Pair filamentation and laser scattering in beam-driven QED cascades.

We report the observation of longitudinal filamentation of an electron-positron pair plasma in a beam-driven QED cascade. The filaments are created in the "pair-reflection" regime, where the generated pairs are partially stopped and reflected in the strong laser field. The density filaments form near the center of the laser pulse and have diameters similar to the laser wavelength. They develop and saturate within a few laser cycles and do not induce sizable magnetostatic fields. We rule out the onset of two-stream instability or Weibel instability and attribute the origin of pair filamentation to laser ponderomotive forces. The small plasma filaments induce strong scattering of laser energy to large angles, serving as a signature of collective QED plasma dynamics.

Blood immunophenotyping identifies distinct kidney histopathology and outcomes in patients with lupus nephritis.

Lupus nephritis (LN) is a frequent manifestation of systemic lupus erythematosus, and fewer than half of patients achieve complete renal response with standard immunosuppressants. Identifying non-invasive, blood-based pathologic immune alterations associated with renal injury could aid therapeutic decisions. Here, we used mass cytometry immunophenotyping of peripheral blood mononuclear cells in 145 patients with biopsy-proven LN and 40 healthy controls to evaluate the heterogeneity of immune activation in patients with LN and to identify correlates of renal parameters and treatment response. Unbiased analysis identified 3 immunologically distinct groups of patients with LN that were associated with different patterns of histopathology, renal cell infiltrates, urine proteomic profiles, and treatment response at one year. Patients with enriched circulating granzyme B+ T cells at baseline showed more severe disease and increased numbers of activated CD8 T cells in the kidney, yet they had the highest likelihood of treatment response. A second group characterized primarily by a high type I interferon signature had a lower likelihood of response to therapy, while a third group appeared immunologically inactive by immunophenotyping at enrollment but with chronic renal injuries. Main immune profiles could be distilled down to 5 simple cytometric parameters that recapitulate several of the associations, highlighting the potential for blood immune profiling to translate to clinically useful non-invasive metrics to assess immune-mediated disease in LN.

Nasal polyp antibody-secreting cells display proliferation signature in aspirin-exacerbated respiratory disease.

BACKGROUND: Chronic rhinosinusitis with nasal polyps (CRSwNP) causes nasal obstruction and olfactory dysfunction. Aspirin-exacerbated respiratory disease (AERD) is the triad of CRSwNP, asthma, and respiratory reactions to COX-1 inhibitors. Patients with AERD have elevated nasal IL-5 levels and high numbers of antibody-secreting cells (ASCs), including plasma cells and plasmablasts, in their polyp tissue; in addition, their nasal polyp (NP) IgE levels are correlated with disease severity and recurrence of nasal polyposis. OBJECTIVE: We sought to explore differences in the transcriptomic profile, activation markers, and IL-5Rα expression and function of NP ASCs from patients with AERD and CRSwNP. METHODS: NP tissue was collected from patients with AERD and CRSwNP and digested into single-cell suspensions. NP cells were analyzed for protein expression by mass cytometry. For IL-5Rα functional studies, plasma cells were purified and cultured in vitro with or without IL-5 and analyzed by bulk RNA sequencing. RESULTS: Compared with polyp tissue from patients with CRSwNP, polyp tissue from patients with AERD contained significantly more ASCs and had increased ASC expression of IL-5Rα. ASCs from patients with AERD expressed higher protein levels of B-cell activation and regulatory markers (CD40, CD19, CD32, and CD38) and the proliferation marker Ki-67. ASCs from patients with AERD also expressed more IL5RA, IGHE, and cell cycle- and proliferation-related transcripts (CCND2, MKI67, CDC25A, and CDC25B) than did ASCs from patients with CRSwNP. Stimulation of plasma cells from patients with AERD with IL-5 induced key cell cycle genes (CCND2 and PTP4A3), whereas IL-5 stimulation of ASCs from patients with CRSwNP induced few transcriptomic changes. CONCLUSION: NP tissue ASCs from patients with AERD express higher levels of functional IL-5Rα and markers associated with cell cycling and proliferation than do ASCs from patients with aspirin-tolerant CRSwNP.

Mesenchymal Stromal Cells Facilitate Neutrophil-Trained Immunity by Reprogramming Hematopoietic Stem Cells.

Novel therapeutics are urgently needed to prevent opportunistic infections in immunocompromised individuals undergoing cancer treatments or other immune-suppressive therapies. Trained immunity is a promising strategy to reduce this burden of disease. We previously demonstrated that mesenchymal stromal cells (MSCs) preconditioned with a class A CpG oligodeoxynucleotide (CpG-ODN), a Toll-like receptor 9 (TLR9) agonist, can augment emergency granulopoiesis in a murine model of neutropenic sepsis. Here, we used a chimeric mouse model to demonstrate that MSCs secrete paracrine factors that act on lineage-negative c-kit+ hematopoietic stem cells (HSCs), leaving them "poised" to enhance emergency granulopoiesis months after transplantation. Chimeric mice developed from HSCs exposed to conditioned media from MSCs and CpG-ODN-preconditioned MSCs showed significantly higher bacterial clearance and increased neutrophil granulopoiesis following lung infection than control mice. By Cleavage Under Targets and Release Using Nuclease (CUT&RUN) chromatin sequencing, we identified that MSC-conditioned media leaves H3K4me3 histone marks in HSCs at genes involved in myelopoiesis and in signaling persistence by the mTOR pathway. Both soluble factors and extracellular vesicles from MSCs mediated these effects on HSCs and proteomic analysis by mass spectrometry revealed soluble calreticulin as a potential mediator. In summary, this study demonstrates that trained immunity can be mediated by paracrine factors from MSCs to induce neutrophil-trained immunity by reprogramming HSCs for long-lasting functional changes in neutrophil-mediated antimicrobial immunity.

Cytomegalovirus durably primes neutrophil oxidative burst.

Cytomegalovirus (CMV) is a ubiquitous herpes virus that infects most humans, thereafter persisting lifelong in tissues of the host. It is a known pathogen in immunosuppressed patients, but its impact on immunocompetent hosts remains less understood. Recent data have shown that CMV leaves a significant and long-lasting imprint in host immunity that may confer some protection against subsequent bacterial infection. Such innate immune activation may come at a cost, however, with potential to cause immunopathology. Neutrophils are central to many models of immunopathology, and while acute CMV infection is known to influence neutrophil biology, the impact of chronic CMV infection on neutrophil function remains unreported. Using our murine model of CMV infection and latency, we show that chronic CMV causes persistent enhancement of neutrophil oxidative burst well after resolution of acute infection. Moreover, this in vivo priming of marrow neutrophils is associated with enhanced formyl peptide receptor expression, and ultimately constitutive c-Jun N-terminal kinase phosphorylation and enhanced CD14 expression in/on circulating neutrophils. Finally, we show that neutrophil priming is dependent on viral load, suggesting that naturally infected human hosts will show variability in CMV-related neutrophil priming. Altogether, these findings represent a previously unrecognized and potentially important impact of chronic CMV infection on neutrophil responsiveness in immunocompetent hosts.

Deep immunophenotyping reveals circulating activated lymphocytes in individuals at risk for rheumatoid arthritis.

Rheumatoid arthritis (RA) is a systemic autoimmune disease with currently no universally highly effective prevention strategies. Identifying pathogenic immune phenotypes in 'At-Risk' populations prior to clinical disease onset is crucial to establishing effective prevention strategies. Here, we applied mass cytometry to deeply characterize the immunophenotypes in blood from At-Risk individuals identified through the presence of serum antibodies to citrullinated protein antigens (ACPA) and/or first-degree relative (FDR) status (n=52), as compared to established RA (n=67), and healthy controls (n=48). We identified significant cell expansions in At-Risk individuals compared with controls, including CCR2+CD4+ T cells, T peripheral helper (Tph) cells, type 1 T helper cells, and CXCR5+CD8+ T cells. We also found that CD15+ classical monocytes were specifically expanded in ACPA-negative FDRs, and an activated PAX5 low naïve B cell population was expanded in ACPA-positive FDRs. Further, we developed an "RA immunophenotype score" classification method based on the degree of enrichment of cell states relevant to established RA patients. This score significantly distinguished At-Risk individuals from controls. In all, we systematically identified activated lymphocyte phenotypes in At-Risk individuals, along with immunophenotypic differences among both ACPA+ and ACPA-FDR At-Risk subpopulations. Our classification model provides a promising approach for understanding RA pathogenesis with the goal to further improve prevention strategies and identify novel therapeutic targets.

Neutrophil heterogeneity and emergence of a distinct population of CD11b/CD18-activated low-density neutrophils after trauma.

INTRODUCTION: Multiple large clinical trauma trials have documented an increased susceptibility to infection after injury. Although neutrophils (polymorphonuclear leukocytes [PMNs]) were historically considered a homogeneous cell type, we hypothesized that injury could alter neutrophil heterogeneity and predispose to dysfunction. To explore whether trauma modifies PMN heterogeneity, we performed an observational mass-spectrometry-based cytometry study on total leukocytes and low-density PMNs found in the peripheral blood mononuclear cell fraction of leukocytes from healthy controls and trauma patients. METHODS: A total of 74 samples from 12 trauma patients, each sampled at 1 or more time points, and matched controls were fractionated and profiled by mass-spectrometry-based cytometry using a panel of 44 distinct markers. After deconvolution and conservative gating on neutrophils, data were analyzed using Seurat, followed by clustering of principal components. RESULTS: Eleven distinct neutrophil populations were resolved in control and trauma neutrophils based on differential protein surface marker expression. Trauma markedly altered the basal heterogeneity of neutrophil subgroups seen in the control samples, with loss of a dominant population of resting neutrophils marked by high expression of C3AR and low levels of CD63, CD64, and CD177 (cluster 1), and expansion of two alternative neutrophil populations, one of which is marked by high expression of CD177 with suppression of CD10, CD16, C3AR, CD63, and CD64 (cluster 6). Remarkably, following trauma, a substantially larger percentage of neutrophils sediment in the monocyte fraction. These low-density neutrophils bear markers of functional exhaustion and form a unique trauma-induced population (cluster 9) with markedly upregulated expression of active surface adhesion molecules (activated CD11b/CD18), with suppression of nearly all other surface markers, including receptors for formyl peptides, leukotrienes, chemokines, and complement. CONCLUSION: Circulating neutrophils demonstrate considerable evidence of functional heterogeneity that is markedly altered by trauma. Trauma induces evolution of a novel, exhausted, low-density neutrophil population with immunosuppressive features.

Perturbing DDR signaling enhances cytotoxic effects of local oncolytic virotherapy and modulates the immune environment in glioma.

CAN-2409 is a replication-deficient adenovirus encoding herpes simplex virus (HSV) thymidine kinase (tk) currently in clinical trials for treatment of glioblastoma. The expression of tk in transduced cancer cells results in conversion of the pro-drug ganciclovir into a toxic metabolite causing DNA damage, inducing immunogenic cell death and immune activation. We hypothesize that CAN-2409 combined with DNA-damage-response inhibitors could amplify tumor cell death, resulting in an improved response. We investigated the effects of ATR inhibitor AZD6738 in combination with CAN-2409 in vitro using cytotoxicity, cytokine, and fluorescence-activated cell sorting (FACS) assays in glioma cell lines and in vivo with an orthotopic syngeneic murine glioma model. Tumor immune infiltrates were analyzed by cytometry by time of flight (CyTOF). In vitro, we observed a significant increase in the DNA-damage marker γH2AX and decreased expression of PD-L1, pro-tumorigenic cytokines (interleukin-1ß [IL-1ß], IL-4), and ligand NKG2D after combination treatment compared with monotherapy or control. In vivo, long-term survival was increased after combination treatment (66.7%) compared with CAN-2409 (50%) and control. In a tumor re-challenge, long-term immunity after combination treatment was not improved. Our results suggest that ATR inhibition could amplify CAN-2409's efficacy in glioblastoma through increased DNA damage while having complex immunological ramifications, warranting further studies to determine the ideal conditions for maximized therapeutic benefit.

Distinct Injury Responsive Regulatory T Cells Identified by Multi-Dimensional Phenotyping.

CD4+ regulatory T cells (Tregs) activate and expand in response to different types of injuries, suggesting that they play a critical role in controlling the immune response to tissue and cell damage. This project used multi-dimensional profiling techniques to comprehensively characterize injury responsive Tregs in mice. We show that CD44high Tregs expand in response to injury and were highly suppressive when compared to CD44low Tregs. T cell receptor (TCR) repertoire analysis revealed that the CD44high Treg population undergo TCRαß clonal expansion as well as increased TCR CDR3 diversity. Bulk RNA sequencing and single-cell RNA sequencing with paired TCR clonotype analysis identified unique differences between CD44high and CD44low Tregs and specific upregulation of genes in Tregs with expanded TCR clonotypes. Gene ontology analysis for molecular function of RNA sequencing data identified chemokine receptors and cell division as the most enriched functional terms in CD44high Tregs versus CD44low Tregs. Mass cytometry (CyTOF) analysis of Tregs from injured and uninjured mice verified protein expression of these genes on CD44high Tregs, with injury-induced increases in Helios, Galectin-3 and PYCARD expression. Taken together, these data indicate that injury triggers the expansion of a highly suppressive CD44high Treg population that is transcriptionally and phenotypically distinct from CD44low Tregs suggesting that they actively participate in controlling immune responses to injury and tissue damage.

Idelalisib reduces regulatory T cells and activates T helper 17 cell differentiation in relapsed refractory patients with chronic lymphocytic leukaemia.

Phosphatidylinositol 3 kinase (PI3K) inhibitors such as idelalisib have been associated with potentially severe autoimmune toxicity. In the present study, we demonstrate that relapsed refractory patients with chronic lymphocytic leukaemia treated with idelalisib rituximab on the phase III registration trial show uniform decrease in regulatory T cells (Tregs) and increase in CD8 T cells with treatment. Patients who do not develop toxicity show enrichment for T cells expressing multiple chemokine receptors, while those who do develop toxicity have an activated CD8 T cell population with T helper 17 cell differentiation at baseline, which then increases, leading to an increased CD8:Treg ratio that likely triggers autoimmune toxicity.

Systemic high-dose dexamethasone treatment may modulate the efficacy of intratumoral viral oncolytic immunotherapy in glioblastoma models.

BACKGROUND: Intratumoral viral oncolytic immunotherapy is a promising new approach for the treatment of a variety of solid cancers. CAN-2409 is a replication-deficient adenovirus that delivers herpes simplex virus thymidine kinase to cancer cells, resulting in local conversion of ganciclovir or valacyclovir into a toxic metabolite. This leads to highly immunogenic cell death, followed by a local immune response against a variety of cancer neoantigens and, next, a systemic immune response against the injected tumor and uninjected distant metastases. CAN-2409 treatment has shown promising results in clinical studies in glioblastoma (GBM). Patients with GBM are usually given the corticosteroid dexamethasone to manage edema. Previous work has suggested that concurrent dexamethasone therapy may have a negative effect in patients treated with immune checkpoint inhibitors in patients with GBM. However, the effects of dexamethasone on the efficacy of CAN-2409 treatment have not been explored. METHODS: In vitro experiments included cell viability and neurosphere T-cell killing assays. Effects of dexamethasone on CAN-2409 in vivo were examined using a syngeneic murine GBM model; survival was assessed according to Kaplan-Meier; analyses of tumor-infiltrating lymphocytes were performed with mass cytometry (CyTOF - cytometry by time-of-flight). Data were analyzed using a general linear model, with one-way analysis of variance followed by Dunnett's multiple comparison test, Kruskal-Wallis test, Dunn's multiple comparison test or statistical significance analysis of microarrays. RESULTS: In a mouse model of GBM, we found that high doses of dexamethasone combined with CAN-2409 led to significantly reduced median survival (29.0 days) compared with CAN-2409 treatment alone (39.5 days). CyTOF analyses of tumor-infiltrating immune cells demonstrated potent immune stimulation induced by CAN-2409 treatment. These effects were diminished when high-dose dexamethasone was used. Functional immune cell characterization suggested increased immune cell exhaustion and tumor promoting profiles after dexamethasone treatment. CONCLUSION: Our data suggest that concurrent high-dose dexamethasone treatment may impair the efficacy of oncolytic viral immunotherapy of GBM, supporting the notion that dexamethasone use should be balanced between symptom control and impact on the therapeutic outcome.

A T cell inflammatory phenotype is associated with autoimmune toxicity of the PI3K inhibitor duvelisib in chronic lymphocytic leukemia.

Several PI3Kδ inhibitors are approved for the therapy of B cell malignancies, but their clinical use has been limited by unpredictable autoimmune toxicity. We have recently reported promising efficacy results in treating chronic lymphocytic leukemia (CLL) patients with combination therapy with the PI3Kδγ inhibitor duvelisib and fludarabine cyclophosphamide rituximab (FCR) chemoimmunotherapy, but approximately one-third of patients develop autoimmune toxicity. We show here that duvelisib FCR treatment in an upfront setting modulates both CD4 and CD8 T cell subsets as well as pro-inflammatory cytokines. Decreases in naive and central memory CD4 T cells and naive CD8 T cells occur with treatment, while activated CD8 T cells, granzyme positive Tregs, and Th17 CD4 and CD8 T cells all increase with treatment, particularly in patients with toxicity. Cytokines associated with Th17 activation (IL-17A and IL-21) are also relatively elevated in patients with toxicity. The only CLL feature associated with toxicity was increased priming for apoptosis at baseline, with a significant decrease during the first week of duvelisib. We conclude that an increase in activated CD8 T cells with activation of Th17 T cells, in the context of lower baseline Tregs and greater CLL resistance to duvelisib, is associated with duvelisib-related autoimmune toxicity.