Evaluating the antitumor role of B cells in patients with non-small cell lung cancer.

2017 ◽  
Vol 35 (7_suppl) ◽  
pp. 75-75 ◽  
Author(s):  
Tullia C. Bruno
Keyword(s):  
B Cells ◽  
Antigen Presentation ◽  
T Regulatory Cells ◽  
Tumor Antigens ◽  
Nsclc Patient ◽  
Tumor Infiltrating Lymphocytes ◽  
Autologous Tumor ◽  
Adjacent Tissue ◽  
Nsclc Patients

75 Background: The focus of immunotherapy has been on CD8 and CD4 tumor infiltrating lymphocytes (TILs), however, tumor infiltrating B cells (TIL-Bs) have been reported in tertiary lymphoid structures (TLS) with CD4 TILs, and both TIL-Bs and TLS correlate with NSCLC patient survival. While TIL-Bs have been identified in NSCLC patients, their function in the tumor microenvironment has been understudied with no focus on their role as antigen presenting cells (APCs) and their influence on CD8 and CD4 TILs. We hypothesize that TIL-Bs help generate potent, long-term immune responses against cancer by presenting tumor antigens to CD4 TILs. Methods: We used freshly isolated B cells from NSCLC patient tumor and tumor adjacent tissue to determine TIL-B function. We generated a specific in vitro antigen presentation assay to determine APC function and immunoregulation by TIL-Bs, and we correlated our findings with tumor-free survival. Results: We observed that the total number of B cells at the site of the tumor versus the tumor-adjacent tissue was increased compared to other immune subsets. Further, we observed three types of CD4 TIL responses when TIL-Bs presented autologous tumor antigens. There were activated responder CD4 TILs that proliferated when combined with TIL-Bs alone, which indicates stimulation with endogenous tumor antigens. There were antigen-associated responders that required exogenous autologous tumor lysate to elicit a CD4 TIL response, and there were patient CD4 TILs that did not respond to antigen presentation by TIL-Bs. Within the activated and antigen-associated responders, the TIL-B phenotype influenced the CD4 TIL phenotype; if the TIL-Bs were activated (CD69+CD27+CD21+), the CD4 TILs were T helper (anti-tumor) CD4 T cells and if the TIL-Bs were exhausted (CD69-CD27-CD21-), the CD4 TILs were T regulatory cells (pro-tumor). These data suggest that TIL-Bs influence the phenotype and function of CD4 TILs in NSCLC patient tumors. Conclusions: In conclusion, determining if TIL-Bs are activated or exhausted in NSCLC patients will determine the extent of their anti-tumor function in cancer. Ultimately, results from this study will help predict how to target TIL-B functions in future immunotherapies for NSCLC patients.

scholarly journals Rapid Identification of the Tumor-Specific Reactive TIL Repertoire via Combined Detection of CD137, TNF, and IFNγ, Following Recognition of Autologous Tumor-Antigens

2021 ◽  
Vol 12 ◽  
Author(s):  
Arianna Draghi ◽  
Christopher Aled Chamberlain ◽  
Shawez Khan ◽  
Krisztian Papp ◽  
Martin Lauss ◽  
...  
Keyword(s):  
Tumor Antigens ◽  
De Novo ◽  
Tumor Infiltrating Lymphocytes ◽  
Rapid Identification ◽  
Autologous Tumor ◽  
Simple Method ◽  
Straightforward Method ◽  
Infiltrating Lymphocytes ◽  
Combined Detection

Detecting the entire repertoire of tumor-specific reactive tumor-infiltrating lymphocytes (TILs) is essential for investigating their immunological functions in the tumor microenvironment. Current in vitro assays identifying tumor-specific functional activation measure the upregulation of surface molecules, de novo production of antitumor cytokines, or mobilization of cytotoxic granules following recognition of tumor-antigens, yet there is no widely adopted standard method. Here we established an enhanced, yet simple, method for identifying simultaneously CD8+ and CD4+ tumor-specific reactive TILs in vitro, using a combination of widely known and available flow cytometry assays. By combining the detection of intracellular CD137 and de novo production of TNF and IFNγ after recognition of naturally-presented tumor antigens, we demonstrate that a larger fraction of tumor-specific and reactive CD8+ TILs can be detected in vitro compared to commonly used assays. This assay revealed multiple polyfunctionality-based clusters of both CD4+ and CD8+ tumor-specific reactive TILs. In situ, the combined detection of TNFRSF9, TNF, and IFNG identified most of the tumor-specific reactive TIL repertoire. In conclusion, we describe a straightforward method for efficient identification of the tumor-specific reactive TIL repertoire in vitro, which can be rapidly adopted in most cancer immunology laboratories.

EXTH-48. NOVEL COMBINATION THERAPY IN PRECLINICAL GLIOMA MODELS USING THE CELL CYCLE STABILIZING COMPOUND ARGYRIN F AND CHECKPOINT INHIBITION

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi174-vi174
Author(s):  
Bianca Walter ◽  
Denis Canjuga ◽  
Simge G Yuez ◽  
Michael Ghosh ◽  
Przemyslaw Bozko ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Ex Vivo ◽  
Tumor Infiltrating Lymphocytes ◽  
Clonogenic Survival ◽  
Autologous Tumor ◽  
Cycle Arrest ◽  
Resected Tissue ◽  
Infiltrating Lymphocytes

Abstract Glioblastoma are incurable aggressive tumors and remain a therapeutic challenge. Glioblastoma frequently harbor alterations in the retinoblastoma pathway with subsequent cell cycle abnormalities. Here, we aimed to investigate the anti-glioma activity of the cell cycle-stabilizing compound Argyrin F and its potential treatment-induced vulnerabilities to exploit possibilities for novel combination therapies. We investigated cell viability, clonogenic survival, cell cycle status and immunoblots of human and murine glioma cells treated with Argyrin F. Moreover, we established an ex vivo glioma model using residual freshly resected tissue from patients, i.e. patient-derived microtumors (PDMs). Additionally, we extracted autologous tumor infiltrating lymphocytes (TILs) to perform co-culturing experiments. We performed mass spectrometry-based immunopeptidomics and used the orthotopic syngeneic SMA560/VM/Dk glioma mouse model. Argyrin F displayed anti-glioma efficacy in glioma cell lines in vitro and in PDM models ex vivo. Moreover, Argyrin F treatment induced cell cycle arrest, reduced clonogenic survival in vitro and prolonged survival in vivo. Argyrin F-treated SMA560 glioma displayed 4.6-fold more glioma-infiltrating CD8+ T cells. We discovered a distinctive treatment-induced immunopeptidome. Combination of Argyrin F plus PD-1 antibody increased cellular toxicity in PDM/TILs co-cultures ex vivo and prolonged overall survival compared with monotherapies in vivo. We conclude that our experimental data suggest a novel combination of Argyrin F plus PD-1 blockade and its clinical translation.

CTIM-22. NIVOLUMAB AND BEVACIZUMAB FOR RECURRENT GLIOBLASTOMA; T-CELL REACTIVITY AGAINST AUTOLOGOUS TUMOR CELLS

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi54-vi55
Author(s):  
Simone Maarup ◽  
Signe Skadborg ◽  
Annie Borch ◽  
Arianna Draghi ◽  
Benedikte Hasselbalch ◽  
...  
Keyword(s):  
Intracellular Cytokine Staining ◽  
Tumor Infiltrating Lymphocytes ◽  
Recurrent Glioblastoma ◽  
Autologous Tumor ◽  
Open Label ◽  
Cell Reactivity ◽  
Translational Study ◽  
Novel Treatments ◽  
Before And After

Abstract INTRODUCTION Glioblastoma is an aggressive brain tumor with a median survival of 14.6 months. We have no standard treatment for relapse and known options have limited effect. Novel treatments are necessary to improve survival and quality of life. METHODS We present our trial; phase II open label, two-armed translational study of Nivolumab and Bevacizumab for recurrent GBM, who have failed Stupp’s regimen. Patients are included in two arms depending on the possibility of salvage neurosurgical resection. Both arms receive Nivolumab and Bevacizumab administrated every second weekend, and the surgical arm also receive Nivolumab 7 days prior surgery. Forty-four patients were included by January 2021; 20 in each arm (four screen-failures). In the surgical arm, 20 fresh tumor samples as well as paired tissue from primary tumor were available. Tumor infiltrating lymphocytes (TILs) and tumor digest were produced in vitro from recurrent settings. Young TILs were expanded from fresh tumor fragments after minimal-culture, whereas rapidly expanded TILs (REP TILs) were obtained after massive expansion. By intracellular cytokine staining, we investigated the TIL reactivity after exposure to autologous tumor digest in order to evaluate whether the TILs were tumor-reactive, non-reactive or bystanders. RNA and whole exome sequencing were available before and after treatment. RESULTS Material from 19 patients was analyzed (one out of the 20 collected biopsies was limited in size, therefore no tumor digest could be produced). Four out of 19 TIL samples showed tumor reactivity after exposure to the autologous tumor digest. Tumor reactivity was ranged between 1,2 to 13,6 tox% in CD8+ TILs and between 2,8 to 10,9 tox% in CD4+ TILs. By flowcytometry we found, IgG4+ CD3+ TILS from tumor biopsies, meaning that Nivolumab were found in the brain. Currently controls are included to evaluate these results. CONCLUSIONS Updated results will be presented at SNO.

scholarly journals Qualitative Analysis of Tumor-Infiltrating Lymphocytes across Human Tumor Types Reveals a Higher Proportion of Bystander CD8+ T Cells in Non-Melanoma Cancers Compared to Melanoma

Cancers ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 3344
Author(s):  
Aishwarya Gokuldass ◽  
Arianna Draghi ◽  
Krisztian Papp ◽  
Troels Holz Borch ◽  
Morten Nielsen ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
T Cells ◽  
T Cell ◽  
Tumor Infiltrating Lymphocytes ◽  
Autologous Tumor ◽  
Epithelial Cancers ◽  
Single Cell Rna Sequencing ◽  
Tumor Types ◽  
Infiltrating Lymphocytes

Background: Human intratumoral T cell infiltrates can be defined by quantitative or qualitative features, such as their ability to recognize autologous tumor antigens. In this study, we reproduced the tumor-T cell interactions of individual patients to determine and compared the qualitative characteristics of intratumoral T cell infiltrates across multiple tumor types. Methods: We employed 187 pairs of unselected tumor-infiltrating lymphocytes (TILs) and autologous tumor cells from patients with melanoma, renal-, ovarian-cancer or sarcoma, and single-cell RNA sequencing data from a pooled cohort of 93 patients with melanoma or epithelial cancers. Measures of TIL quality including the proportion of tumor-reactive CD8+ and CD4+ TILs, and TIL response polyfunctionality were determined. Results: Tumor-specific CD8+ and CD4+ TIL responses were detected in over half of the patients in vitro, and greater CD8+ TIL responses were observed in melanoma, regardless of previous anti-PD-1 treatment, compared to renal cancer, ovarian cancer and sarcoma. The proportion of tumor-reactive CD4+ TILs was on average lower and the differences less pronounced across tumor types. Overall, the proportion of tumor-reactive TILs in vitro was remarkably low, implying a high fraction of TILs to be bystanders, and highly variable within the same tumor type. In situ analyses, based on eight single-cell RNA-sequencing datasets encompassing melanoma and five epithelial cancers types, corroborated the results obtained in vitro. Strikingly, no strong correlation between the proportion of CD8+ and CD4+ tumor-reactive TILs was detected, suggesting the accumulation of these responses in the tumor microenvironment to follow non-overlapping biological pathways. Additionally, no strong correlation between TIL responses and tumor mutational burden (TMB) in melanoma was observed, indicating that TMB was not a major driving force of response. No substantial differences in polyfunctionality across tumor types were observed. Conclusions: These analyses shed light on the functional features defining the quality of TIL infiltrates in cancer. A significant proportion of TILs across tumor types, especially non-melanoma, are bystander T cells. These results highlight the need to develop strategies focused on the tumor-reactive TIL subpopulation.

In Vitro Expansion of Autologous Follicular Lymphoma-Specific T Cells for Adoptive Transfer.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1482-1482
Author(s):  
Seung-Tae Lee ◽  
Yun Fang Jiang ◽  
Soung-Chul Cha ◽  
Hong Qin ◽  
Larry W. Kwak ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Follicular Lymphoma ◽  
Tumor Cells ◽  
Cd4 T Cells ◽  
Cd40 Ligand ◽  
Autologous Tumor ◽  
T Cell Lines

Abstract Advanced stage follicular lymphoma remains an incurable disease with a median survival of 8 to 10 years that has not significantly changed over the last four decades. Therefore, novel treatment options are necessary to improve the clinical outcome in these patients. The observation of spontaneous regressions in a small percentage of patients suggested that augmenting the host immune response could potentially control this malignancy. Strategies using active specific immunotherapy with idiotype vaccines led to induction of clinical and molecular responses in a few patients but have met with only limited success possibly due to the low frequency of antigen-specific T cells induced in the patients. In contrast to active immunization, T cells of a given specificity and function may be selected and expanded in vitro to the desired number for adoptive cell transfer. Towards this goal, we stimulated tumor infiltrating lymphocytes (TILs) or peripheral blood mononuclear cells (PBMCs) from five follicular lymphoma patients with CD40 ligand-activated autologous tumor cells at approximately ten-day intervals in the presence of IL-2 and IL-15. After four rounds of stimulations, T cell lines generated from 3/5 patients recognized autologous unmodified tumor cells by producing significant amounts of TNF-α, GM-CSF and/or IFN-γ. By phenotypic analysis, the T cell lines were predominantly CD4+ T cells (> 70%), and intracellular cytokine assay showed that up to 40% of the CD4+ T cells were tumor-reactive. The inhibition of cytokine production by anti-HLA class II but not class I blocking antibodies confirmed that the CD4+ T cells were tumor-reactive. Further characterization revealed that the T cells from one patient recognized autologous tumor but not autologous normal B cells suggesting that they were tumor-specific. While in a second patient CD4+ T cell clones generated from the T cell line by limiting dilution recognized autologous tumor and autologous normal B cells but not autologous monocytes suggesting that they were B cell lineage-specific. We conclude that follicular lymphoma-specific T cells exist and can be efficiently expanded in vitro from both TILs and PBMCs using CD40 ligand-activated autologous tumor cells for adoptive T cell therapy. Additionally, identification of antigens recognized by these T cells could lead to development of novel immunotherapeutic strategies for lymphomas.

Th17 responses to autologous dendritic cell vaccine.

2019 ◽  
Vol 37 (8_suppl) ◽  
pp. 132-132
Author(s):  
Gabriel I. Nistor ◽  
Aleksandra J. Poole ◽  
Candace Hsieh ◽  
Hans S. Keirstead ◽  
Robert O. Dillman
Keyword(s):  
Tumor Cells ◽  
Tumor Markers ◽  
Tumor Antigens ◽  
Ex Vivo ◽  
Advanced Ovarian Cancer ◽  
Dendritic Cell Vaccine ◽  
Cell Vaccine ◽  
Autologous Tumor ◽  
Type Response

132 Background: Dendritic cells (DC) loaded with autologous tumor antigens in vitro, can induce a Th17 phenotype preceding a Th1 type response. We examined this in patients with metastatic melanoma who had been treated in a randomized trial with either DC loaded with autologous tumor antigens (ATA) from self-renewing tumor cells (DCV) or irradiated tumor cells (TCV). Methods: Blood samples were obtained at baseline, one week before three weekly vaccinations (week-0), and at week-4, (one week after the third vaccination). They were analyzed for growth factors, tumor markers and biomarkers using a quantitative, multiplex ELISA. Monocytes, lymphocytes, loaded and unloaded DC were analyzed for cytokine production and intercellular responses using mixed lymphocyte co-cultures (MLC). Results: When compared to baseline or the TCV arm, DCV-treated patients showed reductions in tumor markers, inflammation and angiogenesis in parallel with Th17 pathway activation, IL12 p70 heterodimer expression was detected in monocytes and DC only after antigen loading. The IL12 p40 fraction was present after differentiation and was enhanced after antigen loading. Similarly, the p19 component of IL23 was expressed before DC loading and later enhanced. MLC showed induction of Th17, Th1 and CD8 subpopulations. In the presence of antigen-loaded DCs, the FOXp3 cells were reduced in number, an effect that was canceled by neutralizing the IL12p40 subunit or by the adding tumor cells alone in the MLC. Conclusions: DC produced ex vivo can activate multiple cytotoxic pathways depending on IL12 subunits. Expression of IL23p19 and the shared IL12p40 fraction are responsible for Th1 priming (IL12 incomplete) and Th17 induction (IL23 complete), determinants of the reactive phenotype of naïve DCs. IL12 p70 heterodimer expression after DC loading enables a targeted Th1 type response while neutralizing the IL12 subunit p40, enhances Foxp3 positive T-regs. DC vaccines produced ex vivo could induce a Th1/Th17 cytotoxic response against tumors. In the absence of antigens, however, The Th17 phenotype can become tolerizing in the microenvironment. The findings are important for applications in settings in which tumor infiltration with Th17 correlates with survival, such as in advanced ovarian cancer. Clinical trial information: NCT00436930.

Generation and characterization of antitumor infiltrating lymphocytes from patients with breast cancer for adoptive cell transfer therapy.

2017 ◽  
Vol 35 (7_suppl) ◽  
pp. 145-145
Author(s):  
Juhua Zhou ◽  
Yin Zhong ◽  
Zhongjun Hou ◽  
Jianzhong Zhang ◽  
Yanmin Li ◽  
...  
Keyword(s):  
Breast Cancer ◽  
T Cells ◽  
Tumor Cells ◽  
Tumor Infiltrating Lymphocytes ◽  
Adoptive Cell Transfer ◽  
Autologous Tumor ◽  
Cell Transfer ◽  
Adoptive Cell Transfer Therapy ◽  
Infiltrating Lymphocytes

145 Background: Clinical trials have shown that adoptive cell transfer therapy is a promising method for cancer treatment. In the current study, we aim to generate and characterize anti-tumor tumor-infiltrating lymphocytes from patients with breast cancer for adoptive cell transfer therapy. Methods: In vitro culture method was used to generate anti-tumor, tumor-infiltrating lymphocytes from patients with breast cancer. FACS analysis, ELISA, and Elispot assay were used to characterize tumor-infiltrating lymphocytes. Autologous anti-tumor tumor-infiltrating lymphocytes from patients with breast cancer were used in adoptive cell transfer therapy. Results: FACS analysis indicated that tumor-infiltrating lymphocytes were present in the tumor tissues, but not detectable in the normal breast tissues from patients with breast cancer. Tumor-infiltrating lymphocytes could be generated in vitro from fresh tumor specimens of patients with breast cancer. Both CD4 T cells and CD8 T cells were detected in tumor-infiltrating lymphocytes. Autologous tumor cells could also generate in vitro from fresh tumor tissue samples of patients with breast cancer. Among 22 samples screened, 6 samples (25%) of tumor-infiltrating lymphocytes are tumor-reactive. Anti-tumor, tumor-infiltrating lymphocytes could recognize autologous tumor cells and allogenic tumor cells. After a large scale T cell expansion, anti-tumor reactivity was maintained in tumor-infiltrating lymphocytes. All of tumor-infiltrating lymphocytes were NK cells in some samples from patients with breast cancer, and these NK cells could recognize autologous tumor cells and a panel of allogenic tumor cells. T cell cloning assay demonstrated that some of the tumor-reactive, tumor-infiltrating lymphocytes were CD4 T cells. Conclusions: The results suggest that anti-tumor, tumor-infiltrating lymphocytes may be generated from patients with breast cancer, which may be used in clinical applications of adoptive cell transfer therapy for patients with breast cancer. The clinical trial of adoptive cell transfer therapy using autologous anti-tumor tumor-infiltrating lymphocytes for patients with breast is under way.

Generation of Antigen-Specific Cytotoxic T Lymphocytes with Peripheral B Cells Activated By α-Galactosylceramide

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3838-3838
Author(s):  
Sun Ok Yun ◽  
Hee Young Ju ◽  
Che Ry Hong ◽  
Ji Won Lee ◽  
Hyery Kim ◽  
...  
Keyword(s):  
B Cells ◽  
T Lymphocytes ◽  
Cytotoxic T Lymphocytes ◽  
Plasmid Dna ◽  
Cell Activation ◽  
Tumor Antigens ◽  
Conflicts Of Interest ◽  
Killing Activity ◽  
Significant Difference

Abstract Dendritic cells (DCs) are well known as the most potent professional antigen presenting cells (APCs). Nonetheless, the use of these cells in immunotherapy has been limited due to the time consuming and laborious steps required to generate DCs from monocytes in vitro. Therefore, alternative APCs has drawn much attention because of their relative convenience in manipulation. In this study, the efficacy of B cells as APCs, as compared to DCs, in induction of cytotoxic T lymphocytes (CTLs) against cytomegalovirus (CMV)-specific antigens was evaluated. B cells were isolated by depletion of peripheral blood mononuclear cell (PBMCs) from healthy individuals with MACS system, loaded with α-galactosylceramide (α-GalCer) for inducing B cell activation, and nucleofected with CMV-antigen coding plasmid DNA, pCK-pp65-IRES-IE1. As other APCs, monocyte-derived DCs were induced with various cytokines (GM-CSF, IL-4, IL-1b, TNF-a), for 6 days and nucleofected with the same plasmid DNA. Ag-nucleofected B cells or DCs were cocultured with T cells for 14 days in vitro. The cells were harvested and subsequently immunoassayed. Proliferation of cells was more expanded by about 25~32% in CMV-CTLs induced by DCs compared to of B cells, but there was no significant difference in immunogenicity between CMV-CTLs induced with B cells and DCs. Compared to CMV-CTLs induced by DCs, the CTLs induced by α-GalCer-loaded B cells induced similar cytotoxicity against CMV antigen (Ag) in vitro. The CMV-CTLs by α-GalCer-loaded B cells recognized CMV antigen pp65 (median 88 SFC/105) and IE-1 (median 86 SFC/105) in donor 1, and CMV antigen pp65 (median 31 SFC/105) and IE-1 (median 37 SFC/105) in donor 2. Similarly, the CMV-CTLs by DCs recognized CMV antigen pp65 (median 133 SFC/105) and IE-1 (median 32 SFC/105) in donor 1, and CMV antigen pp65 (median 37 SFC/105) and IE-1 (median 43 SFC/105) in donor 2. Immunogenicities of both CTLs were similar not only on IFN-γ ELISPOT (Enzyme-linked immunospot) assay but also on cytotoxicity assay. The CMV-CTLs by α-GalCer-loaded B cells have killing activity against CMV antigen pp65 (100%, at E:T ratio 10:1) and IE1 (85%, at E:T ratio 10:1) in donor 1, and CMV antigen pp65 (69%, at E:T ratio 10:1) and IE1 (27%, at E:T ratio 10:1) in donor 2. Also, the CMV-CTLs by DCs show killing activity against CMV antigen pp65 (100%, at E:T ratio 10:1) and IE1 (42%, at E:T ratio 10:1) in donor 1, and CMV antigen pp65 (88%, at E:T ratio 10:1) and IE1 (64%, at E:T ratio 10:1) in donor 2. These observations suggest that α-GalCer-loaded B cells could be used in general as APCs instead of DCs. Using the B cells as APCs have several benefits such as cost-effectiveness, less time-consuming, and less laborious compared to when DCs are used. Furthermore, nucleofection technique might be useful in delivering antigen-coding DNA, not only for virus antigens but also for tumor antigens, directly into the nucleus. Our results demonstrate that α-GalCer-loaded B cells could be potent APCs in generating antigen-specific CTLs for cellular vaccines and adoptive immunotherapy. Acknowledgment: This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. 2012R1A1A2008316) and we thank Ann M. Leen, Helen E. Heslop and Malcomn K. Brenner from Center for Cell and Gene Therapy, Baylor College of Medicine Center for their kind help. Disclosures No relevant conflicts of interest to declare.

Ibrutinib Treatment Reduces Both T-Regulatory Cells and B-Regulatory Cell Phenotype in Malignant B Cells in Chronic Lymphocytic Leukemia Patients

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2940-2940 ◽  
Author(s):  
Meixiao Long ◽  
Kyle A. Beckwith ◽  
Kami J. Maddocks ◽  
Carolyn Cheney ◽  
Jennifer A. Woyach ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
Research Funding ◽  
T Regulatory Cells ◽  
Surface Expression ◽  
Lymphocytic Leukemia ◽  
Regulatory Cells ◽  
Reduced Frequency ◽  
Fold Reduction

Abstract Introduction: Chronic lymphocytic leukemia (CLL) has multiple mechanisms of active immune suppression including expansion of T-regulatory cells which increases with progression of the disease. In addition, the malignant CLL cells were found to produce IL-10 in vitro and functionally recapitulate the phenotype of regulatory B cells. These regulatory "B10" (capable of producing IL10 after hours in vitro stimulation) or "B10 pro" (capable of producing IL10 after 2 days in vitro conditioning) are generally rare in healthy individuals, and play an important role in regulating inflammatory and autoimmune process. Similarly, CLL cells can exert tumor specific as well as global immune suppressive effect via IL-10 production. Ibrutinib, the first in class irreversible BTK inhibitor has been proved to be a safe and effective therapy for CLL. Recently we and others have demonstrated favorable cellular immune modulatory effects of ibrutinib through inhibition of an alternative target interleukin-2 induced T-cell kinase (ITK) that promotes Th1 CD4 polarization. Herein, we explore influence of ibrutinib on other immune suppressive features including T-regulatory cells and the B-regulatory phenotype associated with CLL cells. Methods: PBMCs were collected from nine previously treated CLL patients treated with 420mg of ibrutinib daily per clinical trial OSU-11133 (NCT01589302) at the time of pretreatment, cycle 3 day 1 and cycle 6 day 1. For Brief stimulation (B10 condition), cryopreserved PBMCs were thawed and stimulated with PMA/Ionomycin/Golgi-stop plus CpG for 5 hours. For prolonged stimulation (B10-Pro condition), PBMCs were stimulated with CpG plus CD40L for 48 hours, PMA / Ionomycin / Golgi-stop were added for final 5 hours. The cells were then fixed / permeabilized and stained for intracellular IL-10. For FOXP3 staining, PBMCs were permeabilized and fixed with Foxp3 Buffer Set from eBioscience, and were stained with stained with PE conjugated anti-human Foxp3 antibody (clone 259D/C7). Results: Significant IL-10 production was detected in 8 out 9 patient's CLL cells after 48 hours in vitro stimulation. Interestingly, CLL cells collected from patients treated with ibrutinib in vivo were significantly impaired in their capacity to make IL-10 in 7 out of the 8 patients whose CLL cells were capable of producing IL-10. On average, there is more than 4 fold reduction( P< 0.01) in the frequency of cells producing IL-10 by cycle 3, more than 5 fold reduction (P< 0.01) by cycle 6. (Figure 1 A, upper panel). IL-10 production after a brief 5 hour in vitro stimulation was observed in 4 out of the 9 patients studied, though the frequencies of IL-10 producing cells were low (Figure 1 A, lower panel). Samples collected post-ibrutinib treatment showed a trend towards reduced frequency of IL-10 producing CLL cells after 5 hour stimulation. We have also shown that during the first two cycles of ibrutinib, patients' plasma levels of IL-10 decreased. Analysis of potential immunosuppressive molecules revealed a dramatic reduction in surface expression of CD200, BTLA and PD-1 in CLL cells collected post ibrutinib treatment compared to pre-treatment samples (Figure 1B). We also found that for all the patients analyzed, the percentage of CD4+/Foxp3+ and CD4+/CD25+/Foxp3+ regulatory T cells were significantly reduced in samples collected after ibrutinib treatment. The difference is more dramatic for CD25+Foxp3+ cells (figure 1C). Conclusion: Here we demonstrate a significant decrease in the frequency of T-regulatory cells and IL-10 competent "B-reg" like leukemia cells in CLL patients after ibrutinib treatment. Ability of CLL cells to produce IL-10 and their regulatory B cell like features are considered to play a major role in mediating both global and tumor specific immunosuppression in CLL patients. Ibrutinib has been reported to enhance the immune response against B cell lymphoma in a mouse model. Our findings provide potential mechanisms by which ibrutinib treatment relieve the immunosuppressive effect of malignant B cells, thus enhancing global as well as tumor specific immunity. The main mechanisms likely include impaired IL-10 production capability and reduced surface expression of immunosuppressive molecules by CLL cells, as well as reduced frequency of regulatory T cells and IL-10 producing T cells. Figure 1. Figure 1. Figure 2. Figure 2. Figure 3. Figure 3. Disclosures Maddocks: Novartis: Research Funding; Janssen: Research Funding; Pharmacyclics: Consultancy, Research Funding. Byrd:Acerta Pharma BV: Research Funding; Acerta Pharma BV: Research Funding.

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