Cell Populations In Peripheral Blood Research Articles

Does the upper respiratory tract microbiome change in dairy calves fed milk replacer containing probiotics?

Abstract Our overall hypothesis is that feeding probiotics to dairy calves will alter overall immunity and may additionally change the microbiome in the respiratory tract as well as the gut. A group of 20 dairy calves were split into two treatment groups: Control (N=10, milk replacer), and Probiotic (N=10, milk replacer + 0.5 g/day Bovamine Dairy). On day 0 birth weight was obtained and the calves were provided colostrum as per the dairy SOP. On day 2, probiotics were added to the milk replacer of the treated group. Blood was collected along with nasal and tonsil swabs on days 0, 7, 14, 21, 28, 42, and 48. Calves were monitored daily for fecal, nasal, and ocular discharge scores. Our preliminary data showed no significant differences (P<0.5) between the Control and Probiotic groups in immune cell populations in peripheral blood. DNA was extracted from nasal and tonsil swabs to evaluate the bacterial populations in the upper respiratory microbiome. Hypervariable regions 1 through 3 along the 16S ribosomal RNA gene were amplified by PCR and sequenced by Illumina MiSeq to determine the bacterial taxa present. Evaluation of these samples revealed a greater percentage of operational taxonomy units (OTU) were classified at the genus level in the tonsil compared to the nasal samples. The microbiome of the tonsils was more stable over time points compared to the nasal samples. Evaluation of each timepoint for nasal and tonsil samples identified bacterial taxa that changed in relative abundance with the addition of the probiotic compared to the control diet. Addition of probiotic changed the relative abundance of OTU (P<0.01) in the nasal and tonsil sampling sites and at multiple timepoints. Analyses are ongoing. Project supported by fund from the USDA Agricultural Research Service.

The immune system contributes to the effectiveness of vaccine therapy in patients with metastatic melanoma

The aim of the study was to identify differences in the immune system parameters between metastatic melanoma patients who responded and did not respond to dendritic cell vaccination.Material and Methods. The study group included 20 patients with stage III–IV metastatic melanoma, who received vaccine therapy with dendritic cells (DC) in a prophylactic mode. The control groups included 13 patients who had symptoms of disease progression at the time of starting vaccine therapy, and 5 healthy donors. The DC-vaccine was prepared in the form of a suspension of the patient’s autologous dendritic cells loaded with tumor antigens in vitro. A single dose had 2 million dendritic cells in 1 ml of phosphate buffer solution, which was administered intradermally in the nearest site to the regional lymphatic collectors. The immune system status was assessed before starting vaccination. The immune system status was evaluated according to the indices of 25 peripheral blood cell populations using multicolor flow cytometry and integral characteristic in the form of the visual image generated by the visualization method of multidimensional data (NovoSpark, Canada).Results. The immune status in patients with metastatic melanoma at the start of DC-vaccination differed and was associated with the effectiveness of subsequent vaccine therapy. The response to vaccination was observed in patients whose immune system status was similar to that of healthy individuals. Low efficacy of DC-vaccine therapy was shown in patients whose immune system status corresponded to that of patients with disease progression. Alterations of the immune system in patients with metastatic melanoma were registered both at the level of individual immunological parameters and at the level of visualized integral characteristics. The integral characteristics of the immune system associated with the patient’s immunocompromised status can be considered as a criterion for stratification of patients with metastatic melanoma for the effective DC-vaccine therapy.Conclusion. The effectiveness of vaccine therapy with dendritic cells in patients with metastatic melanoma is associated with the immune system state before starting this therapy.

Combined space stressors induce independent behavioral deficits predicted by early peripheral blood monocytes

Interplanetary space travel poses many hazards to the human body. To protect astronaut health and performance on critical missions, there is first a need to understand the effects of deep space hazards, including ionizing radiation, confinement, and altered gravity. Previous studies of rodents exposed to a single such stressor document significant deficits, but our study is the first to investigate possible cumulative and synergistic impacts of simultaneous ionizing radiation, confinement, and altered gravity on behavior and cognition. Our cohort was divided between 6-month-old female and male mice in group, social isolation, or hindlimb unloading housing, exposed to 0 or 50 cGy of 5 ion simplified simulated galactic cosmic radiation (GCRsim). We report interactions and independent effects of GCRsim exposure and housing conditions on behavioral and cognitive performance. Exposure to GCRsim drove changes in immune cell populations in peripheral blood collected early after irradiation, while housing conditions drove changes in blood collected at a later point. Female mice were largely resilient to deficits observed in male mice. Finally, we used principal component analysis to represent total deficits as principal component scores, which were predicted by general linear models using GCR exposure, housing condition, and early blood biomarkers.

Type 2 and type 17 effector cells are increased in the duodenal mucosa but not peripheral blood of patients with functional dyspepsia.

Functional dyspepsia is characterised by chronic symptoms of post-prandial distress or epigastric pain not associated with defined structural pathology. Increased peripheral gut-homing T cells have been previously identified in patients. To date, it is unknown if these T cells were antigen-experienced, or if a specific phenotype was associated with FD. This study aimed to characterise T cell populations in the blood and duodenal mucosa of FD patients that may be implicated in disease pathophysiology. We identified duodenal T cell populations from 23 controls and 49 Rome III FD patients by flow cytometry using a surface marker antibody panel. We also analysed T cell populations in peripheral blood from 37 controls and 61 patients. Where available, we examined the number of duodenal eosinophils in patients and controls. There was a shift in the duodenal T helper cell balance in FD patients compared to controls. For example, patients had increased duodenal mucosal Th2 populations in the effector (13.03 ± 16.11, 19.84 ± 15.51, p=0.038), central memory (23.75 ± 18.97, 37.52 ± 17.51, p=0.007) and effector memory (9.80±10.50 vs 20.53±14.15, p=0.001) populations. Th17 populations were also increased in the effector (31.74±24.73 vs 45.57±23.75, p=0.03) and effector memory (11.95±8.42 vs 18.44±15.63, p=0.027) subsets. Peripheral T cell populations were unchanged between FD and control. Our findings identify an association between lymphocyte populations and FD, specifically a Th2 and Th17 signature in the duodenal mucosa. The presence of effector and memory cells suggest that the microinflammation in FD is antigen driven.

Vitamin D induced microbicidal activity against Mycobacterium bovis BCG is dependent on the synergistic activity of bovine peripheral blood cell populations

A growing appreciation is emerging of the beneficial role of vitamin D for health and resistance against infectious diseases, including tuberculosis. However, research has predominantly focused on murine and human species and functional data in bovines is limited. Therefore, the objective of this study was to assess the microbicidal activity and immunoregulatory effect of the vitamin D metabolite 1,25(OH)2D3 on bovine peripheral blood leukocytes (PBL) in response to Mycobacterium bovis BCG (BCG) infection using a combination of functional assays and gene expression profiling. Blood from Holstein-Friesian bull calves with low circulating levels of 25(OH)D was stimulated with 1,25(OH)2D3 for 2 h, and then infected with M. bovis BCG. Results showed that 1,25(OH)2D3 supplementation significantly increased BCG killing by on average 16 %, although responses varied between 1 % and 38 % killing. Serial cell subset depletion was then performed on PBL prior to 1,25(OH)2D3 incubation and BCG infected as before to analyse the contribution of major cell types to mycobacterial growth control. Specific antibodies and either magnetic cell separation or density gradient centrifugation of monocytes, granulocytes, CD3+, CD4+, and CD8+ T lymphocytes were used to capture each cell subset. Results showed that depletion of granulocytes had the greatest impact on BCG growth, leading to a significant enhancement of bacterial colonies. In contrast, depletion of CD4+ or CD8+ T cells individually, or in combination (CD3+), had no impact on mycobacterial growth control. In agreement with our previous data, 1,25(OH)2D3 significantly increased bacterial killing in PBL, in monocyte depleted samples, and a similar trend was observed in the granulocyte depleted subset. In addition, specific analysis of sorted neutrophils treated with 1,25(OH)2D3 showed an enhanced microbicidal activity against both BCG and a virulent strain of M. bovis. Lastly, data showed that 1,25(OH)2D3 stimulation increased reactive oxygen species (ROS) production and the expression of genes encoding host defence peptides (HDP) and pathogen recognition receptors (PRRs), factors that play an important role in the microbicidal activity against mycobacteria. In conclusion, the vitamin D metabolite 1,25(OH)2D3 improves antimycobacterial killing in bovine PBLs via the synergistic activity of monocytes and granulocytes and enhanced activation of innate immunity.

Circulating CD22+/CD19-/CD24- progenitors and CD22+/CD19+/CD24- mature B cells: Diagnostic pitfalls for minimal residual disease detection in B-lymphoblastic leukemia.

Multiparametric flow cytometry (MFC) has become a powerful tool in minimal residual disease (MRD) detection in B-lymphoblastic leukemia/lymphoma (B-ALL). In the setting of targeted immunotherapy, B-ALL MRD detection often relies on alterative gating strategies, such as the utilization of CD22 and CD24. It is important to depict the full diversity of normal cell populations included in the alternative B-cell gating methods to avoid false-positive results. We describe two CD22-positive non-neoplastic cell populations in the peripheral blood (PB), including one progenitor population of uncertain lineage and one mature B-cell population, which are immunophenotypic mimics of B-ALL. Using MFC, we investigated the prevalence and phenotypic profiles of both CD22-positive populations in 278 blood samples from 52 patients with B-ALL; these were obtained pre- and post-treatment with CD19 and/or CD22 CAR-T therapies. We further assessed whether these two populations in the blood were exclusively associated with B-ALL or recent anticancer therapies, by performing the same analysis on patients diagnosed with other hematological malignancies but in long-term MRD remission. The progenitor population and mature B-cell population were detected at low levels in PB of 61.5% and 44.2% of B-ALL patients, respectively. Both cell types showed distinctive and highly consistent antigen expression patterns that are reliably distinguishable from B-ALL. Furthermore, their presence is not restricted solely to B-ALL or recent therapy. Our findings aid in building a complete immunophenotypic profile of normal cell populations in PB, thereby preventing misdiagnosis of B-ALL MRD and inappropriate management.

Novel First-in-Class Drug ONC201 As a Post-Transplant Maintenance for AML and MDS: A Phase I Trial in Progress

Background: Relapses of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) after allogeneic hematopoietic cell transplantation represent an area of significant unmet clinical need and a research priority identified by the National Cancer Institute. For patients with high-risk AML and MDS, post-transplant relapses remain the principal cause of mortality. The role of chemotherapy agents outside of FLT3 inhibitors or TKIs, and post-transplant cellular therapy to prevent relapse of leukemia are not well established. However, chemotherapy agents are frequently utilized off-label in clinical practice, thus signifying an urgent need to develop effective therapeutic strategies. ONC201 is a novel first-in-class small molecule imipridone, that antagonizes G protein-coupled receptor, DRD2 resulting in AKT/ERK inactivation. ONC201 is also an allosteric agonist of mitochondrial caseinolytic protease P that activates an integrated stress responses. In preclinical studies, leukemia cells demonstrate sensitivity to ONC201 regardless of their genetic and mutation profiles. Leukemia cells with genetic and mutation changes such as TP53 mutation and complex karyotype that confer resistance to conventional therapy are sensitive to ONC201. ONC201 is also toxic to leukemia stem cells while sparing normal bone marrow cells [Sci Signal. 2016 Feb 16;9(415):ra17]. Several clinical trials in solid and hematologic malignancies have demonstrated that oral ONC201 has a low toxicity profile and is well tolerated. The profile of ONC201 is well suited for further development to prevent post-transplant relapses of AML and MDS. Here, we report the design of the first trial to use ONC201 as post-transplant maintenance (ClinicalTrials.gov ID: NCT03932643). Study Design and Methods: The study will follow a 3+3 phase 1 trial design. The primary objective is to determine the rate of dose-limiting toxicities (DLT) during the first cycle and grade ≥3 toxicities during the first 3 cycles. Key inclusion criteria include adult recipients of allogeneic hematopoietic stem cell transplant within 6-20 weeks for a history of high-risk AML or MDS; <5% bone marrow blasts; Karnofsky Performance Status of ≥70; absolute neutrophil count (ANC) greater than 1000/μL, and platelet count ³50,000/µL. Key exclusion criteria include a history of acute graft-versus-host disease (GVHD) grade III/IV; initiation of any new systemic immunosuppressive agent for GVHD within 4 weeks of enrollment, current use of prednisone at a dose of ≥0.25 mg/kg/day; uncontrolled serious infection; active ischemic heart disease, heart failure or arrhythmia; severe chronic obstructive pulmonary disease; resting O2 saturation <90%; aspartate transaminase, alanine transaminase or bilirubin >2 times the upper limit of normal; and creatinine clearance <30 mL/min. Patients will receive oral weekly ONC201 at a starting dose of 250 mg and dose escalation by 125 mg up to a maximum dose of 625 mg weekly, in the absence of DLT. Patients will be monitored for toxicities (using Common Terminology Criteria for Adverse Events, CTCAE version 5.0), quality of life (QOL) (Functional Assessment of Cancer Therapy-Bone Marrow Transplant, FACT-BMT), changes in functional status (KPS, instrumental activities of daily living and short physical performance battery), rates of disease relapse and mortality (See Study Schema). We will enroll a total of 20 patients; the first 6-9 patients are expected to receive escalating doses of ONC 201, and the remaining patients will receive the maximum tolerated doses. Clinical studies in solid malignancies have indicated that ONC201 can expand and activate NK cells in peripheral blood, or recruit CD8+ T-cells to the tumor. Hence, the study will assess expansion and activation of NK and T cell populations in peripheral blood and utilize transcriptomic analyses of NK and T cells to explore the mechanisms behind the immunologic changes. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

No Evidence of BCMA Expression Loss or Systemic Immune Impairment after Treatment with the BCMA-Targeted Antibody-Drug Conjugate (ADC) Belantamab Mafodotin (Belamaf) in the DREAMM-1 and DREAMM-2 Trials of Patients with Relapsed/Refractory Multiple Myeloma (RRMM)

Introduction: Belamaf, a first-in-class humanized ADC, binds BCMA expressed on differentiated B cells and myeloma (MM) cells. Belamaf monotherapy demonstrated deep and durable responses in the DREAMM-2 trial of patients (pts) with RRMM. Belamaf eliminates MM cells by a multimodal mechanism of action (MoA): direct cell killing and anti-MM immune response. We explored whether complete target loss and/or immune impairment were observed in pts who received belamaf in the DREAMM-1 and -2 trials. Methods: DREAMM-1 was a phase 1 dose-escalation study to investigate the safety, PK/PD, immunogenicity, and clinical activity of belamaf. DREAMM-2 was an open-label, two-arm, phase 2 study of belamaf 2.5 mg/kg or 3.4 mg/kg IV Q3W. Both trials enrolled adult pts with RRMM with ≥3 prior lines of therapy including an immunomodulatory drug, proteasome inhibitor, and an anti-CD38 monoclonal antibody (DREAMM-2 only). Free sBCMA, a shed form of BCMA that circulates in blood after cleavage from the membrane, was measured using an electrochemiluminescence assay on the MesoScale Discovery platform (Alliance Pharma, Malvern, PA; detection range 1.95-1000 ng/mL). Absolute concentrations of sBCMA were examined at baseline (BL), with absolute concentration and fold-change from BL assessed at best achieved response and latest progression. After the first post-infusion timepoint, samples were taken >4 days post-infusion to minimize belamaf interference. In this post hoc analysis, association of sBCMA with time-on-study was modeled using a linear mixed model to adjust for longitudinal categorical response, with time modeled as a quadratic function of square-root-time-on-study. A generalized additive mixed model was used to adjust the time-association for M-protein among pts with apparent secretory disease. Immune cell populations in peripheral blood were analyzed using flow cytometry and clinical hematology tests. Neutrophil-to-lymphocyte ratio and total lymphocyte counts were modeled by categorical pt visit adjusted for continuous or count-based biomarkers; T cell subpopulation counts were modeled using negative binomial mixed effects models. Results: At progression, sBCMA levels were detectable in 98% (50/51) of eligible pts in DREAMM-1 and 98.9% (181/183) of eligible pts in DREAMM-2, regardless of response status. Decreased sBCMA vs predose levels was observed immediately post-infusion in all response groups. In nonresponders, sBCMA returned to predose levels within 1 cycle. Responders (≥partial response) had a quantitatively lower but measurable sBCMA level, suggesting BCMA loss is not commonly observed in pts receiving belamaf. In 97% (64/66) of pts who responded but later progressed in DREAMM-2, sBCMA levels showed a pronounced drop during response but returned to near BL upon progression (Fig A). This suggests membrane BCMA expression is not lost following belamaf treatment and complete target loss is not the primary mechanism driving tumor escape in these pts. Previously, sBCMA levels have been shown to correlate with disease burden markers (e.g., M-protein) and ISS stage; we modeled sBCMA correcting for these associations to evaluate on-treatment dynamics, predicting a decrease in sBCMA with increasing time-on-treatment. After correcting for lower average BL and on-treatment sBCMA levels in responders, sBCMA levels tended to decrease over time. Longitudinal monitoring of pts' systemic immune cell populations showed no change in immune cell ratios (Fig B) or major cell populations regardless of response status. Immune cell profiles of responders and nonresponders were similar and consistent over time suggesting belamaf does not detrimentally affect total lymphocyte numbers while mediating anti-MM activity. Conclusions: Belamaf resistance and immune escape mechanisms are not well understood. We found no evidence that BCMA expression is completely lost after belamaf treatment. When sBCMA decreased after belamaf treatment, levels returned to BL upon progression. Regardless of response status, belamaf does not appear to negatively impact total lymphocyte numbers. These data may help inform clinical sequencing of belamaf with other BCMA-targeted therapies. Funding: GSK (205678/NCT03525678; 117159/NCT02064387). Drug linker technology licensed from Seagen, Inc; monoclonal antibody produced using POTELLIGENT Technology licensed from BioWa. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

CNSC-13. TUMOR INFILTRATION OF GAMMA-DELTA T CELLS IN GLIOBLASTOMA

Abstract Gamma-delta (γδ) T cells are innate immunity effector lymphocytes with known prominent anti-tumor reactivity against aggressive glioblastoma (GBM). However, therapeutic approaches have had limited success due to the protective blood-brain-barrier and the immunosuppressive GBM tumor microenvironment. In this study, we determined Vδ1 a Vδ2 γδ T cell populations in peripheral blood and paired tumor tissue samples in patients (n=40) following the resection and throughtout the therapy follow-up. Tumor samples were processed using enzymatic kits and gentleMACSTM Dissociator (Miltenyi Biotec Inc.) and tumor-infiltrating γδ T lymphocytes (TILs) were analyzed by flow cytometry.We found infiltration of both intratumoral CD3+ γδ T cell subsets in 68% tumor samples. We detected Vδ1 γδ T cells in the range 0-0.8% (median 0.26%). Majority of GBM patients presented the Vδ2 subset among TILs in the range 0-13.8% (median 1.5%). Functional studies showed prominent cytotoxicity of magnetically sorted Vδ1 a Vδ2 γδ T cells against GBM cell lines and more importantly against primary tumors. Detailed phenotypic profiling and single-cell sequencing of Vδ2 γδ T cells is currently underway. Next, we identified the EphA2 receptor as one of the targets for tumor-reactive Vδ1 γδ T cells. Specifically, we found that blocking of EphA2 expression resulted in significant inhibition of GBM killing mediated by Vδ1 γδ T cells. Furthermore, Luminex xMAP technology identified significantly elevated levels of stress ligand MICA and check-point inhibitor ligands PD-L1 (B7-H1, CD274) and B7-H3 (CD276) and galectin-9 in patients‘ plasma samples at diagnosis compared to age-matched controls.The patient’s clinical course and therapeutic protocols will be discussed.This study was supported by Ministry of Health, Czech Republic (grant NV19-05-00410 to AK) by Ministry of Health, Czech Republic-conceptual development of research organization (FNBr, 65269705). All rights reserved.

Characterization of Blood Mucosal-Associated Invariant T Cells in Patients With Axial Spondyloarthritis and of Resident Mucosal-Associated Invariant T Cells From the Axial Entheses of Non-Axial Spondyloarthritis Control Patients.

The importance of interleukin-17A (IL-17A) in the pathogenesis of axial spondyloarthritis (SpA) has been demonstrated by the success of IL-17A blockade. However, the nature of the cell populations that produce this important proinflammatory cytokine remains poorly defined. We undertook this study to characterize the major IL-17A-producing blood cell populations in the peripheral blood of patients with axial SpA, with a focus on mucosal-associated invariant T (MAIT) cells, a population known to be capable of producing IL-17. We evaluated IL-17A production from 5 sorted peripheral blood cell populations, namely, MAIT cells, γδ T cells, CD4+ T cells, CD8+ T cells, and neutrophils, before and after stimulation with phorbol myristate acetate, the calcium ionophore A23187, and β-1,3-glucan. Expression of IL-17A transcripts and protein were determined using nCounter and ultra-sensitive Simoa technology, respectively. MAIT cells from the axial entheses of non-axial SpA control patients (n=5) were further characterized using flow cytometric immunophenotyping and quantitative polymerase chain reaction, and the production of IL-17 was assessed following stimulation. On a per-cell basis, MAIT cells from peripheral blood produced the most IL-17A compared to CD4+ T cells (P < 0.01), CD8+ T cells (P < 0.0001), and γδ T cells (P < 0.0001). IL-17A was not produced by neutrophils. Gene expression analysis also revealed significantly higher expression of IL17A and IL23R in MAIT cells. Stimulation of peripheral blood MAIT cells with anti-CD3/CD28 and IL-7 and/or IL-18 induced strong expression of IL17F. MAIT cells were present in the normal, unaffected entheses of control patients who did not have axial SpA and showed elevated AHR, JAK1, STAT4, and TGFB1 transcript expression with inducible IL-17A protein. IL-18 protein expression was evident in spinal enthesis digests. Both peripheral blood MAIT cells and resident MAIT cells in normal axial entheses contribute to the production of IL-17 and may play important roles in the pathogenesis of axial SpA.

High baseline expression of IL-6 and IL-10 decreased CCR7 B cells in individuals with previous SARS-CoV-2 infection during BNT162b2 vaccination.

The current pandemic generated by SARS-CoV-2 has led to mass vaccination with different biologics that have shown wide variations among human populations according to the origin and formulation of the vaccine. Studies evaluating the response in individuals with a natural infection before vaccination have been limited to antibody titer analysis and evaluating a few humoral and cellular response markers, showing a more rapid and intense humoral response than individuals without prior infection. However, the basis of these differences has not been explored in depth. In the present work, we analyzed a group of pro and anti-inflammatory cytokines, antibody titers, and cell populations in peripheral blood of individuals with previous SARS-CoV-2 infection using BNT162b2 biologic. Our results suggest that higher antibody concentration in individuals with an earlier disease could be generated by higher production of plasma cells to the detriment of the presence of memory B cells in the bloodstream, which could be related to the high baseline expression of cytokines (IL-6 and IL-10) before vaccination.

Effect of intravenous clarithromycin in patients with sepsis, respiratory and multiple organ dysfunction syndrome: a randomized clinical trial

BackgroundClarithromycin may act as immune-regulating treatment in sepsis and acute respiratory dysfunction syndrome. However, clinical evidence remains inconclusive. We aimed to evaluate whether clarithromycin improves 28-day mortality among patients with sepsis, respiratory and multiple organ dysfunction syndrome.MethodsWe conducted a multicenter, randomized, clinical trial in patients with sepsis. Participants with ratio of partial oxygen pressure to fraction of inspired oxygen less than 200 and more than 3 SOFA points from systems other than the respiratory function were enrolled between December 2017 and September 2019. Patients were randomized to receive 1 gr of clarithromycin or placebo intravenously once daily for 4 consecutive days. The primary endpoint was 28-day all-cause mortality. Secondary outcomes were 90-day mortality; sepsis response (defined as at least 25% decrease in SOFA score by day 7); sepsis recurrence; and differences in peripheral blood cell populations and leukocyte transcriptomics.ResultsFifty-five patients were allocated to each arm. By day 28, 27 (49.1%) patients in the clarithromycin and 25 (45.5%) in the placebo group died (risk difference 3.6% [95% confidence interval (CI) − 15.7 to 22.7]; P = 0.703, adjusted OR 1.03 [95%CI 0.35–3.06]; P = 0.959). There were no statistical differences in 90-day mortality and sepsis response. Clarithromycin was associated with lower incidence of sepsis recurrence (OR 0.21 [95%CI 0.06–0.68]; P = 0.012); significant increase in monocyte HLA-DR expression; expansion of non-classical monocytes; and upregulation of genes involved in cholesterol homeostasis. Serious and non-serious adverse events were equally distributed.ConclusionsClarithromycin did not reduce mortality among patients with sepsis with respiratory and multiple organ dysfunction. Clarithromycin was associated with lower sepsis recurrence, possibly through a mechanism of immune restoration.Clinical trial registration clinicaltrials.gov identifier NCT03345992 registered 17 November 2017; EudraCT 2017-001056-55.

Abstract 1256: B-cell infiltration is associated with survival outcomes in nivolumab-treated head and neck squamous cell carcinoma (HNSCC): NCT 03652142

Abstract Background: Efficacy of Programmed Death1 inhibitor pembrolizumab in HNSCC demonstrates a positive correlation with PD-L1 expression, and PD-L1 positivity (CPS ≥1) is the only clinically approved biomarker for patient (pt) selection to this day. The primary objective of the current study is to prospectively explore candidate biomarkers from the tumor and tumor microenvironment for associations with clinical response to nivolumab in patients with recurrent/metastatic HNSCC treated with nivolumab. Methods: Longitudinal tissue biopsies were collected from recurrent and/or metastatic HNSCC patients treated with nivolumab. Biopsies were taken at baseline, 24-72 hours after the second cycle of nivolumab and at progression. Biomarker analysis employing IHC/IF and flow cytometry was conducted. PD-L1 CPS was assessed for 47 cases. Utilizing IF staining, immune cell populations (CD3+/CD8+/CD20+/FOXP3+), were quantified in stromal and tumor segments of 44 evaluable baseline, 29 post-treatment and 9 PD biopsies. Flow cytometry for immune sub-phenotypes was also performed on 57, 29 and 13 samples at the same timepoints, respectively. Results were correlated with response by RECIST 1.1 and survival outcomes. Results: High baseline CD20+ B cell density in stroma was associated with prolonged progression-free survival (PFS) (p=0.011) and the same trend was observed for overall survival (OS) (p=0.072). Intra-tumoral CD20+ did not demonstrate the same effect. None of the other immune-cell subtypes, in neither tissue compartment, were correlated with outcome. Increase in the post-treatment unswitched memory IgD/IgM+ B cell population in peripheral blood was also linked to improved OS (p=0.017). PD-L1 CPS ≥1 in baseline biopsies was associated with longer OS (p=0.001) but not PFS (p=0.12) and CD20+ B cell density was independent of PD-L1 positivity (p=0.121). When cases were stratified into 3 groups based on PD-L1 positivity and CD20+ B cell density, the “PD-L1/CD20+ double +/high” subgroup was associated with both improved PFS (p=0.013) and OS (p=0.028) in contrast with the “PD-L1/CD20+ single +/high” and “PD-L1/CD20+ double -/low” subgroups. Conclusions: Overall, our findings uncover the implication of infiltrating CD20+ B cells in HNSCC nivolumab outcomes and underscore their ability to enhance the predictive value of PD-L1 CPS and thus further refine patient selection. Citation Format: Niki Gavrielatou, Ekaterina Fortis, Aris Spathis, Maria Anastasiou, Panagiota Economopoulou, Maria Gkotzamanidou, Sylvie Rusakiewics, Ioannis Vathiotis, Thazin Nwe Aung, Ioannis Kotsantis, Elena Mihal Vagia, Ioannis Panayiotides, David Rimm, George Coukos, Periklis Foukas, Amanda Psyrri. B-cell infiltration is associated with survival outcomes in nivolumab-treated head and neck squamous cell carcinoma (HNSCC): NCT 03652142 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1256.

Abstract 5623: CD137 humanized mice for preclinical efficacy evaluation of therapeutic antibodies

Abstract CD137 (41-BB, tumor necrosis factor receptor superfamily 9), expressed on activated T cells and NK cells, has gained increased attention as an anti-tumor target because of remarkable clinical efficacy using either a single agent or in combination with other immune checkpoint inhibitors. We constructed CD137 humanized mice based on two different backgrounds, C57BL6 and BALB/c mice, as well as dual-target and triple-target mice crossbred with PD1/PDL1 humanized mice to create an ideal model to study CD137 target drugs. The normal immune cell population in peripheral blood were not affected in CD137 humanized mice, and hCD137 was expressed on T cells after the stimulation with a anti CD3 antibody. We verified the efficacy of anti-CD137 drugs in BALB/c-hCD137 mice inoculated with different cancer cell lines (i.e. CT26, EMT6 and 4T1). The drug Urelumab showed a dramatic response in the hCD137 mice model inoculated with CT26 and EMT6 but not with 4T1 cell line. In a rechallenge study, no tumors grew in tumor-free mice previously treated with Urelumab, with effector memory T cells increased in peripheral blood, indicating Urelumab has a relatively long-lasting anti-tumor effect. The combination of Urelumab and anti-PD1 has a stronger tumor growth inhibition effect than that of anti-CD137 or anti-PD1 monotherapy on dual-target or triple target mice. These data show that the humanized CD137 mouse is a suitable model for evaluating the monotherapies or combination therapies targeting CD137. Citation Format: Yunlong Jiang, Tingting Gu, Weiwei Yu, Hongyan Sun, Mingkun Zhang, Juan Liang, Shuai Li, Cunxiang Ju, Jing Zhao, Xiang Gao, Mark W. Moore. CD137 humanized mice for preclinical efficacy evaluation of therapeutic antibodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5623.

Global characterization of megakaryocytes in bone marrow, peripheral blood, and cord blood by single-cell RNA sequencing.

Megakaryocytes (MK) are mainly derived from bone marrow and are mainly involved in platelet production. Studies have shown that MK derived from bone marrow may have immune function, and that MK from peripheral blood are associated with prostate cancer. Single-cell transcriptome sequencing can help us better understand the heterogeneity and potential function of MK cell populations in bone marrow (BM), peripheral Blood (PB), and cord blood (CB) of healthy and diseased people.We integrated more than 1.2 million single-cell transcriptome data from 132 samples of PB, BM, and CB from healthy individuals and patients from different dataset. We examined the MK (including MK and product of MK) by single-cell RNA sequencing data analysis methods and identification of MK-related protein expression by the Human Protein atlas. We investigate the relationship between the MK subtype and Non-Small Cell Lung Cancer (NSCLC) in 77 non-cancer and 402 NSCLC. We found that MK were widely distributed and the amount of MK in peripheral blood was more than that in bone marrow and there were specificity MK subtypes in peripheral blood. We found classical MK1 with typical MK characteristics and non-classical MK2 closely related to immunity which was the most common subtype in bone marrow and cord blood. Classical MK1 was closely related to Non-Small Cell Lung Cancer (NSCLC) and can be used as a diagnostic marker. MK2 may have potential adaptive immune function and play a role in tumor NSCLC and autoimmune diseases Systemic Lupus Erythematosus. MK have 14 subtypes and are widely distributed in PB, CB, and BM. MK subtypes are closely related to immunity and have potential to be a diagnostic indicator of NSCLC.

First report of safety/tolerability and preliminary antitumor activity of CAN-2409 in inadequate responders to immune checkpoint inhibitors for stage III/IV NSCLC.

9037 Background: Immune checkpoint inhibitors (ICI) are standard of care for advanced NSCLC. Even among patients with initial response, a majority ultimately progress, and rational combination approaches are needed to improve outcomes. CAN-2409 is a replication-deficient adenoviral gene construct that delivers the thymidine kinase gene, resulting in local conversion of a prodrug (valacyclovir) into a toxic metabolite. This leads to tumor cell lysis and immunization against the injected tumor and uninjected metastases. We have previously shown that monotherapy intra-tumoral (IT) delivery of CAN-2409 followed by oral valacyclovir in NSCLC patients is safe and results in CD8+ T cell infiltration in the injected tumor and activation of this cell population in tissue and peripheral blood. Methods: This open-label Ph2 experimental medicine clinical trial evaluates safety and clinical activity of IT CAN-2409 combined with ICI (± chemo) for stage III/IV NSCLC. Three cohorts are defined based on response to ICI at enrollment: stable disease (SD; Cohort 1; C1), progressive disease (PD) after ≥18 weeks (w) of ICI (Cohort 2; C2), or ICI refractory disease (RD; Cohort 3; C3). Two doses of CAN-2409 (5x1011 vp) are given 5-7w apart via bronchoscopic or percutaneous injection into a lung tumor, disease-positive lymph node or peripheral metastasis, followed by valacyclovir. Patients are assessed for safety, immunologic biomarkers (analysis in progress), and clinical response. Results: As of data cutoff (10Jan22), 28 patients received ≥1 dose of CAN-2409 (safety population). Median age was 70 years; 86% stage IV; 32% squamous; 11% PD-L1 &gt;50%; 82% receiving pembrolizumab and 18% nivolumab. Study treatment and procedures were generally well tolerated. The most common TRAEs were Gr1/2, with fatigue, fever, and chills in 18-39% of patients; 1 patient had Gr3 fever. Twenty-two patients are alive and 6 patients died due to disease. Of the 14 RECIST evaluable patients who received 2 doses of CAN-2409, clinical response was seen in 4 patients (Table 1). Two PRs are ongoing (6w, 24w) and reduction in tumor size was observed in non-injected lesions. In C2, 6 of 7 patients achieving SD are ongoing with median duration of 13w (range 10-40w). Conclusions: The addition of CAN-2409 for patients with advanced NSCLC and inadequate response to front-line ICI (± chemo) appears to be well tolerated. Preliminary clinical data suggest that CAN-2409 induced a clinical response in 4/14 evaluable patients and produced disease stabilization in most patients entering the trial with PD, with evidence of abscopal effect in a subset of patients. Clinical trial information: NCT04495153. [Table: see text]

Inside the Joint of Inflammatory Arthritis Patients: Handling and Processing of Synovial Tissue Biopsies for High Throughput Analysis.

Inflammatory arthritis is a chronic systemic autoimmune disease of unknown etiology, which affects the joints. If untreated, these diseases can have a detrimental effect on the patient's quality of life, leading to disabilities, and therefore, exhibit a significant socioeconomic impact and burden. While studies of immune cell populations in arthritis patient's peripheral blood have been informative regarding potential immune cell dysfunction and possible patient stratification, there are considerable limitations in identifying the early events that lead to synovial inflammation. The joint, as the site of inflammation and the local microenvironment, exhibit unique characteristics that contribute to disease pathogenesis. Understanding the contribution of immune and stromal cell interactions within the inflamed joint has been met with several technical challenges. Additionally, the limited availability of synovial tissue biopsies is a key incentive for the utilization of high-throughput techniques in order to maximize information gain. This review aims to provide an overview of key methods and novel techniques that are used in the handling, processing and analysis of synovial tissue biopsies and the potential synergy between these techniques. Herein, we describe the utilization of high dimensionality flow cytometric analysis, single cell RNA sequencing, ex vivo functional assays and non-intrusive metabolic characterization of synovial cells on a single cell level based on fluorescent lifetime imaging microscopy. Additionally, we recommend important points of consideration regarding the effect of different storage and handling techniques on downstream analysis of synovial tissue samples. The introduction of new powerful techniques in the study of synovial tissue inflammation, brings new challenges but importantly, significant opportunities. Implementation of novel approaches will accelerate our path toward understanding of the mechanisms involved in the pathogenesis of inflammatory arthritis and lead to the identification of new avenues of therapeutic intervention.

Single-cell immune profiling reveals functional diversity of T cells in tuberculous pleural effusion.

Orchestration of an effective T lymphocyte response at infection sites is critical for protection against Mycobacterium tuberculosis (Mtb) infection. However, the local T cell immunity landscape in human tuberculosis is poorly defined. Tuberculous pleural effusion (TPE), caused by Mtb, is characterized by an influx of leukocytes to the pleural space, providing a platform suitable for delineating complex tissue responses to Mtb infection. Using single-cell transcriptomics and T cell receptor sequencing, we analyzed mononuclear cell populations in paired pleural fluid and peripheral blood of TPE patients. While all major cell clusters were present in both tissues, their relative proportions varied significantly by anatomic location. Lineage tracking analysis revealed subsets of CD8 and CD4 T cell populations with distinct effector functions specifically expanded at pleural sites. Granzyme K-expressing CD8 T cells were preferentially enriched and clonally expanded in pleural fluid from TPE, suggesting that they are involved in the pathogenesis of the disease. The findings collectively reveal the landscape of local T cell immunity in tuberculosis.

Effects of IFIH1 rs1990760 variants on systemic inflammation and outcome in critically ill COVID-19 patients in an observational translational study.

Variants in IFIH1, a gene coding the cytoplasmatic RNA sensor MDA5, regulate the response to viral infections. We hypothesized that IFIH1 rs199076 variants would modulate host response and outcome after severe COVID-19. Patients admitted to an intensive care unit (ICU) with confirmed COVID-19 were prospectively studied and rs1990760 variants determined. Peripheral blood gene expression, cell populations, and immune mediators were measured. Peripheral blood mononuclear cells from healthy volunteers were exposed to an MDA5 agonist and dexamethasone ex-vivo, and changes in gene expression assessed. ICU discharge and hospital death were modeled using rs1990760 variants and dexamethasone as factors in this cohort and in-silico clinical trials. About 227 patients were studied. Patients with the IFIH1 rs1990760 TT variant showed a lower expression of inflammation-related pathways, an anti-inflammatory cell profile, and lower concentrations of pro-inflammatory mediators. Cells with TT variant exposed to an MDA5 agonist showed an increase in IL6 expression after dexamethasone treatment. All patients with the TT variant not treated with steroids survived their ICU stay (hazard ratio [HR]: 2.49, 95% confidence interval [CI]: 1.29-4.79). Patients with a TT variant treated with dexamethasone showed an increased hospital mortality (HR: 2.19, 95% CI: 1.01-4.87) and serum IL-6. In-silico clinical trials supported these findings. COVID-19 patients with the IFIH1 rs1990760 TT variant show an attenuated inflammatory response and better outcomes. Dexamethasone may reverse this anti-inflammatory phenotype. Centro de Investigación Biomédica en Red (CB17/06/00021), Instituto de Salud Carlos III (PI19/00184 and PI20/01360), and Fundació La Marató de TV3 (413/C/2021).

Phase 1/2 Study of Nexi-001 Donor-Derived Multi-Antigen Specific CD8+ T Cells for the Treatment of Relapsed Acute Myeloid Leukemia (AML) after Allogeneic Hematopoietic Transplantation

Phase 1/2 Study of Nexi-001 Donor-Derived Multi-Antigen Specific CD8+ T Cells for the Treatment of Relapsed Acute Myeloid Leukemia (AML) after Allogeneic Hematopoietic Transplantation