Resident memory CD8+ T cells in regional lymph nodes mediate immunity to metastatic melanoma.

The nature of the anti-tumor immune response changes as primary tumors progress and metastasize. We investigated the role of resident memory (Trm) and circulating memory (Tcirm) cells in anti-tumor responses at metastatic locations using a mouse model of melanoma-associated vitiligo. We found that the transcriptional characteristics of tumor-specific CD8+ T cells were defined by the tissue of occupancy. Parabiosis revealed that tumor-specific Trm and Tcirm compartments persisted throughout visceral organs, but Trm cells dominated lymph nodes (LNs). Single-cell RNA-sequencing profiles of Trm cells in LN and skin were distinct, and T cell clonotypes that occupied both tissues were overwhelmingly maintained as Trm in LNs. Whereas Tcirm cells prevented melanoma growth in the lungs, Trm afforded long-lived protection against melanoma seeding in LNs. Expanded Trm populations were also present in melanoma-involved LNs from patients, and their transcriptional signature predicted better survival. Thus, tumor-specific Trm cells persist in LNs, restricting metastatic cancer.

Validation of Ligand Tetramers for the Detection of Antigen-Specific Lymphocytes.

The study of Ag-specific lymphocytes has been a key advancement in immunology over the past few decades. The development of multimerized probes containing Ags, peptide:MHC complexes, or other ligands was one innovation allowing the direct study of Ag-specific lymphocytes by flow cytometry. Although these types of study are now common and performed by thousands of laboratories, quality control and assessment of probe quality are often minimal. In fact, many of these types of probe are made in-house, and protocols vary between laboratories. Although peptide:MHC multimers can often be obtained from commercial sources or core facilities, few such services exist for Ag multimers. To ensure high quality and consistency with ligand probes, we have developed an easy and robust multiplexed approach using commercially available beads able to bind Abs specific for the ligand of interest. Using this assay, we have sensitively assessed the performance of peptide:MHC and Ag tetramers and have found considerable batch-to-batch variability in performance and stability over time more easily than using murine or human cell-based assays. This bead-based assay can also reveal common production errors such as miscalculation of Ag concentration. This work could set the stage for the development of standardized assays for all commonly used ligand probes to limit laboratory-to-laboratory technical variation and experimental failure caused by probe underperformance.

Memory CD8+ T cell responses to cancer.

Long-lived memory CD8+ T cells play important roles in tumor immunity. Studies over the past two decades have identified four subsets of memory CD8+ T cells - central, effector, stem-like, and tissue resident memory - that either circulate through blood, lymphoid and peripheral organs, or reside in tissues where cancers develop. In this article, we will review studies from both pre-clinical mouse models and human patients to summarize the phenotype, distribution and unique features of each memory subset, and highlight specific roles of each subset in anti-tumor immunity. Moreover, we will discuss how stem-cell like and resident memory CD8+ T cell subsets relate to exhausted tumor-infiltrating lymphocytes (TIL) populations. These studies reveal how memory CD8+ T cell subsets together orchestrate durable immunity to cancer.

CD39 is expressed on functional effector and tissue resident memory CD8+ T cells.

The ecto-ATPase CD39 is expressed on exhausted CD8+ T cells in chronic viral infection and has been proposed as a marker of tumor-specific CD8+ T cells in cancer, but the role of CD39 in an effector and memory T cell response has not been clearly defined. We report that CD39 is expressed on antigen-specific CD8+ short-lived effector cells (SLECs), while it's co-ecto-enzyme, CD73, is found on memory precursor effector cells (MPEC) in vivo . Inhibition of CD39 enzymatic activity during in vitro T cell priming enhances MPEC differentiation in vivo after transfer and infection. The enriched MPEC phenotype is associated with enhanced tissue resident memory (T RM ) establishment in the brain and salivary gland following an acute intranasal viral infection, suggesting that CD39 ATPase activity plays a role in memory CD8+ T cell differentiation. We also show that CD39 is expressed on human and murine T RM across several non-lymphoid tissues and melanoma, while CD73 is expressed on both circulating and resident memory subsets in mice. In contrast to exhausted CD39+ T cells in chronic infection, CD39+ T RM are fully functional when stimulated ex vivo with cognate antigen. This work further expands the identity of CD39 beyond a T cell exhaustion marker.

The Effect of Lung Resection for NSCLC on Circulating Immune Cells: A Pilot Study.

This pilot study sought to evaluate the circulating levels of immune cells, particularly regulatory T-cell (Treg) subsets, before and after lung resection for non-small cell lung cancer. Twenty-five patients consented and had specimens collected. Initially, peripheral blood of 21 patients was collected for circulating immune cell studies. Two of these patients were excluded due to technical issues, leaving 19 patients for the analyses of circulating immune cells. Standard gating and high-dimensional unsupervised clustering flow cytometry analyses were performed. The blood, tumors and lymph nodes were analyzed via single-cell RNA and TCR sequencing for Treg analyses in a total of five patients (including four additional patients from the initial 21 patients). Standard gating flow cytometry revealed a transient increase in neutrophils immediately following surgery, with a variable neutrophil-lymphocyte ratio and a stable CD4-CD8 ratio. Unexpectedly, the total Treg and Treg subsets did not change with surgery with standard gating in short- or long-term follow-up. Similarly, unsupervised clustering of Tregs revealed a dominant cluster that was stable perioperatively and long-term. Two small FoxP3hi clusters slightly increased following surgery. In the longer-term follow-up, these small FoxP3hi Treg clusters were not identified, indicating that they were likely a response to surgery. Single-cell sequencing demonstrated six CD4+FoxP3+ clusters among the blood, tumors and lymph nodes. These clusters had a variable expression of FoxP3, and several were mainly, or only, present in tumor and lymph node tissue. As such, serial monitoring of circulating Tregs may be informative, but not completely reflective of the Tregs present in the tumor microenvironment.

Resident and circulating memory T cells persist for years in melanoma patients with durable responses to immunotherapy.

While T-cell responses to cancer immunotherapy have been avidly studied, long-lived memory has been poorly characterized. In a cohort of metastatic melanoma survivors with exceptional responses to immunotherapy, we probed memory CD8+ T-cell responses across tissues, and across several years. Single-cell RNA sequencing revealed three subsets of resident memory T (TRM) cells shared between tumors and distant vitiligo-affected skin. Paired T-cell receptor sequencing further identified clonotypes in tumors that co-existed as TRM in skin and as effector memory T (TEM) cells in blood. Clonotypes that dispersed throughout tumor, skin, and blood preferentially expressed a IFNG / TNF-high signature, which had a strong prognostic value for melanoma patients. Remarkably, clonotypes from tumors were found in patient skin and blood up to nine years later, with skin maintaining the most focused tumor-associated clonal repertoire. These studies reveal that cancer survivors can maintain durable memory as functional, broadly-distributed TRM and TEM compartments.