by arrested T-lymphocyte production and by B-lymphocyte dysfunction, which result in life-threatening infections. Early diagnosis of SCID through population-based screening of newborns can aid clinical management and help improve outcomes; it also permits the identification of previously unknown factors that are essential for lymphocyte development in humans.SCID was detected in a newborn before the onset of infections by means of screening of T-cell-receptor excision circles, a biomarker for thymic output. On confirmation of the condition, the affected infant was treated with allogeneic hematopoietic stem-cell transplantation. Exome sequencing in the patient and parents was followed by functional analysis of a prioritized candidate gene with the use of human hematopoietic stem cells and zebrafish embryos.The infant had "leaky" SCID (i.e., a form of SCID in which a minimal degree of immune function is preserved), as well as craniofacial and dermal abnormalities and the absence of a corpus callosum; his immune deficit was fully corrected by hematopoietic stem-cell transplantation. Exome sequencing revealed a heterozygous de novo missense mutation, p.N441K, in BCL11B. The resulting BCL11B protein had dominant negative activity, which abrogated the ability of wild-type BCL11B to bind DNA, thereby arresting development of the T-cell lineage and disrupting hematopoietic stem-cell migration; this revealed a previously unknown function of BCL11B. The patient's abnormalities, when recapitulated in bcl11ba-deficient zebrafish, were reversed by ectopic expression of functionally intact human BCL11B but not mutant human BCL11B.Newborn screening facilitated the identification and treatment of a previously unknown cause of human SCID. Coupling exome sequencing with an evaluation of candidate genes in human hematopoietic stem cells and in zebrafish revealed that a constitutional BCL11B mutation caused human multisystem anomalies with SCID and also revealed a prethymic role for BCL11B in hematopoietic progenitors. (Funded by the National Institutes of Health and others.).
Costimulatory and coinhibitory receptors on T-cells are key mediators of the overall immune response, and the clinical success of checkpoint inhibitor therapies has proven the therapeutic benefits of targeting these pathways. Viable, cryopreserved dissociated tissue provides an opportunity to understand the dynamic expression patterns of these receptor/ligand sets at the single-cell level, providing insights into the differential expression of each receptor/ligand pair across different cancer indications as a means to target new therapeutic interventions.
Methods
The study compared expression patterns from two cohorts composed of indications that are clinically approved for checkpoint inhibitor therapy versus indications that are not currently approved for checkpoint therapy. We performed bulk RNA sequencing on both cohorts to understand the global expression of costimulatory and coinhibitory receptors, and their ligands, within the tumor microenvironment. Furthermore, we developed a flow cytometry panel that allows for efficient screening of these receptor/ligand sets on the major cellular subsets (tumor cells, T-cells, B-cells, myeloid cells) within the tumor microenvironment. These data sets were initially evaluated for the expression patterns of PD1/PDL1/PDL2 as the standard for immunomodulatory receptors, and to evaluate the accuracy and sensitivity of the methods for the evaluation of receptor/ligand expression. In addition, we evaluated the expression patterns of two less well-characterized immunomodulatory receptors sets: TIGIT/PVR/PVRL2/CD226 and CD160/HVEM/BTLA/LIGHT to provide new insight into the expression patterns of these potential receptor/ligand sets and to better understand their value as therapeutic targets for specific cancer indications.
Results
The protocols developed for this study were able to provide a clear and detailed picture of the receptor-ligand expression patterns across different cancer indications. Consistent with previous reports, PD1 expression was high on T-cell subsets, but low on the other lymphoid subsets within the tumor microenvironment. PDL1, on the other hand, was more differentially expressed on tumor cells across patient samples and was also expressed on myeloid cell populations. The expression patterns for the TGIT and CD160 receptors and their associated ligands demonstrate the distinct differences in expression patterns across cancer indications.
Conclusions
Given the varied and distinct expression patterns observed in different cancer indications, it is clear that the successful targeting of immune modulatory therapeutics will require an understanding of the expression of individual receptors and ligands at the single-cell level. This study demonstrates the utility of dissociated tumor cells as a tool to provide detailed insight into, and an understanding of the receptor-ligand expression of different cancer indications.
Unlike αβ-T lineage cells, where the role of ligand in intrathymic selection is well established, the role of ligand in the development of γδ-T cells remains controversial. Here we provide evidence for the role of a bona fide selecting ligand in shaping the γδ-T cell-receptor (TCR) repertoire. Reactivity of the γδ-TCR with the major histocompatibility complex (MHC) Class Ib ligands, H2-T10/22, is critically dependent upon the EGYEL motif in the complementarity determining region 3 (CDR3) of TCRδ. In the absence of H2-T10/22 ligand, the commitment of H2-T10/22 reactive γδ-T cells to the γδ fate is diminished, and the specification of those γδ committed cells to the IFN-γ or interleukin-17 effector fate is altered. Furthermore, those cells that do adopt the γδ fate and mature exhibit a profound alteration in the γδTCR repertoire, including depletion of the EGYEL motif and reductions in both CDR3δ length and charge. Taken together, these data suggest that ligand plays an important role in shaping the TCR repertoire of γδ-T cells.
The c-Myb transcription factor is required for normal adult hematopoiesis. However, the embryonic lethality of Myb-null mutations has been an impediment to identifying roles for c-Myb during lymphocyte development. We have used tissue-specific inactivation of the Myb locus in early progenitor cells to demonstrate that c-Myb is absolutely required for the differentiation of CD19(+) B-lineage cells and B cell differentiation is profoundly blocked beyond the pre-pro-B cell stage in Myb(f/f) Mb1-cre mice. We demonstrate that c-Myb is required for the intrinsic survival of CD19(+) pro-B cells as well as the proper expression of the alpha-chain of the IL-7 receptor (CD127) and Ebf1. However, survival of c-Myb-deficient CD19(+) pro-B cells cannot be rescued by transduction with CD127-producing retrovirus, suggesting that c-Myb controls a survival pathway independent of CD127. Furthermore, c-Myb-deficient progenitor cells inefficiently generate CD19(+) B-lineage cells during stromal cell culture but this process can be partially rescued with exogenous Ebf1. Thus, c-Myb does not appear to be required for commitment to B cell differentiation but is crucial for B cell differentiation to the CD19(+) pro-B cell stage as well as survival of CD19(+) pro-B cells. Surprisingly, forced c-Myb expression in lymphoid-primed multipotent progenitors favors differentiation toward the myeloid lineage, suggesting that proper c-Myb expression is crucial for B-lineage development.
Abstract c-Myb is abundantly expressed in lymphocyte progenitors yet little is known about c-Myb function during lymphocyte development due to the embryonic lethality of Myb null mutations. We have targeted the murine Myb locus with loxP sites for conditional deletion and crossed Mybff mice to the Mb1Cre and CD19Cre strains to ablate c-Myb expression during different stages of B cell development. Mb1Cre mediated deletion begins in pre-pro-B cells and is complete by the pro-B cell stage. We detect a no decrease in the number pre-pro-B cells but <1% the normal number of CD19+ B-lineage cells at any subsequent stage of development, demonstrating that c-Myb is essential for the development past the point of commitment to the B cell lineage. In Mybf/f CD19Cre mice, B-lineage cells are reduced in number by 80% and blocked in transition from pro-B to pre-B cells, resulting in about 10% the normal number of IgM+ bone marrow B cells. c-Myb deficient B cells are hyporesponsive to IL-7 in methylcellulose cloning assays and in vitro proliferation assays, suggesting that at least part of the defect in pro-B to pre-B cell transition is due to a defect in the IL-7 signaling pathway.
