BackgroundAdoptive transfer of CD19-specific chimeric antigen receptor (CD19CAR) T cells can induce dramatic disease regression in patients with B cell malignancies. CD19CAR T cell therapy may be ...limited by insufficient engraftment and persistence, resulting in tumor relapse. We previously demonstrated a proof of principle that cytomegalovirus (CMV)-specific T cells can be isolated and enriched prior to CD19CAR transduction to produce CMV-CD19CAR T cells, and that these CMV-CD19CAR T cells can be expanded in vivo through CMV vaccination, resulting in better tumor control in a murine model. Here we developed a clinical platform for generating CMV-CD19CAR T cells.MethodsPeripheral blood mononuclear cells (PBMCs) collected from CMV-seropositive healthy donors were stimulated with a good manufacturing practices-grade PepTivator overlapping CMVpp65 peptide pool and enriched for CMV-responsive interferon γ (IFNγ)+T cells using IFNγ Catchmatrix, within the CliniMACS Prodigy Cytokine Capture System (Miltenyi Biotec). Resulting CMV-specific T cells were transduced with a lentiviral vector encoding a second generation CD19R:CD28:ζ/EGFRt CAR and expanded with interleukin 2 (IL-2) and IL-15 for 15 days before characterization.ResultsCMV-specific T cells were enriched from 0.8%±0.5 of input PBMC to 76.3%±11.6 in nine full-scale qualification runs (absolute yield of 4.2±3.3×106 IFNγ+T cells from an input of 1×109 PBMCs). Average CD19CAR transduction efficiency of CMV-specific T cells was 27.0%±14.2 in the final products, which underwent rapid expansion, resulting in a total cell dose of 6.2±0.9 × 106 CD19CAR-tranduced T cells with CMV specificity (ie, functionally bispecific). CMV-CD19CAR T cells were polyclonal, expressed memory markers but had low expression of exhaustion markers, responded to both CD19 and CMVpp65 stimulation with rapid proliferation and exhibited antigen-specific effector functions against both CD19-expressing tumors and CMVpp65 antigen. The final products passed release criteria for clinical use.ConclusionsWe demonstrated the feasibility of our large-scale platform for generating CMV-CD19CAR T cells for clinical application. We plan to initiate a clinical trial at City of Hope using CMV-CD19CAR T cells for patients with intermediate/high-grade B cell non-Hodgkin’s lymphoma immediately after autologous hematopoietic cell transplantation followed by vaccination with a novel CMV vaccine based on Modified Vaccinia Ankara (Triplex) 28 days and 56 days post-T cell infusion.
Osteopenia and periarticular bony erosion are consequences of chronic inflammatory autoimmune disease due to an imbalance of osteoclast activity relative to new bone formation. Osteoclasts, which are ...specialized as the only bone resorbing cell type, are differentiated from hematopoietic myeloid precursor cells. Inflammatory signals mediated by multiple types of immune cells and cytokines have significant influence over osteoclast differentiation and function through direct effects on osteoclast precursors and indirect effects via osteoblasts and other cells in the bony microenvironment including synovial cells, stromal cells, osteocytes and chondrocytes. Recent studies have demonstrated that osteoclasts themselves express a number of immune receptors and are regulated similarly to macrophages and dendritic cells, closely related cells in the innate immune system. Though we are only beginning to understand the roles of innate immune receptors in osteoclasts, some of these receptors have been shown to be critical regulators of differentiation and function of osteoclasts. Osteoclasts likely function as the innate immune cells of the bone, thus are highly regulated to appropriately respond to stress and inflammatory changes in their microenvironment.
By homology to triggering receptor expressed by myeloid cells-2, we screened the mouse expressed sequence tag database and isolated a new single Ig domain receptor, which we have expressed and ...characterized. The receptor is most similar in sequence to the human CMRF-35 receptor, and thus we have named it CMRF-35-like molecule (CLM)-1. By screening the mouse genome, we determined that CLM-1 was part of a multigene family located on a small segment of mouse chromosome 11. Each contains a single Ig domain, and they are expressed mainly in cells of the myeloid lineage. CLM-1 contains multiple cytoplasmic tyrosine residues, including two that lie in consensus immunoreceptor tyrosine-based inhibitory motifs, and we demonstrate that CLM-1 can associate with Src-homology 2 containing phosphatase-1. Expression of CLM-1 mRNA is down-regulated by treatment with receptor activator of NF-kappaB ligand (RANKL), a cytokine that drives osteoclast formation. Furthermore, expression of CLM-1 in the osteoclastogenic cell line RAW (RAW.CLM-1) prevents osteoclastogenesis induced by RANKL and TGF-beta. RAW.CLM-1 cells fail to multinucleate and do not up-regulate calcitonin receptor, but they express tartrate-resistant acid phosphatase, cathepsin K, and beta(3) integrin, suggesting that osteoclastogenesis is blocked at a late-intermediate stage. Thus, we define a new family of myeloid receptors, and demonstrate that the first member of this family, CLM-1, is an inhibitory receptor, able to block osteoclastogenesis.
BackgroundPeople living with human immunodeficiency virus (HIV) are at significantly higher risk than the general population of developing cancer, with a lifetime prevalence of cancer diagnosis of ...25–40%. Non-Hodgkin lymphomas (NHL) are the most common type of AIDS-defining malignancy and predominantly manifests as Burkitt lymphoma (25%) or diffuse large B cell lymphoma (75%). Although CD19-directed (CD19) CAR T cell therapies are promising in treating B cell NHL, clinical trials have excluded HIV-positive patients due to concerns about safety (infectious complications), viral control, and limited CAR efficacy.Recent reports have demonstrated the safety and feasibility of using CD19CAR T cell therapy to treat NHL in patients with HIV, indicating a move toward addressing the unmet need of this patient population. We previously designed mono-specific CAR constructs targeting either lymphoma (CD19) or HIVgp120 (N6) that show efficacy against their respective diseases in clinical and preclinical testing, respectively. We hypothesized that a dual construct targeting both antigens could simultaneously target both lymphoma cells and HIV-infected cells in the same individual. Thus, we developed a bi-specific CD19/N6 CAR T cell platform that can target both antigens in a single therapeutic product.MethodsWe generated 3 bi-specific CAR constructs (2 tandem and 1 loop) (figure 1A) that incorporate a humanized (hu)CD19 single-chain variable fragment (scFv) and an N6 scFv from an anti-HIV broadly neutralizing antibody (bNAb) into a 2nd-generation CAR backbone. The tandem CARs consisted of N6 and huCD19 scFvs fused with a G4S linker in either huCD19:N6 or N6:huCD19 orientation. The loop CAR was generated by fusing huCD19(VL):N6(VH):N6(VL):huCD19(VH) with a Whitlow linker. All constructs included the CD4 transmembrane domain, a double-mutated IgG4 Fc spacer, 4–1BB co-stimulatory and CD3ζ signaling domains, and EGFRt separated by a T2A ribosomal skip sequence. We tested the 3 bi-specific constructs in healthy-donor T cells using cytotoxicity co-culture assay (figure 1B) against either Raji (CD19+) or 8E5 (gp120+) target cells and confirmed functionality in HIV-positive donors.ResultsAlthough all 3 bi-specific CAR constructs were functional against both CD19 and HIVgp120 antigens, the N6:huCD19CAR tandem CAR demonstrated equivalent or better efficacy against both antigens and could serially target single or alternating antigens (figure 1C,D). Moreover, we successfully generated N6:huCD19CAR tandem CAR T cells using HIV-positive donors and confirmed functionality.ConclusionsThe development of the N6:huCD19CAR bi-specific CAR represents a novel CAR T cell therapy that could potentially provide life-saving treatment for patients with HIV-associated NHL through simultaneous targeting of tumor and viral suppression.Ethics ApprovalThis study was approved by the City of Hope IRB 09025.Abstract 314 Figure 1N6:huCD19 bi-specific CAR-mediated elimination of HIVgp120- and CD19-expressing cells. (A) Schema of three tandem designs: N6-huCD19 scfv, huCD19-N6 scfv, huCD19-N6 loop design. (B) Serial killing assay schema. On day 0, healthy donor N6-huCD19 CAR bi-specific T cells were co-cultured with gp120-expressing cells (8E5). After a 48-hour incubation period, the cells were examined to determine the survival of gp120-expressing cells. Simultaneously, CD19-expressing cells were introduced into the co-culture. Following an additional 48-hour incubation, the survival of CD19-expressing cells was quantified. (C) Cytotoxic analysis was performed on Day 2 to evaluate the targeting of HIV gp120. (D) On Day 4, the cytotoxic analysis was conducted to assess CD19 targeting. huCD19 and N6 monospecific CARs were included as control groups.
Lateral flow assays (LFA) are sensitive for detecting antibodies to SARS-CoV-2 proteins within weeks after infection. This study tested samples from immunocompetent adults, and those receiving ...treatments for chronic inflammatory diseases (CID), before and after mRNA SARS-CoV-2 vaccination.
We compared results obtained with the COVIBLOCK Covid-19 LFA to those obtained by anti-spike (S) ELISA.
The LFA detected anti-S antibodies in 29 of 29 (100%) of the immunocompetent and 110 of 126 (87.3%) of the CID participants after vaccination. Semiquantitative LFA scores were statistically significantly lower in samples from immunosuppressed participants, and were significantly correlated with anti-S antibody levels measured by ELISA.
This simple LFA test is a practical alternative to laboratory-based assays for detecting anti-S antibodies after infection or vaccination. This type of test may be most useful for testing people in outpatient or resource-limited settings.
Macrophage recognition of Salmonella enterica serovar Typhimurium leads to a cascade of signaling events, including the activation of Src family and Syk kinases and the production of reactive oxygen ...species (ROS), which are critical for host innate defense during early stages of bacterial infection. ROS production depends on the NADPH oxidase, but little is known about the innate immune receptors and proximal adapters that regulate Salmonella-induced ROS. Herein, we demonstrate that serovar Typhimurium induces ROS through a pathway that requires both triggering receptor expressed on myeloid cells 2 (TREM2) and DAP12. This pathway is highly analogous to the pathways utilized by Fc receptors and integrins to regulate ROS production. Oral infection of mice with serovar Typhimurium demonstrates that the DAP12-dependent pathway regulates cecal colonization during early stages of Salmonella infection. Thus, DAP12 is an important regulator of Salmonella-induced ROS production in macrophages, and TREM2 is essential for linking DAP12 to the innate response to serovar Typhimurium.
Rheumatoid arthritis (RA) management lean toward achieving remission or low-disease activity. In this study, we conducted single-cell RNA sequencing (scRNAseq) of peripheral blood mononuclear cells ...(PBMCs) from 36 individuals (18 RA patients and 18 matched controls, accounting for age, sex, race, and ethnicity), to identify disease-relevant cell subsets and cell type-specific signatures associated with disease activity. Our analysis revealed 18 distinct PBMC subsets, including an IFITM3 overexpressing Interferon-activated (IFN-activated) monocyte subset. We observed an increase in CD4+ T effector memory cells in patients with moderate to high disease activity (DAS28-CRP ≥ 3.2), and a decrease in non-classical monocytes in patients with low disease activity or remission (DAS28-CRP < 3.2). Pseudobulk analysis by cell type identified 168 differentially expressed genes between RA and matched controls, with a downregulation of pro-inflammatory genes in the gamma-delta T cells subset, alteration of genes associated with RA predisposition in the IFN-activated subset, and non-classical monocytes. Additionally, we identified a gene signature associated with moderate-high disease activity, characterized by upregulation of pro-inflammatory genes such as TNF, JUN, EGR1, IFIT2, MAFB, G0S2, and downregulation of genes including HLA-DQB1, HLA-DRB5, TNFSF13B. Notably, cell-cell communication analysis revealed an upregulation of signaling pathways, including VISTA, in both moderate-high and remission-low disease activity contexts. Our findings provide valuable insights into the systemic cellular and molecular mechanisms underlying RA disease activity.Rheumatoid arthritis (RA) management lean toward achieving remission or low-disease activity. In this study, we conducted single-cell RNA sequencing (scRNAseq) of peripheral blood mononuclear cells (PBMCs) from 36 individuals (18 RA patients and 18 matched controls, accounting for age, sex, race, and ethnicity), to identify disease-relevant cell subsets and cell type-specific signatures associated with disease activity. Our analysis revealed 18 distinct PBMC subsets, including an IFITM3 overexpressing Interferon-activated (IFN-activated) monocyte subset. We observed an increase in CD4+ T effector memory cells in patients with moderate to high disease activity (DAS28-CRP ≥ 3.2), and a decrease in non-classical monocytes in patients with low disease activity or remission (DAS28-CRP < 3.2). Pseudobulk analysis by cell type identified 168 differentially expressed genes between RA and matched controls, with a downregulation of pro-inflammatory genes in the gamma-delta T cells subset, alteration of genes associated with RA predisposition in the IFN-activated subset, and non-classical monocytes. Additionally, we identified a gene signature associated with moderate-high disease activity, characterized by upregulation of pro-inflammatory genes such as TNF, JUN, EGR1, IFIT2, MAFB, G0S2, and downregulation of genes including HLA-DQB1, HLA-DRB5, TNFSF13B. Notably, cell-cell communication analysis revealed an upregulation of signaling pathways, including VISTA, in both moderate-high and remission-low disease activity contexts. Our findings provide valuable insights into the systemic cellular and molecular mechanisms underlying RA disease activity.
To identify predictors of bone remodelling in children and young adults with SLE.
Ninety subjects with SLE aged 8-22 years underwent yearly measurements of height, bone age, bone turnover markers, ...serum Type I IFNs, SLEDAI and BMD. Predictors of bone turnover were examined using serum osteocalcin as a marker of bone formation and both serum tartrate-resistant acid phosphatase (TRAP) and urine N-telopeptide (NTx) as markers of bone resorption.
Subjects demonstrated short stature, high BMI and bone age delay. A spine BMD Z-score of less than -2.0 was seen in 16.1% of subject visits. Serum osteocalcin was negatively correlated with glucocorticoid dose (Spearman rank correlation coefficient R = -0.34, P < 0.0001) but was not associated with SLEDAI after adjustment for confounders. Serum TRAP was negatively associated with SLEDAI, even after controlling for confounders (P = 0.04). Similar results were obtained for urine NTx. There was a negative association between TRAP and serum IFN-β (P = 0.03).
In this population of children and young adults with moderate lupus disease activity, glucocorticoid dose was a negative predictor of bone formation, whereas lupus disease activity was not. Interestingly, lupus disease activity was a negative predictor of bone resorption, suggesting that lupus disease activity is not the primary factor contributing to the bone deficits of childhood-onset SLE. The potential protective role of IFN-β and the effects of SLE treatment on bone loss require further study.
Fisheries are highly complex social-ecological systems that often face ‘wicked’ problems from unsustainable resource management to climate change. Addressing these challenges requires ...transdisciplinary approaches that integrate perspectives across scientific disciplines and knowledge systems. Despite widespread calls for transdisciplinary fisheries research (TFR), there are still limitations in personal and institutional capacity to conduct and support this work to the highest potential. The viewpoints of early career researchers (ECRs) in this field can illuminate challenges and promote systemic change within fisheries research. This paper presents the perspectives of ECRs from across the globe, gathered through a virtual workshop held during the 2021 World Fisheries Congress, on goals, challenges, and future potential for TFR. Big picture goals for TFR were guided by principles of co-production and included (i) integrating transdisciplinary thinking at all stages of the research process, (ii) ensuring that research is inclusive and equitable, (iii) co-creating knowledge that is credible, relevant, actionable, and impactful, and (iv) consistently communicating with partners. Institutional inertia, lack of recognition of the extra time and labour required for TFR, and lack of skill development opportunities were identified as three key barriers in conducting TFR. Several critical actions were identified to help ECRs, established researchers, and institutions reach these goals. We encourage ECRs to form peer-mentorship networks to guide each other along the way. We suggest that established researchers ensure consistent mentorship while also giving space to ECR voices. Actions for institutions include retooling education programs, developing and implementing new metrics of impact, and critically examining individualism and privilege in academia. We suggest that the opportunities and actions identified here, if widely embraced now, can enable research that addresses complex challenges facing fishery systems contributing to a healthier future for fish and humans alike.
Post-transplantation cyclophosphamide (PTCy) is a safe and efficacious graft-versus-host-disease (GVHD) prophylaxis following hematopoietic cell transplantation (HCT) from a haploidentical (haplo) ...donor. Cytokine release syndrome (CRS) is a common complication of this platform. Early fever post-haplo-HCT using bone marrow grafts is associated with higher CD3
cell dose and CRS. However, the impact of CD3
and CD34
cell dose on CRS post-haplo-HCT using peripheral blood stem cell (PBSC) grafts is unknown. Our goals were to evaluate the incidence of CRS following PBSC transplantation (PBSCT) and to identify factors that can be modified to prevent the development of severe CRS in this setting. In 271 patients, we investigated factors associated with the development of CRS following haplo-PBSCT and examined the impact of CRS on clinical outcomes. Ninety-three percent of the patients developed CRS of any grade post-haplo-PBSCT. In multivariate analysis, severe CRS (grade 3-4 versus grade 0-1) was associated with higher nonrelapse mortality (hazard ratio HR, 6.42; 95% confidence interval CI, 2.68 to 15.39; P < .001), worse 1-year overall survival (HR, 3.40; 95% CI, 1.63 to 7.08; P = .005), and worse disease-free survival (HR, 4.02; 95% CI, 1.99 to 8.08; P < .001). Moderate to severe CRS (grade 2-4) did not impact 1-year relapse or acute GVHD (grade II-IV and III-IV) at 100 days (P = .71 and .19, respectively). Importantly, higher CD3
cell dose, but not CD34
cell dose, predicted a higher incidence of grade 2-4 CRS (HR, 1.20; 95% CI,1.07 to 1.36; P = .003) and grade 3-4 CRS (HR, 1.40; 95% CI, 1.05 to 1.86; P = .022). Both older age (HR, 8.57; 95% CI, 1.73 to 42.36; P < .001) and non-total body irradiation-based reduced-intensity conditioning with fludarabine/melphalan (HR, 15.38; 955 CI, 2.06 to 114.67; P < .001) were predictive of grade 3-4 CRS. Overall, we observed that severe CRS (grade 3-4) negatively affected transplantation outcome, and that higher CD3 cell dose was associated with the development of any grade CRS and severe CRS.
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