T cell receptor (TCR) binding strength to peptide-MHC antigen complex influences numerous T cell functions. However, the vast diversity of a polyclonal T cell repertoire for even a single antigen greatly increases the complexity of studying the impact TCR affinity has on T cell function. Here, we determined how TCR binding strength affected the protein and transcriptional profile of an endogenous, polyclonal T cell response to a known tumor-associated antigen (TAA) within the tumor microenvironment (TME). We confirmed that flow and CITE-Seq counts of MHC-tetramer labeling were reliable surrogates for the TCR-peptide-MHC steady-state binding affinity. We further demonstrated by single-cell RNA sequencing that tumor-infiltrating lymphocytes (TILs) with high and low binding affinity for a TAA can differentiate into cells with many antigen-specific transcriptional profiles within an established TME. However, more progenitor-like phenotypes were significantly biased towards lower affinity T cells, and proliferating phenotypes showed significant bias towards high-affinity TILs. Additionally, we found in a progressing TME that higher affinity T cells advanced more rapidly to terminal phases of T cell exhaustion and exhibited better tumor control. We confirmed the polyclonal TIL results using a TCR transgenic mouse possessing a single low-affinity TCR targeting the same TAA. These T cells maintained a progenitor-exhausted phenotype and exhibited impaired tumor control. We propose that high-affinity TCR interactions drive T cell fate decisions more rapidly than low-affinity interactions and that these cells differentiate faster. These findings illustrate divergent forms of T cell dysfunction based on TCR affinity which may impact TIL therapies and antitumor responses.

CITE-Seq enables simultaneous single cell transcriptome and proteome analysis via combining single cell RNA-seq with oligo-labeled antibodies. Conventional techniques such as flow or mass cytometry have caveats including the number of epitopes accurately detected or an inability to recover samples for transcriptome analysis. These limitations are prohibitive for multivariate analysis of limited clinical samples. We developed a CITE-seq assay that enables comprehensive immune cell profiling of Sarcoidosis. Sarcoidosis is a granulomatous lung disease characterized by abnormal CD4+ T cell Th1 activity. However, the disease etiology and course are variable and the underlying molecular drivers remain unknown. The long-term goal of this study is to utilize CITE-seq to identify immune molecular pathways of Sarcoidosis pathogenesis. In our initial studies, we analyzed PBMC’s by CITE-seq vs. flow cytometry and observed similar cell profiles. However, the synergy of protein detection coupled with transcriptome analysis via CITE-seq enhanced cell subset identification vs. flow or scRNA-seq alone. We utilized CITE-seq in an ongoing longitudinal study of Sarcoidosis subjects to enhance resolution of the immune components contributing to disease. We compared CITE-seq to a flow cytometry panel analyzing the differential contributions of various CD4+ T cell lineages. The enhanced granularity provided by CITE-seq elucidated molecular pathways associated with disease pathogenesis. Thus, moving forward CITE-seq can provide the resolution and multivariate data collection required to identify the inflammatory drivers of Sarcoidosis.

IntroductionSarcoidosis is a multiorgan granulomatous disorder thought to be triggered and influenced by gene–environment interactions. Sarcoidosis affects 45–300/100 000 individuals in the USA and has an increasing mortality rate. The greatest gap in knowledge about sarcoidosis pathobiology is a lack of understanding about the underlying immunological mechanisms driving progressive pulmonary disease. The objective of this study is to define the lung-specific and blood-specific longitudinal changes in the adaptive immune response and their relationship to progressive and non-progressive pulmonary outcomes in patients with recently diagnosed sarcoidosis.Methods and analysisThe BRonchoscopy at Initial sarcoidosis diagnosis Targeting longitudinal Endpoints study is a US-based, NIH-sponsored longitudinal blood and bronchoscopy study. Enrolment will occur over four centres with a target sample size of 80 eligible participants within 18 months of tissue diagnosis. Participants will undergo six study visits over 18 months. In addition to serial measurement of lung function, symptom surveys and chest X-rays, participants will undergo collection of blood and two bronchoscopies with bronchoalveolar lavage separated by 6 months. Freshly processed samples will be stained and flow-sorted for isolation of CD4 +T helper (Th1, Th17.0 and Th17.1) and T regulatory cell immune populations, followed by next-generation RNA sequencing. We will construct bioinformatic tools using this gene expression to define sarcoidosis endotypes that associate with progressive and non-progressive pulmonary disease outcomes and validate the tools using an independent cohort.Ethics and disseminationThe study protocol has been approved by the Institutional Review Boards at National Jewish Hospital (IRB# HS-3118), University of Iowa (IRB# 201801750), Johns Hopkins University (IRB# 00149513) and University of California, San Francisco (IRB# 17-23432). All participants will be required to provide written informed consent. Findings will be disseminated via journal publications, scientific conferences, patient advocacy group online content and social media platforms.

The contribution and regulation of various CD4+ T cell lineages that occur with remitting vs progressive courses in sarcoidosis are poorly understood. We developed a multiparameter flow cytometry panel to sort these CD4+ T cell lineages followed by measurement of their functional potential using RNA-sequencing analysis at six-month intervals across multiple study sites. To obtain good quality RNA for sequencing, we relied on chemokine receptor expression to identify and sort lineages. To minimize gene expression changes induced by perturbations of T cells and avoid protein denaturation caused by freeze/thaw cycles, we optimized our protocols using freshly isolated samples at each study site. To accomplish this study, we had to overcome significant standardization challenges across multiple sites. Here, we detail standardization considerations for cell processing, flow staining, data acquisition, sorting parameters, and RNA quality control analysis that were performed as part of the NIH-sponsored, multi-center study, BRonchoscopy at Initial sarcoidosis diagnosis Targeting longitudinal Endpoints (BRITE). After several rounds of iterative optimization, we identified the following aspects as critical for successful standardization: 1) alignment of PMT voltages across sites using CS&T/rainbow bead technology; 2) a single template created in the cytometer program that was used by all sites to gate cell populations during data acquisition and cell sorting; 3) use of standardized lyophilized flow cytometry staining cocktails to reduce technical error during processing; 4) development and implementation of a standardized Manual of Procedures. After standardization of cell sorting, we were able to determine the minimum number of sorted cells necessary for next generation sequencing through analysis of RNA quality and quantity from sorted T cell populations. Overall, we found that implementing a multi-parameter cell sorting with RNA-seq analysis clinical study across multiple study sites requires iteratively tested standardized procedures to ensure comparable and high-quality results.

<p>Flow cytometry panels, gating strategy and comparison of TILs to other gene signatures.</p>