key: cord-0829601-ob5bs4wr authors: Ansari, Asgar; Sachan, Shilpa; Jit, Bimal Prasad; Sharma, Ashok; Coshic, Poonam; Sette, Alessandro; Weiskopf, Daniela; Gupta, Nimesh title: An efficient immunoassay for the B-cell help function of SARS-CoV-2 specific memory CD4+ T cells date: 2022-05-10 journal: Cell Rep Methods DOI: 10.1016/j.crmeth.2022.100224 sha: 7d98b81cd7d3417219e4197e48095d1379cba13c doc_id: 829601 cord_uid: ob5bs4wr The B cell “help” function of CD4+ T cells is an important mechanism of adaptive immunity. Here, we describe improved antigen-specific T-B co-cultures for quantitative measurement of T cell-dependent B cell responses, with as few as ∼90 T cells. Utilizing Mtb, we show that early priming and activation of CD4+ T cells is important for productive interaction between T and B cells, and that similar effects are achieved by supplementing co-cultures with monocytes. We find that monocytes promote survivability of B cells via BAFF and SCGF/CLEC11A, but this alone does not fully recapitulate the effects of monocyte-supplementation. Importantly, we demonstrate improved activation and immunological output of SARS-CoV-2 specific memory CD4+ T - B cell co-cultures with the inclusion of monocytes. This method may therefore provide a more sensitive assay to evaluate the B cell help quality of memory CD4+ T cells, for example after vaccination or natural infection. quantified the activated CD4 + T cells in cocultures. We detected a significant number of CD38 + PD-1 + CD4 + 168 T cells as early as 3 days, which peaked at Day 9 (Figure S3 E-F; P=0.001). Additionally, all the CD38 + PD-169 1 + CD4 + T cells expressed high levels of ICOS as activation associated co-stimulatory molecule, which 170 showed better detectability than CD38 (data not shown). Therefore, with the maximal activation of T cells 171 without significant loss in the viability of both T and B cells, we concluded 9 days of coculture as the 172 optimal duration for further experiments. Also, we utilized ICOS + PD-1 + co-expression to distinctly identify 173 the activated CD4 + T cells in subsequent experiments. Altogether, the results clearly suggest that the monocyte supplementation to the T-B coculture enhances 324 the sensitivity for efficient qualitative assessment of the SARS-CoV-2 specific memory CD4 + T cells. Limitations of the study 562 563 There are limitations inherent to this assay. Although monocyte supplementation to the co-culture system 564 can enhance the sensitivity and consistency in T and B cell output, the inter-individual variability in Ag-565 specific T cell and B cell repertoires may influence the assay readouts. The human inter-individual 566 heterogeneity in immune response is an inherent biological variation. Thus, it may be appropriate to apply 567 this assay after confirming the detectable humoral and Ag-specific T cell response. Also, for comparative 568 study, it may be helpful to select the subjects with comparable frequency of Ag-specific CD4 + T cells. In all the experiments, data are represented as the mean±s.e.m. The significance of the differences between 936 the groups was analyzed with two-tailed paired t-test, two-tailed Mann-Whitney test, two tailed Wilcoxon 937 matched-pairs signed rank test, one-way ANOVA followed by Bonferroni's multiple comparisons test, 938 multiple t tests corrected using specified methods. P values < 0.05 were considered statistically significant. CD40 ligand gene defects responsible for 945 X-linked hyper-IgM syndrome Immune Memory in Mild COVID-19 Patients and Unexposed Donors Reveals Persistent 948 In 950 vivo-activated CD4 T cells upregulate CXC chemokine receptor 5 and reprogram their response to 951 lymphoid chemokines IL-21 promotes CD4 T cell responses by phosphatidylinositol 3-kinase-dependent 954 upregulation of CD86 on B cells Distinguishing 956 naive-from memory-derived human B cells during acute responses Molecular cloning of a new 959 secreted sulfated mucin-like protein with a C-type lectin domain that is expressed in lymphoblastic cells BAFF mediates survival of peripheral immature B lymphocytes Comparison of monocytes and B cells for activation of 965 human T helper cell subsets IL-1 acts directly on CD4 T cells to enhance their antigen-driven expansion and 968 differentiation Induction of ICOS+CXCR3+CXCR5+ TH cells correlates 971 with antibody responses to influenza vaccination SARS-CoV-2-reactive T cells in healthy donors and patients with 975 COVID-19 Follicular B 977 helper T cells express CXC chemokine receptor 5, localize to B cell follicles, and support immunoglobulin 978 production Structure, function, and biogenesis of the cell wall of Mycobacterium tuberculosis Cytokine-mediated regulation of human B cell differentiation into Ig-secreting cells: 983 predominant role of IL-21 produced by CXCR5+ T follicular helper cells A CD4(+) T cell population expanded in lupus blood provides B 987 cell help through interleukin-10 and succinate Regulation of memory B-cell survival by the BH3-990 only protein Puma Longitudinal analysis shows durable and broad immune memory after 993 SARS-CoV-2 infection with persisting antibody responses and memory B and T cells Cell-mediated immune responses in tuberculosis BAFF regulates B cell survival by 998 downregulating the BH3-only family member Bim via the ERK pathway Macrophage-and dendritic cell--dependent 1001 regulation of human B-cell proliferation requires the TNF family ligand BAFF Follicular helper CD4 T cells (TFH) CD4+ T Cells in Blood Immunological memory to SARS-CoV-2 assessed for up to 8 months after 1011 infection Mapping the Lineage Relationship between 1014 CXCR5(+) and CXCR5(-) CD4(+) T Cells in HIV-Infected Human Lymph Nodes BAFF augments IgA2 and IL-10 production by TLR7/8 stimulated total peripheral blood B cells Quiescence-inducing and antiapoptotic activities of IL-15 enhance secondary CD4+ T cell responsiveness 1021 to antigen Molecular 1023 mechanism and function of CD40/CD40L engagement in the immune system Monocytes and T cells cooperate to favor normal and follicular lymphoma B-cell growth: role of IL-15 and 1027 CD40L signaling IL-21 induces differentiation of human naive and memory B cells into antibody-1030 secreting plasma cells IL-21 in Conjunction with Anti-1032 CD40 and IL-4 Constitutes a Potent Polyclonal B Cell Stimulator for Monitoring Antigen-Specific Memory 1033 B Cells BAFF-R 1035 promotes cell proliferation and survival through interaction with IKKbeta and NF-kappaB/c-Rel in the 1036 nucleus of normal and neoplastic B-lymphoid cells Visualization 1039 of specific B and T lymphocyte interactions in the lymph node Cytokine-driven proliferation and differentiation of 1042 human naive, central memory, and effector memory CD4(+) T cells Resting human memory B cells are intrinsically 1045 programmed for enhanced survival and responsiveness to diverse stimuli compared to naive B cells Macrophage migration inhibitory factor induces B cell survival by activation of a CD74-CD44 1049 receptor complex Targets of T Cell Responses to SARS-CoV Coronavirus in Humans with COVID-19 Disease and Unexposed Individuals Stem cell growth factor: in situ hybridization analysis on the gene expression, molecular characterization 1056 and in vitro proliferative activity of a recombinant preparation on primitive hematopoietic progenitor cells B Cells Are the Dominant Antigen-Presenting Cells that Activate Naive CD4(+) T Cells upon 1060 Immunization with a Virus-Derived Nanoparticle Antigen The role of B cell antigen presentation in the initiation of CD4+ T cell 1063 response Optimization of a human IgG B-cell ELISpot assay for the analysis of vaccine-induced B-cell responses Assembly of the Mycobacterial Cell Wall Survival of resting mature B 1070 lymphocytes depends on BCR signaling via the Igalpha/beta heterodimer Essential role of IL-21 in B cell activation, expansion, and plasma cell generation during CD4+ T cell-B 1074 cell collaboration Comprehensive mapping of immune 1077 perturbations associated with severe COVID-19 SARS-CoV-2-specific T cell immunity in cases of COVID-19 and SARS, and 1080 uninfected controls A Quantitative Analysis of Complexity of Human 1083 CD4 T Cell Responses in Healthy M. tuberculosis Infected South Africans Cross-reactive memory T cells and herd immunity 1086 to SARS-CoV-2 Human circulating PD-1+CXCR3-CXCR5+ memory Tfh cells are 1089 highly functional and correlate with broadly neutralizing HIV antibody responses Clonal analysis of immunodominance and cross-reactivity of the CD4 T 1093 cell response to SARS-CoV-2 Human blood CXCR5(+)CD4(+) T cells are 1096 counterparts of T follicular cells and contain specific subsets that differentially support antibody secretion Critical role of monocytes to support normal B cell and diffuse large B cell lymphoma survival and 1100 proliferation Critical requirement for BCR, BAFF, and BAFFR in memory B cell 1103 survival Synthesis and release of B-lymphocyte stimulator from 1106 myeloid cells Finding the right niche: B-cell migration in the early 1108 phases of T-dependent antibody responses Expression of LSLCL, a new C-type lectin, is closely restricted, in bone marrow Antigen presentation by monocytes and monocyte-1113 derived cells Pathologically expanded peripheral T helper cell subset drives 1116 B cells in rheumatoid arthritis Crucial role for BAFF-BAFF-R signaling 1118 in the survival and maintenance of mature B cells Comparative analysis of activation induced marker (AIM) 1121 assays for sensitive identification of antigen-specific CD4 T cells New insights into the regulation of T cells by 1124 gamma(c) family cytokines Functional SARS-CoV-2-Specific Immune 1127 Memory Persists after Mild COVID-19 Antigen-Specific Adaptive Immunity to SARS-CoV-2 in 1130 Acute COVID-19 and Associations with Age and Disease Severity The BAFF receptor transduces survival signals by co-opting the B cell receptor signaling pathway Adaptive immunity to SARS-CoV-2 and COVID-19 Integrin alpha11 is an Osteolectin receptor and is required for the maintenance of adult skeletal bone mass Immature 1141 monocytes acquire antigens from other cells in the bone marrow and present them to T cells after maturing 1142 in the periphery ICOS is essential for effective T-helper-cell responses Early induction of functional SARS-CoV-2-specific T cells associates 1148 with rapid viral clearance and mild disease in COVID-19 patients Intrinsic differences in the 1151 proliferation of naive and memory human B cells as a mechanism for enhanced secondary immune 1152 responses Regulation of the germinal center and humoral immunity by interleukin-1154 Human monoclonal antibody combination 1157 against SARS coronavirus: synergy and coverage of escape mutants Monocyte-derived human B-cell growth factor identified as interferon-beta 2 (BSF-2, 1161 IL-6) B cells responses and cytokine production are 1163 regulated by their immune microenvironment Germinal centers SARS-CoV-2-specific T cells in COVID-19 patients with acute respiratory distress syndrome Mice deficient for the CD40 ligand