Tumor reactive γδ T cells contribute to a complete response to PD-1 blockade in a Merkel cell carcinoma patient.

Immunotherapies targeting PD-1/PD-L1 are now widely used in the clinic to treat a variety of malignancies. While most of the research on T cell exhaustion and PD-1 blockade has been focused on conventional αß T cells, the contribution of innate-like T cells such as γδ T cells to anti-PD-1/PD-L1 mediated therapy is limited. Here we show that tumor reactive γδ T cells respond to PD-1 blockade in a Merkel cell carcinoma (MCC) patient experiencing a complete response to therapy. We find clonally expanded γδ T cells in the blood and tumor after pembrolizumab treatment, and this Vγ2Vδ1 clonotype recognizes Merkel cancer cells in a TCR-dependent manner. Notably, the intra-tumoral γδ T cells in the MCC patient are characterized by higher expression of PD-1 and TIGIT, relative to conventional CD4 and CD8 T cells. Our results demonstrate that innate-like T cells could also contribute to an anti-tumor response after PD-1 blockade.

Pre-encoded responsiveness to type I interferon in the peripheral immune system defines outcome of PD1 blockade therapy.

Type I interferons (IFN-Is) are central regulators of anti-tumor immunity and responses to immunotherapy, but they also drive the feedback inhibition underlying therapeutic resistance. In the present study, we developed a mass cytometry approach to quantify IFN-I-stimulated protein expression across immune cells and used multi-omics to uncover pre-therapy cellular states encoding responsiveness to inflammation. Analyzing peripheral blood cells from multiple cancer types revealed that differential responsiveness to IFN-Is before anti-programmed cell death protein 1 (PD1) treatment was highly predictive of long-term survival after therapy. Unexpectedly, IFN-I hyporesponsiveness efficiently predicted long-term survival, whereas high responsiveness to IFN-I was strongly associated with treatment failure and diminished survival time. Peripheral IFN-I responsive states were not associated with tumor inflammation, identifying a disconnect between systemic immune potential and 'cold' or 'hot' tumor states. Mechanistically, IFN-I responsiveness was epigenetically imprinted before therapy, poising cells for differential inflammatory responses and dysfunctional T cell effector programs. Thus, we identify physiological cell states with clinical importance that can predict success and long-term survival of PD1-blocking immunotherapy.

The baseline interferon signature predicts disease severity over the subsequent 5 years in systemic lupus erythematosus.

OBJECTIVES: Type I interferons (IFNs) play an important role in the pathophysiology of systemic lupus erythematosus (SLE). While cross-sectional data suggest an association between IFN-induced gene expression and SLE disease activity, interest in this as a biomarker of flare has been tempered by a lack of fluctuation with disease activity in the majority of patients. This led us to question whether IFN-induced gene expression might instead be a biomarker of overall disease severity, with patients with high levels spending more time in an active disease state. METHODS: Levels of five interferon-responsive genes were measured in the whole peripheral blood at baseline visit for 137 SLE patients subsequently followed for 5 years. Log transformed values were summed to yield a composite IFN5 score, and the correlation with various disease outcomes examined. Receiver operator characteristic analyses were performed for outcomes of interest. Kaplan-Meier curves were generated to compare the proportion of flare-free patients with high and low IFN5 scores over time. RESULTS: The baseline IFN5 score was positively correlated with the adjusted mean SLE disease activity index-2000, number of flares, adjusted mean prednisone dose, and number of new immunosuppressive medications over the subsequent 5 years. Optimal cut-offs for the IFN5 score were determined using Youden's index and predicted more severe outcomes with 57-67% accuracy. A high baseline IFN5 level was associated with a significantly increased risk of subsequent flare. CONCLUSIONS: Measurement of the type I IFN signature is a useful tool for predicting the subsequent disease activity course.

Fatigue severity in anti-nuclear antibody-positive individuals does not correlate with pro-inflammatory cytokine levels or predict imminent progression to symptomatic disease.

BACKGROUND: Fatigue is a common symptom of systemic autoimmune rheumatic disease (SARD). Patients with SARD have a protracted pre-clinical phase during which progressive immunologic derangements occur culminating in disease. In this study, we sought to determine when fatigue develops and whether its presence correlates with inflammatory factors or predicts disease progression. METHODS: Anti-nuclear antibody (ANA)-negative healthy controls (HCs) and ANA-positive participants with no criteria, at least one clinical criteria (undifferentiated connective tissue disease, UCTD), or meeting SARD classification criteria were recruited. Fatigue was assessed using a modified version of the FACIT-F questionnaire and the presence of fibromyalgia determined using a questionnaire based on the modified 2010 ACR criteria. Peripheral blood expression of five IFN-induced genes was quantified by NanoString and the levels of IL-1ß, IL-6, or TNF-α by ELISA. RESULTS: Fatigue was as prevalent and severe in individuals lacking SARD criteria as it was in UCTD and SARD. Overall, ~ 1/3 of ANA+ subjects met fibromyalgia criteria, with no differences between sub-groups. Although fatigue was more severe in these individuals, those lacking fibromyalgia remained significantly more fatigued than ANA- HC. However, even in these subjects, fatigue correlated with the widespread pain index and symptom severity scores on the fibromyalgia questionnaire. Fatigue was not associated with elevated cytokine levels in any of the ANA+ sub-groups and did not predict imminent disease progression. CONCLUSIONS: Fatigue is common in ANA+ individuals lacking sufficient criteria for a SARD diagnosis, correlates with fibromyalgia-related symptoms, and is not associated with inflammation or predictive of disease progression.

Validation of CyTOF Against Flow Cytometry for Immunological Studies and Monitoring of Human Cancer Clinical Trials.

Flow cytometry is a widely applied approach for exploratory immune profiling and biomarker discovery in cancer and other diseases. However, flow cytometry is limited by the number of parameters that can be simultaneously analyzed, severely restricting its utility. Recently, the advent of mass cytometry (CyTOF) has enabled high dimensional and unbiased examination of the immune system, allowing simultaneous interrogation of a large number of parameters. This is important for deep interrogation of immune responses and particularly when sample sizes are limited (such as in tumors). Our goal was to compare the accuracy and reproducibility of CyTOF against flow cytometry as a reliable analytic tool for human PBMC and tumor tissues for cancer clinical trials. We developed a 40+ parameter CyTOF panel and demonstrate that compared to flow cytometry, CyTOF yields analogous quantification of cell lineages in conjunction with markers of cell differentiation, function, activation, and exhaustion for use with fresh and viably frozen PBMC or tumor tissues. Further, we provide a protocol that enables reliable quantification by CyTOF down to low numbers of input human cells, an approach that is particularly important when cell numbers are limiting. Thus, we validate CyTOF as an accurate approach to perform high dimensional analysis in human tumor tissue and to utilize low cell numbers for subsequent immunologic studies and cancer clinical trials.

An interim report on the investigator-initiated phase 2 study of pembrolizumab immunological response evaluation (INSPIRE).

BACKGROUND: Immune checkpoint inhibitors (ICIs) demonstrate unprecedented efficacy in multiple malignancies; however, the mechanisms of sensitivity and resistance are poorly understood and predictive biomarkers are scarce. INSPIRE is a phase 2 basket study to evaluate the genomic and immune landscapes of peripheral blood and tumors following pembrolizumab treatment. METHODS: Patients with incurable, locally advanced or metastatic solid tumors that have progressed on standard therapy, or for whom no standard therapy exists or standard therapy was not deemed appropriate, received 200 mg pembrolizumab intravenously every three weeks. Blood and tissue samples were collected at baseline, during treatment, and at progression. One core biopsy was used for immunohistochemistry and the remaining cores were pooled and divided for genomic and immune analyses. Univariable analysis of clinical, genomic, and immunophenotyping parameters was conducted to evaluate associations with treatment response in this exploratory analysis. RESULTS: Eighty patients were enrolled from March 21, 2016 to June 1, 2017, and 129 tumor and 382 blood samples were collected. Immune biomarkers were significantly different between the blood and tissue. T cell PD-1 was blocked (≥98%) in the blood of all patients by the third week of treatment. In the tumor, 5/11 (45%) and 11/14 (79%) patients had T cell surface PD-1 occupance at weeks six and nine, respectively. The proportion of genome copy number alterations and abundance of intratumoral 4-1BB+ PD-1+ CD8 T cells at baseline (P < 0.05), and fold-expansion of intratumoral CD8 T cells from baseline to cycle 2-3 (P < 0.05) were associated with treatment response. CONCLUSION: This study provides technical feasibility data for correlative studies. Tissue biopsies provide distinct data from the blood and may predict response to pembrolizumab.

The presence of anti-nuclear antibodies alone is associated with changes in B cell activation and T follicular helper cells similar to those in systemic autoimmune rheumatic disease.

BACKGROUND: Diagnosis of systemic autoimmune rheumatic diseases (SARD) relies on the presence of hallmark anti-nuclear antibodies (ANA), many of which can be detected years before clinical manifestations. However, ANAs are also seen in healthy individuals, most of whom will not develop SARD. Here, we examined a unique cohort of asymptomatic ANA+ individuals to determine whether they share any of the cellular immunologic features seen in SARD. METHODS: Healthy ANA- controls and ANA+ (ANA ≥1:160 by immunofluorescence) participants with no SARD criteria, with at least one criterion (undifferentiated connective tissue disease (UCTD)), or meeting SARD classification criteria were recruited. Peripheral blood cellular immunological changes were assessed by flow cytometry and transcript levels of BAFF, interferon (IFN)-induced and plasma cell-expressed genes were quantified by NanoString. RESULTS: A number of the immunologic abnormalities seen in SARD, including changes in peripheral B (switched memory) and T (iNKT, T regulatory, activated memory T follicular helper) subsets and B cell activation, were also seen in asymptomatic ANA+ subjects and those with UCTD. The extent of these immunologic changes correlated with ANA titer or the number of different specific ANAs produced. Principal component analysis of the cellular data indicated that a significant proportion of asymptomatic ANA+ subjects and subjects with UCTD clustered  with patients with early SARD, rather than ANA- healthy controls. CONCLUSIONS: ANA production is associated with altered T and B cell activation even in asymptomatic individuals. Some of the currently accepted cellular features of SARD may be associated with ANA production rather than the immunologic events that cause symptoms in SARD.

Identification of a neutrophil-related gene expression signature that is enriched in adult systemic lupus erythematosus patients with active nephritis: Clinical/pathologic associations and etiologic mechanisms.

Both a lack of biomarkers and relatively ineffective treatments constitute impediments to management of lupus nephritis (LN). Here we used gene expression microarrays to contrast the transcriptomic profiles of active SLE patients with and without LN to identify potential biomarkers for this condition. RNA isolated from whole peripheral blood of active SLE patients was used for transcriptomic profiling and the data analyzed by linear modeling, with corrections for multiple testing. Results were validated in a second cohort of SLE patients, using NanoString technology. The majority of genes demonstrating altered transcript abundance between patients with and without LN were neutrophil-related. Findings in the validation cohort confirmed this observation and showed that levels of RNA abundance in renal remission were similar to active patients without LN. In secondary analyses, RNA abundance correlated with disease activity, hematuria and proteinuria, but not renal biopsy changes. As abundance levels of the individual transcripts correlated strongly with each other, a composite neutrophil score was generated by summing all levels before examining additional correlations. There was a modest correlation between the neutrophil score and the blood neutrophil count, which was largely driven by the dose of glucocorticosteroids and not the proportion of low density and/or activated neutrophils. Analysis of longitudinal data revealed no correlation between baseline neutrophil score or changes over the first year of follow-up with subsequent renal flare or treatment outcomes, respectively. The findings argue that although the neutrophil score is associated with LN, its clinical utility as a biomarker may be limited.

Presence of an interferon signature in individuals who are anti-nuclear antibody positive lacking a systemic autoimmune rheumatic disease diagnosis.

BACKGROUND: Elevated levels of type I interferons (IFNs) are a characteristic feature of the systemic autoimmune rheumatic diseases (SARDs) and are thought to play an important pathogenic role. However, it is unknown whether these elevations are seen in anti-nuclear antibody-positive (ANA+) individuals who lack sufficient criteria for a SARD diagnosis. We examined IFN-induced gene expression in asymptomatic ANA+ individuals and patients with undifferentiated connective tissue disease (UCTD) to address this question. METHODS: Healthy ANA- control subjects and ANA+ titre (≥1:160 by immunofluorescence) participants meeting no criteria, meeting at least one criterion (UCTD) or meeting SARD classification criteria were recruited. Whole peripheral blood IFN-induced and BAFF gene expression were quantified using NanoString technology. The normalized levels of five IFN-induced genes were summed to produce an IFN5 score. RESULTS: The mean IFN5 scores were increased in all ANA+ participant subsets as compared with healthy control subjects. We found that 36.8% of asymptomatic ANA+ and 50% of UCTD participants had IFN5 scores >2 SD above the mean for healthy control subjects. In all ANA+ subsets, the IFN5 score correlated with the presence of anti-Ro/La antibodies. In the asymptomatic ANA+ subset, this score also correlated with the ANA titre, whereas in the other ANA+ subsets, it correlated with the number of different ANA specificities. Development of new SARD criteria was seen in individuals with normal and high IFN5 scores. CONCLUSIONS: An IFN signature is seen in a significant proportion of ANA+ individuals and appears to be associated with ANA titre and type of autoantibodies, rather than with the presence or development of clinical SARD symptoms.

A discrete cluster of urinary biomarkers discriminates between active systemic lupus erythematosus patients with and without glomerulonephritis.

BACKGROUND: Management of lupus nephritis (LN) would be greatly aided by the discovery of biomarkers that accurately reflect changes in disease activity. Here, we used a proteomics approach to identify potential urinary biomarkers associated with LN. METHODS: Urine was obtained from 60 LN patients with paired renal biopsies, 25 active non-LN SLE patients, and 24 healthy controls. Using Luminex, 128 analytes were quantified and normalized to urinary creatinine levels. Data were analyzed by linear modeling and non-parametric statistics, with corrections for multiple comparisons. A second cohort of 33 active LN, 16 active non-LN, and 30 remission LN SLE patients was used to validate the results. RESULTS: Forty-four analytes were identified that were significantly increased in active LN as compared to active non-LN. This included a number of unique proteins (e.g., TIMP-1, PAI-1, PF4, vWF, and IL-15) as well as known candidate LN biomarkers (e.g., adiponectin, sVCAM-1, and IL-6), that differed markedly (>4-fold) between active LN and non-LN, all of which were confirmed in the validation cohort and normalized in remission LN patients. These proteins demonstrated an enhanced ability to discriminate between active LN and non-LN patients over several previously reported biomarkers. Ten proteins were found to significantly correlate with the activity score on renal biopsy, eight of which strongly discriminated between active proliferative and non-proliferative/chronic renal lesions. CONCLUSIONS: A number of promising urinary biomarkers that correlate with the presence of active renal disease and/or renal biopsy changes were identified and appear to outperform many of the existing proposed biomarkers.

Lack of Interferon and Proinflammatory Cyto/chemokines in Serologically Active Clinically Quiescent Systemic Lupus Erythematosus.

OBJECTIVE: Serologically active clinically quiescent (SACQ) patients with systemic lupus erythematosus (SLE) remain clinically quiescent for prolonged periods despite anti-dsDNA antibodies and/or low complements, indicating the presence of immune complexes. The immune mechanisms leading to this quiescence are unknown. However, in addition to activating complement, immune complex uptake by various cells leads to the production of interferon (IFN)-α and other proinflammatory factors that are also involved in tissue damage. Here we investigate whether production of these factors is reduced in SACQ patients. METHODS: The levels of 5 IFN-induced genes and 19 cyto/chemokines were measured in SACQ patients and were compared with those in serologically and clinically active (SACA) and serologically and clinically quiescent (SQCQ) patients. SACQ and SQCQ were defined as ≥ 2 years without clinical activity, with/without persistent serologic activity, respectively, and off corticosteroids/immunosuppressives. SACA was defined as disease activity compelling immunosuppression. Levels of OAS1, IFIT1, MX1, LY6E, and ISG15 were measured by quantitative real-time polymerase chain reaction (PCR) and a composite score (IFN-5) derived from this. Plasma cyto/chemokines were measured by Luminex assay. Nonparametric univariate and logistic regression analyses were conducted. RESULTS: There were no differences in gene expression or cyto/chemokine levels between SACQ and SQCQ patients. The SACQ IFN-5 score was significantly lower than that of SACA (p = 0.003) and was driven by SACQ status, not by autoantibody profile or disease duration. Levels of granulocyte-macrophage colony-stimulating factor, interleukin (IL) 6, IL-10, IFN-γ-inducible protein 10, monocyte chemoattractant protein 1, and tumor necrosis factor-α were significantly lower in SACQ than SACA. CONCLUSION: The levels of proinflammatory factors in SACQ mirror those of SQCQ patients, indicating reduced production of these factors despite the presence of immune complexes.

T cell and dendritic cell abnormalities synergize to expand pro-inflammatory T cell subsets leading to fatal autoimmunity in B6.NZBc1 lupus-prone mice.

We have previously shown that B6 congenic mice with a New Zealand Black chromosome 1 (c1) 96-100 cM interval produce anti-nuclear Abs and that at least two additional genetic loci are required to convert this subclinical disease to fatal glomerulonephritis in mice with a c1 70-100 cM interval (c1(70-100)). Here we show that the number of T follicular helper and IL-21-, IFN-γ-, and IL-17-secreting CD4(+) T cells parallels disease severity and the number of susceptibility loci in these mice. Immunization of pre-autoimmune mice with OVA recapitulated these differences. Differentiation of naïve T cells in-vitro under polarizing conditions and in-vivo following adoptive transfer of OVA-specific TCR transgenic cells into c1(70-100) or B6 recipient mice, revealed T cell functional defects leading to increased differentiation of IFN-γ- and IL-17-producing cells in the 96-100 cM and 88-96 cM intervals, respectively. However, in-vivo enhanced differentiation of pro-inflammatory T cell subsets was predominantly restricted to c1(70-100) recipient mice, which demonstrated altered dendritic cell function, with increased production of IL-6 and IL-12. The data provide support for the role of pro-inflammatory T cells in the conversion of subclinical disease to fatal autoimmunity and highlight the importance of synergistic interactions between individual susceptibility loci in this process.

Dlx5- and Dlx6-mediated chondrogenesis: Differential domain requirements for a conserved function.

During endochondral ossification in the vertebrate limb, multipotent mesenchymal cells first differentiate into chondroblasts (chondrogenesis) that further differentiate (via chondrocyte hypertrophy) to a terminal cellular phenotype. Dlx5 and Dlx6 are functionally redundant regulators of chondrocyte hypertrophy. We now show that Dlx5 and Dlx6 also regulate the earlier step of chondrogenesis in the limb. Limb bud mesenchymal cells from Dlx5/6(-/-) embryos show reduced chondrogenesis compared to wild-type littermates, and expression of either Dlx5 or Dlx6 stimulated differentiation of limb bud mesenchymal cells to chondroblasts. The functional overlap between Dlx5 and Dlx6 occurs despite the fact that the amino- and carboxyl-terminal domains of the encoded proteins are dissimilar. In order to reconcile the disparity between the divergent structures of Dlx5 and Dlx6 with their overlapping biological functions, we investigated the domain requirements and transcriptional activities associated with Dlx5- and Dlx6-mediated chondrogenesis. We find distinct domain requirements for the chondrogenic function of these related homeoproteins, indicating divergent molecular mechanisms of action.

Virulence genes of O149 enterotoxigenic Escherichia coli from outbreaks of postweaning diarrhea in pigs.

The goal of this research was to determine whether isolates of O149 porcine enterotoxigenic Escherichia coli (ETEC) recovered from recent outbreaks of severe diarrhea in weaned pigs in Ontario, Canada, had virulence attributes different from those of isolates of the same serogroup from diarrhea of pigs in the 1970s and 1980s. Polymerase chain reaction amplification was used to determine the distribution of 11 virulence-associated genes in recent (100 isolates) and old (35 isolates) Ontario O149 porcine ETEC. These tests demonstrated that 92% of the recent isolates possessed the estA gene for STa enterotoxin, whereas none of the old isolates had this gene. H antigen determination showed that all the isolates which lacked the estA gene (all 35 old isolates plus 8 recent isolates) were H43, whereas isolates which had the estA gene were H10. The astA gene for enteroaggregative heat-stable enterotoxin (EAST1) and the K88ac antigen were present in all 135 isolates. Plasmid analyses identified a cryptic 5.1kb plasmid in 99% of recent and 60% of old isolates. Suppressive subtractive hybridization associated several types of DNA fragments with the recent O149 ETEC, namely, fragments with no homology to DNA in databases, fragments of LPS biosynthesis genes, and F plasmid DNA. We conclude that the recent outbreaks of PWD in Ontario pigs were associated primarily with a new serotype of O149 ETEC and that isolates of this serotype possessed the estA gene that was not present in old O149 ETEC isolated from pigs in Ontario.