scholarly journals SAT0013 PIM-1 KINASE IS A MEASURABLE MEDIATOR OF CD4+ T CELL DYSREGULATION AND THERAPEUTIC TARGET IN EARLY RHEUMATOID ARTHRITIS

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 936.1-937
Author(s):  
N. Maney ◽  
H. De Paula-Lemos ◽  
B. Barron-Millar ◽  
A. Mellor ◽  
J. D. Isaacs ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
T Cells ◽  
T Cell ◽  
Therapeutic Target ◽  
Cd4 T Cells ◽  
Kinase Inhibitors ◽  
Early Arthritis ◽  
Early Ra ◽  
Sat 1

Background:As well as being an established oncoprotein and a therapeutic target in cancer,Proviral Integration site for murine Moloney leukemia virus-1(pim-1) has been implicated in human autoimmunity. We previously confirmed this serine-threonine protein kinase to be strikingly upregulated in circulating CD4+ T cells of untreated rheumatoid arthritis (RA) patients as a consequence of IL-6 signalling1-2. Evidence for the relevance of pim-1 signalling in the disruption of RA synovial fibroblast (RASF) homeostasis3further supports its candidacy as a therapeutic target.Objectives:To investigate PIM1 and its family members (PIM2 and PIM3) as potential candidates for drug repurposing in RA.Methods:A flow cytometric assay for PIM1 transcript measurement in circulating CD4+ T cells of early arthritis patients was validated against real-time PCR in paired cells isolated by bead selection. Synovial protein expression in tissue from the same cohort of untreated RA patients and disease controls was determined by quantitative multiplex immunofluorescence. The functional consequences of manipulating pim kinase family expression in freshly purified T cell receptor (TCR)-stimulated CD4+ T cells from early RA patients was explored. The impact of pim-1 specific and pim-1-3 (pan-pim) kinase inhibition on progression of the IL-6 dependent collagen-induced arthritis (CIA) model was assessed.Results:The percentage of circulating CD4+ T cells positive forPIM1transcript by flow cytometry proved a faithful surrogate for gene expression in early arthritis (Figure 1A), distinguishing RA from other pathologies (Figure 1B). Pim-1 protein expression was increased in the synovium of untreated RA compared with disease controls, including amongst infiltrating CD4+ T cells (Figure 1C-D).In vitro, exposure of TCR-stimulated early RA CD4+ T cells to pim kinase inhibitors restrained their activation and proliferative capacity; diminished pro-inflammatory cytokine production (IFN-g and IL-17) and an expanded CD25hiFoxP3+ regulatory T cell (Treg) fraction were also observed in treatedversusun-treated cells. Finally, administration of pim inhibitors robustly attenuated clinical scores of arthritis in the CIA model, with reduced cartilage loss observed in animals treated with a pan-PIM inhibitor compared with vehicle control (Figure 2).Conclusion:Our data highlight pim kinases as plausible therapeutic targets for a subgroup of early RA patients that may be identifiable using tractable in vitro assays. Pim kinase inhibitors could simultaneously target immune inflammation and RASF dysregulation; consideration should now be given to their repurposing for this condition.References:[1] Anderson AE et al Annals of the Rheumatic Diseases 2016; 75:466-73.[2] Anderson AE et al Rheumatology 2019; 58:1250-1258[3] Ha YJ et al Rheumatology 2019; 58:154-64Disclosure of Interests:Nicola Maney Consultant of: Current employee of Eli Lilly, Henrique De Paula-Lemos: None declared, Ben Barron-Millar: None declared, Andrew Mellor Shareholder of: NewLink Genetics PLC, and has received patent licensing income from this source., John D Isaacs Consultant of: AbbVie, Bristol-Myers Squibb, Eli Lilly, Gilead, Janssen, Merck, Pfizer, Roche, Amy Anderson: None declared, Arthur Pratt Grant/research support from: Pfizer, GlaxoSmithKlein

scholarly journals THU0046 A PIPELINE TO STUDY ANTIGEN-SPECIFIC CD4+ T CELLS IN RHEUMATOID ARTHRITIS

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 235.1-236
Author(s):  
R. Kumar ◽  
N. Yoosuf ◽  
C. Gerstner ◽  
S. Turcinov ◽  
K. Chemin ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
T Cells ◽  
T Cell ◽  
Cd4 T Cells ◽  
Mononuclear Cells ◽  
Tenascin C ◽  
Tetramer Staining ◽  
Tcr Repertoire ◽  
Citrullinated Antigen

Background:Autoimmunity to citrullinated autoantigens forms a critical component of disease pathogenesis in rheumatoid arthritis (RA). Presence of anti-citrullinated protein antibodies (ACPAs) in patients has high diagnostic value. Recently, several citrullinated antigen specific CD4+T cells have been described. However, detailed studies of their T-cell receptor usage and in-vivo profile suffer from the disadvantage that these cells are present at very low frequencies. In this context, we here present a pipeline for TCR repertoire analysis of antigen-specific CD4+T cells from RA patients, including both citrulline and influenza (control) specificities using in-vitro peptide challenge induced-cell expansion.Objectives:To enable studies of the T cell repertoire of citrullinated antigen-specific CD4+T cells in rheumatoid arthritisMethods:Peripheral blood mononuclear cells (PBMCs) (n=7) and synovial fluid mononuclear cells (SFMCs) (n=5) from HLA-DR*0401-postive RA patients were cultured in the presence of citrullinated Tenascin C peptide cocktails or influenza peptides (positive control). Citrulline reactive cells were further supplemented with recombinant human IL-15 and IL-7 on day 2. All cultures were replenished with fresh medium on day 6 and rIL-2 was added every 2 days from then. Assessment of proportion of peptide-HLA-tetramer positive cells was performed using flow cytometry whereby individual antigen-specific CD4+T cells were sorted into 96-well plates containing cell lysis buffer, followed by PCR-based alpha/beta TCR sequencing. TCR sequencing data was demultiplexed and aligned for TCR gene usage using MiXCR. Some tetramer positive cells were sorted into complete medium containing human IL-2 and PHA for expansion of antigen-specific cells. Cells were supplemented with irradiated allogenic PBMCs (30 times number of antigen specific cells). Clones of antigen specific CD4+T cells were further subjected to tetramer staining to confirm expansion of cells.Results:As evidenced by increase in frequency of tetramer positive CD4+T cells, in vitro peptide stimulation resulted in expansion of both influenza specific (Fig. 1a) and citrullinated antigen specific (Fig. 1b) CD4+T cells. Polyclonal in-vitro expansion of tenascin C tetramer positive sorted cells followed by tetramer staining further confirmed antigen specificity and enrichment for antigen specific CD4+T cells after polyclonal stimulation (Fig.1c). TCR repertoire analysis in PB and SF dataset from the first patient showed clonal expansion of influenza specific cells in both sites. Synovial fluid had more diversity of expanding clones as compared to paired PB, with few expanded clones being shared among SF and PB. We observed a more diverse TCR repertoire in citrulline specific CD4+T cells. We also observed sharing of TCR alpha chains among different citrulline specific CD4+T cell clones.Fig. 1In-vitroexpansion of antigen specific CD4+T cells:Conclusion:This method provides a highly suitable approach for investigating TCR specificities of antigen specific CD4+T cells under conditions of low cell yields. Building on this dataset will allow us to assess specific features of TCR usage of autoreactive T cells in RA.PBMCs were cultured in presence of (a) influenza (HA, MP54) and (b) citrullinated tenascin peptides. The proportion of antigen specific CD4+T cells was assessed using HLA-class II tetramer staining. We observed an increase in frequency of (a) Infleunza specific cells (red dots in upper left and lower right quadrants) and (b) citrullinated tenascin C specific cells (red dots in lower right quadrant), at day 13 post culture as compared to day 3. (c) Sorting of citrullinated tenascin specific CD4+T cells, followed by PHA expansion resulted in visible increase in proportion of citrullinated tenascin specific CD4+T cells.Disclosure of Interests:Ravi kumar: None declared, Niyaz Yoosuf: None declared, Christina Gerstner: None declared, Sara Turcinov: None declared, Karine Chemin: None declared, Vivianne Malmström Grant/research support from: VM has had research grants from Janssen Pharmaceutica

scholarly journals AB0045 ULTRASOUND GUIDED BIOPSIES OF RA JOINTS AT TIME OF CLINICAL DIAGNOSIS CONTAIN PROFOUND T CELL CLONALITIES

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 1325.1-1325
Author(s):  
S. Turcinov ◽  
E. Af Klint ◽  
A. De Bondt ◽  
M. S. Mia ◽  
A. Catrina ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Cd4 T Cells ◽  
Clonal Expansion ◽  
Ultrasound Guided ◽  
Alpha Beta

Background:Rheumatoid arthritis (RA) is a disease characterized by synovial joint inflammation, mainly affecting small joints. Histological findings in synovial biopsies ranges from inflammatory infiltration including ectopic lymphoid structures, to a cell sparse fibroid phenotype. T cells in affected joints are non-naïve and have by flow cytometry approaches been shown to have a wide TCR-beta chain gene usage. New technologies allow for analyses of paired TCR sequences and their antigen-specificities.Objectives:To study the alpha/beta-T cell receptor repertoire in single sorted T cells from synovial biopsies at time of RA-diagnosis.Meth ods:Synovial biopsies were taken, primarily using an ultrasound guided technique, from seventeen patients (12 ACPA+, 5 ACPA-) with rheumatoid arthritis. Fresh biopsies were enzymatically digested, followed by mild mechanical treatment, prior to flow cytometry cell sorting. Single cell index sorting of T cells was made into 384-well plates with PCR-buffer followed by a nested PCR and deep sequencing of the TCR amplicons. TCR-receptor sequences showing clonal expansion from four ACPA+ HLA-DRB1*0401 patients were further cloned into SKW3 cells for studies of their reactivity byin vitrostimulation with peptides of viral and citrullinated origin from the literature. A positive response, as measured by CD69-up regulation or IL-2 production, was used to define specificity.Results:Fourteen of the assessed joints were small (1 MTP, 4 MCP and 8 wrists), whereas the remaining three were large joints (2 knees and 1 ankle), table 1. Individual T cells could be isolated from all of these biopsies, with a variating CD4:CD8 ratio. Based on the flow cytometry phenotyping we could identify CD4 T cells of both Treg and T peripheral helper phenotype already at this early time point. Productive alpha/beta-TCR sequences could be retrieved from 16 out of 17 patients and clonal expansion (>1 copy/TCR) was seen in all but one of these patients, with clone sizes ranging between 2 – 34 copies of each TCR.Table 1.Patient characteristics.PatientsGender(F/M)HLA-SE allelesJointsJoint swelling prior to biopsy (months)Stiffness specific joint (median VAS)Pain specific joint (median VAS)ACPA+ (n = 12)9/3*0401, *0404, *0408, *01 and *101 MTP, 4 MCP, 6 wrists, 1 knee4 (1-12)a46 (0-84)45 (22-99)ACPA- (n = 5)3/2*04011 MCP, 2 wrists, 1 ankle, 1 knee5 (0.25-7)59 (15-73)47 (33-81)SKW3 cell lines(patients n = 4)4/0*0401/0404 n=2*0401 n=21 MTP, 3 wrists2 (1-6)50.5 (42-84)50 (40-99)aData not available for one patient. One patient with prior RA-diagnosis, but after 9 months of treatment remission lasting for 20 years.Artificial T cell lines were generated from the expanded clones of HLA-DRB1*04:01 RA subjects. Ourin vitrostimulation protocol identified virus specific CD4 T cells in all samples. So far, no citrulline reactivity has been found. HCMV, followed by HHV were the most commonly found viral reactivities, whereas others were found only in one donor (e.g. JCV, EBV). The majority of clones are thus “orphans”, to which we are still seeking the driving antigen.Conclusion:Clonally expanded T cells are found in the synovium of early RA patients and include virus-specific CD4+ T cells. Our data show that the local T cell repertoire is broad already at the time of RA diagnosisDisclosure of Interests:Sara Turcinov: None declared, Erik af Klint Paid instructor for: Abbvie (courses and lectures), An De Bondt Employee of: Janssen., Muhammad Sohel Mia: None declared, Anca Catrina: None declared, Frederik Stevenaert Employee of: Janssen, Vivianne Malmström Grant/research support from: VM has had research grants from Janssen Pharmaceutica

Comparison of In Vitro and In Vivo Effects of Infliximab on Cytokine Production in Proliferating CD4+ T Cells in Patients with Rheumatoid Arthritis

2015 ◽  
Vol 1 (2) ◽  
pp. 122-128
Author(s):  
Syuichi Koarada ◽  
Yuri Sadanaga ◽  
Natsumi Nagao ◽  
Satoko Tashiro ◽  
Rie Suematsu ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
T Cells ◽  
Cd4 T Cells ◽  
Cytokine Production

scholarly journals Cd40-Independent Pathways of T Cell Help for Priming of Cd8+ Cytotoxic T Lymphocytes

2000 ◽  
Vol 191 (3) ◽  
pp. 541-550 ◽  
Author(s):  
Zhengbin Lu ◽  
Lingxian Yuan ◽  
Xianzheng Zhou ◽  
Eduardo Sotomayor ◽  
Hyam I. Levitsky ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd4 T Cells ◽  
Cytotoxic T Lymphocyte ◽  
Cell Communication ◽  
Cd8 T Cell ◽  
Cd4 Help ◽  
T Cell Help

In many cases, induction of CD8+ CTL responses requires CD4+ T cell help. Recently, it has been shown that a dominant pathway of CD4+ help is via antigen-presenting cell (APC) activation through engagement of CD40 by CD40 ligand on CD4+ T cells. To further study this three cell interaction, we established an in vitro system using dendritic cells (DCs) as APCs and influenza hemagglutinin (HA) class I and II peptide–specific T cell antigen receptor transgenic T cells as cytotoxic T lymphocyte precursors and CD4+ T helper cells, respectively. We found that CD4+ T cells can provide potent help for DCs to activate CD8+ T cells when antigen is provided in the form of either cell lysate, recombinant protein, or synthetic peptides. Surprisingly, this help is completely independent of CD40. Moreover, CD40-independent CD4+ help can be documented in vivo. Finally, we show that CD40-independent T cell help is delivered through both sensitization of DCs and direct CD4+–CD8+ T cell communication via lymphokines. Therefore, we conclude that CD4+ help comprises at least three components: CD40-dependent DC sensitization, CD40-independent DC sensitization, and direct lymphokine-dependent CD4+–CD8+ T cell communication.

scholarly journals CD4+ T Cells from Glutamic Acid Decarboxylase (GAD)65-specific T Cell Receptor Transgenic Mice Are Not Diabetogenic and Can Delay Diabetes Transfer

2002 ◽  
Vol 196 (4) ◽  
pp. 481-492 ◽  
Author(s):  
Kristin V. Tarbell ◽  
Mark Lee ◽  
Erik Ranheim ◽  
Cheng Chi Chao ◽  
Maija Sanna ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Transgenic Mice ◽  
T Cell Receptor ◽  
Glutamic Acid Decarboxylase ◽  
Cd4 T Cells ◽  
Cell Receptor ◽  
Acid Decarboxylase ◽  
Gad 65

Glutamic acid decarboxylase (GAD)65 is an early and important antigen in both human diabetes mellitus and the nonobese diabetic (NOD) mouse. However, the exact role of GAD65-specific T cells in diabetes pathogenesis is unclear. T cell responses to GAD65 occur early in diabetes pathogenesis, yet only one GAD65-specific T cell clone of many identified can transfer diabetes. We have generated transgenic mice on the NOD background expressing a T cell receptor (TCR)-specific for peptide epitope 286–300 (p286) of GAD65. These mice have GAD65-specific CD4+ T cells, as shown by staining with an I-Ag7(p286) tetramer reagent. Lymphocytes from these TCR transgenic mice proliferate and make interferon γ, interleukin (IL)-2, tumor necrosis factor (TNF)-α, and IL-10 when stimulated in vitro with GAD65 peptide 286–300, yet these TCR transgenic animals do not spontaneously develop diabetes, and insulitis is virtually undetectable. Furthermore, in vitro activated CD4 T cells from GAD 286 TCR transgenic mice express higher levels of CTL-associated antigen (CTLA)-4 than nontransgenic littermates. CD4+ T cells, or p286-tetramer+CD4+ Tcells, from GAD65 286–300-specific TCR transgenic mice delay diabetes induced in NOD.scid mice by diabetic NOD spleen cells. This data suggests that GAD65 peptide 286–300-specific T cells have disease protective capacity and are not pathogenic.

scholarly journals CCL21 mediates CD4+ T-cell costimulation via a DOCK2/Rac-dependent pathway

Blood ◽  
2009 ◽  
Vol 114 (3) ◽  
pp. 580-588 ◽  
Author(s):  
Kathrin Gollmer ◽  
François Asperti-Boursin ◽  
Yoshihiko Tanaka ◽  
Klaus Okkenhaug ◽  
Bart Vanhaesebroeck ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cd4 T Cells ◽  
Cell Activation ◽  
Early Time ◽  
Cell Receptor ◽  
Tcr Signaling ◽  
Activation Markers

Abstract CD4+ T cells use the chemokine receptor CCR7 to home to and migrate within lymphoid tissue, where T-cell activation takes place. Using primary T-cell receptor (TCR)–transgenic (tg) CD4+ T cells, we explored the effect of CCR7 ligands, in particular CCL21, on T-cell activation. We found that the presence of CCL21 during early time points strongly increased in vitro T-cell proliferation after TCR stimulation, correlating with increased expression of early activation markers. CCL21 costimulation resulted in increased Ras- and Rac-GTP formation and enhanced phosphorylation of Akt, MEK, and ERK but not p38 or JNK. Kinase-dead PI3KδD910A/D910A or PI3Kγ-deficient TCR-tg CD4+ T cells showed similar responsiveness to CCL21 costimulation as control CD4+ T cells. Conversely, deficiency in the Rac guanine exchange factor DOCK2 significantly impaired CCL21-mediated costimulation in TCR-tg CD4+ T cells, concomitant with impaired Rac- but not Ras-GTP formation. Using lymph node slices for live monitoring of T-cell behavior and activation, we found that G protein-coupled receptor signaling was required for early CD69 expression but not for Ca2+ signaling. Our data suggest that the presence of CCL21 during early TCR signaling lowers the activation threshold through Ras- and Rac-dependent pathways leading to increased ERK phosphorylation.

scholarly journals Response of naive antigen-specific CD4+ T cells in vitro: characteristics and antigen-presenting cell requirements.

1992 ◽  
Vol 176 (5) ◽  
pp. 1431-1437 ◽  
Author(s):  
M Croft ◽  
D D Duncan ◽  
S L Swain
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
Cd4 T Cells ◽  
Cd4 Cells ◽  
Peptide Antigen ◽  
Naive T Cells ◽  
Naïve T Cells ◽  
Antigen Presenting

Because of the low frequency of T cells for any particular soluble protein antigen in unprimed animals, the requirements for naive T cell responses in specific antigens have not been clearly delineated and they have been difficult to study in vitro. We have taken advantage of mice transgenic for the V beta 3/V alpha 11 T cell receptor (TCR), which can recognize a peptide of cytochrome c presented by IEk. 85-90% of CD4+ T cells in these mice express the transgenic TCR, and we show that almost all such V beta 3/V alpha 11 receptor-positive cells have a phenotype characteristic of naive T cells, including expression of high levels of CD45RB, high levels of L-selectin (Mel-14), low levels of CD44 (Pgp-1), and secretion of interleukin 2 (IL-2) as the major cytokine. Naive T cells, separated on the basis of CD45RB high expression, gave vigorous responses (proliferation and IL-2 secretion) to peptide antigen presented in vitro by a mixed antigen-presenting cell population. At least 50% of the T cell population appeared to respond, as assessed by blast transformation, entry into G1, and expression of increased levels of CD44 by 24 h. Significant contributions to the response by contaminating memory CD4+ cells were ruled out by demonstrating that the majority of the CD45RB low, L-selectin low, CD44 high cells did not express the V beta 3/V alpha 11 TCR and responded poorly to antigen. We find that proliferation and IL-2 secretion of the naive CD4 cells is minimal when resting B cells present peptide antigen, and that both splenic and bone marrow-derived macrophages are weak stimulators. Naive T cells did respond well to high numbers of activated B cells. However, dendritic cells were the most potent stimulators of proliferation and IL-2 secretion at low cell numbers, and were far superior inducers of IL-2 at higher numbers. These studies establish that naive CD4 T cells can respond vigorously to soluble antigen and indicate that maximal stimulation can be achieved by presentation of antigen on dendritic cells. This model should prove very useful in further investigations of activation requirements and functional characteristics of naive helper T cells.

scholarly journals Dual T cell– and B cell–intrinsic deficiency in humans with biallelic RLTPR mutations

2016 ◽  
Vol 213 (11) ◽  
pp. 2413-2435 ◽  
Author(s):  
Yi Wang ◽  
Cindy S. Ma ◽  
Yun Ling ◽  
Aziz Bousfiha ◽  
Yildiz Camcioglu ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Cd4 T Cells ◽  
Ex Vivo ◽  
Cell Phenotype ◽  
Loss Of Function ◽  
Inborn Errors

Combined immunodeficiency (CID) refers to inborn errors of human T cells that also affect B cells because of the T cell deficit or an additional B cell–intrinsic deficit. In this study, we report six patients from three unrelated families with biallelic loss-of-function mutations in RLTPR, the mouse orthologue of which is essential for CD28 signaling. The patients have cutaneous and pulmonary allergy, as well as a variety of bacterial and fungal infectious diseases, including invasive tuberculosis and mucocutaneous candidiasis. Proportions of circulating regulatory T cells and memory CD4+ T cells are reduced. Their CD4+ T cells do not respond to CD28 stimulation. Their CD4+ T cells exhibit a "Th2" cell bias ex vivo and when cultured in vitro, contrasting with the paucity of "Th1," "Th17," and T follicular helper cells. The patients also display few memory B cells and poor antibody responses. This B cell phenotype does not result solely from the T cell deficiency, as the patients’ B cells fail to activate NF-κB upon B cell receptor (BCR) stimulation. Human RLTPR deficiency is a CID affecting at least the CD28-responsive pathway in T cells and the BCR-responsive pathway in B cells.

scholarly journals BDC12-4.1 T-Cell Receptor Transgenic Insulin-Specific CD4 T Cells Are Resistant to In Vitro Differentiation into Functional Foxp3+ T Regulatory Cells

PLoS ONE ◽  
2014 ◽  
Vol 9 (11) ◽  
pp. e112242 ◽  
Author(s):  
Ghanashyam Sarikonda ◽  
Georgia Fousteri ◽  
Sowbarnika Sachithanantham ◽  
Jacqueline F. Miller ◽  
Amy Dave ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
Cd4 T Cells ◽  
T Regulatory Cells ◽  
Cell Receptor ◽  
Regulatory Cells ◽  
In Vitro Differentiation

BTK Inhibitors, Irrespective of ITK Inhibition, Increase Efficacy of a CD19/CD3 Bispecific Antibody in CLL

Blood ◽  
2021 ◽  
Author(s):  
Maissa Mhibik ◽  
Erika M. Gaglione ◽  
David Eik ◽  
Ellen K Kendall ◽  
Amy Blackburn ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cytotoxic Activity ◽  
Kinase Inhibitors ◽  
Bispecific Antibody ◽  
Mononuclear Cells ◽  
Cell Function ◽  
Lymphocytic Leukemia ◽  
Peripheral Blood Mononuclear

Bruton Tyrosine Kinase inhibitors (BTKis) are a preferred treatment for patients with chronic lymphocytic leukemia (CLL). Indefinite therapy with BTKis, while effective, presents clinical challenges. Combination therapy can deepen responses, shorten treatment duration, and possibly prevent or overcome drug resistance. We previously reported on a CD19/CD3 bispecific antibody (bsAb) that recruits autologous T cell cytotoxicity against CLL cells in vitro. Compared to observations with samples from treatment-naïve patients, T cells from patients being treated with ibrutinib expanded more rapidly and exerted superior cytotoxic activity in response to the bsAb. In addition to BTK, ibrutinib also inhibits IL2 inducible T cell Kinase (ITK). In contrast, acalabrutinib, does not inhibit ITK. Whether ITK inhibition contributes to the observed immune effects is unknown. To better understand how BTKis modulate T-cell function and cytotoxic activity, we cultured peripheral blood mononuclear cells (PBMCs) from BTKi-naive, and ibrutinib- or acalabrutinib-treated CLL patients with CD19/CD3 bsAb in vitro. T-cell expansion, activation, differentiation, and cytotoxicity were increased in PBMCs from patients on treatment with either BTKi compared to that observed for BKTi-naïve patients. BTKi therapy transcriptionally downregulated immunosuppressive effectors expressed by CLL cells, including CTLA-4 and CD200. CTLA-4 blockade with ipilimumab in vitro increased the cytotoxic activity of the bsAb in BTKi-naïve but not BTKi-treated PBMCS. Taken together, BTKis enhance bsAb induced cytotoxicity by relieving T cells of immunosuppressive restraints imposed by CLL cells. The benefit of combining bsAb immunotherapy with BTKis needs to be confirmed in clinical trials.

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