Age-associated Senescent - T Cell Signaling Promotes Type 3 Immunity that Inhibits the Biomaterial Regenerative Response.

Aging is associated with immunological changes that compromise response to infections and vaccines, exacerbate inflammatory diseases and can potentially mitigate tissue repair. Even so, age-related changes to the immune response to tissue damage and regenerative medicine therapies remain unknown. Here, it is characterized how aging induces changes in immunological signatures that inhibit tissue repair and therapeutic response to a clinical regenerative biological scaffold derived from extracellular matrix. Signatures of inflammation and interleukin (IL)-17 signaling increased with injury and treatment both locally and regionally in aged animals, and computational analysis uncovered age-associated senescent-T cell communication that promotes type 3 immunity in T cells. Local inhibition of type 3 immune activation using IL17-neutralizing antibodies improves healing and restores therapeutic response to the regenerative biomaterial, promoting muscle repair in older animals. These results provide insights into tissue immune dysregulation that occurs with aging that can be targeted to rejuvenate repair.

Transfer learning in a biomaterial fibrosis model identifies in vivo senescence heterogeneity and contributions to vascularization and matrix production across species and diverse pathologies.

Cellular senescence is a state of permanent growth arrest that plays an important role in wound healing, tissue fibrosis, and tumor suppression. Despite senescent cells' (SnCs) pathological role and therapeutic interest, their phenotype in vivo remains poorly defined. Here, we developed an in vivo-derived senescence signature (SenSig) using a foreign body response-driven fibrosis model in a p16-CreERT2;Ai14 reporter mouse. We identified pericytes and "cartilage-like" fibroblasts as senescent and defined cell type-specific senescence-associated secretory phenotypes (SASPs). Transfer learning and senescence scoring identified these two SnC populations along with endothelial and epithelial SnCs in new and publicly available murine and human data single-cell RNA sequencing (scRNAseq) datasets from diverse pathologies. Signaling analysis uncovered crosstalk between SnCs and myeloid cells via an IL34-CSF1R-TGFßR signaling axis, contributing to tissue balance of vascularization and matrix production. Overall, our study provides a senescence signature and a computational approach that may be broadly applied to identify SnC transcriptional profiles and SASP factors in wound healing, aging, and other pathologies.

Helminth egg derivatives as proregenerative immunotherapies.

The immune system is increasingly recognized as an important regulator of tissue repair. We developed a regenerative immunotherapy from the helminth Schistosoma mansoni soluble egg antigen (SEA) to stimulate production of interleukin (IL)-4 and other type 2-associated cytokines without negative infection-related sequelae. The regenerative SEA (rSEA) applied to a murine muscle injury induced accumulation of IL-4-expressing T helper cells, eosinophils, and regulatory T cells and decreased expression of IL-17A in gamma delta (γδ) T cells, resulting in improved repair and decreased fibrosis. Encapsulation and controlled release of rSEA in a hydrogel further enhanced type 2 immunity and larger volumes of tissue repair. The broad regenerative capacity of rSEA was validated in articular joint and corneal injury models. These results introduce a regenerative immunotherapy approach using natural helminth derivatives.

Lung Cancer Recurrence Risk Prediction through Integrated Deep Learning Evaluation.

Background: Prognostic risk factors for completely resected stage IA non-small-cell lung cancers (NSCLCs) have advanced minimally over recent decades. Although several biomarkers have been found to be associated with cancer recurrence, their added value to TNM staging and tumor grade are unclear. Methods: Features of preoperative low-dose CT image and histologic findings of hematoxylin- and eosin-stained tissue sections of resected lung tumor specimens were extracted from 182 stage IA NSCLC patients in the National Lung Screening Trial. These features were combined to predict the risk of tumor recurrence or progression through integrated deep learning evaluation (IDLE). Added values of IDLE to TNM staging and tumor grade in progression risk prediction and risk stratification were evaluated. Results: The 5-year AUC of IDLE was 0.817 ± 0.037 as compared to the AUC = 0.561 ± 0.042 and 0.573 ± 0.044 from the TNM stage and tumor grade, respectively. The IDLE score was significantly associated with cancer recurrence (p < 0.0001) even after adjusting for TNM staging and tumor grade. Synergy between chest CT image markers and histological markers was the driving force of the deep learning algorithm to produce a stronger prognostic predictor. Conclusions: Integrating markers from preoperative CT images and pathologist's readings of resected lung specimens through deep learning can improve risk stratification of stage 1A NSCLC patients over TNM staging and tumor grade alone. Our study suggests that combining markers from nonoverlapping platforms can increase the cancer risk prediction accuracy.

Inflammation, microbiome and colorectal cancer disparity in African-Americans: Are there bugs in the genetics?

Dysregulated interactions between host inflammation and gut microbiota over the course of life increase the risk of colorectal cancer (CRC). While environmental factors and socio-economic realities of race remain predominant contributors to CRC disparities in African-Americans (AAs), this review focuses on the biological mediators of CRC disparity, namely the under-appreciated influence of inherited ancestral genetic regulation on mucosal innate immunity and its interaction with the microbiome. There remains a poor understanding of mechanisms linking immune-related genetic polymorphisms and microbiome diversity that could influence chronic inflammation and exacerbate CRC disparities in AAs. A better understanding of the relationship between host genetics, bacteria, and CRC pathogenesis will improve the prediction of cancer risk across race/ethnicity groups overall.

Human Colon Cancer-Derived Clostridioides difficile Strains Drive Colonic Tumorigenesis in Mice.

Defining the complex role of the microbiome in colorectal cancer and the discovery of novel, protumorigenic microbes are areas of active investigation. In the present study, culturing and reassociation experiments revealed that toxigenic strains of Clostridioides difficile drove the tumorigenic phenotype of a subset of colorectal cancer patient-derived mucosal slurries in germ-free ApcMin/+ mice. Tumorigenesis was dependent on the C. difficile toxin TcdB and was associated with induction of Wnt signaling, reactive oxygen species, and protumorigenic mucosal immune responses marked by the infiltration of activated myeloid cells and IL17-producing lymphoid and innate lymphoid cell subsets. These findings suggest that chronic colonization with toxigenic C. difficile is a potential driver of colorectal cancer in patients. SIGNIFICANCE: Colorectal cancer is a leading cause of cancer and cancer-related deaths worldwide, with a multifactorial etiology that likely includes procarcinogenic bacteria. Using human colon cancer specimens, culturing, and murine models, we demonstrate that chronic infection with the enteric pathogen C. difficile is a previously unrecognized contributor to colonic tumorigenesis. See related commentary by Jain and Dudeja, p. 1838. This article is highlighted in the In This Issue feature, p. 1825.

Murine fecal microbiota transfer models selectively colonize human microbes and reveal transcriptional programs associated with response to neoadjuvant checkpoint inhibitors.

Human gut microbial species found to associate with clinical responses to immune checkpoint inhibitors (ICIs) are often tested in mice using fecal microbiota transfer (FMT), wherein tumor responses in recipient mice may recapitulate human responses to ICI treatment. However, many FMT studies have reported only limited methodological description, details of murine cohorts, and statistical methods. To investigate the reproducibility and robustness of gut microbial species that impact ICI responses, we performed human to germ-free mouse FMT using fecal samples from patients with non-small cell lung cancer who had a pathological response or nonresponse after neoadjuvant ICI treatment. R-FMT mice yielded greater anti-tumor responses in combination with anti-PD-L1 treatment compared to NR-FMT, although the magnitude varied depending on mouse cell line, sex, and individual experiment. Detailed investigation of post-FMT mouse microbiota using 16S rRNA amplicon sequencing, with models to classify and correct for biological variables, revealed a shared presence of the most highly abundant taxa between the human inocula and mice, though low abundance human taxa colonized mice more variably after FMT. Multiple Clostridium species also correlated with tumor outcome in individual anti-PD-L1-treated R-FMT mice. RNAseq analysis revealed differential expression of T and NK cell-related pathways in responding tumors, irrespective of FMT source, with enrichment of these cell types confirmed by immunohistochemistry. This study identifies several human gut microbial species that may play a role in clinical responses to ICIs and suggests attention to biological variables is needed to improve reproducibility and limit variability across experimental murine cohorts.

Pathways of immune exclusion in metastatic osteosarcoma are associated with inferior patient outcomes.

BACKGROUND: Current therapy for osteosarcoma pulmonary metastases (PMs) is ineffective. The mechanisms that prevent successful immunotherapy in osteosarcoma are incompletely understood. We investigated the tumor microenvironment of metastatic osteosarcoma with the goal of harnessing the immune system as a therapeutic strategy. METHODS: 66 osteosarcoma tissue specimens were analyzed by immunohistochemistry (IHC) and immune markers were digitally quantified. Tumor-infiltrating lymphocytes (TILs) from 25 specimens were profiled by functional cytometry. Comparative transcriptomic studies of distinct tumor-normal lung 'PM interface' and 'PM interior' regions from 16 PMs were performed. Clinical follow-up (median 24 months) was available from resection. RESULTS: IHC revealed a statistically significantly higher concentration of TILs expressing immune checkpoint and immunoregulatory molecules in PMs compared with primary bone tumors (including programmed cell death 1 (PD-1), programmed death ligand 1 (PD-L1), lymphocyte-activation gene 3 (LAG-3), T-cell immunoglobulin and mucin domain-containing protein 3 (TIM-3), and indoleamine 2,3-dioxygenase (IDO1). Remarkably, these lymphocytes are excluded at the PM interface compared with PM interior. TILs from PMs exhibited significantly higher amounts of PD-1 and LAG-3 and functional cytokines including interferon-γ (IFNγ) by flow cytometry. Gene expression profiling further confirmed the presence of CD8 and CD4 lymphocytes concentrated at the PM interface, along with upregulation of immunoregulatory molecules and IFNγ-driven genes in the same region. We further discovered a strong alternatively activated macrophage signature throughout the entire PMs along with a polymorphonuclear myeloid-derived suppressor cell signature focused at the PM interface. Expression of PD-L1, LAG-3, and colony-stimulating factor 1 receptor (CSF1R) at the PM interface was associated with significantly worse progression-free survival (PFS), while gene sets indicative of productive T cell immune responses (CD8 T cells, T cell survival, and major histocompatibility complex class 1 expression) were associated with significantly improved PFS. CONCLUSIONS: Osteosarcoma PMs exhibit immune exclusion characterized by the accumulation of TILs at the PM interface. These TILs produce effector cytokines, suggesting their capability of activation and recognition of tumor antigens. Our findings suggest cooperative immunosuppressive mechanisms in osteosarcoma PMs including immune checkpoint molecule expression and the presence of immunosuppressive myeloid cells. We identify cellular and molecular signatures that are associated with patient outcomes, which could be exploited for successful immunotherapy.

G-protein coupled receptor 35 (GPR35) regulates the colonic epithelial cell response to enterotoxigenic Bacteroides fragilis.

G protein-coupled receptor (GPR)35 is highly expressed in the gastro-intestinal tract, predominantly in colon epithelial cells (CEC), and has been associated with inflammatory bowel diseases (IBD), suggesting a role in gastrointestinal inflammation. The enterotoxigenic Bacteroides fragilis (ETBF) toxin (BFT) is an important virulence factor causing gut inflammation in humans and animal models. We identified that BFT signals through GPR35. Blocking GPR35 function in CECs using the GPR35 antagonist ML145, in conjunction with shRNA knock-down and CRISPRcas-mediated knock-out, resulted in reduced CEC-response to BFT as measured by E-cadherin cleavage, beta-arrestin recruitment and IL-8 secretion. Importantly, GPR35 is required for the rapid onset of ETBF-induced colitis in mouse models. GPR35-deficient mice showed reduced death and disease severity compared to wild-type C57Bl6 mice. Our data support a role for GPR35 in the CEC and mucosal response to BFT and underscore the importance of this molecule for sensing ETBF in the colon.

Type 2 immunity induced by bladder extracellular matrix enhances corneal wound healing.

The avascular nature of cornea tissue limits its regenerative potential, which may lead to incomplete healing and formation of scars when damaged. Here, we applied micro- and ultrafine porcine urinary bladder matrix (UBM) particulate to promote type 2 immune responses in cornea wounds. Results demonstrated that UBM particulate substantially reduced corneal haze formation as compared to the saline-treated group. Flow cytometry and gene expression analysis showed that UBM particulate suppressed the differentiation of corneal stromal cells into α-smooth muscle actin-positive (αSMA+) myofibroblasts. UBM treatments up-regulated interleukin-4 (IL-4) produced primarily by eosinophils in the wounded corneas and CD4+ T cells in draining lymph nodes, suggesting a cross-talk between local and peripheral immunity. Gata1-/- mice lacking eosinophils did not respond to UBM treatment and had impaired wound healing. In summary, stimulating type 2 immune responses in the wounded cornea can promote proregenerative environments that lead to improved wound healing for vision restoration.

Bacterial-Driven Inflammation and Mutant BRAF Expression Combine to Promote Murine Colon Tumorigenesis That Is Sensitive to Immune Checkpoint Therapy.

Colorectal cancer is multifaceted, with subtypes defined by genetic, histologic, and immunologic features that are potentially influenced by inflammation, mutagens, and/or microbiota. Colorectal cancers with activating mutations in BRAF are associated with distinct clinical characteristics, although the pathogenesis is not well understood. The Wnt-driven multiple intestinal neoplasia (MinApcΔ716/+) enterotoxigenic Bacteroides fragilis (ETBF) murine model is characterized by IL17-dependent, distal colon adenomas. Herein, we report that the addition of the BRAF V600E mutation to this model results in the emergence of a distinct locus of midcolon tumors. In ETBF-colonized BRAF V600E Lgr5 CreMin (BLM) mice, tumors have similarities to human BRAF V600E tumors, including histology, CpG island DNA hypermethylation, and immune signatures. In comparison to Min ETBF tumors, BLM ETBF tumors are infiltrated by CD8+ T cells, express IFNγ signatures, and are sensitive to anti-PD-L1 treatment. These results provide direct evidence for critical roles of host genetic and microbiota interactions in colorectal cancer pathogenesis and sensitivity to immunotherapy. SIGNIFICANCE: Colorectal cancers with BRAF mutations have distinct characteristics. We present evidence of specific colorectal cancer gene-microbial interactions in which colonization with toxigenic bacteria drives tumorigenesis in BRAF V600E Lgr5 CreMin mice, wherein tumors phenocopy aspects of human BRAF-mutated tumors and have a distinct IFNγ-dominant immune microenvironment uniquely responsive to immune checkpoint blockade.This article is highlighted in the In This Issue feature, p. 1601.

Gutting it Out: Developing Effective Immunotherapies for Patients With Colorectal Cancer.

Risk factors for colorectal cancer (CRC) include proinflammatory diets, sedentary habits, and obesity, in addition to genetic syndromes that predispose individuals to this disease. Current treatment relies on surgical excision and cytotoxic chemotherapies. There has been a renewed interest in immunotherapy as a treatment option for CRC given the success in melanoma and microsatellite instable (MSI) CRC. Immunotherapy with checkpoint inhibitors only plays a role in the 4%-6% of patients with MSIhigh tumors and even within this subpopulation, response rates can vary from 30% to 50%. Most patients with CRC do not respond to this modality of treatment, even though colorectal tumors are frequently infiltrated with T cells. Tumor cells limit apoptosis and survive following intensive chemotherapy leading to drug resistance and induction of autophagy. Pharmacological or molecular inhibition of autophagy improves the efficacy of cytotoxic chemotherapy in murine models. The microbiome clearly plays an etiologic role, in some or most colon tumors, realized by elegant findings in murine models and now investigated in human clinical trials. Recent results have suggested that cancer vaccines may be beneficial, perhaps best as preventive strategies. The search for therapies that can be combined with current approaches to increase their efficacy, and new knowledge of the biology of CRC are pivotal to improve the care of patients suffering from this disease. Here, we review the basic immunobiology of CRC, current "state-of-the-art" immunotherapies and define those areas with greatest therapeutic promise for the future.

Comparative Analysis of Colon Cancer-Derived Fusobacterium nucleatum Subspecies: Inflammation and Colon Tumorigenesis in Murine Models.

Fusobacteria are commonly associated with human colorectal cancer (CRC), but investigations are hampered by the absence of a stably colonized murine model. Further, Fusobacterium nucleatum subspecies isolated from human CRC have not been investigated. While F. nucleatum subspecies are commonly associated with CRC, their ability to induce tumorigenesis and contributions to human CRC pathogenesis are uncertain. We sought to establish a stably colonized murine model and to understand the inflammatory potential and virulence genes of human CRC F. nucleatum, representing the 4 subspecies, animalis, nucleatum, polymorphum, and vincentii. Five human CRC-derived and two non-CRC derived F. nucleatum strains were tested for colonization, tumorigenesis, and cytokine induction in specific-pathogen-free (SPF) and/or germfree (GF) wild-type and ApcMin/+ mice, as well as in vitro assays and whole-genome sequencing (WGS). SPF wild-type and ApcMin/+ mice did not achieve stable colonization with F. nucleatum, whereas certain subspecies stably colonized some GF mice but without inducing colon tumorigenesis. F. nucleatum subspecies did not form in vivo biofilms or associate with the mucosa in mice. In vivo inflammation was inconsistent across subspecies, whereas F. nucleatum induced greater cytokine responses in a human colorectal cell line, HCT116. While F. nucleatum subspecies displayed genomic variability, no distinct virulence genes associated with human CRC strains were identified that could reliably distinguish these strains from non-CRC clinical isolates. We hypothesize that the lack of F. nucleatum-induced tumorigenesis in our model reflects differences in human and murine biology and/or a synergistic role for F. nucleatum in concert with other bacteria to promote carcinogenesis. IMPORTANCE Colon cancer is a leading cause of cancer morbidity and mortality, and it is hypothesized that dysbiosis in the gut microbiota contributes to colon tumorigenesis. Fusobacterium nucleatum, a member of the oropharyngeal microbiome, is enriched in a subset of human colon tumors. However, it is unclear whether this genetically varied species directly promotes tumor formation, modulates mucosal immune responses, or merely colonizes the tumor microenvironment. Mechanistic studies to address these questions have been stymied by the lack of an animal model that does not rely on daily orogastric gavage. Using multiple murine models, in vitro assays with a human colon cancer cell line, and whole-genome sequencing analysis, we investigated the proinflammatory and tumorigenic potential of several F. nucleatum clinical isolates. The significance of this research is development of a stable colonization model of F. nucleatum that does not require daily oral gavages in which we demonstrate that a diverse library of clinical isolates do not promote tumorigenesis.

IL-17 and immunologically induced senescence regulate response to injury in osteoarthritis.

Senescent cells (SnCs) are implicated in the pathogenesis of age-related diseases including osteoarthritis (OA), in part via expression of a senescence-associated secretory phenotype (SASP) that includes immunologically relevant factors and cytokines. In a model of posttraumatic OA (PTOA), anterior cruciate ligament transection (ACLT) induced a type 17 immune response in the articular compartment and draining inguinal lymph nodes (LNs) that paralleled expression of the senescence marker p16INK4a (Cdkn2a) and p21 (Cdkn1a). Innate lymphoid cells, γδ+ T cells, and CD4+ T cells contributed to IL-17 expression. Intra-articular injection of IL-17-neutralizing antibody reduced joint degeneration and decreased expression of the senescence marker Cdkn1a. Local and systemic senolysis was required to attenuate tissue damage in aged animals and was associated with decreased IL-17 and increased IL-4 expression in the articular joint and draining LNs. In vitro, we found that Th17 cells induced senescence in fibroblasts and that SnCs skewed naive T cells toward Th17 or Th1, depending on the presence of TGF-ß. The SASP profile of the inflammation-induced SnCs included altered Wnt signaling, tissue remodeling, and cell-cycle pathways not previously implicated in senescence. These findings provide molecular targets and mechanisms for senescence induction and therapeutic strategies to support tissue healing in an aged environment.

Interleukin 17 and senescent cells regulate the foreign body response to synthetic material implants in mice and humans.

Medical devices and implants made of synthetic materials can induce an immune-mediated process when implanted in the body called the foreign body response, which results in formation of a fibrous capsule around the implant. To explore the immune and stromal connections underpinning the foreign body response, we analyzed fibrotic capsules surrounding surgically excised human breast implants from 12 individuals. We found increased numbers of interleukin 17 (IL17)-producing γδ+ T cells and CD4+ T helper 17 (TH17) cells as well as senescent stromal cells in the fibrotic capsules. Further analysis in a murine model demonstrated an early innate IL17 response to implanted synthetic material (polycaprolactone) particles that was mediated by innate lymphoid cells and γδ+ T cells. This was followed by a chronic adaptive CD4+ TH17 cell response that was antigen dependent. Synthetic materials with varying chemical and physical properties implanted either in injured muscle or subcutaneously induced similar IL17 responses in mice. Mice deficient in IL17 signaling established that IL17 was required for the fibrotic response to implanted synthetic materials and the development of p16INK4a senescent cells. IL6 produced by senescent cells was sufficient for the induction of IL17 expression in T cells. Treatment with a senolytic agent (navitoclax) that killed senescent cells reduced IL17 expression and fibrosis in the mouse implant model. Discovery of a feed-forward loop between the TH17 immune response and the senescence response to implanted synthetic materials introduces new targets for therapeutic intervention in the foreign body response.

Host responses to mucosal biofilms in the lung and gut.

The impact of the human microbiome on health and disease is of utmost importance and has been studied intensively in recent years. Microbes promote immune system development and are essential to the production and absorption of nutrients for the host but are also implicated in disease pathogenesis. Particularly, bacterial biofilms have long been recognized as contributors to chronic infections and diseases in humans. However, our understanding of how the host responds to the presence of biofilms, specifically the immune response to biofilms, and how this contributes to disease pathogenesis is limited. This review aims to highlight what is known about biofilm formation and in vivo models available for the biofilm study. We critique the contribution of biofilms to human diseases, focusing on the lung diseases, cystic fibrosis and chronic obstructive pulmonary disease, and the gut diseases, inflammatory bowel disease and colorectal cancer.

Epigenetic therapy inhibits metastases by disrupting premetastatic niches.

Cancer recurrence after surgery remains an unresolved clinical problem1-3. Myeloid cells derived from bone marrow contribute to the formation of the premetastatic microenvironment, which is required for disseminating tumour cells to engraft distant sites4-6. There are currently no effective interventions that prevent the formation of the premetastatic microenvironment6,7. Here we show that, after surgical removal of primary lung, breast and oesophageal cancers, low-dose adjuvant epigenetic therapy disrupts the premetastatic microenvironment and inhibits both the formation and growth of lung metastases through its selective effect on myeloid-derived suppressor cells (MDSCs). In mouse models of pulmonary metastases, MDSCs are key factors in the formation of the premetastatic microenvironment after resection of primary tumours. Adjuvant epigenetic therapy that uses low-dose DNA methyltransferase and histone deacetylase inhibitors, 5-azacytidine and entinostat, disrupts the premetastatic niche by inhibiting the trafficking of MDSCs through the downregulation of CCR2 and CXCR2, and by promoting MDSC differentiation into a more-interstitial macrophage-like phenotype. A decreased accumulation of MDSCs in the premetastatic lung produces longer periods of disease-free survival and increased overall survival, compared with chemotherapy. Our data demonstrate that, even after removal of the primary tumour, MDSCs contribute to the development of premetastatic niches and settlement of residual tumour cells. A combination of low-dose adjuvant epigenetic modifiers that disrupts this premetastatic microenvironment and inhibits metastases may permit an adjuvant approach to cancer therapy.

Compartmental Analysis of T-cell Clonal Dynamics as a Function of Pathologic Response to Neoadjuvant PD-1 Blockade in Resectable Non-Small Cell Lung Cancer.

PURPOSE: Neoadjuvant PD-1 blockade is a promising treatment for resectable non-small cell lung cancer (NSCLC), yet immunologic mechanisms contributing to tumor regression and biomarkers of response are unknown. Using paired tumor/blood samples from a phase II clinical trial (NCT02259621), we explored whether the peripheral T-cell clonotypic dynamics can serve as a biomarker for response to neoadjuvant PD-1 blockade. EXPERIMENTAL DESIGN: T-cell receptor (TCR) sequencing was performed on serial peripheral blood, tumor, and normal lung samples from resectable NSCLC patients treated with neoadjuvant PD-1 blockade. We explored the temporal dynamics of the T-cell repertoire in the peripheral and tumoral compartments in response to neoadjuvant PD-1 blockade by using the TCR as a molecular barcode. RESULTS: Higher intratumoral TCR clonality was associated with reduced percent residual tumor at the time of surgery, and the TCR repertoire of tumors with major pathologic response (MPR; <10% residual tumor after neoadjuvant therapy) had a higher clonality and greater sharing of tumor-infiltrating clonotypes with the peripheral blood relative to tumors without MPR. Additionally, the posttreatment tumor bed of patients with MPR was enriched with T-cell clones that had peripherally expanded between weeks 2 and 4 after anti-PD-1 initiation and the intratumoral space occupied by these clonotypes was inversely correlated with percent residual tumor. CONCLUSIONS: Our study suggests that exchange of T-cell clones between tumor and blood represents a key correlate of pathologic response to neoadjuvant immunotherapy and shows that the periphery may be a previously underappreciated originating compartment for effective antitumor immunity.See related commentary by Henick, p. 1205.

Interleukin-36γ-producing macrophages drive IL-17-mediated fibrosis.

Biomaterials induce an immune response and mobilization of macrophages, yet identification and phenotypic characterization of functional macrophage subsets in vivo remain limited. We performed single-cell RNA sequencing analysis on macrophages sorted from either a biologic matrix [urinary bladder matrix (UBM)] or synthetic biomaterial [polycaprolactone (PCL)]. Implantation of UBM promotes tissue repair through generation of a tissue environment characterized by a T helper 2 (TH2)/interleukin (IL)-4 immune profile, whereas PCL induces a standard foreign body response characterized by TH17/IL-17 and fibrosis. Unbiased clustering and pseudotime analysis revealed distinct macrophage subsets responsible for antigen presentation, chemoattraction, and phagocytosis, as well as a small population with expression profiles of both dendritic cells and skeletal muscle after UBM implantation. In the PCL tissue environment, we identified a CD9hi+IL-36γ+ macrophage subset that expressed TH17-associated molecules. These macrophages were virtually absent in mice lacking the IL-17 receptor, suggesting that they might be involved in IL-17-dependent immune and autoimmune responses. Identification and comparison of the unique phenotypical and functional macrophage subsets in mouse and human tissue samples suggest broad relevance of the new classification. These distinct macrophage subsets demonstrate previously unrecognized myeloid phenotypes involved in different tissue responses and provide targets for potential therapeutic modulation in tissue repair and pathology.

Immunopathologic Stratification of Colorectal Cancer for Checkpoint Blockade Immunotherapy.

Mismatch-repair deficiency in solid tumors predicts their response to PD-1 blockade. Based on this principle, pembrolizumab is approved as standard of care for patients with unresectable or metastatic microsatellite instability-high (MSI-H) cancer. Despite this success, a large majority of metastatic colorectal cancer patients are not MSI-H and do not benefit from checkpoint blockade treatment. Predictive biomarkers to develop personalized medicines and guide clinical trials are needed for these patients. We, therefore, asked whether immunohistologic stratification of metastatic colorectal cancer based on primary tumor PD-L1 expression associated with the presence or absence of extracellular mucin defines a subset of metastatic colorectal cancer patients who exhibit a preexisting antitumor immune response and who could potentially benefit from the checkpoint blockade. To address this, we studied 26 advanced metastatic colorectal cancer patients treated with pembrolizumab (NCT01876511). To stratify patients, incorporation of histopathologic characteristics (percentage of extracellular mucin) and PD-L1 expression at the invasive front were used to generate a composite score, the CPM (composite PD-L1 and mucin) score, which discriminated patients who exhibited clinical benefit (complete, partial, or stable disease) from those patients with progressive disease. When validated in larger cohorts, the CPM score in combination with MSI testing may guide immunotherapy interventions for colorectal cancer patient treatment.