Setting an intrinsic brake on T cell division number: Roles for TCR affinity and IL-2 concentration

AbstractThe quality of T cell responses depends on the lymphocytes’ ability to undergo clonal expansion, acquire effector functions and traffic to the site of infection. Although TCR signal strength is thought to dominantly shape the T cell response, by using TCR transgenic CD4+ T cells with different pMHC binding affinity, we reveal that TCR affinity does not control Th1 effector function acquisition nor the functional output of individual effectors following mycobacterial infection. Rather, TCR affinity calibrates the rate of cell division to synchronize the distinct processes of T cell proliferation, differentiation and trafficking. By timing cell division-dependent IL-12R expression, TCR affinity controls when T cells become receptive to Th1-imprinting IL-12 signals, determining the emergence and magnitude of the Th1 effector pool. These findings reveal a distinct yet cooperative role for IL-12 and TCR signalling in Th1 differentiation and suggests that the temporal activation of clones with different TCR affinity is a major strategy to coordinate immune surveillance against persistent pathogens.

Download Full-text

Detection of the potentiality before the actuality: Measurement of T‐cell proliferation before cell division occurs

Cytometry Part A ◽

10.1002/cyto.a.24312 ◽

2021 ◽

Author(s):

Daniele Lilleri ◽

Chiara Fornara

Keyword(s):

Cell Proliferation ◽

Cell Division ◽

T Cell ◽

T Cell Proliferation

Download Full-text

AB0144 STRATIFICATION OF PATIENTS WITH PRIMARY SJÖGREN’S SYNDROME BY MEASURING B-CELL HEALTH

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2020-eular.3507 ◽

2020 ◽

Vol 79 (Suppl 1) ◽

pp. 1372-1373

Author(s):

G. M. Verstappen ◽

J. C. Tempany ◽

H. Cheon ◽

A. Farchione ◽

S. Downie-Doyle ◽

...

Keyword(s):

Cell Division ◽

Cell Differentiation ◽

T Cell ◽

B Cells ◽

B Cell ◽

B Cell Differentiation ◽

Research Support ◽

Differentiation Capacity ◽

Cell Responses ◽

Healthy Donors

Background:Primary Sjögren’s syndrome (pSS) is a heterogeneous immune disorder with broad clinical phenotypes that can arise from a large number of genetic, hormonal, and environmental causes. B-cell hyperactivity is considered to be a pathogenic hallmark of pSS. However, whether B-cell hyperactivity in pSS patients is a result of polygenic, B cell-intrinsic factors, extrinsic factors, or both, is unclear. Despite controversies about the efficacy of rituximab, new B-cell targeting therapies are under investigation with promising early results. However, for such therapies to be successful, the etiology of B-cell hyperactivity in pSS needs to be clarified at the individual patient level.Objectives:To measure naïve B-cell function in pSS patients and healthy donors using quantitative immunology.Methods:We have developed standardised, quantitative functional assays of B-cell responses that measure division, death, differentiation and isotype switching, to reveal the innate programming of B cells in response to T-independent and dependent stimuli. This novel pipeline to measure B-cell health was developed to reveal the sum total of polygenic defects and underlying B-cell dysfunction at an individual level. For the current study, 25 pSS patients, fulfilling 2016 ACR-EULAR criteria, and 15 age-and gender-matched healthy donors were recruited. Standardized quantitative assays were used to directly measure B cell division, death and differentiation in response to T cell-independent (anti-Ig + CpG) and T-cell dependent (CD40L + IL-21) stimuli. Naïve B cells (IgD+CD27-) were sorted from peripheral blood mononuclear cells and were labeled with Cell Trace Violet at day 0 to track cell division until day 6. B cell differentiation was measured at day 5.Results:Application of our standardized assays, and accompanying parametric models, allowed us to study B cell-intrinsic defects in pSS patients to a range of stimuli. Strikingly, we demonstrated a hyperresponse of naïve B cells to combined B cell receptor (BCR) and Toll-like receptor (TLR)-9 stimulation in pSS patients. This hyperresponse was revealed by an increased mean division number (MDN) at day 5 in pSS patients compared with healthy donors (p=0.021). A higher MDN in pSS patients was observed at the cohort level and was likely attributed to an increased division burst (division destiny) time. The MDN upon BCR/TLR-9 stimulation correlated with serum IgG levels (rs=0.52; p=0.011). No difference in MDN of naïve B cells after T cell-dependent stimulation was observed between pSS patients and healthy donors. B cell differentiation capacity (e.g., plasmablast formation and isotype switching) after T cell-dependent stimulation was also assessed. At the cohort level, no difference in differentiation capacity between groups was observed, although some pSS patients showed higher plasmablast frequencies than healthy donors.Conclusion:Here, we demonstrate defects in B-cell responses both at the cohort level, as well as individual signatures of defective responses. Personalized profiles of B cell health in pSS patients reveal a group of hyperresponsive patients, specifically to combined BCR/TLR stimulation. These patients may benefit most from B-cell targeted therapies. Future studies will address whether profiles of B cell health might serve additional roles, such as prediction of disease trajectories, and thus accelerate early intervention and access to precision therapies.Disclosure of Interests:Gwenny M. Verstappen: None declared, Jessica Catherine Tempany: None declared, HoChan Cheon: None declared, Anthony Farchione: None declared, Sarah Downie-Doyle: None declared, Maureen Rischmueller Consultant of: Abbvie, Bristol-Meyer-Squibb, Celgene, Glaxo Smith Kline, Hospira, Janssen Cilag, MSD, Novartis, Pfizer, Roche, Sanofi, UCB, Ken R. Duffy: None declared, Frans G.M. Kroese Grant/research support from: Unrestricted grant from Bristol-Myers Squibb, Consultant of: Consultant for Bristol-Myers Squibb, Speakers bureau: Speaker for Bristol-Myers Squibb, Roche and Janssen-Cilag, Hendrika Bootsma Grant/research support from: Unrestricted grants from Bristol-Myers Squibb and Roche, Consultant of: Consultant for Bristol-Myers Squibb, Roche, Novartis, Medimmune, Union Chimique Belge, Speakers bureau: Speaker for Bristol-Myers Squibb and Novartis., Philip D. Hodgkin Grant/research support from: Medimmune, Vanessa L. Bryant Grant/research support from: CSL

Download Full-text

TCR Affinity Controls the Dynamics but Not the Functional Specification of the Antimycobacterial CD4+ T Cell Response

The Journal of Immunology ◽

10.4049/jimmunol.2001271 ◽

2021 ◽

pp. ji2001271

Author(s):

Nayan D. Bhattacharyya ◽

Claudio Counoupas ◽

Lina Daniel ◽

Guoliang Zhang ◽

Stuart J. Cook ◽

...

Keyword(s):

T Cell ◽

Cell Response ◽

T Cell Response ◽

Cd4 T Cell ◽

Functional Specification ◽

Tcr Affinity

Download Full-text

T Cell Effector Function and Anergy Avoidance Are Quantitatively Linked to Cell Division

The Journal of Immunology ◽

10.4049/jimmunol.165.5.2432 ◽

2000 ◽

Vol 165 (5) ◽

pp. 2432-2443 ◽

Cited By ~ 78

Author(s):

Andrew D. Wells ◽

Matthew C. Walsh ◽

David Sankaran ◽

Laurence A. Turka

Keyword(s):

Cell Division ◽

T Cell ◽

Effector Function ◽

Cell Effector Function ◽

Cell Effector

Download Full-text

Imaging Asymmetric T Cell Division

The Immune Synapse - Methods in Molecular Biology ◽

10.1007/978-1-4939-6881-7_23 ◽

2017 ◽

pp. 383-398 ◽

Cited By ~ 4

Author(s):

Mirren Charnley ◽

Sarah M. Russell

Keyword(s):

Cell Division ◽

T Cell

Download Full-text

P01.13 MERTK signaling is critical for T cell proliferation and memory

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-itoc7.26 ◽

2020 ◽

Vol 8 (Suppl 2) ◽

pp. A14.2-A15

Author(s):

RM Powell ◽

MJW Peeters ◽

A Rachbech ◽

PT Straten

Keyword(s):

T Cells ◽

Cell Division ◽

T Cell ◽

Cd8 T Cells ◽

Central Memory ◽

T Cell Proliferation ◽

Flow Cytometric ◽

Donor T Cells ◽

Memory Differentiation

BackgroundOverexpression of TAM receptors, including MERTK, in some cancers are integral for chemoresistance, proliferation and metastasis.1 Our group has previously demonstrated that T cells also express MERTK and engagement of MERTK signaling is responsible for increased proliferation, functional capacity and metabolic fitness.2 It is therefore important to further study the effect of MERTK inhibition on T cell function in the context of cancer treatments where MERTK inhibitors may play a role. Here we provide evidence that MERTK inhibition impacts greatly on T cell proliferation, specifically reducing phosphorylated mTOR. We have also demonstrated that MERTK expression is increased on CD8 central memory subsets during longterm expansion providing evidence that this signaling pathway may be important for sustaining T memory responses.Materials and MethodsFlow cytometric analysis was used to investigate the effect of titration of MERTK small molecule inhibitor UNC2025 on healthy donor T cells activated with CD3/CD28 dynabeads. Cell trace dye was used to track proliferation of CD4 and CD8 T cells along with markers of memory differentiation (CCR7 and CD45RO), activation (CD137) and function (IFNy, Tnfa and IL-2). MERTK signaling was assessed using phospho flow cytometric methodology of phosphorylated mTOR, AKT, ERK1/2, p38-MAPK and STAT5. Long term cultures of donor T cells of up to 28 days were investigated for MERTK expression alongside memory differentiation.ResultsWe demonstrated that moderate concentrations of MERTK inhibitor reduced proliferation of activated T cells. Despite inhibition of cell division, cell size still increased 2 fold compared to resting cells and cell viability remained unchanged. Additionally, the proportion of central memory to effector memory populations and intracellular cytokine production was not impacted. Analysis of molecules involved in MERTK signaling revealed that phosphorylated mTOR was significantly modulated following the addition of MERTK inhibitor. Long term culture of CD8 T cells demonstrated MERTK was significantly increased following early and late re-stimulation, and expression of MERTK was strongly associated with central memory subsets.ConclusionsOur results demonstrate that inhibition of MERTK signaling on T cells reduces cell division where mTOR is significantly impacted. Despite this, other functional aspects, such as intracellular cytokine production remain unchanged. Therefore, interruption of MERTK signaling on T cells has a specific effect on cell division rather than cytotoxic function on a cell by cell basis. This has potential ramifications on the use of MERTK inhibitors to treat tumors where the ability to form substantial cytotoxic T cell populations might be reduced. In addition, increased MERTK expression on central memory subsets during long term culture suggests this signaling pathway could be critical for generating memory pools of T cells and provide new avenues for the improvement of adoptive T cell therapy protocols.ReferencesCummings CT, Deryckere D, Earp HS, Graham DK. Molecular pathways: MERTK signaling in cancer. Clin Cancer Res 2013;19(19):5275–5280.Peeters MJW, Dulkeviciute D, Draghi A, et al. MERTK Acts as a Costimulatory Receptor on Human CD8+T Cells. Cancer Immunol Res 2019;7(9):1472–1484.Disclosure InformationR.M. Powell: None. M.J.W. Peeters: None. A. Rachbech: None. P.T. Straten: None.

Download Full-text

A Human Hematopoietic Environment and Autologous T-Cell Growth Support Proliferation of Chronic Lymphocytic Leukemia B Cells in Immune-Deficient Mice

Blood ◽

10.1182/blood.v112.11.26.26 ◽

2008 ◽

Vol 112 (11) ◽

pp. 26-26

Author(s):

Davide Bagnara ◽

Matthew Kaufman ◽

Xiao J. Yan ◽

Kanti Rai ◽

Nicholas Chiorazzi

Keyword(s):

Cell Division ◽

T Cell ◽

B Cells ◽

Cell Growth ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Hematopoietic Microenvironment ◽

Cd34 Cells ◽

T Cell Growth

Abstract B-cell type chronic lymphocytic leukemia (B-CLL), an incurable disease of unknown etiology, results from the clonal expansion of a CD5+CD19+ B lymphocyte. Progress into defining the cell of origin of the disease and identifying a stem cell reservoir has been impeded because of the lack of reproducible models for growing B-CLL cells in vitro and in vivo. To date, attempts to adoptively transfer B-CLL cells into immune deficient mice and achieve engraftment and growth are sub-optimal. At least one possible cause for this is the murine microenvironment’s inability to support B-CLL survival and proliferation. We have attempted to overcome this barrier by creating a human hematopoietic microenvironment by reconstituting the tibiae of NOD/SCID/γcnull mice by intrabone (ib) injection of 1–3 × 105 CD34+ cord blood cells along with ~106 bone marrow-derived human mesenchymal stem cells (hMSCs). When human engraftment of 1–10% CD45+ cells was documented in the blood by immunofluorescence using flow cytometry, a total of 108 PBMCs from individual B-CLL patients were injected into the same bones. Before injection, B-CLL PBMCs were labeled with CFSE to permit distinction of leukemic B cells from normal B cells that might arise from the injected CD34+ cells. CFSE labeling also permitted tracking initial rounds of cell division in vivo. Every two weeks after B-CLL injection, peripheral blood from the mice was examined for the presence of cells bearing human CD45, CFSE, and various human lineage markers by flow cytometry. In the presence of a human hematopoietic microenvironment, CD5+CD19+ leukemic cells underwent at least 6 cell doublings, after which CFSE fluorescence was no longer detectable. Timing of B-CLL cell division varied among patients, occurring between 2 to 6 weeks after the injection of PBMC. In contrast, leukemic cells injected into mice that were not reconstituted by ib injection with hCD34+ cells and hMSCs or were reconstituted with only hMSCs failed to proliferate. Moreover the number of CFSE+CD5+CD19+ cells detected in the blood of mice with a human hematopoietic microenvironment far exceeded that in mice receiving only hMSC. Robust T-cell expansion occurred in several mice receiving CD34+ cells; in some instances T-cell growth was also found without hCD34+ cell injection, although in these cases it was usually less extensive. Based on genome-wide SNP analyses, the T cells were of B-CLL patient origin and not from hCD34+ cells. Furthermore, most of the mice with significant T-cell overexpansion died within 6 weeks of B-CLL cell injection from apparent graft vs. host disease. Therefore in subsequent experiments, we eliminated T cells by injecting an anti-CD3 antibody (OKT3); this treatment led to an inhibition of B-CLL cell proliferation. Moreover the percentage of CD38+ cells in the CFSE+CD5+CD19+ cell fraction was similar to that in the donor patient inoculum only in the mice in which T-cell-mediated B-CLL cell proliferation occurred. The percentage and intensity of CD38− expressing B-CLL cells was higher in the spleen and bone marrow (BM) of mice not treated with OKT3 antibody. Finally, the percentage of CFSE+CD5+CD19+ cells in the spleen far exceeded that in the blood, BM, liver and peritoneum, even when leukemic cells were no longer present in the blood and other organs; these findings suggest that the spleen is better at supporting B-CLL cell viability and proliferation than the other anatomic sites. These studies demonstrate conditions making adoptive xenogeneic transfer and clonal expansion of B-CLL cells into a mouse model possible. Factors conferring an advantage in this model include both a human hematopoietic environment and autologous T cell growth. Increased numbers of CD38+ B-CLL cells, similar to those in the patient, were only found when leukemic B cell division occurred. The optimal site for B-CLL cell growth was murine spleen. Since non-genetic factors promoting B-CLL expansion are not completely known, this model will be useful in discovering these as well as for studying the basic biology of this disease, such as if leukemic stem cells exist and also to conduct preclinical tests on possible therapeutics.

Download Full-text

Turning Tregs into class I suppressors

Blood ◽

10.1182/blood-2012-02-410969 ◽

2012 ◽

Vol 119 (15) ◽

pp. 3373-3374 ◽

Cited By ~ 2

Author(s):

Terrence L. Geiger

Keyword(s):

T Cells ◽

T Cell ◽

Regulatory T Cells ◽

Mhc Class I ◽

T Cell Receptors ◽

Class I ◽

Cell Receptors ◽

Tcr Affinity

In this issue of Blood, Plesa et al demonstrate that human Foxp3+ regulatory T cells can be redirected using MHC class I–restricted T-cell receptors (TCRs), showing a surprising lack of correlation of TCR affinity and their suppressive potency.1

Download Full-text