Replicative history marks transcriptional and functional disparity in the CD8+ T cell memory pool.

Clonal expansion is a core aspect of T cell immunity. However, little is known with respect to the relationship between replicative history and the formation of distinct CD8+ memory T cell subgroups. To address this issue, we developed a genetic-tracing approach, termed the DivisionRecorder, that reports the extent of past proliferation of cell pools in vivo. Using this system to genetically 'record' the replicative history of different CD8+ T cell populations throughout a pathogen-specific immune response, we demonstrate that the central memory T (TCM) cell pool is marked by a higher number of prior divisions than the effector memory T cell pool, owing to the combination of strong proliferative activity during the acute immune response and selective proliferative activity after pathogen clearance. Furthermore, by combining DivisionRecorder analysis with single-cell transcriptomics and functional experiments, we show that replicative history identifies distinct cell pools within the TCM compartment. Specifically, we demonstrate that lowly divided TCM cells display enriched expression of stem-cell-associated genes, exist in a relatively quiescent state, and are superior in eliciting a proliferative recall response upon activation. These data provide the first evidence that a stem-cell-like memory T cell pool that reconstitutes the CD8+ T cell effector pool upon reinfection is marked by prior quiescence.

ITPK1 Sensitizes Tumor Cells to IgA-dependent Neutrophil Killing In Vivo.

Neutrophils can efficiently trigger cytotoxicity toward tumor cells and other target cells upon engagement of the IgA receptor CD89. However, the cell-intrinsic factors that influence the induction of cell death upon exposure to neutrophil effector mechanisms in vivo remain largely unknown. To uncover genetic regulators that influence target cell sensitivity to IgA-induced neutrophil-mediated killing, we used a human CD89 (hCD89) transgenic mouse model in which IgA-mediated killing of Her2-positive CD47-deficient murine target cells is mediated by neutrophils. Using a genome-wide in vivo screening approach, we demonstrate that deletion of the gene encoding inositol-tetrakisphosphate 1 kinase (ITPK1) increases survival of target cells in anti-Her2 IgA-treated mice. Moreover, we show that this effect depends on neutrophil activity and on the ITPK1 kinase domain. Notably, ITPK1 deficiency did not measurably impact survival of IgA-opsonized target cells in in vitro systems, underscoring the importance of in vivo screening systems to uncover physiologically relevant regulators of neutrophil killing.

What's in a name: the multifaceted function of DNA- and RNA-binding proteins in T cell responses.

Cellular differentiation allows cells to transition between different functional states and adapt to various environmental cues. The diversity and plasticity of this process is beautifully exemplified by T cells responding to pathogens, which undergo highly specialized differentiation tailored to the ongoing infection. Such antigen-induced T cell differentiation is regulated at the transcriptional level by DNA-binding proteins and at the post-transcriptional level by RNA-binding proteins. Although traditionally defined as separate protein classes, a growing body of evidence indicates an overlap between these two groups of proteins, collectively coined DNA/RNA-binding proteins (DRBPs). In this review, we describe how DRBPs might bind both DNA and RNA, discuss the putative functional relevance of this dual binding, and provide an exploratory analysis into characteristics that are associated with DRBPs. To exemplify the significance of DRBPs in T cell biology, we detail the activity of several established and putative DRBPs during the T cell response. Finally, we highlight several methodologies that allow untangling of the distinct functionalities of DRBPs at the DNA and RNA level, including key considerations to take into account when applying such methods.

Gene and protein sequence features augment HLA class I ligand predictions.

The sensitivity of malignant tissues to T cell-based immunotherapies depends on the presence of targetable human leukocyte antigen (HLA) class I ligands. Peptide-intrinsic factors, such as HLA class I affinity and proteasomal processing, have been established as determinants of HLA ligand presentation. However, the role of gene and protein sequence features as determinants of epitope presentation has not been systematically evaluated. We perform HLA ligandome mass spectrometry to evaluate the contribution of 7,135 gene and protein sequence features to HLA sampling. This analysis reveals that a number of predicted modifiers of mRNA and protein abundance and turnover, including predicted mRNA methylation and protein ubiquitination sites, inform on the presence of HLA ligands. Importantly, integration of such "hard-coded" sequence features into a machine learning approach augments HLA ligand predictions to a comparable degree as experimental measures of gene expression. Our study highlights the value of gene and protein features for HLA ligand predictions.

QPCTL regulates macrophage and monocyte abundance and inflammatory signatures in the tumor microenvironment.

The enzyme glutaminyl-peptide cyclotransferase-like protein (QPCTL) catalyzes the formation of pyroglutamate residues at the NH2-terminus of proteins, thereby influencing their biological properties. A number of studies have implicated QPCTL in the regulation of chemokine stability. Furthermore, QPCTL activity has recently been shown to be critical for the formation of the high-affinity SIRPα binding site of the CD47 "don't-eat-me" protein. Based on the latter data, interference with QPCTL activity -and hence CD47 maturation-may be proposed as a means to promote anti-tumor immunity. However, the pleiotropic activity of QPCTL makes it difficult to predict the effects of QPCTL inhibition on the tumor microenvironment (TME). Using a syngeneic mouse melanoma model, we demonstrate that QPCTL deficiency alters the intra-tumoral monocyte-to-macrophage ratio, results in a profound increase in the presence of pro-inflammatory cancer-associated fibroblasts (CAFs) relative to immunosuppressive TGF-ß1-driven CAFs, and leads to an increased IFN and decreased TGF-ß transcriptional response signature in tumor cells. Importantly, the functional relevance of the observed TME remodeling is demonstrated by the synergy between QPCTL deletion and anti PD-L1 therapy, sensitizing an otherwise refractory melanoma model to anti-checkpoint therapy. Collectively, these data provide support for the development of strategies to interfere with QPCTL activity as a means to promote tumor-specific immunity.

A committed tissue-resident memory T cell precursor within the circulating CD8+ effector T cell pool.

An increasing body of evidence emphasizes the role of tissue-resident memory T cells (TRM) in the defense against recurring pathogens and malignant neoplasms. However, little is known with regard to the origin of these cells and their kinship to other CD8+ T cell compartments. To address this issue, we followed the antigen-specific progeny of individual naive CD8+ T cells to the T effector (TEFF), T circulating memory (TCIRCM), and TRM pools by lineage-tracing and single-cell transcriptome analysis. We demonstrate that a subset of T cell clones possesses a heightened capacity to form TRM, and that enriched expression of TRM-fate-associated genes is already apparent in the circulating TEFF offspring of such clones. In addition, we demonstrate that the capacity to generate TRM is permanently imprinted at the clonal level, before skin entry. Collectively, these data provide compelling evidence for early stage TRM fate decisions and the existence of committed TRM precursor cells in the circulatory TEFF compartment.

A mouse model that is immunologically tolerant to reporter and modifier proteins.

Reporter proteins have become an indispensable tool in biomedical research. However, exogenous introduction of these reporters into mice poses a risk of rejection by the immune system. Here, we describe the generation, validation and application of a multiple reporter protein tolerant 'Tol' mouse model that constitutively expresses an assembly of shuffled reporter proteins from a single open reading frame. We demonstrate that expression of the Tol transgene results in the deletion of CD8+ T cells specific for a model epitope, and substantially improves engraftment of reporter-gene transduced T cells. The Tol strain provides a valuable mouse model for cell transfer and viral-mediated gene transfer studies, and serves as a methodological example for the generation of poly-tolerant mouse strains.

Comprehensive analysis of cutaneous and uveal melanoma liver metastases.

BACKGROUND: The profound disparity in response to immune checkpoint blockade (ICB) by cutaneous melanoma (CM) and uveal melanoma (UM) patients is not well understood. Therefore, we characterized metastases of CM and UM from the same metastatic site (liver), in order to dissect the potential underlying mechanism in differential response on ICB. METHODS: Tumor liver samples from CM (n=38) and UM (n=28) patients were analyzed at the genomic (whole exome sequencing), transcriptional (RNA sequencing) and protein (immunohistochemistry and GeoMx Digital Spatial Profiling) level. RESULTS: Comparison of CM and UM metastases from the same metastatic site revealed that, although originating from the same melanocyte lineage, CM and UM differed in somatic mutation profile, copy number profile, tumor mutational burden (TMB) and consequently predicted neoantigens. A higher melanin content and higher expression of the melanoma differentiation antigen MelanA was observed in liver metastases of UM patients. No difference in B2M and human leukocyte antigen-DR (HLA-DR) expression was observed. A higher expression of programmed cell death ligand 1 (PD-L1) was found in CM compared with UM liver metastases, although the majority of CM and UM liver metastases lacked PD-L1 expression. There was no difference in the extent of immune infiltration observed between CM and UM metastases, with the exception of a higher expression of CD163 (p<0.0001) in CM liver samples. While the extent of immune infiltration was similar for CM and UM metastases, the ratio of exhausted CD8 T cells to cytotoxic T cells, to total CD8 T cells and to Th1 cells, was significantly higher in UM metastases. CONCLUSIONS: While TMB was different between CM and UM metastases, tumor immune infiltration was similar. The greater dependency on PD-L1 as an immune checkpoint in CM and the identification of higher exhaustion ratios in UM may both serve as explanations for the difference in response to ICB. Consequently, in order to improve current treatment for metastatic UM, reversal of T cell exhaustion beyond programmed cell death 1 blockade should be considered.

High-throughput identification of antigen-specific TCRs by TCR gene capture.

The transfer of T cell receptor (TCR) genes into patient T cells is a promising approach for the treatment of both viral infections and cancer. Although efficient methods exist to identify antibodies for the treatment of these diseases, comparable strategies to identify TCRs have been lacking. We have developed a high-throughput DNA-based strategy to identify TCR sequences by the capture and sequencing of genomic DNA fragments encoding the TCR genes. We establish the value of this approach by assembling a large library of cancer germline tumor antigen-reactive TCRs. Furthermore, by exploiting the quantitative nature of TCR gene capture, we show the feasibility of identifying antigen-specific TCRs in oligoclonal T cell populations from either human material or TCR-humanized mice. Finally, we demonstrate the ability to identify tumor-reactive TCRs within intratumoral T cell subsets without knowledge of antigen specificities, which may be the first step toward the development of autologous TCR gene therapy to target patient-specific neoantigens in human cancer.