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.

Pharmacodynamic measures within tumors expose differential activity of PD(L)-1 antibody therapeutics.

Macromolecules such as monoclonal antibodies (mAbs) are likely to experience poor tumor penetration because of their large size, and thus low drug exposure of target cells within a tumor could contribute to suboptimal responses. Given the challenge of inadequate quantitative tools to assess mAb activity within tumors, we hypothesized that measurement of accessible target levels in tumors could elucidate the pharmacologic activity of a mAb and could be used to compare the activity of different mAbs. Using positron emission tomography (PET), we measured the pharmacodynamics of immune checkpoint protein programmed-death ligand 1 (PD-L1) to evaluate pharmacologic effects of mAbs targeting PD-L1 and its receptor programmed cell death protein 1 (PD-1). For PD-L1 quantification, we first developed a small peptide-based fluorine-18-labeled PET imaging agent, [18F]DK222, which provided high-contrast images in preclinical models. We then quantified accessible PD-L1 levels in the tumor bed during treatment with anti-PD-1 and anti-PD-L1 mAbs. Applying mixed-effects models to these data, we found subtle differences in the pharmacodynamic effects of two anti-PD-1 mAbs (nivolumab and pembrolizumab). In contrast, we observed starkly divergent target engagement with anti-PD-L1 mAbs (atezolizumab, avelumab, and durvalumab) that were administered at equivalent doses, correlating with differential effects on tumor growth. Thus, we show that measuring PD-L1 pharmacodynamics informs mechanistic understanding of therapeutic mAbs targeting PD-L1 and PD-1. These findings demonstrate the value of quantifying target pharmacodynamics to elucidate the pharmacologic activity of mAbs, independent of mAb biophysical properties and inclusive of all physiological variables, which are highly heterogeneous within and across tumors and patients.

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.

High-dimensional Cytometry (ExCYT) and Mass Spectrometry of Myeloid Infiltrate in Clinically Localized Clear Cell Renal Cell Carcinoma Identifies Novel Potential Myeloid Targets for Immunotherapy.

Renal Cell Carcinoma (RCC) is one of the most commonly diagnosed cancers worldwide with research efforts dramatically improving understanding of the biology of the disease. To investigate the role of the immune system in treatment-naïve clear cell Renal Cell Carcinoma (ccRCC), we interrogated the immune infiltrate in patient-matched ccRCC tumor samples, benign normal adjacent tissue (NAT) and peripheral blood mononuclear cells (PBMCs isolated from whole blood, focusing our attention on the myeloid cell infiltrate. Using flow cytometric, MS, and ExCYT analysis, we discovered unique myeloid populations in PBMCs across patient samples. Furthermore, normal adjacent tissues and ccRCC tissues contained numerous myeloid populations with a unique signature for both tissues. Enrichment of the immune cell (CD45+) fraction and subsequent gene expression analysis revealed a number of myeloid-related genes that were differentially expressed. These data provide evidence, for the first time, of an immunosuppressive and pro-tumorigenic role of myeloid cells in early, clinically localized ccRCC. The identification of a number of immune proteins for therapeutic targeting provides a rationale for investigation into the potential efficacy of earlier intervention with single-agent or combination immunotherapy for ccRCC.

Cyclin-dependent kinase 5 activity is required for allogeneic T-cell responses after hematopoietic cell transplantation in mice.

Molecular intermediates in T-cell activation pathways are crucial targets for the therapy and prevention of graft-versus-host disease (GVHD) following allogeneic hematopoietic cell transplantation (allo-HCT). We recently identified an essential role for cyclin-dependent kinase 5 (Cdk5) in T-cell activation and effector function, but the contribution of Cdk5 activity to the development of GVHD has not been explored. Using an established, preclinical, murine, GVHD model, we reveal that Cdk5 activity is increased in key target organs early after allo-HCT. We then generated chimeric mice (Cdk5+/+C or Cdk5-/-C) using hematopoietic progenitors from either embryonic day 16.5 Cdk5+/+ or Cdk5-/- embryos to enable analyses of the role of Cdk5 in GVHD, as germ line Cdk5 gene deletion is embryonically lethal. The immunophenotype of adult Cdk5-/-C mice is identical to control Cdk5+/+C mice. However, transplantation of donor Cdk5-/-C bone marrow and T cells dramatically reduced the severity of systemic and target organ GVHD. This phenotype is attributed to decreased T-cell migration to secondary lymphoid organs (SLOs), reduced in vivo proliferation within these organs, and fewer cytokine-producing donor T cells during GVHD development. Moreover, these defects in Cdk5-/- T-cell function are associated with altered CCR7 signaling following ligation by CCL19, a receptor:ligand interaction critical for T-cell migration into SLOs. Although Cdk5 activity in donor T cells contributed to graft-versus-tumor effects, pharmacologic inhibition of Cdk5 preserved leukemia-free survival. Collectively, our data implicate Cdk5 in allogeneic T-cell responses after HCT and as an important new target for therapeutic intervention.

Donor CD4+ Foxp3+ regulatory T cells are necessary for posttransplantation cyclophosphamide-mediated protection against GVHD in mice.

Posttransplantation cyclophosphamide (PTCy) is an effective prophylaxis against graft-versus-host disease (GVHD). However, it is unknown whether PTCy works singularly by eliminating alloreactive T cells via DNA alkylation or also by restoring the conventional (Tcon)/regulatory (Treg) T-cell balance. We studied the role of Tregs in PTCy-mediated GVHD prophylaxis in murine models of allogeneic blood or marrow transplantation (alloBMT). In 2 distinct MHC-matched alloBMT models, infusing Treg-depleted allografts abrogated the GVHD-prophylactic activity of PTCy. Using allografts in which Foxp3(+) Tregs could be selectively depleted in vivo, either pre- or post-PTCy ablation of donor thymus-derived Tregs (tTregs) abolished PTCy protection against GVHD. PTCy treatment was associated with relative preservation of donor Tregs. Experiments using combinations of Foxp3(-) Tcons and Foxp3(+) Tregs sorted from different Foxp3 reporter mice indicated that donor Treg persistence after PTCy treatment was predominantly caused by survival of functional tTregs that retained Treg-specific demethylation and also induction of peripherally derived Tregs. Finally, adoptive transfer of tTregs retrieved from PTCy-treated chimeras rescued PTCy-treated, Treg-depleted recipients from lethal GVHD. Our findings indicate that PTCy-mediated protection against GVHD is not singularly dependent on depletion of donor alloreactive T cells but also requires rapidly recovering donor Tregs to initiate and maintain alloimmune regulation.

Human mesenchymal stromal cells attenuate graft-versus-host disease and maintain graft-versus-leukemia activity following experimental allogeneic bone marrow transplantation.

We sought to define the effects and underlying mechanisms of human, marrow-derived mesenchymal stromal cells (hMSCs) on graft-versus-host disease (GvHD) and graft-versus-leukemia (GvL) activity. Irradiated B6D2F1 mice given C57BL/6 BM and splenic T cells and treated with hMSCs had reduced systemic GvHD, donor T-cell expansion, and serum TNFα and IFNγ levels. Bioluminescence imaging demonstrated that hMSCs redistributed from lungs to abdominal organs within 72 hours, and target tissues harvested from hMSC-treated allogeneic BMT (alloBMT) mice had less GvHD than untreated controls. Cryoimaging more precisely revealed that hMSCs preferentially distributed to splenic marginal zones and regulated T-cell expansion in the white pulp. Importantly, hMSCs had no effect on in vitro cytotoxic T-cell activity and preserved potent GvL effects in vivo. Mixed leukocyte cultures containing hMSCs exhibited decreased T-cell proliferation, reduced TNFα, IFNγ, and IL-10 but increased PGE2 levels. Indomethacin and E-prostanoid 2 (EP2) receptor antagonisms both reversed while EP2 agonism restored hMSC-mediated in vitro T-cell suppression, confirming the role for PGE2 . Furthermore, cyclo-oxygenase inhibition following alloBMT abrogated the protective effects of hMSCs. Together, our data show that hMSCs preserve GvL activity and attenuate GvHD and reveal that hMSC biodistribute to secondary lymphoid organs wherein they attenuate alloreactive T-cell proliferation likely through PGE2 induction.

CD8(+)IL-17(+) T Cells Mediate Neutrophilic Airway Obliteration in T-bet-Deficient Mouse Lung Allograft Recipients.

Acute cellular rejection is a known risk factor for the development of obliterative bronchiolitis, which limits the long-term survival of lung transplant recipients. However, the T cell effector mechanisms in both of these processes remain incompletely understood. Using the mouse orthotopic lung transplant model, we investigated whether C57BL/6 T-bet(-/-) recipients of major histocompatibility complex (MHC)-mismatched BALB/c lung grafts develop rejection pathology and allospecific cytokine responses that differ from wild-type mice. T-bet(-/-) recipients demonstrated vigorous allograft rejection at 10 days, characterized by neutrophilic inflammation and predominantly CD8(+) T cells producing allospecific IL-17 and/or IFN-γ, in contrast to IFN-γ-dominant responses in WT mice. CD4(+) T cells produced IL-17 but not IFN-γ responses in T-bet(-/-) recipients, in contrast to WT controls. Costimulation blockade using anti-CD154 Ab significantly reduced allospecific CD8(+)IFN-γ(+) responses in both T-bet(-/-) and WT mice but had no attenuating effect on lung rejection pathology in T-bet(-/-) recipients or on the development of obliterative airway inflammation that occurred only in T-bet(-/-) recipients. However, neutralization of IL-17A significantly attenuated costimulation blockade-resistant rejection pathology and airway inflammation in T-bet(-/-) recipients. In addition, CXCL1 (neutrophil chemokine) was increased in T-bet(-/-) allografts, and IL-17 induced CXCL1 from mouse lung epithelial cells in vitro. Taken together, our data show that T-bet-deficient recipients of complete MHC-mismatched lung allografts develop costimulation blockade-resistant rejection characterized by neutrophilia and obliterative airway inflammation that is predominantly mediated by CD8(+)IL-17(+) T cells. Our data support T-bet-deficient mouse recipients of lung allografts as a viable animal model to study the immunopathogenesis of small airway injury in lung transplantation.

Radiation Therapy and Myeloid-Derived Suppressor Cells: Breaking Down Their Cancerous Partnership.

Radiation therapy (RT) has been a primary treatment modality in cancer for decades. Increasing evidence suggests that RT can induce an immunosuppressive shift via upregulation of cells such as tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs). MDSCs inhibit antitumor immunity through potent immunosuppressive mechanisms and have the potential to be crucial tools for cancer prognosis and treatment. MDSCs interact with many different pathways, desensitizing tumor tissue and interacting with tumor cells to promote therapeutic resistance. Vascular damage induced by RT triggers an inflammatory signaling cascade and potentiates hypoxia in the tumor microenvironment (TME). RT can also drastically modify cytokine and chemokine signaling in the TME to promote the accumulation of MDSCs. RT activation of the cGAS-STING cytosolic DNA sensing pathway recruits MDSCs through a CCR2-mediated mechanism, inhibiting the production of type 1 interferons and hampering antitumor activity and immune surveillance in the TME. The upregulation of hypoxia-inducible factor-1 and vascular endothelial growth factor mobilizes MDSCs to the TME. After recruitment, MDSCs promote immunosuppression by releasing reactive oxygen species and upregulating nitric oxide production through inducible nitric oxide synthase expression to inhibit cytotoxic activity. Overexpression of arginase-1 on subsets of MDSCs degrades L-arginine and downregulates CD3ζ, inhibiting T-cell receptor reactivity. This review explains how radiation promotes tumor resistance through activation of immunosuppressive MDSCs in the TME and discusses current research targeting MDSCs, which could serve as a promising clinical treatment strategy in the future.

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.

Autologous Protein Solution processing alters lymphoid and myeloid cell populations and modulates gene expression dependent on cell type.

Osteoarthritis (OA) is a degenerative disease associated with cartilage degradation, osteophyte formation, and fibrillation. Autologous Protein Solution (APS), a type of autologous anti-inflammatory orthobiologic, is used for pain management and treatment of OA. Various compositions of autologous PRP formulations are in clinical use for musculoskeletal pathologies, by nature of their minimal processing and source of bioactive molecules. Currently, there is no consensus on the optimal composition of the complex mixture. In this study, we focused on elucidating the immune cell subtypes and phenotypes in APS. We identified the immune cell types in APS from healthy donors and investigated phenotypic changes in the immune cells after APS processing. Based on flow cytometric analysis, we found that neutrophils and T cells are the most abundant immune cell types in APS, while monocytes experience the largest fold change in concentration compared to WBCs. Gene expression profiling revealed that APS processing results in differential gene expression changes dependent on immune cell type, with the most significantly differentially regulated genes occurring in the monocytes. Our results demonstrate that the mechanical processing of blood, whose main purpose is enrichment and separation, can alter its protein and cellular composition, as well as cellular phenotypes in the final product.

Therapeutic Targeting of Checkpoint Receptors within the DNAM1 Axis.

Therapeutic antibodies targeting the CTLA4/PD-1 pathways have revolutionized cancer immunotherapy by eliciting durable remission in patients with cancer. However, relapse following early response, attributable to primary and adaptive resistance, is frequently observed. Additional immunomodulatory pathways are being studied in patients with primary or acquired resistance to CTLA4 or PD-1 blockade. The DNAM1 axis is a potent coregulator of innate and adaptive immunity whose other components include the immunoglobulin receptors TIGIT, PVRIG, and CD96, and their nectin and nectin-like ligands. We review the basic biology and therapeutic relevance of this family, which has begun to show promise in cancer clinical trials. SIGNIFICANCE: Recent studies have outlined the immuno-oncologic ascendancy of coinhibitory receptors in the DNAM1 axis such as TIGIT and PVRIG and, to a lesser extent, CD96. Biological elucidation backed by ongoing clinical trials of single-agent therapy directed against TIGIT or PVRIG is beginning to provide the rationale for testing combination regimens of DNAM1 axis blockers in conjunction with anti-PD-1/PD-L1 agents.

Effects of B cell-activating factor on tumor immunity.

Immunotherapies that modulate T cell function have been firmly established as a pillar of cancer therapy, whereas the potential for B cells in the antitumor immune response is less established. B cell-activating factor (BAFF) is a B cell-activating cytokine belonging to the TNF ligand family that has been associated with autoimmunity, but little is known about its effects on cancer immunity. We find that BAFF upregulates multiple B cell costimulatory molecules; augments IL-12a expression, consistent with Be-1 lineage commitment; and enhances B cell antigen-presentation to CD4+ Th cells in vitro. In a syngeneic mouse model of melanoma, BAFF upregulates B cell CD40 and PD-L1 expression; it also modulates T cell function through increased T cell activation and TH1 polarization, enhanced expression of the proinflammatory leukocyte trafficking chemokine CCR6, and promotion of a memory phenotype, leading to enhanced antitumor immunity. Similarly, adjuvant BAFF promotes a memory phenotype of T cells in vaccine-draining lymph nodes and augments the antitumor efficacy of whole cell vaccines. BAFF also has distinct immunoregulatory functions, promoting the expansion of CD4+Foxp3+ Tregs in the spleen and tumor microenvironment (TME). Human melanoma data from The Cancer Genome Atlas (TCGA) demonstrate that BAFF expression is positively associated with overall survival and a TH1/IFN-γ gene signature. These data support a potential role for BAFF signaling as a cancer immunotherapy.

Transcriptional profiling of tumor associated macrophages in human renal cell carcinoma reveals significant heterogeneity and opportunity for immunomodulation.

Among the more notable immunotherapies are checkpoint inhibitors, which prevent suppressive signaling on T cells, thereby (re)activating them to kill tumor cells. Despite remarkable treatment responses to immune checkpoint blockade, with a subset of patients achieving complete responses, a large population have little-to-no response, dictating the necessity of further research in this field. Myeloid derived cells heavily infiltrate the tumor microenvironment (TME) of many cancers and are believed to have a number of potent anti-inflammatory effects. Here we use primary non-metastatic renal cell carcinoma to interrogate the gene expression profiles of M2-tumor associated macrophages (M2-TAMs). We performed Fluorescent Activated Cell (FACS) sorting on monocytes from the peripheral blood and tumors of fresh clear cell renal cell carcinoma (ccRCC) samples obtained after patients underwent a partial (7 patients-87.5%) or radical (1 patient-12.5%) nephrectomy. We then utilized NanoString gene expression profiling to show that TAMs express a heterogeneous transcriptional profile that does not cleanly fit into the traditional M1-M2 TAM paradigm. We identified expression of M1 associated costimulatory molecules, a multitude of diverse chemokines, canonical M2 associated molecules, as well as factors involved in the Complement system and checkpoint receptors. Our data are in agreement with other published literature investigating TAMs in various non-ccRCC TMEs, and support the growing literature concerning expression of Complement factors and checkpoint receptors on TAMs.

High-throughput screening of amastigotes of Leishmania donovani clinical isolates against drugs using a colorimetric beta-lactamase assay.

A simple colorimetric beta-lactamase assay for quantifying Leishmania amastigotes in macrophages grown in microtiter plates has been reported. The beta-lactamase gene was integrated into the rRNA region of the genome, thereby allowing for high-level stable expression of the enzyme. Both visceral leishmaniasis (VL) and post-kala azar dermal leishmaniasis (PKDL) isolates were transfected with beta-Lactamase gene. These beta-lactamase-expressing promastigotes were used for infecting intracellular J774A.1 macrophages in vitro. Quantification was done by a colorimetric readout with CENTA beta-lactamase as substrate and with an optical density plate reader. The assay was carried out in 96-well plates. Results obtained demonstrate that this methodology could be a valuable high-throughput screening assay for checking efficacy of anti-leishmanial drugs in the clinical isolates.

ExCYT: A Graphical User Interface for Streamlining Analysis of High-Dimensional Cytometry Data.

With the advent of flow cytometers capable of measuring an increasing number of parameters, scientists continue to develop larger panels to phenotypically explore characteristics of their cellular samples. However, these technological advancements yield high-dimensional data sets that have become increasingly difficult to analyze objectively within traditional manual-based gating programs. In order to better analyze and present data, scientists partner with bioinformaticians with expertise in analyzing high-dimensional data to parse their flow cytometry data. While these methods have been shown to be highly valuable in studying flow cytometry, they have yet to be incorporated in a straightforward and easy-to-use package for scientists who lack computational or programming expertise. To address this need, we have developed ExCYT, a MATLAB-based Graphical User Interface (GUI) that streamlines the analysis of high-dimensional flow cytometry data by implementing commonly employed analytical techniques for high-dimensional data including dimensionality reduction by t-SNE, a variety of automated and manual clustering methods, heatmaps, and novel high-dimensional flow plots. Additionally, ExCYT provides traditional gating options of select populations of interest for further t-SNE and clustering analysis as well as the ability to apply gates directly on t-SNE plots. The software provides the additional advantage of working with either compensated or uncompensated FCS files. In the event that post-acquisition compensation is required, the user can choose to provide the program a directory of single stains and an unstained sample. The program detects positive events in all channels and uses this select data to more objectively calculate the compensation matrix. In summary, ExCYT provides a comprehensive analysis pipeline to take flow cytometry data in the form of FCS files and allow any individual, regardless of computational training, to use the latest algorithmic approaches in understanding their data.

Divergent immune responses to synthetic and biological scaffolds.

The immune system plays a critical role in wound healing and the response to biomaterials. Biomaterials-directed regenerative immunology is an immunoengineering strategy that targets the immune system to promote tissue repair. Biomaterial scaffolds employed in regenerative medicine can be broadly classified as biological (such as those derived from the tissue extracellular matrix) or synthetic. Here, we show in depth the divergent myeloid response to biological versus synthetic biomaterial scaffolds. While neutrophil depletion and changes in physical properties such as shape and mechanics can modulate the pro-inflammatory myeloid immune response to synthetic materials to a degree, the overall general divergent myeloid responses persist. Biologic scaffolds elicit a type-2-like immune response with upregulation of genes such as Il4, Cd163, Mrc1 and Chil3, as well as genes associated with damage-associated molecular patterns providing another possible mechanism by which ECM scaffolds promote wound healing via amplification of endogenous wound-associated signaling pathways. Synthetic materials recruit a high proportion of neutrophils which is compounded by material stiffness and by the presence of an injury. Understanding the complex immune response to biomaterial classes will help in the efficient design of immunoengineering strategies and optimizing regenerative and reducing foreign body fibrotic responses to scaffolds.

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.

A biologic scaffold-associated type 2 immune microenvironment inhibits tumor formation and synergizes with checkpoint immunotherapy.

Biomaterials in regenerative medicine are designed to mimic and modulate tissue environments to promote repair. Biologic scaffolds (derived from decellularized tissue extracellular matrix) promote a wound-healing (proregenerative) immune phenotype and are used clinically to treat tissue loss, including in the context of tumor resection. It is unknown whether a biomaterial microenvironment that encourages tissue formation may also promote tumor development. We implanted a urinary bladder matrix (UBM) scaffold, which is used clinically for wound management, with syngeneic cancer cell lines in mice to study how wound-healing immune responses affect tumor formation and sensitivity to immune checkpoint blockade. The UBM scaffold created an immune microenvironment that inhibited B16-F10 melanoma tumor formation in a CD4+ T cell-dependent and macrophage-dependent manner. In-depth immune characterization revealed an activated type 2-like immune response that was distinct from the classical tumor microenvironment, including activated type 2 T helper T cells, a unique macrophage phenotype, eosinophil infiltration, angiogenic factors, and complement. Tumor growth inhibition by PD-1 and PD-L1 checkpoint blockade was potentiated in the UBM scaffold immune microenvironment. Engineering the local tumor microenvironment to promote a type 2 wound-healing immune signature may serve as a therapeutic target to improve immunotherapy efficacy.

Mouse PVRIG Has CD8+ T Cell-Specific Coinhibitory Functions and Dampens Antitumor Immunity.

A limitation to antitumor immunity is the dysfunction of T cells in the tumor microenvironment, in part due to upregulation of coinhibitory receptors such as PD-1. Here, we describe that poliovirus receptor-related immunoglobulin domain protein (PVRIG) acts as a coinhibitory receptor in mice. Murine PVRIG interacted weakly with poliovirus receptor (PVR) but bound poliovirus receptor-like 2 (PVRL2) strongly, making the latter its principal ligand. As in humans, murine NK and NKT cells constitutively expressed PVRIG. However, when compared with humans, less PVRIG transcript and surface protein was detected in murine CD8+ T cells ex vivo However, activated CD8+ T cells upregulated PVRIG expression. In the mouse tumor microenvironment, infiltrating CD8+ T cells expressed PVRIG whereas its ligand, PVRL2, was detected predominantly on myeloid cells and tumor cells, mirroring the expression pattern in human tumors. PVRIG-deficient mouse CD8+ T cells mounted a stronger antigen-specific effector response compared with wild-type CD8+ T cells during acute Listeria monocytogenes infection. Furthermore, enhanced CD8+ T-cell effector function inhibited tumor growth in PVRIG-/- mice compared with wild-type mice and PD-L1 blockade conferred a synergistic antitumor response in PVRIG-/- mice. Therapeutic intervention with antagonistic anti-PVRIG in combination with anti-PD-L1 reduced tumor growth. Taken together, our results suggest PVRIG is an inducible checkpoint receptor and that targeting PVRIG-PVRL2 interactions results in increased CD8+ T-cell function and reduced tumor growth.See related article on p. 257.