Neutrophils utilize immunoglobulins (Igs) to clear antigen, but their role in Ig production is unknown. Here we identified neutrophils around the marginal zone (MZ) of the spleen, a B cell area specialized in T-independent Ig responses to circulating antigen. Neutrophils colonized peri-MZ areas after post-natal mucosal colonization by microbes and enhanced their B-helper function upon receiving reprogramming signals from splenic sinusoidal endothelial cells, including interleukin 10 (IL-10). Splenic neutrophils induced Ig class switching, somatic hypermutation and antibody production by activating MZ B cells through a mechanism involving the cytokines BAFF, APRIL and IL-21. Neutropenic patients had fewer and hypomutated MZ B cells and less preimmune Igs to T-independent antigens, which indicates that neutrophils generate an innate layer of antimicrobial Ig defense by interacting with MZ B cells.

A dense mucous layer in the large intestine prevents inflammation by shielding the underlying epithelium from luminal bacteria and food antigens. This mucous barrier is organized around the hyperglycosylated mucin MUC2. Here we show that the small intestine has a porous mucous layer, which permitted the uptake of MUC2 by antigen-sampling dendritic cells (DCs). Glycans associated with MUC2 imprinted DCs with anti-inflammatory properties by assembling a galectin-3-Dectin-1-FcγRIIB receptor complex that activated β-catenin. This transcription factor interfered with DC expression of inflammatory but not tolerogenic cytokines by inhibiting gene transcription through nuclear factor-κB. MUC2 induced additional DC-conditioning signals via intestinal epithelial cells. Thus, mucus does not merely form a nonspecific physical barrier, but also constraints the immunogenicity of gut antigens by delivering tolerogenic signals.

Assessment of tumor infiltrating lymphocytes (TILs) in histopathological specimens can provide important prognostic information in diverse solid tumor types, and may also be of value in predicting response to treatments. However, implementation as a routine clinical biomarker has not yet been achieved. As successful use of immune checkpoint inhibitors and other forms of immunotherapy become a clinical reality, the need for widely applicable, accessible and reliable immuno-oncology biomarkers is clear. In Part 1 of this review we briefly discuss the host immune response to tumors and different approaches to TIL assessment. We propose a standardized methodology to assess TILs in solid tumors on H&E sections, in both primary and metastatic settings, based on the International Immuno-Oncology Biomarker Working Group guidelines for TIL assessment in invasive breast carcinoma. A review of the literature regarding the value of TIL assessment in different solid tumor types follows in Part 2. The method we propose is reproducible, affordable, easily applied, and has demonstrated prognostic and predictive significance in invasive breast carcinoma. This standardized methodology may be used as a reference against which other methods are compared, and should be evaluated for clinical validity and utility. Standardization of TIL assessment will help to improve consistency and reproducibility in this field, enrich both the quality and quantity of comparable evidence, and help to thoroughly evaluate the utility of TILs assessment in this era of immunotherapy.

Assessment of the immune response to tumors is growing in importance as the prognostic implications of this response are increasingly recognized, and as immunotherapies are evaluated and implemented in different tumor types. However, many different approaches can be used to assess and describe the immune response, which limits efforts at implementation as a routine clinical biomarker. In part 1 of this review, we have proposed a standardized methodology to assess tumor infiltrating lymphocytes (TILs) in solid tumors, based on the International Immuno-Oncology Biomarkers Working Group guidelines for invasive breast carcinoma. In part 2 of this review, we discuss the available evidence for the prognostic and predictive value of TILs in common solid tumors, including carcinomas of the lung, gastrointestinal tract, genitourinary system, gynecological system, and head and neck, as well as primary brain tumors, mesothelioma and melanoma. The particularities and different emphases in TIL assessment in different tumor types are discussed. The standardized methodology we propose can be adapted to different tumor types and may be used as a standard against which other approaches can be compared. Standardization of TIL assessment will help clinicians, researchers and pathologists to conclusively evaluate the utility of this simple biomarker in the current era of immunotherapy.

Antibody targeting of the immune checkpoint receptor PD1 produces therapeutic activity in a variety of solid tumors, but most patients exhibit partial or complete resistance to treatment for reasons that are unclear. In this study, we evaluated tumor specimens from 65 patients with melanoma, lung nonsquamous, squamous cell lung or head and neck cancers who were treated with the approved PD1-targeting antibodies pembrolizumab or nivolumab. Tumor RNA before anti-PD1 therapy was analyzed on the nCounter system using the PanCancer 730-Immune Panel, and we identified 23 immune-related genes or signatures linked to response and progression-free survival (PFS). In addition, we evaluated intra- and interbiopsy variability of PD1, PD-L1, CD8A, and CD4 mRNAs and their relationship with tumor-infiltrating lymphocytes (TIL) and PD-L1 IHC expression. Among the biomarkers examined, PD1 gene expression along with 12 signatures tracking CD8 and CD4 T-cell activation, natural killer cells, and IFN activation associated significantly with nonprogressive disease and PFS. These associations were independent of sample timing, drug used, or cancer type. TIL correlated moderately (∼0.50) with PD1 and CD8A mRNA levels and weakly (∼0.35) with CD4 and PD-L1. IHC expression of PD-L1 correlated strongly with PD-L1 (0.90), moderately with CD4 and CD8A, and weakly with PD1. Reproducibility of gene expression in intra- and interbiopsy specimens was very high (total SD <3%). Overall, our results support the hypothesis that identification of a preexisting and stable adaptive immune response as defined by mRNA expression pattern is reproducible and sufficient to predict clinical outcome, regardless of the type of cancer or the PD1 therapeutic antibody administered to patients. .

Innate lymphoid cells (ILCs) regulate stromal, epithelial and immune cells, but their impact on B cells remains unclear. We identified RORγt+ ILCs nearby the marginal zone (MZ), a splenic compartment containing innate-like B cells that respond to circulating T cell-independent (TI) antigens. Spenic ILCs established a bidirectional crosstalk with MAdCAM-1+ marginal reticular cells by providing tumor necrosis factor (TNF) and lymphotoxin, and activated MZ B cells via BAFF, CD40 ligand and the Notch ligand, Delta-like 1. Splenic ILCs further helped MZ B cells and their plasma cell progeny by co-opting neutrophils through the release of GM-CSF. Consequently, ILC depletion impaired both pre- and post-immune TI antibody responses. Thus, ILCs integrate stromal and myeloid signals to orchestrate innate-like antibody production at the interface between the immune and circulatory systems.

SummarySecretory immunoglobulin A (SIgA) enhances host-microbiota symbiosis, whereas SIgM remains poorly understood. We found that gut IgM+ plasma cells (PCs) were more abundant in humans than mice and clonally related to a large repertoire of memory IgM+ B cells disseminated throughout the intestine but rare in systemic lymphoid organs. In addition to sharing a gut-specific gene signature with memory IgA+ B cells, memory IgM+ B cells were related to some IgA+ clonotypes and switched to IgA in response to T cell-independent or T cell-dependent signals. These signals induced abundant IgM which, together with SIgM from clonally affiliated PCs, recognized mucus-embedded commensals. Bacteria recognized by human SIgM were dually coated by SIgA and showed increased richness and diversity compared to IgA-only-coated or uncoated bacteria. Thus, SIgM may emerge from pre-existing memory rather than newly activated naive IgM+ B cells and could help SIgA to anchor highly diverse commensal communities to mucus.

Stromal tumor-infiltrating lymphocytes (sTILs) are important prognostic and predictive biomarkers in triple-negative (TNBC) and HER2-positive breast cancer. Incorporating sTILs into clinical practice necessitates reproducible assessment. Previously developed standardized scoring guidelines have been widely embraced by the clinical and research communities. We evaluated sources of variability in sTIL assessment by pathologists in three previous sTIL ring studies. We identify common challenges and evaluate impact of discrepancies on outcome estimates in early TNBC using a newly-developed prognostic tool. Discordant sTIL assessment is driven by heterogeneity in lymphocyte distribution. Additional factors include: technical slide-related issues; scoring outside the tumor boundary; tumors with minimal assessable stroma; including lymphocytes associated with other structures; and including other inflammatory cells. Small variations in sTIL assessment modestly alter risk estimation in early TNBC but have the potential to affect treatment selection if cutpoints are employed. Scoring and averaging multiple areas, as well as use of reference images, improve consistency of sTIL evaluation. Moreover, to assist in avoiding the pitfalls identified in this analysis, we developed an educational resource available at www.tilsinbreastcancer.org/pitfalls.