scholarly journals Simultaneous analysis of antigen-specific B and T cells after SARS-CoV-2 infection and vaccination

2021 ◽  
Author(s):  
Krista L Newell ◽  
Mitchell J Waldran ◽  
Stephen J Thomas ◽  
Timothy P Endy ◽  
Adam Tully Waickman
Keyword(s):  
Flow Cytometry ◽  
T Cell ◽  
B Cells ◽  
Simultaneous Detection ◽  
T Cell Subsets ◽  
Cell Populations ◽  
T And B Cells ◽  
Simultaneous Identification ◽  
Immune Profiling ◽  
Insight Into

Conventional methods for quantifying and phenotyping antigen-specific lymphocytes can rapidly deplete irreplaceable specimens. This is due to the fact that antigen-specific T and B cells have historically been analyzed in independent assays each requiring millions of cells. A technique that facilitates the simultaneous detection of antigen-specific T and B cells would allow for more thorough immune profiling with significantly reduced sample requirements. To this end, we developed the B And T cell Tandem Lymphocyte Evaluation (BATTLE) assay, which allows for the simultaneous identification of SARS-CoV-2 Spike reactive T and B cells using an optimized Activation Induced Marker (AIM) T cell assay and dual-color B cell antigen probes. Using this assay, we demonstrate that antigen-specific B and T cell subsets can be identified simultaneously using conventional flow cytometry platforms and provide insight into the differential effects of mRNA vaccination on B and T cell populations following natural SARS-CoV-2 infection.

Improved T Cell Recovery After Transplantation of CD3/CD19 depleted Haploidentical Stem Cell Grafts in Pediatric Patients.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4652-4652
Author(s):  
Heiko-Manuel Teltschik ◽  
Tobias Feuchtinger ◽  
Matthias Pfeiffer ◽  
Michael Schumm ◽  
Ingo Mueller ◽  
...  
Keyword(s):  
T Cell ◽  
B Cells ◽  
Conflicts Of Interest ◽  
Negative Impact ◽  
Conditioning Regimen ◽  
T Cell Subsets ◽  
Control Group ◽  
T And B Cells ◽  
Haploidentical Transplantation ◽  
Significant Difference

Abstract Abstract 4652 Profound depletion of T and B cells is a fundamental prerequisite for haploidentical transplantation and allows to minimize GvHD despite HLA incompatibility. However, posttransplant recovery of donor derived T cells is delayed after various graft manipulation procedures and may result in severe infections. Methods to improve this recovery are of great importance. Here we present immune reconstitution data in patients who received CD3/CD19 depleted stem cells in combination with melphalan based or standard conditioning regimens. 32 patients with ALL (n=14), AML/MDS (n=17), CML (n=1) were included. T and B cells were directly depleted using antiCD3/antiCD19 coated magnetic microbeads and the CliniMACSTM device. The patients received either TBI or Bu i.v. and OKT3 (n=9) or a reduced intensity conditioning (“RIC”: Mel 140mg/m2, Flud 160mg/m2, TT 10mg/kg, OKT3, n= 23). Absolute numbers of lymphocyte subsets per microliter on day 90 were compared within these both groups and with a historical control group (patients with leukemias who received CD34 selected grafts and TBI or Bu based standard conditioning regimen in combination with ATG, n=28). CD3+4+, CD3+8+ and total numbers of CD3+ of patients after CD3/CD19 depletion were significantly higher in the RIC-group than in the TBI/Bu-group (mean numbers: 85.83 vs. 38.84; 133.46 vs. 19.69; 270.27 vs. 63.99; p<0.05, unpaired t-test). Comparison with the whole CD34 historical group showed a faster recovery of CD3+4+ in patients with CD3/19 depletion and RIC (104.26 vs. 54.22; p= 0,034) but no significant difference in CD3+8+ and CD3+. Furthermore, subgroups of the CD34 historical population were compared: patients with CD3/19 depletion and RIC had a significantly faster recovery of CD3+4+, CD3+8+ and CD3+ than CD34 patients with TBI (104.26vs. 25.48; 133.46vs. 43.17; 270.27 vs. 65.86; p<0.05) but had no advantage over CD34 patients with non-TBI conditioning. Conclusions: the type of graft manipulation appeared to have an influence on the speed of CD4+ recovery (CD3/19 depletion > CD34 selection). Moreover, the use of TBI had a clear negative impact on all T cell subsets: patients with TBI had a slower recovery than patients with non-TBI conditioning, independent from graft manipulation procedures and probably due to thymic damage. Thus, the use of RIC-protocols in combination with CD3/CD19 depletion may help to speed up the immune recovery after haploidentical transplantation. Further studies are warranted to evaluate the risk of relapse. Disclosures: No relevant conflicts of interest to declare.

Massive, Clinical Grade Expansion Of Polyclonal T Cells Using Blinatumomab For Adoptive Autologous Cellular Therapy Of CLL Patients

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3272-3272 ◽  
Author(s):  
Josée Golay ◽  
Anna D’amico ◽  
Gianmaria Borleri ◽  
Maria Chiara Finazzi ◽  
Giulia Quaresmini ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Peripheral Blood ◽  
T Cell Subsets ◽  
Th2 Cells ◽  
Adoptive Therapy ◽  
Specific Patient

Abstract Background The combined use of chemotherapy and monoclonal antibodies has proved highly effective for the treatment of CLL but often results in severe life threatening immunosuppression. The development of adoptive therapy with autologous T cells could be clinically relevant to overcome these problems. Methods We have devised a novel, simple and efficient method for ex vivo expansion of normal autologous T cells from the peripheral blood of CLL patients for adoptive therapy, using blinatumomab (CD3xCD19) and rhIL-2 in serum-free medium. The complete phenotype of in vitro expanded T cells was analyzed by flow cytometry and their cytotoxic activity by calcein release assays. Results We performed 18 expansions of T cells, starting from a very small volume of peripheral blood from untreated CLL patients (mean 10.3 ml, range 2-30 ml) that contained a mean of 9.2x106 T cells (range 0.4 to 51x106)(Fig.1). This method allowed reproducible expansion in about 21 days of a mean 410x106 CD3+ T cells (range 71 to 2184x106). The mean fold expansion of T cells in about 3 weeks of in vitro culture was 224 (range 4.4-1326). The only significant contaminant in final Blinatumomab Expanded T cell cultures (BET) were NK cells (mean 18.5%). Indeed addition of blinatumomab and rhIL-2 to the cultures led to a rapid decrease in CLL B cells, which took place from days 7 to 14 onwards and resulted in their complete depletion within 3 weeks (mean 0.2% CLL B cells at days 18-25). Only in one case, a significant percentage of CLL B cells could be observed at the end of culture, but this was due to the particularly high percentage neoplastic cells in the starting population in this patient (98%), resulting in relatively late depletion of these cells, which took place between days 14 and 21, and therefore remained detectable in this case at day 24 (3.8% CLL B cells at day 24). Despite the very low percentage of starting T cells in this specific patient (1.2%), 152x106 T cells could be obtained, equivalent to a 42 fold expansion. In the 18 expansions performed, the resulting BET cells contained both CD4+ and CD8+ cells in varying proportions (median 46.2% and 44.4% respectively). Only in two cases the final product was composed predominantly of CD4+ cells (95%). Expanded T cells were polyclonal, as shown by TCR Vβ expression which was within the normal range by flow cytometry. Indeed CMV specific clones, detected by CMV peptide (pp65495-503)-loaded HLA-A*0201 tetramer, were expanded using this method and detected in equivalent proportion before and after expansion. Final T cells were composed predominantly of the effector and central memory subsets. Th1 were slightly prevalent over Th2 cells (means 20% and 10%, respectively), whereas Th17 and Treg were less than 1%. Since CLL derived T cells have been shown previously to have enhanced expression of the synapse regulators CD272 and CD279 compared to normal T cells, leading to impaired immunological synapse formation, we have analyzed these markers in both starting and BET cells from 4 patients. We observed that CD272 and CD279 diminished in BET compared to the starting CLL T populations (from 73% to 19% and 61% to 18%, respectively). These data suggest that stimulation and expansion with blinatumomab and rhIL-2 has normalized expression of these regulators on CLL T cells. Indeed BET were highly cytotoxic against CD19+ targets cell lines or primary CLL cells, with 70-90% lysis at a 3:1 effector target ratio in presence of blinatumomab. Finally BET were compared to Xcellerated cells expanded using anti-CD3/CD28 Dynabeads and rhIL-2. The expansion protocols using either blinatumomab or anti-CD3/CD28 Dynabeads showed equivalent efficiency and comparable cell composition at the end of culture. Further comparison of the T cell subsets present in BET or CD3/CD28 cultures is in progress. Conclusions These data altogether suggest that the use of blinatumomab and rhIL-2 provides a reproducible, simple and GMP-compliant protocol, allowing expansion of large numbers of autologous polyclonal T cells depleted of CLL cells, from relatively small volumes of peripheral blood from CLL patients. This approach is an attractive option for adoptive therapy in these patients after immunosuppressive treatments. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Comparison of DNA repair and radiosensitivity of different blood cell populations

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Daniel Heylmann ◽  
Viviane Ponath ◽  
Thomas Kindler ◽  
Bernd Kaina
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Blood Cell ◽  
Peripheral Blood ◽  
Cell Populations ◽  
T And B Cells ◽  
Myeloid Lineage ◽  
Γh2ax Foci

AbstractDespite the frequent use of ionising radiation (IR) in therapy and diagnostics and the unavoidable exposure to external radiation sources, our knowledge regarding the radiosensitivity of human blood cell populations is limited and published data, obtained under different experimental conditions, are heterogeneous. To compare the radiosensitivity of different hematopoietic cell populations, we set out to determine the responses of cells obtained from peripheral blood of healthy volunteers under identical conditions (resting, non-stimulated cells). First, we measured the radiation response of T cells (Treg, Th, CTL), B cells, NK cells, CD34+ progenitor cells and monocytes obtained from peripheral blood and monocyte-derived macrophages (Mph) and immature dendritic cells (iDC) ex vivo and show that T and B cells are highly sensitive, starting to undergo apoptosis following IR with a dose as low as 0.125 Gy. Importantly, there was no clear threshold dose and cell death/apoptosis increased up to a saturation level with a dose of 2 Gy. The sensitivity decreased in the order of T cells > NK and B cells > monocytes > macrophages and iDC. The data confirm a previous report that Mph and iDC are radiation-resistant compared to their progenitor monocytes. Although non-stimulated T and B cells were highly radiation-sensitive compared to monocytes and macrophages, they were competent in the repair of DNA double-strand breaks, as shown by a decline in γH2AX foci in the post-exposure period. CD34+ cells obtained from peripheral blood also showed γH2AX decline post-exposure, indicating they are repair competent. Granulocytes (CD15+) did not display any γH2AX staining following IR. Although peripheral blood lymphocytes, the main fraction are T cells, were significantly more radiation-sensitive than monocytes, they displayed the expression of the repair proteins XRCC1, ligase III and PARP-1, which were nearly non-expressed in monocytes. To assess whether monocytes are depleted in vivo following IR, we measured the amount of T cells and monocytes in cancer patients who received total-body radiation (TBR, 6 × 2 Gy). We observed that the number of T cells in the peripheral blood significantly declined already after the first day of TBR and remained at a low level, which was accompanied by an increase in the number of γH2AX foci in the surviving CD3+ T cell fraction. In contrast, the number of monocytes did not decline extensively, reflecting their radiation resistance compared to T cells. Monocytes also showed an accumulation of γH2AX foci in vivo, but the levels were significantly lower than in T cells. CD56+ NK cells displayed a response similar to T cells. The data support the notion that unstimulated T cell subfractions are nearly equally radiation sensitive. There are, however, remarkable differences in the radiation sensitivity between the lymphoid and the myeloid lineage, with lymphoid cells being significantly more sensitive than cells of the myeloid lineage. In the myeloid lineage, macrophages and iDCs were the most radio-resistant cell types.

HLA-E restricted CD8+ T cell subsets are phenotypically altered in multiple sclerosis patients

2013 ◽  
Vol 20 (7) ◽  
pp. 790-801 ◽  
Author(s):  
Kim Pannemans ◽  
Bieke Broux ◽  
An Goris ◽  
Bénédicte Dubois ◽  
Tom Broekmans ◽  
...  
Keyword(s):  
Multiple Sclerosis ◽  
T Cells ◽  
T Cell ◽  
B Cells ◽  
T Cell Subsets ◽  
Regulatory Role ◽  
T And B Cells ◽  
Autoreactive T Cell ◽  
Multiple Sclerosis Patients

Background: The importance of Qa-1 restricted CD8+ T cells in regulating autoreactive T cell responses has been demonstrated in animal models for autoimmune disorders, including multiple sclerosis (MS). Objective: We hypothesize that their human variant, HLA-E restricted CD8+ T cells, fulfills a similar regulatory role in man and that these cells are of importance in MS. Methods: A large cohort of MS patients and healthy controls was genotyped for the two known HLA-E polymorphisms. Flow cytometry was used to determine HLA-E expression kinetics and to phenotype HLA-E restricted CD8+ T cells. Immunohistochemistry was performed to investigate HLA-E expression in the central nervous system (CNS) of MS patients. Results: HLA-E is upregulated on immune cells upon in vitro activation and this upregulation is polymorphism-dependent for T and B cells. T and B cells in lesions of MS patients show enhanced HLA-E expression. Furthermore, NKG2C+CD8+ T cells of MS patients have a significantly lower Foxp3 expression, while NKG2A+CD8+ T cells of MS patients produce higher levels of pro-inflammatory cytokines compared to those of healthy individuals. Conclusion: Our study indicates that the HLA-E system is altered in MS and could play a regulatory role in disease.

Impact of Labor on Peripheral Blood Maternal T-Cell Subsets and on Regulatory T and B Cells

2016 ◽  
Vol 24 (2) ◽  
pp. 276-284 ◽  
Author(s):  
Jorge Lima ◽  
Catarina Martins ◽  
Glória Nunes ◽  
Maria-José Sousa ◽  
Jorge C. Branco ◽  
...  
Keyword(s):  
T Cell ◽  
B Cells ◽  
Peripheral Blood ◽  
T Cell Subsets ◽  
T And B Cells

scholarly journals Comparison of Lethal and Nonlethal Mouse Models of Orientia tsutsugamushi Infection Reveals T-Cell Population-Associated Cytokine Signatures Correlated with Lethality and Protection

2021 ◽  
Vol 6 (3) ◽  
pp. 121
Author(s):  
Alison Luce-Fedrow ◽  
Suchismita Chattopadhyay ◽  
Teik-Chye Chan ◽  
Gregory Pearson ◽  
John B. Patton ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cell ◽  
Host Response ◽  
Cd8 T Cell ◽  
Cd4 T Cell ◽  
Cell Populations ◽  
Murine Models ◽  
Orientia Tsutsugamushi ◽  
Multicolor Flow Cytometry ◽  
Interstrain Difference

The antigenic diversity of Orientia tsutsugamushi as well as the interstrain difference(s) associated with virulence in mice impose the necessity to dissect the host immune response. In this study we compared the host response in lethal and non-lethal murine models of O. tsutsugamushi infection using the two strains, Karp (New Guinea) and Woods (Australia). The models included the lethal model: Karp intraperitoneal (IP) challenge; and the nonlethal models: Karp intradermal (ID), Woods IP, and Woods ID challenges. We monitored bacterial trafficking to the liver, lung, spleen, kidney, heart, and blood, and seroconversion during the 21-day challenge. Bacterial trafficking to all organs was observed in both the lethal and nonlethal models of infection, with significant increases in average bacterial loads observed in the livers and hearts of the lethal model. Multicolor flow cytometry was utilized to analyze the CD4+ and CD8+ T cell populations and their intracellular production of the cytokines IFNγ, TNF, and IL2 (single, double, and triple combinations) associated with both the lethal and nonlethal murine models of infection. The lethal model was defined by a cytokine signature of double- (IFNγ-IL2) and triple-producing (IL2-TNF-IFNγ) CD4+ T-cell populations; no multifunctional signature was identified in the CD8+ T-cell populations associated with the lethal model. In the nonlethal model, the cytokine signature was predominated by CD4+ and CD8+ T-cell populations associated with single (IL2) and/or double (IL2-TNF) populations of producers. The cytokine signatures associated with our lethal model will become depletion targets in future experiments; those signatures associated with our nonlethal model are hypothesized to be related to the protective nature of the nonlethal challenges.

scholarly journals OP0083 INFECTIOUS AND AUTOINFLAMMATORY MODIC TYPE 1 CHANGES HAVE DIFFERENT PATHOMECHANISMS

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 45.2-45
Author(s):  
I. Heggli ◽  
R. Schüpbach ◽  
N. Herger ◽  
T. A. Schweizer ◽  
A. Juengel ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Immune Response ◽  
T Cell ◽  
Cell Populations ◽  
Gene Set ◽  
Go Enrichment ◽  
Modic Type ◽  
And Control ◽  
Infectious Etiology

Background:Modic type 1 changes (MC1) are vertebral bone marrow (BM) edema that associate with non-specific low back pain (LBP). Two etiologies have been described. In the infectious etiology the anaerobic aerotolerant Cutibacterium acnes (C. acnes) invades damaged intervertebral discs (IVDs) resulting in disc infection and endplate damage, which leads to the evocation of an immune response. In the autoinflammatory etiology disc and endplate damage lead to the exposure of immune privileged disc cells and matrix to leukocytes, thereby evoking an immune response in the BM. Different etiologies require different treatment strategies. However, it is unknown if etiology-specific pathological mechanisms exist.Objectives:The aim of this study was to identify etiology-specific dysregulated pathways of MC1 and to perform in-depth analysis of immune cell populations of the autoinflammatory etiology.Methods:BM aspirates and biopsies were obtained from LBP patients with MC1 undergoing spinal fusion. Aspirates/biopsies were taken prior screw insertion through the pedicle screw trajectory. From each patient, a MC1 and an intra-patient control aspiration/biopsy from the adjacent vertebral level was taken. If C. acnes in IVDs adjacent to MC1 were detected by anaerobic bacterial culture, patients were assigned to the infectious, otherwise to the autoinflammatory etiology.Total RNA was isolated from aspirates and sequenced (Novaseq) (infectious n=3 + 3, autoinflammatory n=5 + 5). Genes were considered as differentially expressed (DEG) if p-value < 0.01 and log2fc > ± 0.5. Gene ontology (GO) enrichment was performed in R (GOseq), gene set enrichment analysis (GSEA) with GSEA software.Changes in cell populations of the autoinflammatory etiology were analyzed with single cell RNA sequencing (scRNAseq): Control and MC1 biopsies (n=1 + 1) were digested, CD45+CD66b- mononuclear cells isolated with fluorescence activated cell sorting (FACS), and 10000 cells were sequenced (10x Genomics). Seurat R toolkit was used for quality-control, clustering, and differential expression analysis.Transcriptomic changes (n=5 + 5) of CD45+CD66b+ neutrophils isolated with flow cytometry from aspirates were analyzed as for total bulk RNAseq. Neutrophil activation (n=3 + 3) was measured as CD66b+ expression with flow cytometry. CD66bhigh and CD66blow fractions in MC1 and control neutrophils were compared with paired t-test.Results:Comparing MC1 to control in total bulk RNAseq, 204 DEG in the autoinflammatory and 444 DEG in the infectious etiology were identified with only 67 shared genes (Fig. 1a). GO enrichment revealed “T-cell activation” (p = 2.50E-03) in the autoinflammatory and “complement activation, classical pathway” (p=1.1E-25) in the infectious etiology as top enriched upregulated biological processes (BP) (Fig 1b). ScRNAseq of autoinflammatory MC1 showed an overrepresentation of T-cells (p= 1.00E-34, OR=1.54) and myelocytes (neutrophil progenitor cells) (p=4.00E-05, OR=2.27) indicating an increased demand of these cells (Fig. 1c). Bulk RNAseq analysis of neutrophils from the autoinflammatory etiology revealed an activated, pro-inflammatory phenotype (Fig 1d), which was confirmed with more CD66bhigh neutrophils in MC1 (+11.13 ± 2.71%, p=0.02) (Fig. 1e).Figure 1.(a) Venn diagram of DEG from total bulk RNAseq (b) Top enriched upregulated BP of autoinflammatory (left) and infectious (right) etiology (c) Cell clustering of autoinflammatory MC1 BM (d) Enrichment of “inflammatory response” gene set in autoinflammatory MC1 neutrophils (e) Representative histogram of CD66b+ expression in MC1 and control neutrophils.Conclusion:Autoinflammatory and infectious etiologies of MC1 have different pathological mechanisms. T-cell and neutrophil activation seem to be important in the autoinflammatory etiology. This has clinical implication as it could be explored for diagnostic approaches to distinguish the two MC1 etiologies and supports developing targeted treatments for both etiologies.Disclosure of Interests:None declared

scholarly journals SELECTIVE TRIGGERING OF HUMAN T AND B LYMPHOCYTES IN VITRO BY POLYCLONAL MITOGENS

1974 ◽  
Vol 140 (1) ◽  
pp. 1-18 ◽  
Author(s):  
Melvyn Greaves ◽  
George Janossy ◽  
Michael Doenhoff
Keyword(s):  
B Cells ◽  
B Lymphocytes ◽  
Human Lymphocytes ◽  
Culture Conditions ◽  
Pokeweed Mitogen ◽  
Cell Populations ◽  
T And B Cells ◽  
Activated T Cells ◽  
Lymphocyte Responses

Human lymphocytes from spleen and tonsils have been cultured with a variety of polyclonal mitogens. Cultures consisted of either unseparated T and B cells or alternatively purified T or B lymphocytes. The purity of the starting cell populations and the origin of activated lymphoblasts was analyzed with a panel of seven markers which discriminate between T and B cells. The selectivity of the lymphocyte responses was influenced by cell populations in a given culture, the mitogen used, and to a limited extent on culture conditions. Purified T lymphocytes from tonsil and spleen responded to phytohemagglutinin (PHA), pokeweed mitogen (PWM), and staphylococcal enterotoxin B (SEB). Purified B cells from spleen responded well to PWM, weakly to SEB and lipopolysaccharide, but not at all to PHA. Tonsil B cells responded weakly to PWM and SEB but not to PHA. Some B lymphocytes do respond to PHA in the presence of activated T cells. These results are discussed in relation to previously reported selective responses of human cells and parallel studies in animal species.

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