Paired Peripheral Blood Mononuclear Cells Research Articles

BRAF-V600E mutations in plasma and peripheral blood mononuclear cells correlate with prognosis of pediatric Langerhans cell histiocytosis treated with first-line therapy.

The clinical relevance of BRAF-V600E alleles in peripheral blood mononuclear cells (PBMCs) and the prognostic impact of the mutants in cell-free (cf) and PBMC DNAs of Langerhans cell histiocytosis (LCH) have not been fully clarified in pediatric LCH. We retrospectively determined the levels of BRAF-V600E mutation in paired plasma and PBMC samples at the time of diagnosis of LCH. Subsequently, we performed a separate or combined analysis of the clinical and prognostic impact of the mutants. We assessed BRAF-V600E mutation in peripheral blood from 94 patients of childhood LCH. Our data showed that cfBRAF-V600E was related to young age, multiple-system (MS) disease, involvements of organs with high risk, increased risk of relapse, and worse progression-free survival (PFS) of patients. We also observed that the presence of BRAF-V600E in PBMCs at baseline was significantly associated with MS LCH with risk organ involvement, younger age, and disease progression or relapse. The coexisting of plasma(+)/PBMC(+) identified 36.2% of the patients with the worst outcome, and the hazard ratio was more significant than either of the two alone or neither, indicating that combined analysis of the mutation in plasma and PBMCs was more accurate to predict relapse than evaluation of either one. Concurrent assessment of BRAF-V600E mutation in plasma and PBMCs significantly impacted the prognosis of children with LCH. Further prospective studies with larger cohorts need to validate the results of this study.

BCG vaccination alters the epigenetic landscape of progenitor cells in human bone marrow to influence innate immune responses

Although the Bacille-Calmette-Guérin (BCG) vaccine is used to prevent tuberculosis, it also offers protection against a diverse range of non-mycobacterial infections. However, the underlying protective mechanisms in humans are not yet fully understood. Here, we surveyed at single-cell resolution the gene expression and chromatin landscape of human bone marrow, aspirated before and 90days after BCG vaccination or placebo. We showed that BCG alters both the gene expression and epigenetic profiles of human hematopoietic stem and progenitor cells (HSPCs). Changes in gene expression occurred primarily within uncommitted stem cells. By contrast, changes in chromatin accessibility were most prevalent within differentiated progenitor cells at sites influenced by Kruppel-like factor (KLF) and early growth response (EGR) transcription factors and were highly correlated (r>0.8) with the interleukin (IL)-1β secretion capacity of paired peripheral blood mononuclear cells (PBMCs). Our findings shed light on BCG vaccination's profound and lasting effects on HSPCs and its influence on innate immune responses and trained immunity.

Expansion of effector regulatory T cells in steroid responders of severe alcohol-associated hepatitis.

While steroid therapy is the preferred treatment for severe alcohol-associated hepatitis, the role of effector regulatory T (eTreg) cells and their association with steroid response and clinical outcomes in these patients remains to be elucidated. We prospectively enrolled 47 consecutive patients with alcohol-associated hepatitis, consisting of severe alcohol-associated hepatitis treated with steroids (n=18; steroid-treated group) and mild alcohol-associated hepatitis (n=29; nontreated group). After isolating peripheral blood mononuclear cells from the patients at enrollment and again 7 days later, the frequency of eTreg cells was examined using flow cytometry. Single-cell RNA sequencing analysis was conducted using paired peripheral blood mononuclear cells. In vitro experiments were also performed to assess phenotype changes and the suppressive function of Treg cells following steroid treatment. The steroid-treated group exhibited significantly higher Model for End-Stage Liver Disease scores than the nontreated group ( p < 0.01). Within the steroid-treated group, the proportion of eTreg cells significantly expanded in the steroid responders (n=13; p = 0.01). Furthermore, a significant positive correlation was observed between the decrease in the Model for End-Stage Liver Disease score and the increase in eTreg cells ( p < 0.05). Single-cell RNA sequencing using paired peripheral blood mononuclear cells (pre-steroid and post-steroid therapy) from a steroid responder revealed gene expression changes in T cells and monocytes, suggesting enhancement of Treg cell function. In vitro results showed an elevation in the proportion of eTreg cells after steroid therapy. In conclusion, our findings suggest that the efficacy of steroid therapy in patients with severe alcohol-associated hepatitis is mediated by an increase in the number of eTreg cells.

Immune features revealed by single-cell RNA and single-cell TCR/BCR sequencing in patients with rheumatoid arthritis receiving COVID-19 booster vaccination.

Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, have profoundly affected human health. Booster COVID-19 vaccines have demonstrated significant efficacy in reducing infection and severe cases. However, the effects of booster COVID-19 vaccines on key immune cell subsets and their responses in rheumatoid arthritis (RA) are not well understood. By using single-cell RNA sequencing (scRNA-seq) combined with scTCR/BCR-seq analysis, a total of 8 major and 27 minor cell clusters were identified from paired peripheral blood mononuclear cells (PBMCs) which were collected 1 week before and 4 weeks after booster vaccination in stable RA patients. Booster vaccination only had limited impact on the composition and proportions of PBMCs cell clusters. CD8+ cytotoxic T cells (CD8+T_CTL) showed a trend toward an increase after vaccination, while naive B cells and conventional dendritic cells (cDCs) showed a trend toward a decrease. Transcriptomic changes were observed after booster vaccination, primarily involving T/B cell receptor signaling pathways, phagosome, antigen processing and presenting, and viral myocarditis pathways. Interferon (IFN) and pro-inflammatory response gene sets were slightly upregulated across most major cell subpopulations in COVID-19 booster-vaccinated RA individuals. Plasma neutralizing antibody titers significantly increased after booster COVID-19 vaccination (p = 0.037). Single-cell TCR/BCR analysis revealed increased B cell clone expansion and repertoire diversity postvaccination, with no consistent alterations in T cells. Several clonotypes of BCRs and TCRs were identified to be significantly over-represented after vaccination, such as IGHV3-15 and TRBV28. Our study provided a comprehensive single-cell atlas of the peripheral immune response and TCR/BCR immune repertoire profiles to inactivated SARS-CoV-2 booster vaccination in RA patients, which helps us to understand vaccine-induced immune responses better.

Abstract 2419: Personalized longitudinal ctDNA monitoring is useful for biomarker in head and neck squamous cell carcinoma

Abstract Introduction: There is no established and useful tumor biomarker to evaluate treatment response and predict early locoregional and distant metastatic relapse in head and neck squamous cell carcinoma (HNSCC). Circulating tumor DNA (ctDNA) has a potential for detecting minimal residual diseases (MRDs). However, the absence of specific hotspot mutations in most HNSCCs makes ctDNA-based studies difficult in HNSCC. As a novel biomarker, we investigated whether individualized ctDNA analysis could help monitor and predict treatment response and relapse in HNSCC. Methods: Thirty-two HNSCC patients were enrolled in this study. Twenty-two patients received radical resection and 10 patients were received radical irradiation as initial curative treatment. We collected tumor tissues and paired PBMCs (peripheral blood mononuclear cells) before treatment. Furthermore, we collected serial plasma samples for ctDNA analysis. To identify tumor specific individualized mutation, we isolated DNA from tumor tissue and PBMC and performed mutation analysis using custom SCC panel with frequent altered 31 genes (all exon coverage). We defined tumor-specific mutated genes per patient with variant allele frequency (VAF) &amp;gt;10 % and total coverage &amp;gt;100 as ctDNA candidate genes and performed ctDNA analysis using dPCR. We generated mutation specific primer-probe for droplet digital PCR (ddPCR), quantified the amount of ctDNA in plasma and compared ctDNA level with clinical data. Results: For mutation analysis, most frequent mutated gene was TP53 (21/32, 65.6%). Longitudinal ctDNA was monitored in a total of 24 cases. In 13 cases, ctDNA tested positive again or did not test negative, and all 13 cases had a residual tumor or relapsed after initial treatment. In 11 cases, after initial treatment, ctDNA remained negative and patients were alive without recurrence. Our cohort included 6 patients in whom it was possible to monitor ctDNA during chemotherapy in recurrent metastatic HNSCC cases. ctDNA alterations correlated well with clinical treatment response in four cases, but not in two cases. Patients who remained negative for ctDNA during follow-up after initial treatment (n=11) had a significantly better prognosis than those who reverted to ctDNA positivity (n=13, P &amp;lt; 0.0001). Conclusion: Custom SCC panel is useful to detect more tumor specific mutation in HNSCC patients. Detection of ctDNA using next generation sequencing and digital PCR is promising strategy capable of monitoring in HNSCC patients. However, this method is not useful in all HNSCC, and there is room for improvement in the selection of ctDNA candidate genes. Citation Format: Ryunosuke Kogo, Tomomi Manako, Takashi Nakagawa. Personalized longitudinal ctDNA monitoring is useful for biomarker in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2419.

Abstract 493: Unlocking solid tumors to nano-immunotherapy

Abstract Despite the remarkable efficiency of cancer immunotherapies, only a low percentage of patients achieve long-lasting clinical responses. Non-tumor cells within the tumor microenvironment (TME), including tumor vasculature and immune stromal cells, dictate therapeutic efficacy. The presence of germinal centers (GC) within the TME of cancer patients, including infiltrating T follicular helper (Tfh) cells and B cells, has been considered predictive of response to immunotherapies. We are developing nano-immunotherapies to induce immunological memory to control tumor relapse without any follow-up treatment.Nanomaterials co-incorporated tumor-associated antigens, clinically relevant toll-like receptor ligands, and regulators of tumor progression, namely the PD-L1/PD-1 expression and the secretion of TGF-β. Nanoparticle (NP) physicochemical properties were fully addressed. The immunotherapeutic potential of this nano-immunotherapy was addressed in melanoma, colorectal cancer (CRC), pancreatic cancer, and triple-negative breast cancer (TNBC) 3D models. These were created using patient-derived tumor cells, cancer-associated fibroblasts, and endothelial cells, to which paired patients' peripheral blood mononuclear cells were added to assess T-cell infiltration and tumor sprouting to identify the best nano-immunotherapy dosage/schedule. Our in vivo studies evaluated tumor volume, and animals’ survival, and characterized the tumor-infiltrating immune cells within TME, including the differentiation of Tfh cells and primary GC by flow cytometry.Our nano-immunotherapy remodeled the TME of B16F10 melanoma, MC38 CRC, 4T1 and E0771 TNBC, and KPC PDAC. Different TME subpopulations were identified as major blockers of anti-tumor immunity, and this knowledge guided the selection of combination approaches using modulators of those tumor immune evasion-related pathways (e.g., PD-L1, FAK). Our combination nano-immunotherapies tailored to address the tumor immune suppression profile found in vaccinated animals, significantly delayed tumor development, and increased disease-free survival rates. We found that the adjuvants delivered by our NP led to a ~4-fold increase in antibody production, being far more effective in generating GC responses. Thus, the effectiveness of combinational immunotherapy is at least partially related to the modulation of Tfh cells driving the formation of GC responses.Our nano-immunotherapy is a promising clinically translatable approach to defeating solid tumors. Acknowledgments: This work was supported by PTDC/BTM-SAL/4350/2021 and LCF/PR/HR22/52420016. Citation Format: Barbara Carreira, Rita C. Acúrcio, Ana I. Matos, Liane M. Moura, Ana C. Santos, Raquel Gouveia, Jéssica Cordeiro, Carina Peres, Daniela Vaskovich-Koubi, Ron Kleiner, Sabina Pozzi, Ronit Satchi-Fainaro, Helena F. Florindo. Unlocking solid tumors to nano-immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 493.

Abstract 971: Validation of a tumor-naïve circulating tumor DNA (ctDNA) response monitoring panel in advanced non-small cell lung cancer (aNSCLC)

Abstract Introduction: Monitoring of ctDNA can be a minimally invasive complement to tumor imaging for assessing treatment effect. Technical limitations require methods to distinguish tumor signal from clonal hematopoiesis (CH), often including non-tumor sequencing. We developed a tumor-naïve panel (FoundationOne® Monitor) to quantify ctDNA tumor fraction (TF). TF analytical validation used peripheral blood mononuclear cell (PBMC) sequencing. We then leveraged plasma collected serially from patients with aNSCLC in the real-world (rw) Prospective Clinico-Genomic (PCG; NCT04180176) study to investigate the utility of TF for monitoring therapy (tx) response. Methods: TF was quantified using a combination of aneuploidy and variant allele frequencies of genomic alterations (GAs), while excluding CH mutations and aneuploidy using fragmentomic signal from cfDNA. In the PCG study, data from consenting patients were collected from electronic health records from 23 participating Flatiron Health Research Network sites. We analyzed plasma collected 6-15 weeks after start of tx in exploratory and validation cohorts per prespecified criteria. We defined molecular response (MR) as undetectable TF on tx regardless of baseline TF. Hazard ratios (HR) and 95% confidence Interval (CI) were calculated with univariate Cox proportional hazard regression. Results: For TF validation, 1135 samples separate from the PCG study with paired PBMC results were included. Overall, 24/4274 (0.56%) CH derived non-aneuploidy GAs detected in 1134 samples were falsely classified as somatic. Of these, 4 were detected among 317 samples with no tumor signal, resulting in a false positive TF value (specificity = 98.7%). CH derived aneuploidy was observed in 27 PBMCs and was appropriately filtered during TF estimation in all cases. Assessing the impact of CH aneuploidy filtering on sensitivity, we only identified 1/320 (0.31%) samples where non-CH derived aneuploidy was omitted. To assess clinical validity, 222 patients were analyzed from the PCG study. MR was assessed after a median of 10.8 weeks of tx (IQR 8.9-12.0). In a subset of 152 patients treated with physicians’ choice, MR was associated with favorable rw progression free survival (rwPFS: 9.4 v 2.8 months [mo]; HR = 0.30; 95% CI: [0.21-0.44]) and rw overall survival (rwOS: 22.0 v 7.5 mo; HR = 0.33 [0.22-0.49]). We validated this finding on 70 patients receiving immunotherapy (50 with chemo). Again, MR was associated with favorable rwPFS (9.0 v 2.8 mo; HR = 0.28 [0.16-0.51]) and rwOS (20.2 v 9.4 mo; HR = 0.42 [0.23-0.78]). Conclusions: We describe a highly specific tumor naïve algorithmic filtration of non-tumor signal to enable high confidence ctDNA quantification and MR assessment. On tx MR is associated with favorable outcomes. These findings may enable personalized tx approaches tailored to a patient’s risk of progression and downstream cancer morbidity. Citation Format: Anne C. Chiang, Russell W. Madison, Yanmei Huang, Alexander Fine, Dexter X. Jin, Geoffrey R. Oxnard, Jason Hughes, Zoe June Assaf, Yi Cao, Vladan Antic, Ole Gjoerup, Amanda Young, David Fabrizio, Shaily Lakhanpal, Richard Zuniga, Katja Schulze, Lincoln W. Pasquina. Validation of a tumor-naïve circulating tumor DNA (ctDNA) response monitoring panel in advanced non-small cell lung cancer (aNSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 971.

Ruxolitinib Promotes Clinical Responses in Large Granular Lymphocytic Leukemia Via Suppression of JAK/STAT-Dependent Inflammatory Cascades

Background: Large granular lymphocytic leukemias (LGL) are T or NK-cell lymphoproliferative disorders that are characterized by myelosuppression and frequent mutations in STAT3. Commonly used agents include low doses of methotrexate or cyclophosphamide. Among 5 patients (pts) with LGL treated on our phase II study of ruxolitinib in T-cell lymphoma (Moskowitz, et al. Blood 2021), 2 achieved partial response (PR) and 2 achieved stable disease, suggesting oncogenic dependence on JAK/STAT pathway activity in LGL. Methods: To further explore the efficacy and mechanism of JAK1/2 blockade in LGL, we expanded our phase II study to include 23 pts with relapsed/refractory LGL. This sample size was chosen to allow for narrowing of the 95% confidence interval to 54-87% (Wilson score method) if similar efficacy was observed. Pts received ruxolitinib 20mg PO BID continuously. Responses were assessed after cycles 2, 5, and every 3 cycles thereafter using the response assessment from the ECOG-5998 study for LGL. Furthermore, stable disease (SD) was defined as improvement in cytopenia(s) by 1 grade (without meeting criteria for PR) for a minimum of 6 months. Overall response rate (ORR) included complete response (CR) plus PR; clinical benefit rate (CBR) included ORR plus SD. In parallel, we performed single-cell RNA sequencing (scRNA-seq) on paired C1D1 and C1D8 peripheral blood mononuclear cells (PBMCs) from 7 patients (5 PR, 1 SD, and 1 non-responder) and plasma proteomic profiling on 5 patients (3 PR, 1 SD, and 1 POD). Results: Among 23 pts, median age was 58 (31-86), 13 (57%) were male, 19 (83%) were white, 1 (4%) was black. Median number of prior therapies was 2 (range 0-5). 12/22 (54%) had STAT3 mutations (of whom 1 also had a JAK1 mutation); 1 had a JAK2 mutation, 1 pt was missing sequencing. Baseline cytopenias included anemia (n=14, 61%), thrombocytopenia (n=12, 52%), and/or neutropenia (n=16, 70%). Among 20 pts evaluable for response (3 too early), there were 5 (25%) CRs, 6 (30%) PRs, and 4 (20%) SD. ORR and CBR were 55% and 75% respectively. Median event free survival (EFS) was not reached and was 68% (95% CI: 50-93) at 14 months ( Fig. 1A). STAT3 mutation status predicted for improved EFS (14-month EFS 100% vs 40%, p=0.007). After a median duration of treatment of 13.1 months, there were no unexpected toxicities. Severe adverse events included febrile neutropenia (n=3, 13%) and herpes zoster (n=1, 4%). To understand the mechanism by which ruxolitinib improved hematologic parameters in LGL, we performed unbiased proteomic analysis of peripheral blood plasma samples using the OLink 96 Inflammation and Immune Response Platforms. Ruxolitinib led to a decrease in several soluble mediators known to be driven by interferon-driven JAK/STAT activation, including CXCL9, CXCL10, and PD-L1, as well as TNF ( Fig. 1B). Several of these factors have been shown to suppress hematopoietic stem cell function, suggesting a JAK/STAT-dependent paracrine mechanism by which LGL leads to myelosuppression. ScRNA-seq of PBMCs identified LGL cells based on expression of cytotoxic genes, including NKG7, CST7, CCL5, GZMB and PRF1. Analysis of cell type proportions within the peripheral blood showed that patients achieving a PR to Ruxolitinib showed a decrease in the proportion of circulating myeloid cells. Gene set enrichment analysis (GSEA) of LGL cells revealed substantial downregulation of both JAK/STAT signaling and TNF signaling in LGL cells in PR but not SD patients, whereas persistently elevated TNF signaling was seen not only in LGL cells but also circulating myeloid and naïve T-cells in SD patients, suggesting that LGL cells might promote bone marrow suppression via paracrine effects on the bystander host immune system. Network analysis of LGL cells and non-malignant peripheral blood immune cells is ongoing. Conclusions: Ruxolitinib induced durable responses in pts with LGL and therefore may represent an option for pts requiring treatment. STAT3 mutation status was predictive of EFS and responses were associated with JAK/STAT pathway down-regulation, demonstrating on-target effects. Integrated immune profiling suggests that ruxolitinib may interrupt JAK/STAT-dependent paracrine inflammatory signals that promote bone marrow suppression. An ongoing phase II study of ruxolitinib in LGL will aid in clarifying optimal dosing and confirming both efficacy and mechanism of action (NCT05592015).

Distinct Patterns of CD4+ and CD8+ T-Cell Clonal Expansion Enable Broad Clinical Responses to Pembrolizumab + GVD in Patients with Relapsed Hodgkin Lymphoma

Introduction: Classical Hodgkin lymphoma (cHL) is characterized by immune dysregulation, with recurrent amplifications of the 9p24 locus encoding several genes including CD274 (PD-L1), PDCD1LG2 (PD-L2), and JAK2, frequent genetic and epigenetic downregulation of major histocompatibility complex (MHC)-I and (MHC)-II, and a tumor microenvironment (TME) defined by T-cell infiltration and high rates of PD-L1 expression. PD-1 blockade is highly active both as single agent and in combination with chemotherapeutic agents in relapsed/refractory (RR) cHL. However, the mechanism by which PD-1 blockade promotes clinical responses in combination with chemotherapy, and whether the mechanism of action varies based on tumor or TME-specific expression of either MHC or PD-L1 remains unclear. Methods: We previously conducted a phase II study in patients with relapsed or refractory cHL evaluating pembrolizumab in combination with gemcitabine, vinorelbine and liposomal doxorubicin (P-GVD) before proceeding to high-dose therapy and autologous hematopoietic cell transplantation (AHCT) (Moskowitz AJ et al, JCO 2021). Treatment consisted of 2 to 4 cycles of P-GVD followed by AHCT. We performed combined single-cell surface protein identification, RNA sequencing, and (T cell receptor) TCR sequencing using the 10x Genomics Single Cell Immune Profiling Platform on paired C1D1 and C3D1 peripheral blood mononuclear cells (PBMCs) from 23 patients who received P-GVD and 5 control patients with relapsed cHL treated with standard chemotherapy regimens. Expression of MHC-I, MHC-II, and PD-L1 on both Hodgkin Reed Sternberg (HRS) cells and within the TME was assessed by immunohistochemistry. Results:Of 38 evaluable pts, the complete response (CR) rate after 2 or 4 cycles of P-GVD was 95%. After a median follow-up of 40 months (range: 2-56), only 1 pt experienced relapse 23 months after AHCT and another pt died 41 months after AHCT while in remission due to unrelated causes. The estimated 40-month progression-free survival (PFS) is 96% (95% CI: 91-100). CITE-Sequencing of PBMCs across 56 samples from 28 patients revealed predominantly CD4+ T, CD8+ T, NK, and myeloid cells within the peripheral blood, with smaller proportions of hematopoietic progenitors and plasmacytoid dendritic cells. Combined RNA and TCR analysis of the T-cell compartment revealed significant pre-treatment clonal expansion of both CD4+ and CD8+ T-cells exhibiting a cytotoxic phenotype, including expression of GZMH, KLRG1, and CCL5 by CD8+ T-cells and expression of PRF1, NKG7, GNLY, FGFBP2, and GZMH by CD4+ T-cells ( Fig. 1A). Correlation of pre-treatment clonal expansion with tumor-specific MHC status demonstrated that clonal expansion of CD8+ T-cells was more robust in patients whose tumors expressed either MHC-I or MHC-II; conversely, clonal expansion of CD4+ T-cells was most robust in tumors expressing MHC-II but lacking MHC-I, suggesting cytotoxic CD4+ T-cell responses may promote anti-tumor immunity in MHC-I-deficient tumors ( Fig. 1B). Both CD4+ and CD8+ T-cell clonal expansion was highest in patients whose tumors expressed high levels of PD-L1 on stromal cells. Treatment with P-GVD did not result in further clonal expansion of either CD4+ or CD8+ T-cells but did increase expression of cytotoxic genes in pre-treatment expanded CD4+ T-cells, including NKG7 and PLEK, while downregulating negative genes of T-cell function in pre-treatment expanded CD8+ T-cells, including TNFAIP3 and DUSP1, suggesting that P-GVD promotes anti-tumor immunity in cHL by enhancing the cytotoxicity of tumor-reactive expanded CD4+ and CD8+ T-cells. Conclusions: Treatment of relapsed cHL with P- GVD resulted in durable remissions in patients undergoing AHCT. Distinct patterns of peripheral blood T-cell expansion were observed based on MHC-I and MHC-II expression on tumor cells, with CD4+ T-cell clonal expansion uniquely expanding in patients with MHC-I-deficient tumors. Further analysis of the impact of P-GVD on T-cell differentiation and function is ongoing. AcknowledgementT: Supported in part by a research grant from Investigator-Initiated Studies Program of Merck Sharp &amp; Dohme LLC. The opinions expressed in this paper are those of the authors and do not necessarily represent those of Merck Sharp &amp; Dohme LLC.

Single-Cell Transcriptomics Reveals Crucial Cell Subsets and Functional Heterogeneity Associated With Carotid Atherosclerosis and Cerebrovascular Events.

Carotid atherosclerosis is a chronic inflammatory disorder and is responsible for the vast majority of ischemic strokes. Inappropriate innate and adaptive immune responses synergize with malfunctional vascular wall cells to cause atherosclerotic lesions. Yet, functional characteristics of specific immune and endothelial cell subsets associated with atherosclerosis and cerebrovascular events are poorly understood. Here, using single-cell RNA sequencing, the unprecedentedly largest data set from 20 patients' carotid artery plaques and paired peripheral blood mononuclear cells was generated, with which an ultra-high-precision cellular landscape of the atherosclerotic microenvironment involving 372 070 cells was depicted. Compared with peripheral blood mononuclear cells, 3 plaque-specific T-cell subsets exhibiting proatherogenic features of both activation and exhaustion were identified. Strikingly, usually antiatherogenic, CD4+FOXP3+ regulatory T cells from plaques of patients with symptomatic disease acquired proinflammatory properties by probably converting to T helper 17 and T helper 9 cells, while CD4+NR4A1+/C0 and CD8+SLC4A10+ T cells related to cerebrovascular events possessed atherogenic attributes including proinflammation, polarization, and exhaustion. In addition, monocyte-macrophage dynamics dominated innate immune response. Two plaque-specific monocyte subsets performed diametrically opposed functions, EREG+ monocytes promoted cerebrovascular events while C3+ monocytes are anti-inflammatory. Similarly, IGF1+ and HS3ST2+ macrophages with classical proinflammatory M1 macrophage features were annotated and contributed to cerebrovascular events. Moreover, SULF1+ (sulfatase-1) endothelial cells were also found to participate in cerebrovascular events through affecting plaque vulnerability. This compendium of single-cell transcriptome data provides valuable insights into the cellular heterogeneity of the atherosclerotic microenvironment and the development of more precise cardiovascular immunotherapies.

Three doses of COVID-19 mRNA vaccine induce class-switched antibody responses in inflammatory arthritis patients on immunomodulatory therapies.

Patients with inflammatory arthritis (IA) are at increased risk of severe COVID-19 due to medication-induced immunosuppression that impairs host defenses. The aim of this study was to assess antibody and B cell responses to COVID-19 mRNA vaccination in IA patients receiving immunomodulatory therapies. Adults with IA were enrolled through the Johns Hopkins Arthritis Center and compared with healthy controls (HC). Paired plasma and peripheral blood mononuclear cell (PBMC) samples were collected prior to and 30 days or 6 months following the first two doses of mRNA vaccines (D2; HC=77 and IA=31 patients), or 30 days following a third dose of mRNA vaccines (D3; HC=11 and IA=96 patients). Neutralizing antibody titers, total binding antibody titers, and B cell responses to vaccine and Omicron variants were analyzed. Anti-Spike (S) IgG and S-specific B cells developed appropriately in most IA patients following D3, with reduced responses to Omicron variants, and negligible effects of medication type or drug withholding. Neutralizing antibody responses were lower compared to healthy controls after both D2 and D3, with a small number of individuals demonstrating persistently undetectable neutralizing antibody levels. Most IA patients respond as well to mRNA COVID-19 vaccines as immunocompetent individuals by the third dose, with no evidence of improved responses following medication withholding. These data suggest that IA-associated immune impairment may not hinder immunity to COVID-19 mRNA vaccines in most individuals.

Gut Inflammation in axial Spondyloarthritis patients is characterized by a marked Type 17 skewed mucosal Innate-like T cell signature.

Patients with spondyloarthritis (SpA) often present with microscopic signs of gut inflammation, a risk factor for progressive disease. We investigated whether mucosal innate-like T cells are involved in dysregulated interleukin-23 (IL-23)/IL-17 responses in the gut-joint axis in SpA. Ileal and colonic intraepithelial lymphocytes (IELs), lamina propria lymphocytes (LPLs), and paired peripheral blood mononuclear cells (PBMCs) were isolated from treatment-naive patients with nonradiographic axial SpA with (n=11) and without (n=14) microscopic gut inflammation and healthy controls (n=15) undergoing ileocolonoscopy. The presence of gut inflammation was assessed histopathologically. Immunophenotyping of innate-like T cells and conventional T cells was performed using intracellular flow cytometry. Unsupervised clustering analysis was done by FlowSOM technology. Serum IL-17A levels were measured via Luminex. Microscopic gut inflammation in nonradiographic axial SpA was characterized by increased ileal intraepithelial γδ-hi T cells, a γδ-T cell subset with elevated γδ-T cell receptor expression. γδ-hi T cellswere also increased in PBMCs of patients with nonradiographic axial SpA versus healthy controls and were strongly associated with Ankylosing Spondylitis Disease Activity Score. The abundance of mucosal-associated invariant T cells and invariant natural killer T cells was unaltered. Innate-like T cells in the inflamed gut showed increased RORγt, IL-17A, and IL-22 levels with loss of T-bet, a signature that was less pronounced in conventional T cells. Presence of gut inflammation was associated with higher serum IL-17A levels. In patients treated with tumor necrosis factor blockade, the proportion of γδ-hi cells and RORγt expression in blood was completely restored. Intestinal innate-like T cells display marked type 17 skewing in the inflamed gut mucosa of patients with nonradiographic axial SpA. γδ-hi T cells are linked to intestinal inflammation and disease activity in SpA.

Intracellular Islatravir-Triphosphate in Dried Blood Spots and Peripheral Blood Mononuclear Cells from Pig-Tailed Macaques.

The purpose of this study was to evaluate the relationship between intracellular islatravir-triphosphate (ISL-TP) in paired peripheral blood mononuclear cells (PBMCs) and dried blood spots (DBS). Three pig-tailed macaques (PMs) were dosed with a single intravaginal extended-release ISL-etonogestrel film for a period of 31 days. After extraction and quantification, repeated measures correlation (rrm) was assessed between log-transformed DBS and PBMC ISL-TP concentrations. Twenty-six paired PBMC/DBS samples were included. Peak ISL-TP concentrations in DBS ranged from 262 to 913 fmol/punches, PBMC Cmax ranged from 427 to 857 fmol/106 cells. Repeated measures correlation yielded an rrm value of 0.96 (95% confidence interval 0.92-0.98; p < .0001). Importantly, ISL-TP was quantifiable in DBS and its pharmacokinetics were similar to PBMC in PMs. Human studies should evaluate DBS applications in clinical pharmacokinetic studies to help define ISL's place in the antiretroviral drug armamentarium.

Impact of shipping temperature on cell viability and T cell responses to bacterial antigens.

Background: Interferon-γ (IFN-γ) secretion by T cells is a key correlate of immune protection against many pathogens including tuberculosis and the neglected tropical disease melioidosis. Clinical studies in tropical regions of immune responses to pathogens and vaccine monitoring studies require the collection of samples in resource-limited rural areas and subsequent shipment to central laboratories for downstream assays and long-term storage. Here, we studied the impact of two different shipping temperatures on the viability, composition and function of peripheral blood mononuclear cells (PBMC) using multi-colour flow cytometry and IFN-γ enzyme-linked immunospot assay (IFN-γ ELISpot), in order to provide guidance on sample shipment conditions for future clinical studies. Methods: Paired peripheral blood mononuclear cell (PBMC) samples from recovered melioidosis patients were stored in liquid nitrogen (-196°C) and then shipped from Bangkok, Thailand to Oxford, UK at either -80°C (dry ice) or -196°C (dry shipper). After thawing, cell viability and composition were assessed by flow cytometry and antigen specific responses to Burkholderia pseudomallei (BP) were measured using IFN-γ ELISpot. Results: We observed modest lowering of viability in the majority of samples and a reduction in IFN-γ responses to BP which correlated to a decrease of monocytes and natural killer cells in samples shipped at -80°C compared to -196°C. Despite being lower in magnitude antigen-specific responses remained detectable in the majority of samples. Conclusions: Here we demonstrate that shipment of cryopreserved PBMC at -196°C has a benefit on cell viability, recovery and T cell responses to bacterial antigens, although useful information can still be obtained from samples shipped at -80°C, thus providing important guidance for sample management in future clinical trials.

Single-cell immune profiling reveals immune responses in oral lichen planus.

Oral lichen planus (OLP) is a common chronic inflammatory disorder of the oral mucosa with an unclear etiology. Several types of immune cells are involved in the pathogenesis of OLP. We used single-cell RNA sequencing and immune repertoire sequencing to characterize the mucosal immune microenvironment of OLP. The presence of tissue-resident memory CD8+ T cells are validated by multiplex immunofluorescence. We generated a transcriptome atlas from four OLP biopsy samples and their paired peripheral blood mononuclear cells (PBMCs), and compared them with two healthy tissues and three healthy PBMCs samples. Our analysis revealed activated tissue-resident memory CD8+ T cells in OLP tissues. T cell receptor repertoires displayed apperant clonal expansion and preferrential gene pairing in OLP patients. Additionally, obvious BCR clonal expansion was observed in OLP lesions. Plasmacytoid dendritic cells, a subtype that can promote dendritic cell maturation and enhance lymphocyte cytotoxicity, were identified in OLP. Conventional dendritic cells and macrophages are also found to exhibit pro-inflammatory activity in OLP. Cell-cell communication analysis reveals that fibroblasts might promote the recruitment and extravasation of immune cells into connective tissue. Our study provides insights into the immune ecosystem of OLP, serving as a valuable resource for precision diagnosis and therapy of OLP.

Mature Red Blood Cells Contain Long DNA Fragments and Could Acquire DNA from Lung Cancer Tissue.

Red blood cells (RBC) are commonly known as cells with no nucleus or mitochondria and are assumed to be a transportation vehicle. This study confirms that RBC contain long DNA fragments inside with stain by both microscope and flow cytometry, which covers most nuclear and mitochondrial genome regions by next-generation sequencing (NGS). Such characteristics demonstrate a significant difference compared with A549 cell line or paired peripheral blood mononuclear cell as nucleated cells. To further explore the characteristics of RNA DNA, DNA from 20 RBC samples is sequenced by NGS. Interestingly, several gaps and multiple regions with copy number variation are observed significantly different between different samples, which could be used to distinguish samples with different health status accurately. Using an in vitro co-culture system, it is shown that RBC could absorb DNA-bearing tumorigenic mutations from cancer cell lines but requires cell-to-cell contact. Finally, based on a small scale clinical trial, it is confirmed that common genetic mutations of cancer tissues could be detected in RBC from patients with early-stage non-small-cell lung cancer. This study highlights a new biological phenomenon involving RBC and its translational potential as a novel liquid biopsy technology platform for early cancer screening and diagnosis of malignancy.

Circulating Tumor DNA Clearance during Treatment As a Predictive and Prognostic Biomarker for Diffuse Large B-Cell Lymphoma Patients Receiving First-Line Therapy

Background: Circulating tumor DNA (ctDNA) dynamics has been proven to be a reliable outcome prediction biomarker in a plenty of solid tumors. However, its clinical utility in response assessment and prognosis prediction for diffuse large B-cell lymphoma (DLBCL) has not been well established. This study was aimed to explore the association between ctDNA dynamics and clinical benefits in a cohort of DLBCL patients. Methods: A total of 37 newly diagnosed DLBCL patients treated with first-line therapy were enrolled retrospectively in this study. Tumor biopsy tissues were collected at diagnosis (n = 17) and peripheral blood samples were obtained before treatment (n = 31) as well as after receiving the initial two treatment cycles (C2, n = 37). Tumor genomic DNA and cell-free DNA (cfDNA) were extracted from tumor tissues and plasma samples, respectively. Targeted next generation sequencing of 188 lymphoma associated genes (Onco-LymScan, Genetron Health) was performed in all of the tumor samples to identify disease-associated mutations. Genomic DNA extracted from the paired peripheral blood mononuclear cells were also sequenced and analyzed to filter out germline variants for each patient. Results: All of the 37 patients were identified at least one somatic mutation from their pretreatment tumor and/or plasma samples. We sought whether these basal mutations could be traced in the C2 plasma of each patient. Mutations that were only detected in C2 plasma were considered uninformative and were removed from analysis. Accordingly, 12 patients were identified at least one basal mutation at C2 and 25 patients showed a rapid clearance of these basal mutations. Based on the ctDNA clearance status at C2, patients were classified into two groups: the ctDNA clearance group (n = 25) and the ctDNA persistence group (n = 12). There were no significant difference in serval clinical factors such as age, B symptoms, LDH (Lactate Dehydrogenase) level, stage and IPI (International prognostic Index) between the two groups. In response to the first-line treatment, patients with a ctDNA clearance showed a significantly higher complete remission rate (62.5% vs. 23.1%, p = 0.038; Figure A) than the patients with a persistence of ctDNA. With a median follow-up visit period of 26.0 mouths, patients with a ctDNA clearance at C2 presented a favorable progression-free survival (median PFS, unreached vs. 5.45 months; HR 2.919; p = 0.0039; Figure B) and overall survival (median OS, 43.6 vs. 25.87 months; HR 2.758; p = 0.0393; Figure C) compared with those without a ctDNA clearance. These results indicated that patients achieving an early ctDNA clearance during treatment were more likely to achieve a superior later clinical benefit. Conclusions: Early ctDNA clearance during treatment could effectively serve as a predictive and prognostic biomarker for DLBCL patients receiving first-line therapy. Integrating ctDNA profiling into clinical disease management may help to identify patients at high-risk and contribute to individualized treatment. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Circulating Tumor DNA Correlation with Lymphoma Response and Survival Outcomes at Multiple Time Points of Anti - CD19 CAR T Cell Therapy

Background Chimeric antigen receptor T (CAR T) cells targeting CD19 have clinical activity against relapsed or refractory (r/r) non-Hodgkin lymphoma (NHL), with high response rates. However, approximately a third of patients fail to respond to CAR T cells and more than half will present disease progression within one year. Circulating tumor DNA (ctDNA) is emerging as a powerful tool for prediction of response in hematologic malignancies. Here, we present the results of ctDNA analysis of 28 patients participating in a phase I clinical trial of point of care manufactured antiCD19 CAR T cells with TNFRSF19 transmembrane domain using CliniMACS Prodigy device (Caimi PF, et al. Blood 2021; 138(S1):3833) (NCT03434769). Methods Twenty-eight relapsed or refractory NHL patients treated with CAR T cells were included in the ctDNA analysis. Blood samples were collected at baseline (prior to lymphodepletion), on the day of infusion (T = 0) and 30 days after CAR T cell infusion and profiled using the CAPP-Seq strategy (Rossi, 2017). A targeted resequencing panel optimized to include the coding exons and splice sites of 154 genes (370 Kb) recurrently mutated in B cell NHL were used on cfDNA from plasma and on paired germline DNA isolated from paired peripheral blood mononuclear cells. Sequencing was performed using the Nextseq 550 platform (Illumina) to obtain a depth of coverage >2000x in >80% of the target region in all samples. A stringent and completely automated bioinformatic pipeline was applied to call non-synonymous somatic mutations, using the somatic function of VarScan2. Disease response was measured on day 30 and 90 with PET/CT scan. Progression free survival (PFS) and overall survival (OS) were measured from the time of CAR T cell infusion and estimated using Kaplan-Meier analysis. Statistical analyses were done using R and its packages. Comparisons between groups were done using Fisher exact test between categorical variables, optimal ctDNA cutpoints for categorical endpoints were done using the cutpointr R package, and using the maximally selected rank statistics for time to event endpoints. Results 75 plasma samples were available for analysis (T-6, n = 26; T0, n = 22; T30, n = 27). The median ctDNA load reported as haploid genome equivalents per ml (hGE/mL) was 75.81 at T-6 (range: 0-8373). There was a statistically significant decrease in ctDNA load on T0 (0,33 hGE/mL [range: 0-48126], p = 0.02) and T+30 (0,38 hGE/ml [range: 0-2855], p < 0.001)(Figure 1). Six patients (23%) had undetectable ctDNA on T-6, despite having measurable disease. The optimal cutoff value for T-6 ctDNA was determined at 250 hGE/mL, patients with lower values (20/26) had higher rates of complete response (CR) (95% vs. 33%, p < 0.01) as well as longer median PFS (26 vs. 2 months, p < 0.001) and OS (NR vs. 20 months, p <0.01). We did not find an association between higher T-6 ctDNA levels and CRS or ICANS. Genes recurrently affected by non-synonymous somatic variants in 26 patients at T-6 were BCL2 and KMT2D (27%), IGLL5 (23%), BCL6 and CARD11 (19%), MYC and TP53 (15%). Lymphodepletive chemotherapy resulted in a decrease in ctDNA in 17/20 patients with paired samples, 13 patients decreased ≥1 log and 8 by ≥ 2 log. Sixteen patients had undetectable ctDNA levels at T0, which includes 9 patients with previous measurable ctDNA. Patients with ctDNA under 10 hGE/ml had higher CR rates (88% vs. 33%, p = 0.03) as well as longer median PFS (26 vs. 1 month, p = 0.03) and OS (NR vs. 13 months, p = 0.005). On day T30, 21/26 patients with paired preCAR T samples had decreased ctDNA by >1 log, 19 (70%) had undetectable levels. Among patients with undetectable ctDNA on day 30, best response was CR for 18 (94%), 4 of whom had PR on PET/CT on day 30 and went on to convert to CR. Patients with undetectable ctDNA had longer median PFS (37 vs. 2 months, p = 0.002) (Figure 2) and OS (NR vs. 13 months, p < 0.001). Conclusions Detection of ctDNA in r/r NHL patients treated with CAR T cells has predictive value at different timepoints. Lower ctDNA burden prior to lymphodepletion is associated with improved outcomes, likely a reflection of lower disease burden. In addition, ctDNA concentration decreases both after lymphodepletive chemotherapy and after CAR T cells correlate with disease response, PFS and OS. Mutational profiling of ctDNA can provide information regarding recurrent mutations in cases with and without disease response to CAR T cells. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Identification of Differentially Expressed Mirna by Next Generation Sequencing in Locally Advanced Breast Cancer Patients of South Indian Origin.

miRNAs are known to be aberrantly expressed in the serum, tissue, and Peripheral Blood Mononuclear Cells (PBMC) of cancer patients and could serve as potential noninvasive diagnostic markers for breast cancer. The aim of this study was to identify the differentially expressed miRNA using next-generation sequencing (NGS) from the paired PBMC samples from breast cancer patients and age-matched healthy individuals and explore their functional significance. In this study, PBMCs were employed for the detection of miRNAs by NGS in locally advanced breast cancer (LABC) women of South Indian origin who were divided into three age groups, (a) 40yrs-50yrs (b) 50yrs-60yrs and (c) 60yrs-70yrs, compared with age-matched control groups. Four miRNAs (hsa-miR-192-5p, hsa-miR-24-2-2p, hsa-miR-3609, and hsa-miR-664b-3p) were found to be differentially expressed among LABC patients compared with age matched healthy women of the South Indian population. While miR-24-2-5p, miR3609, and miR-664b-3p were down-regulated, miR-192-5p was up-regulated. Gene Ontology (GO) annotations implicated miRNA with signaling pathways in peripheral nerve synapses, glutamatergic synapse, and cell morphogenesis, all of which play a pivotal role in the manifestation of cancer. Four miRNAs- 3 (While miR-24-2-5p, miR3609, and miR-664b-3p) downregulated and one upregulated (miR-192-5p) were identified as potential biomarkers for patients with locally advanced breast cancer. These markers could be validated in studies with a larger sample size.

Copy number variations across the blood–brain barrier in multiple sclerosis

ObjectiveMultiple sclerosis (MS) is a neuroinflammatory disease where immune cells cross the blood–brain barrier (BBB) into the central nervous system (CNS). What predisposes these immune cells to cross the BBB is still unknown. Here, we examine the possibility that genomic rearrangements could predisposespecific immune cells in the peripheral blood to cross the BBB and form sub‐populations of cells involved in the inflammatory process in the CNS.MethodsWe compared copy number variations in paired peripheral blood mononuclear cells (PBMCs) and cerebrospinal fluid (CSF) cells from MS patients. Thereafter, using next generation sequencing, we studied the T‐cell receptor beta (TRB) locus rearrangements and profiled the αβ T cell repertoire in peripheral CD4+ and CD8+ T cells and in the CSF.ResultsWe identified deletions in the T‐cell receptor alpha/delta (TRA/D), gamma (TRG), and TRB loci in CSF cells compared to PBMCs. Further characterization revealed diversity of the TRB locus which was used to describe the character and clonal expansion of T cells in the CNS. T‐cell repertoire profiling from either side of the BBB concluded that the most frequent clones in the CSF samples are unique to an individual. Furthermore, we observed a difference in the proportion of expanded T‐cell clones when comparing samples from MS patients in relapse and remission with opposite trends in CSF and peripheral blood.InterpretationThis study provides a characterization of the T cells in the CSF and might indicate a role of expanded clones in MS pathogenicity.