Stereotactic ablative radiotherapy (SABR) is a standard-of-care for medically-inoperable-early-stage non-small cell lung cancer (NSCLC). One third of patients progress and chemotherapy is rarely used ...in this population. We questioned if addition of the immune-checkpoint-inhibitor (ICI) atezolizumab to standard-of-care SABR can improve outcomes. We initiated a multi-institutional single-arm phase I study (NCT02599454) enrolling twenty patients with the primary endpoint of maximum tolerated dose (MTD); secondary endpoints of safety and efficacy; and exploratory mechanistic correlatives. Treatment is well tolerated and full dose atezolizumab (1200 mg) is the MTD. Efficacy signals include early responses (after 2 cycles of ICI, before initiation of SABR) in 17% of patients. Biomarkers of functional adaptive immunity, including T cell activation in the tumor and response to ex-vivo stimulation by circulating T cells, are highly predictive of benefit. These results require validation and are being tested in a phase III randomized trial.
Analysis of serum protein glycovariants has the potential to identify new biomarkers of human disease. However, the inability to rapidly quantify glycans in a site-specific fashion remains the major ...barrier to applying such biomarkers clinically. Advancements in sample preparation and glycopeptide quantification are thus needed to better bridge glycoscience with biomarker discovery research. We present here the successful utilization of several sample preparation techniques, including multienzyme digestion and glycopeptide enrichment, to increase the repertoire of glycopeptides that can be generated from serum glycoproteins. These techniques combined with glycopeptide retention time prediction and UHPLC-QqQ conditions optimization were then used to develop a dynamic multiple-reaction monitoring (dMRM)-based strategy to simultaneously monitor over 100 glycosylation sites across 50 serum glycoproteins. In total, the abundances of over 600 glycopeptides were simultaneously monitored, some of which were identified by utilizing theoretically predicted ion products and presumed m/z values. The dMRM method was found to have good sensitivity. In the targeted dMRM mode, the limit of quantitation (LOQ) of nine standard glycoproteins reached femtomole levels with dynamic ranges spanning 3–4 orders of magnitude. The dMRM-based strategy also showed high reproducibility with regards to both instrument and sample preparation performance. The high coverage of the serum glycoproteins that can be quantitated to the glycopeptide level makes this method especially suitable for the biomarker discovery from large sample sets. We predict that, in the near future, biomarkers, such as these, will be deployed clinically, especially in the fields of cancer and autoimmunity.
Objective
This study was undertaken to identify the mechanistic role of γδ T cells in the pathogenesis of experimental psoriatic arthritis (PsA).
Methods
In this study, we performed interleukin‐23 ...(IL‐23) gene transfer in wild‐type (WT) and T cell receptor δ–deficient (TCRδ−/−) mice and conducted tissue phenotyping in the joint, skin, and nails to characterize the inflammatory infiltrate. We further performed detailed flow cytometry, immunofluorescence staining, RNA sequencing, T cell repertoire analysis, and in vitro T cell polarization assays to identify regulatory mechanisms of γδ T cells.
Results
We demonstrated that γδ T cells support systemic granulopoiesis, which is critical for murine PsA‐like pathology. Briefly, γδ T cell ablation inhibited the expression of neutrophil chemokines CXCL1 and CXCL2 and neutrophil CD11b+Ly6G+ accumulation in the aforementioned PsA‐related tissues. Although significantly reduced expression of granulocyte–macrophage colony‐stimulating factor (GM‐CSF) and IL‐17A was detected systemically in TCRδ−/− mice, no GM‐CSF+/IL‐17A+ γδ T cells were detected locally in the inflamed skin or bone marrow in WT mice. Our data showed that nonresident γδ T cells regulate the expansion of an CD11b+Ly6G+ neutrophil population and their recruitment to joint and skin tissues, where they develop hallmark pathologic features of human PsA.
Conclusion
Our findings do not support the notion that tissue‐resident γδ T cells initiate the disease but demonstrate a novel role of γδ T cells in neutrophil regulation that can be exploited therapeutically in PsA patients.
Prospective human data are lacking regarding safety, efficacy, and immunologic impacts of different radiation doses administered with combined PD-L1/CTLA-4 blockade.
We performed a multicenter phase ...II study randomly assigning patients with metastatic microsatellite stable colorectal cancer to repeated low-dose fractionated radiation (LDFRT) or hypofractionated radiation (HFRT) with PD-L1/CTLA-4 inhibition. The primary endpoint was response outside the radiation field. Correlative samples were analyzed using multiplex immunofluorescence (IF), IHC, RNA/T-cell receptor (TCR) sequencing, cytometry by time-of-flight (CyTOF), and Olink.
Eighteen patients were evaluable for response. Median lines of prior therapy were four (range, 1-7). Sixteen patients demonstrated toxicity potentially related to treatment (84%), and 8 patients had grade 3-4 toxicity (42%). Best response was stable disease in 1 patient with out-of-field tumor shrinkage. Median overall survival was 3.8 months (90% confidence interval, 2.3-5.7 months). Correlative IF and RNA sequencing (RNA-seq) revealed increased infiltration of CD8
and CD8
/PD-1
/Ki-67
T cells in the radiation field after HFRT. LDFRT increased foci of micronuclei/primary nuclear rupture in two subjects. CyTOF and RNA-seq demonstrated significant declines in multiple circulating immune populations, particularly in patients receiving HFRT. TCR sequencing revealed treatment-associated changes in T-cell repertoire in the tumor and peripheral blood.
We demonstrate the feasibility and safety of adding LDFRT and HFRT to PD-L1/CTLA-4 blockade. Although the best response of stable disease does not support the use of concurrent PD-L1/CTLA-4 inhibition with HFRT or LDFRT in this population, biomarkers provide support that both LDFRT and HFRT impact the local immune microenvironment and systemic immunogenicity that can help guide future studies.
The efficacy of allogeneic hematopoietic stem cell transplantation (allo-HSCT) is limited by the occurrence of acute and chronic graft-
-host disease (GVHD). We have recently demonstrated that ...obesity results in exacerbated acute gastrointestinal GVHD in both mouse models and clinical outcomes due to increased pro-inflammatory cytokine responses and microbiota alterations. We therefore wanted to delineate the role of the various parameters in obesity, adiposity, effects of high-fat (HF) diet, and the role of microbiome on GVHD pathogenesis, by taking advantage of a mouse strain resistant to diet-induced obesity (DIO). Female BALB/c mice are resistant to DIO phenotype with approximately 50% becoming DIO under HF diets. The DIO-susceptible recipients rapidly succumb to acute gut GVHD, whereas the DIO-resistant recipient littermates, which do not become obese, are partially protected from GVHD, indicating that being on HF diet alone contributes to but is not the primary driver of GVHD. Microbiome assessment revealed restricted diversity in both cohorts of mice, but coprophagy normalizes the microbiota in mice housed together. We then individually housed DIO-resistant, DIO-susceptible, and lean control mice. Notably, each of the individually housed groups demonstrates marked restricted diversity that has been shown to occur from the stress of single housing. Despite the restricted microbiome diversity, the GVHD pathogenesis profile remains consistent in the group-housed mice, with the lean control single-housed mice exhibiting no acute GVHD and DIO-resistant recipients showing again partial protection. These results demonstrate that the deleterious effects of obesity on acute gut GVHD are critically dependent on adiposity with the HF diet also playing a lesser role, and the microbiome alterations with obesity instead appear to fuel ongoing acute GVHD processes.
IgE induced by type 2 immune responses in atopic dermatitis is implicated in the progression of atopic dermatitis to other allergic diseases, including food allergies, allergic rhinitis, and asthma. ...However, the keratinocyte-derived signals that promote IgE and ensuing allergic diseases remain unclear. Herein, in a mouse model of atopic dermatitis-like skin inflammation induced by epicutaneous Staphylococcus aureus exposure, keratinocyte release of IL‑36α along with IL-4 triggered B cell IgE class-switching, plasma cell differentiation, and increased serum IgE levels-all of which were abrogated in IL-36R-deficient mice or anti-IL‑36R-blocking antibody-treated mice. Moreover, skin allergen sensitization during S. aureus epicutaneous exposure-induced IL-36 responses was required for the development of allergen-specific lung inflammation. In translating these findings, elevated IL‑36 cytokines in human atopic dermatitis skin and in IL‑36 receptor antagonist-deficiency patients coincided with increased serum IgE levels. Collectively, keratinocyte-initiated IL‑36 responses represent a key mechanism and potential therapeutic target against allergic diseases.
Pyoderma gangrenosum (PG) is a debilitating ulcerative skin disease that is one of the most common associated diseases seen in patients with inflammatory bowel disease and rheumatoid arthritis. ...Although PG is classified as a neutrophilic dermatosis, its pathophysiology is poorly understood.
Use data obtained from patient-reported histories, immunohistochemistry, and gene expression analysis to formulate a hypothesis on PG pathophysiology.
Ten PG patients participated and answered questions about new ulcer formation. Skin biopsies of healed prior ulcers and adjacent normal skin were obtained from four patients for immunohistochemistry. Scars from healthy patients and patients with discoid lupus were used as additional controls. New onset PG papules were analyzed using immunohistochemistry and gene expression analysis
quantitative real-time PCR.
All PG patients reported that healed sites of previous ulceration are refractory to re-ulceration. Simultaneous biopsies of healed and uninvolved skin triggered ulceration only in the latter. On immunohistochemistry, healed PG scars showed complete loss of pilosebaceous units, which were present in normal skin, and to a lesser extent in control scars, and discoid scars. Early PG papules showed perivascular and peripilosebaceous T cell infiltrates, rather than neutrophils. These early inflammatory events were dominated by increased gene expression of
, IFNG, and
and transcription factors consistent with Th1 phenotype.
Small sample size was the main limitation.
We put forth the hypothesis that PG is a T cell response resulting in the destruction of pilosebaceous units.
T cell cytokines contribute to immunity against Staphylococcus aureus, but the predominant T cell subsets involved are unclear. In an S. aureus skin infection mouse model, we found that the IL- 17 ...response was mediated by γδ T cells, which trafficked from lymph nodes to the infected skin to induce neutrophil recruitment, proinflammatory cytokines IL-1α, IL-1β, and TNF, and host defense peptides. RNA-seq for TRG and TRD sequences in lymph nodes and skin revealed a single clonotypic expansion of the encoded complementarity-determining region 3 amino acid sequence, which could be generated by canonical nucleotide sequences of TRGV5 or TRGV6 and TRDV4. However, only TRGV6 and TRDV4 but not TRGV5 sequences expanded. Finally, Vγ6⁺ T cells were a predominant γδ T cell subset that produced IL-17A as well as IL-22, TNF, and IFNγ, indicating a broad and substantial role for clonal Vγ6⁺Vδ4⁺ T cells in immunity against S. aureus skin infections.
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