Human papillomavirus-associated head and neck squamous cell carcinomas (HPV-HNSCC) originate in the tonsils, the major lymphoid organ that orchestrates immunity to oral infections. Despite its location, the virus escapes immune elimination during malignant transformation and progression. Here, we provide evidence for the role of the PD-1:PD-L1 pathway in HPV-HNSCC immune resistance. We demonstrate membranous expression of PD-L1 in the tonsillar crypts, the site of initial HPV infection. In HPV-HNSCCs that are highly infiltrated with lymphocytes, PD-L1 expression on both tumor cells and CD68+ tumor associated macrophages (TAMs) is geographically localized to sites of lymphocyte fronts, while the majority of CD8+ tumor infiltrating lymphocytes (TILs) express high levels of PD-1, the inhibitory PD-L1 receptor. Significant levels of mRNA for interferon-γ (IFN-γ), a major cytokine inducer of PD-L1 expression, were found in HPV+ PD-L1(+) tumors. Our findings support the role of the PD-1:PD-L1 interaction in creating an “immune-privileged” site for initial viral infection and subsequent adaptive immune resistance once tumors are established and suggest a rationale for therapeutic blockade of this pathway in patients with HPV-HNSCC.

Summary Regulatory T cells (Tregs) are a barrier to anti-tumor immunity. Neuropilin-1 (Nrp1) is required to maintain intratumoral Treg stability and function but is dispensable for peripheral immune tolerance. Treg-restricted Nrp1 deletion results in profound tumor resistance due to Treg functional fragility. Thus, identifying the basis for Nrp1 dependency and the key drivers of Treg fragility could help to improve immunotherapy for human cancer. We show that a high percentage of intratumoral NRP1+ Tregs correlates with poor prognosis in melanoma and head and neck squamous cell carcinoma. Using a mouse model of melanoma where Nrp1-deficient (Nrp1−/−) and wild-type (Nrp1+/+) Tregs can be assessed in a competitive environment, we find that a high proportion of intratumoral Nrp1−/− Tregs produce interferon-γ (IFNγ), which drives the fragility of surrounding WT Tregs, boosts anti-tumor immunity, and facilitates tumor clearance. We also show that IFNγ-induced Treg fragility is required for response to anti-PD1, suggesting that cancer therapies promoting Treg fragility may be efficacious.

Highlights d Single-cell RNA-seq revealed distinct immune profiles in HPVand HPV + HNSCC d B cells, myeloid cells, and CD4+ Tconv cells were divergent by high-dimensional analysis d Multispectral imaging uncovered immune structures (TLSs) associated with HPV + disease d T follicular helper signature was associated with favorable survival in TCGA patients

Myeloid-derived suppressor cells (MDSC) play a key immunosuppressive role in various types

Lymphocyte activation gene-3 (LAG-3) is a cell-surface molecule with diverse biologic effects on T cell function. We recently showed that LAG-3 signaling is important in CD4 + regulatory T cell suppression of autoimmune responses. Here, we demonstrate that LAG-3 maintains tolerance to self and tumor antigens via direct effects on CD8 + T cells using 2 murine systems. Naive CD8 + T cells express low levels of LAG-3, and expression increases upon antigen stimulation. Our data show increased levels of LAG-3 protein on antigen-specific CD8 + T cells within antigen-expressing organs or tumors. In vivo antibody blockade of LAG-3 or genetic ablation of the Lag-3 gene resulted in increased accumulation and effector function of antigen-specific CD8 + T cells within organs and tumors that express their cognate antigen. Most notably, combining LAG-3 blockade with specific antitumor vaccination resulted in a significant increase in activated CD8 + T cells in the tumor and disruption of the tumor parenchyma. A major component of this effect was CD4 independent and required LAG-3 expression by CD8 + T cells. Taken together, these data demonstrate a direct role for LAG-3 on CD8 + T cells and suggest that LAG-3 blockade may be a potential cancer treatment.

). We also found that a preponderance of T H 17-mediated inflammation was associated with a lower pathologic Gleason score.These protein level data were reflected at the message level, as analyzed by quantitative reverse transcription-PCR. Microarray analysis of pooled prostate-infiltrating T reg revealed expected T reg -associated transcripts (FoxP3, CTLA-4, GITR, LAG-3) as well as a number of unique cell surface markers that may serve as additional T reg markers.

Regulatory T cells (T reg cells) maintain host self-tolerance but are a major barrier to effective cancer immunotherapy. T reg cells subvert beneficial anti-tumor immunity by modulating inhibitory receptor expression on tumor-infiltrating lymphocytes (TILs); however, the underlying mediators and mechanisms have remained elusive. Here we found that the cytokines IL-10 and IL-35 (Ebi3–IL-12α heterodimer) were divergently expressed by T reg cell subpopulations in the tumor microenvironment (TME) and cooperatively promoted intratumoral T cell exhaustion by modulating multiple inhibitory receptor expression and exhaustion-associated transcriptomic signature of CD8 + TILs. While expression of BLIMP1 (encoded by Prdm1 ) was a common target; IL-10 and IL-35 differentially affected effector T cell versus memory T cell fates, respectively, highlighting their differential, partially overlapping but non-redundant regulation of anti-tumor immunity. Our results reveal previously unappreciated cooperative roles for T reg cell-derived IL-10 and IL-35 in promoting BLIMP1-dependent exhaustion of CD8 + TILs that limits effective anti-tumor immunity.

Rationale: The pulmonary mononuclear phagocyte system is a critical host defense mechanism composed of macrophages, monocytes, monocyte-derived cells, and dendritic cells. However, our current characterization of these cells is limited because it is derived largely from animal studies and analysis of human mononuclear phagocytes from blood and small tissue resections around tumors.Objectives: Phenotypic and morphologic characterization of mononuclear phagocytes that potentially access inhaled antigens in human lungs.Methods: We acquired and analyzed pulmonary mononuclear phagocytes from fully intact nondiseased human lungs (including the major blood vessels and draining lymph nodes) obtained en bloc from 72 individual donors. Differential labeling of hematopoietic cells via intrabronchial and intravenous administration of antibodies within the same lobe was used to identify extravascular tissue-resident mononuclear phagocytes and exclude cells within the vascular lumen. Multiparameter flow cytometry was used to identify mononuclear phagocyte populations among cells labeled by each route of antibody delivery. Measurements and Main Results:We performed a phenotypic analysis of pulmonary mononuclear phagocytes isolated from whole nondiseased human lungs and lung-draining lymph nodes. Five pulmonary mononuclear phagocytes were observed, including macrophages, monocyte-derived cells, and dendritic cells that were phenotypically distinct from cell populations found in blood.Conclusions: Different mononuclear phagocytes, particularly dendritic cells, were labeled by intravascular and intrabronchial antibody delivery, countering the notion that tissue and blood mononuclear phagocytes are equivalent systems. Phenotypic descriptions of the mononuclear phagocytes in nondiseased lungs provide a precedent for comparative studies in diseased lungs and potential targets for therapeutics. Correspondence and requests for reprints should be addressed to Claudia V. Jakubzick, Ph.D., Department of Pediatrics and Immunology, National Jewish Health, 1400 Jackson Street, Denver, CO 80206. E-mail: jakubzickc@njhealth.org This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org The human respiratory tract has a branching structure that terminates in millions of alveoli, whose luminal surface covers an approximate area of 50 to 100 m 2 . In comparison to other barrier surfaces, such as the skin (2 m 2 ) and the gut (10 m 2 ), this surface area is massive, and therefore, comprises the body's largest interface with the ambient environment. Because of normal respiratory function, the average human exchanges 7,000 to 9,000 L of gas each day and inhales billions of particles, allergens, and microbes. Accordingly, the human lung constitutes a major site for the innate and adaptive immune responses. In this context, cells in the mononuclear phagocyte system (MPS), which consists of macrophages, monocytes, monocytederived cells, and dendritic cells (DCs), play critical roles. T...