scholarly journals Ibrutinib Treatment Improves T-Cell Proliferative Ability and Effector Function in Relapsed/Refractory Chronic Lymphocytic Leukemia (CLL) Patients

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3114-3114
Author(s):  
Isabelle G. Solman ◽  
Marlene Taylor ◽  
Hana You ◽  
Susan M. O'Brien ◽  
Stephen P. Mulligan ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
Cell Death ◽  
T Cell ◽  
Research Funding ◽  
T Cell Proliferation ◽  
Healthy Donors ◽  
Equity Ownership ◽  
Proliferative Ability

Abstract Background: CLL is a B-cell malignancy characterized by profound immune dysregulation, including dysfunctional T cells. Ibrutinib (ibr), a first-in-class, once-daily BTK inhibitor, is approved in the US for the treatment of CLL/SLL, and has been reported to additionally inhibit ITK in T cells. Previous studies showed that ibr has a positive impact on T-cell compartments by decreasing abnormally elevated regulatory T cells and pseudo-exhausted effector T cells, while preserving naive T-cell counts post 1 year of treatment (Solman ASH Lymphoma 2016; Solman ASCO 2017). Moreover, lower infection rates have been reported following 6 months of ibr treatment, suggesting that ibr might restore T-cell functions over time (Long JCI 2017). To further assess the effects of ibr on T-cell function, we evaluated the ability of cells to proliferate, degranulate, and release cytokines upon stimulation. Methods: Samples for this study were derived from patients (pts) with relapsed/refractory CLL enrolled in the RESONATE™ trial (Byrd NEJM 2014). In the 2 treatment arms, ibr and ofatumumab (ofa), we compared responders (PR) and non-responders (SD) per investigator assessment at week (wk) 24 - end of ofa treatment - to isolate the treatment effect on T cells. In total, we selected 19 pts wk1 through wk48 from ibr arm (420 mg once daily; 12 PR & 7 SD at wk24) and 21 pts wk1 through wk24 from ofa arm (300 mg once followed by weekly 2000 mg; 10 PR & 11 SD at wk24). Also included in the study for reference were 18 untreated, age-matched healthy donors. Cryopreserved PBMCs were immunophenotyped before and after 4-day in vitro stimulation with anti-CD3/CD28. T-cell proliferation was measured with CFSE staining, while secreted lytic proteins and cytokines were measured by bead-based immunoassays. Unless specified otherwise, median changes at wk24 relative to baseline (wk1) are reported. Results: Compared to reference range (ref) of healthy donors, all CLL samples were found to have higher percentage of apoptotic T cells. We further evaluated the fraction of apoptotic T cells in each treatment arm. In the treated CLL patients, a larger decrease in the apoptotic fraction was observed in vivo with ibr (46%) compared to ofa (24%). To evaluate how well T cells were able to respond to stimulation, we assessed T-cell death post in vitro stimulation. In vitro, both treatments were found to reduce cell death, with ofa exerting an earlier effect on apoptosis (57% for ofa vs 17% for ibr at wk24, down to 37% at wk48), and ibr having a larger effect on non-apoptotic cell death (53% for ibr at wk12-48 vs no change for ofa). T-cell proliferative ability improved significantly with ibr (+28%, P=0.001), while it decreased with ofa (-49%, P=0.003). In both arms, T-cell proliferation was higher in pts with PR compared to SD (+39% vs +6% for ibr and -20% vs -96% for ofa), and for CD8+ cells compared to CD4+ (14% for ibr and 52% for ofa). We confirmed degranulation impairment in CLL pts at baseline with a lower ability to release perforin, granzyme-A, granzyme-B, and granulysin upon stimulation. Lytic protein secretion was improved by both treatments, with a greater overall effect observed in ibr (5-fold) vs ofa pts (3-fold) at wk24. In particular, granulysin increased up to 11-fold at wk48 with ibr treatment, leading to full restoration according to ref. Relative to healthy donors, CLL pts at baseline secreted less IL-4, IL-6, IL-10, and IL-17. Each of these cytokines was increased by 3-4-fold and maintained near ref through treatment with ibr, while maximum 2-fold improvement was observed in ofa pts. IL-2 and TNF-α were elevated in CLL pts, and their secretion remained high with both treatments. Further analyses showed that degranulation and cytokine secretion changes were likely not a direct result of cell counts or disease resolution. Conclusions: Together, these data suggest ibr has a positive impact on T-cell proliferation and effector functions including degranulation and cytokine release. T-cell proliferative ability was found to be associated with treatment response, but improvement was significantly higher in ibr-treated pts, indicating a unique immune reconstitution effect of ibr beyond the effect of disease resolution. Improvement in effector T-cell function at wk24 and beyond may lead to lower infection rates in later cycles of therapy and/or support an adaptive anti-CLL immune response. Disclosures Solman: AbbVie: Equity Ownership; Pharmacyclics LLC, an AbbVie company: Employment, Other: TRAVEL, ACCOMMODATIONS, EXPENSES. Taylor:AbbVie: Equity Ownership; Pharmacyclics LLC, an AbbVie Company: Employment, Other: Travel, accomodations, expenses. You:Pharmacyclics LLC, an AbbVie Company: Employment, Other: Travel, accomodations, expenses; AbbVie: Equity Ownership. O'Brien:Acerta: Research Funding; Regeneron: Research Funding; Kite Pharma: Research Funding; Vaniam Group LLC: Consultancy; TG Therapeutics: Consultancy, Research Funding; Janssen: Consultancy; Amgen: Consultancy; Pharmacyclics: Consultancy, Research Funding; Celgene: Consultancy; Abbvie: Consultancy; Pfizer: Consultancy, Research Funding; Aptose Biosciences Inc.: Consultancy; GlaxoSmithKline: Consultancy; Astellas: Consultancy; Alexion: Consultancy; Sunesis: Consultancy, Research Funding; Gilead: Consultancy, Research Funding. Dean:CTI BioPharma Corp.: Employment, Equity Ownership; Pharmacyclics LLC, an AbbVie Company: Employment; AbbVie: Equity Ownership. James:Pharmacyclics LLC, an AbbVie Company: Employment; AbbVie: Equity Ownership, Other: Spouse's employment and stocks, Patents & Royalties: AbbVie Patent Applications. Mongan:Thermo Fisher Scientific: Patents & Royalties: Patents; AbbVie: Equity Ownership; Pharmacyclics LLC, an AbbVie Company: Employment, Other: Travel, accommodations, expenses.

scholarly journals Ineffective Cellular Immune Response Associated with T-Cell Apoptosis in Susceptible Mycobacterium bovisBCG-Infected Mice

2000 ◽  
Vol 68 (7) ◽  
pp. 4264-4273 ◽  
Author(s):  
Laurent Kremer ◽  
Jérôme Estaquier ◽  
Isabelle Wolowczuk ◽  
Franck Biet ◽  
Jean-Claude Ameisen ◽  
...  
Keyword(s):  
Immune Response ◽  
Cell Proliferation ◽  
T Cells ◽  
Cell Death ◽  
T Cell ◽  
Cell Apoptosis ◽  
T Cell Proliferation ◽  
T Cell Apoptosis ◽  
Tnf Α

ABSTRACT It has previously been reported that inhibition of delayed-type hypersensitivity-mediating functions of T cells during mycobacterial infection in mice is haplotype dependent. In the present study, we show that Mycobacterium bovis BCG infection induced, in susceptible C57BL/6 and BALB/c mice but not in resistant C3H/HeJ and DBA/2 mice, an important splenomegaly. An in vitro defect in T-cell proliferation in response to T-cell receptor (TCR) stimulation with mitogens or anti-CD3 antibodies was associated with enhanced levels of CD4+ and CD8+ T-cell apoptosis in susceptible but not in resistant mice 2 weeks after infection. Further investigations of C57BL/6 and C3H/HeJ mice revealed that in vivo splenomegaly was associated with destruction of the lymphoid tissue architecture, liver cellular infiltrates, and increased numbers of apoptotic cells in both spleen and liver tissue sections. Infection of C57BL/6 mice but not of C3H/HeJ mice induced massive production of tumor necrosis factor alpha (TNF-α) in serum, as well as an increase in Fas and Fas ligand (FasL) expression in T cells. In vitro addition of neutralizing anti-TNF-α antibodies led to a significant reduction in CD3-induced T-cell apoptosis of both CD4+ and CD8+ T cells of C57BL/6 mice, while the blockade of Fas-FasL interactions reduced apoptosis only in CD4+ but not in CD8+ T cells. Together, these results suggest that TNF-α and Fas-FasL interactions play a role in the activation-induced cell death (AICD) process associated with a defect in T-cell proliferation of the susceptible C57BL/6 mice. T-cell death by apoptosis may represent one of the important components of the ineffective immune response against mycobacterium-induced immunopathology in susceptible hosts.

scholarly journals Anti-CD6 Monoclonal Antibody Itolizumab Efficiently Inhibits T Cell Proliferation after in Vitro TCR Stimulation in the Setting of Acute Graft Versus Host Disease

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4517-4517 ◽  
Author(s):  
Benedetta Rambaldi ◽  
Carol Reynolds ◽  
Sharmila Chamling Rai ◽  
Takeru Asano ◽  
Yohei Arihara ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cd8 T Cells ◽  
Peripheral Blood ◽  
Research Funding ◽  
Cell Activation ◽  
T Cell Proliferation

CD6 is a co-stimulatory receptor expressed on T cells that binds activated leukocyte cell adhesion molecule (ALCAM), a ligand expressed on antigen presenting cells and various epithelial and endothelial tissues. The CD6-ALCAM pathway plays an integral role in modulating T cell activation, proliferation and trafficking and is central to inflammation. Early studies by Soiffer et al. demonstrated that ex vivo depletion of CD6+ donor cells prior to hematopoietic cell transplantation (HCT) decreased the incidence of acute graft versus host disease (aGVHD), highlighting the importance of CD6+ cells in GVHD pathogenesis. Itolizumab, a humanized anti-CD6 monoclonal antibody, has been shown to modulate T cell activation and proliferation. The aim of this study was to characterize: (1) expression of CD6 and ALCAM, and (2) activity of itolizumab on T cell responses in peripheral blood from HCT patients pre- and post-aGvHD. We analyzed immune reconstitution in 31 adult patients who underwent HLA matched donor HCT for hematological malignancies. Patients received peripheral blood stem cell grafts and GVHD prophylaxis with tacrolimus and methotrexate. Twelve of 31 patients developed aGVHD at a median of 58 days, range 27-208, after HCT and systemic treatment was started in 83% of these cases. aGVHD grade severity was 25%, 58.3% and 16.7% of grade I, II and IV, respectively. Patient samples were collected at 1, 2 and 3 months after HCT and analyzed using multi-color flow cytometry. Nine healthy donors (HD) were analyzed as controls. Suppressive activity of itolizumab was tested using peripheral blood mononuclear cells (PBMC) obtained from HD and patients before (preGVHD) and after (postGVHD) aGvHD onset (within 30 days). PBMC were stimulated with antiCD3/CD2/CD28 coated beads in the presence of itolizumab or isotype control (cetuximab) for 72 hours. T cell proliferation was measured by CFSE dilution, while T cell activation and maturation was measured by expression of CD25 and CD45RO, respectively. For statistical analysis, non-parametric unpaired (Mann-Whitney) or paired (Wilcoxon matched-pairs signed rank) test were used. CD6+ T cells reconstituted early after transplant, accounting for 95% of positive CD3 T cells, range 57-100 at 1 month. Similar to HD PBMC, in the first 3 months after HCT, CD4 Tcon had the highest CD6 expression, while CD4 Treg had a lower CD6 expression compared to both CD4 Tcon and CD8 T cells (Fig 1A and 1B). To characterize the expression of CD6 on different T cell subsets, we used a t-Distributed Stochastic Neighbor Embedding (t-SNE) algorithm and visualized the data using a viSNE map (Fig 1C). Within the Tcon compartment, there were no differences in expression of CD6 between HD and patients at all 3 time points. Within CD4 Treg and CD8 T cells, CD6 expression was reduced in naïve CD8 T cells and CM Treg after transplant compared to HD. In HD, ALCAM expression was detected in 35% of CD14+ monocytes, 23% of CD19+ B cells, 20% of myeloid (CD11c+ CD123-) DCs and 97% of plasmacytoid (CD11c-CD123+) DCs. After HCT, expression of ALCAM in DC compartments was similar to HD. In functional studies, itolizumab inhibited CD4 and CD8 T cell proliferation in preGVHD samples, similar to HD controls. This effect was less prominent in samples collected from patients who had developed GVHD and were already receiving immunosuppressive medications, potentially confounding the ability to assess the effect of itolizumab in this assay (Fig 2A). Similar results were observed for CD25 (Fig 2B) and CD45RO (Fig 2C) expression pre- and post-aGVHD. Finally, itolizumab did not increase rates of cell death in samples from HCT patients as assessed by Annexin V expression, suggesting that itolizumab-mediated T cell inhibition was not due to increased T cell apoptosis. There was a slight increase in Annexin V expression in HD vs isotype control (21%, range 10-43 vs 15%, range 11-31, p= 0.0273). In conclusion, we demonstrate for the first time that CD6+ T cells reconstitute rapidly in peripheral blood after HCT and that CD6 expression is highest in Tcon while lowest in Treg (Tcon>CD8>Treg). Itolizumab efficiently inhibits T cell proliferation and activation after in vitro TCR stimulation of PBMC from aGvHD patients, thus representing a potential therapeutic for treating aGvHD. A phase I/II study using itolizumab as first line treatment in combination with steroids for patients with aGVHD is currently ongoing (NCT03763318). Disclosures Rambaldi: Equillium: Research Funding. Koreth:Amgen: Consultancy; Cugene: Consultancy; Equillium: Consultancy. Cutler:Pharmacyclics: Consultancy; Omeros: Consultancy; Kadmon: Consultancy; BiolineRx: Other: DSMB; Cellect: Other: DSMB; Kalytera: Other: DSMB; ElsaLys: Consultancy; Genentech: Consultancy; BMS: Consultancy; Jazz: Consultancy; Incyte: Consultancy; Fate Therapeutics: Consultancy. Nikiforow:Kite/Gilead: Honoraria; Novartis: Honoraria; NKarta: Honoraria. Ho:Omeros Corporation: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Research Funding; Jazz Pharmaceuticals: Consultancy. Soiffer:Jazz: Consultancy; Gilead, Mana therapeutic, Cugene, Jazz: Consultancy; Juno, kiadis: Membership on an entity's Board of Directors or advisory committees, Other: DSMB; Cugene: Consultancy; Mana therapeutic: Consultancy; Kiadis: Other: supervisory board. Ampudia:Equillium: Employment. Ng:Equillium: Employment, Equity Ownership. Connelly:Equillium: Employment, Equity Ownership. Ritz:Equillium: Research Funding; Merck: Research Funding; Kite Pharma: Research Funding; Aleta Biotherapeutics: Consultancy; Celgene: Consultancy; Avrobio: Consultancy; LifeVault Bio: Consultancy; TScan Therapeutics: Consultancy; Talaris Therapeutics: Consultancy; Draper Labs: Consultancy.

scholarly journals Predictors of Efficacy for Blinatumomab in BCP-ALL Patients: Non-Responders Show Impaired CD19-BiTE®-Mediated Cytotoxicity in Vitro

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2632-2632 ◽  
Author(s):  
Veit Bücklein ◽  
Michaela Scheurer ◽  
Bettina Brauchle ◽  
Roman Kischel ◽  
Michael von Bergwelt ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Cell Lines ◽  
Research Funding ◽  
Response Rates ◽  
Effector Memory ◽  
T Cell Proliferation

Bispecific T-cell engaging (BiTE®) antibody constructs recruit T cells to target antigens independent of their T-cell receptor specificity. Blinatumomab, a CD19xCD3 BiTE® antibody construct, is approved for the treatment of relapsed/refractory (r/r) B-cell precursor acute lymphoblastic leukemia (BCP-ALL), including patients with persistence or reoccurrence of measurable residual disease (MRD). Despite superior anti-leukemic efficacy compared to cytostatic agents, a majority of patients do not respond to treatment. Biomarkers for the identification of non-responders prior to or early during treatment are unknown. However, the definition of robust biomarkers for efficacy is of high importance for clinical decision-making and might also indicate ways to improve response rates. We therefore established a comprehensive immune-monitoring program for blinatumomab patients. We sequentially analysed peripheral blood of patients with r/r or MRD-positive disease receiving blinatumomab prior to the start of infusion and once weekly over the course of the first 28-day cycle. We determined CD3+ T cell counts and their subset distribution (CD4, CD8, naïve [TN], central memory [TCM], effector memory [TEM], and effector memory CD45RA-positive [TEMRA] T cells) by multiparameter flow cytometry (MPFC). Additionally, patient-derived T cells were cocultered with BCP-ALL cell lines (SEM and REH) at an effector: target ratio of 1:3 in presence of blinatumomab or a control BiTE® (0.5 ng/ml, respectively) for 3 days to assess their cytotoxic capacity. Blinatumomab-mediated cytotoxicity was determined by MPFC. T-cell proliferation was evaluated by MPFC (Far Red Cell tracer) after incubation with CD3/CD28 beads for 6 days. A total of 16 patients were enrolled. Four of these patients suffered from MRD disease, whereas the remaining 12 had overt relapse of BCP-ALL. Response rates for patients with morphological relapse were 50% (4 CRMRD- and 2 CRMRD+ after the first cycle) and 75% for patients with MRD disease (3 MRD conversions, 1 MRD persistence). Absolute lymphocyte counts were not significantly different between responders and non-responders before treatment initiation (0.9 G/l and 0.7 G/l, respectively). Whereas the percentage of CD3+ T cells (of all lymphocytes) did not significantly differ between responders and non-responders on day 0, non-responders had a significantly reduced CD3+ percentage on day 7 (81.2% vs 92.9%, p=0.03). Until day 28 of the first cycle, CD3+ percentages of responders and non-responders re-converged (80.2% and 81.4%, respectively). There were no significant differences for CD4+ and CD8+ T-cell percentages prior to and over the course of the first cycle. This was also true for TN, TCM, TEM and TEMRA subset distributions. Additionally, CD19-BiTE®-mediated cytotoxicity of patient-derived T cells was assessed against CD19 expressing target B cell lines in vitro. Interestingly, specific lysis did not differ between responders and non-responders on day 0 (89.6% vs 79.8%, p=0.89), but decreased for non-responders over the course of the first cycle (normalized AUC for cytotoxicity 149 vs 403 in responders, p=0.03). However, this observation was only true for the patients with refractory morphologic relapse, as the non-responding MRD patient maintained T cell cytotoxicity over the first cycle of therapy. Impaired T cell proliferation (leading to reduced E:T ratios) might contribute to the observed dysfunctional cytotoxicity in non-responders, as the percentage of proliferating T cells after CD3/CD28 bead-based stimulation tended to be reduced for non-responders (17.9% vs 36.1%, p=0.07). In summary, lymphocyte counts, T cell percentage and T cell subset distributions do not allow for a response prediction prior to treatment start for patients receiving blinatumomab. BCP-ALL patients with morphological relapse/persistence who do not achieve a remission with blinatumomab therapy show reduced cytotoxicity in vitro in comparison to patients responding to treatment. As this observation could not be confirmed with one MRD non-responder in our cohort, the burden of disease might contribute to the observed T cell dysfunction, possibly by interfering with T cell proliferation. Evaluation of immune checkpoint expression on effector and target cells over the course of the therapy is currently ongoing, as are analyses of the T cell transcriptome of responders and non-responders. Disclosures Kischel: AMGEN Research (Munich) GmbH: Employment, Equity Ownership. Subklewe:Celgene: Consultancy, Honoraria; Oxford Biotherapeutics: Research Funding; Miltenyi: Research Funding; Roche: Consultancy, Research Funding; Janssen: Consultancy; Gilead: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Morphosys: Research Funding; AMGEN: Consultancy, Honoraria, Research Funding.

scholarly journals Elevated serum soluble CD40 ligand in cancer patients may play an immunosuppressive role

Blood ◽  
2012 ◽  
Vol 120 (15) ◽  
pp. 3030-3038 ◽  
Author(s):  
Jianping Huang ◽  
Caroline Jochems ◽  
Tara Talaie ◽  
Austin Anderson ◽  
Alessandra Jales ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Cancer Patients ◽  
Elevated Serum ◽  
Cd40 Ligand ◽  
T Cell Proliferation ◽  
Soluble Cd40 Ligand ◽  
Healthy Donors

Abstract Tumor cells can induce certain cytokines and soluble receptors that have a suppressive effect on the immune system. In this study, we showed that an extracellular portion of a membrane-bound ligand of CD40 (soluble CD40 ligand; sCD40L) was significantly elevated in the serum of cancer patients compared with healthy donors. In addition, PBMCs from cancer patients had a relatively larger population of myeloid-derived suppressor cells (MDSCs), defined as CD33+HLA-DR− cells, and these cells expressed higher levels of CD40. T-cell proliferation and IFN-γ production decreased when stimulated T cells were cocultured with an increased amount of autologous MDSCs. The addition of recombinant monomeric sCD40L enriched MDSCs and had an additive inhibitory effect on T-cell proliferation. PBMCs cultured in vitro with sCD40L also showed an expansion of regulatory T cells (CD4+CD25highFoxp3+), as well as induction of cytokines, such as IL-10 and IL-6. Moreover, sCD40L-induced enrichment of programmed death-1–expressing T cells was greater in cancer patients than in healthy donors. Preexisting sCD40L also inhibited IL-12 production from monocytes on activation. These data suggest that the higher levels of sCD40L seen in cancer patients may have an immunosuppressive effect. These studies were registered at www.clinicaltrials.gov as NCT00060528, NCT00019695, NCT00179309, NCT00514072, NCT00081848, and NCT00436956.

scholarly journals Siplizumab, an Anti-CD2 Monoclonal Antibody, Induces a Unique Set of Immune Modulatory Effects Compared to Alemtuzumab and Rabbit Anti-Thymocyte Globulin In Vitro

2020 ◽  
Vol 11 ◽  
Author(s):  
Christian Binder ◽  
Felix Sellberg ◽  
Filip Cvetkovski ◽  
Erik Berglund ◽  
David Berglund
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Mixed Lymphocyte Reaction ◽  
T Cell Proliferation ◽  
Allogeneic Mixed Lymphocyte Reaction ◽  
Dependent Cell

Antibodies are commonly used in organ transplant induction therapy and to treat autoimmune disorders. The effects of some biologics on the human immune system remain incompletely characterized and a deeper understanding of their mechanisms of action may provide useful insights for their clinical application. The goal of this study was to contrast the mechanistic properties of siplizumab with Alemtuzumab and rabbit Anti-Thymocyte Globulin (rATG). Mechanistic assay systems investigating antibody-dependent cell-mediated cytotoxicity, antibody-dependent cell phagocytosis and complement-dependent cytotoxicity were used to characterize siplizumab. Further, functional effects of siplizumab, Alemtuzumab, and rATG were investigated in allogeneic mixed lymphocyte reaction. Changes in T cell activation, T cell proliferation and frequency of naïve T cells, memory T cells and regulatory T cells induced by siplizumab, Alemtuzumab and rATG in allogeneic mixed lymphocyte reaction were assessed via flow cytometry. Siplizumab depleted T cells, decreased T cell activation, inhibited T cell proliferation and enriched naïve and bona fide regulatory T cells. Neither Alemtuzumab nor rATG induced the same combination of functional effects. The results presented in this study should be used for further in vitro and in vivo investigations that guide the clinical use of immune modulatory biologics.

A Nanobody Against Cytotoxic T-Lymphocyte Associated Antigen-4 Increases the Anti-Tumor Effects of Specific CD8+ T Cells

2019 ◽  
Vol 15 (11) ◽  
pp. 2229-2239 ◽  
Author(s):  
Zhuoran Tang ◽  
Fengzhen Mo ◽  
Aiqun Liu ◽  
Siliang Duan ◽  
Xiaomei Yang ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
Cytotoxic T Lymphocyte ◽  
Ex Vivo ◽  
T Cell Proliferation ◽  
Tumor Cell Apoptosis ◽  
Xenograft Models

Adoptive cell-based immunotherapy typically utilizes cytotoxic T lymphocytes (CTLs), expanding these cells ex vivo. Such expansion is traditionally accomplished through the use of autologous APCs that are capable of interactions with T cells. However, incidental inhibitory program such as CTLA-4 pathway can impair T cell proliferation. We therefore designed a nanobody which is specific for CTLA-4 (CTLA-4 Nb 16), and we then used this molecule to assess its ability to disrupt CTLA-4 signaling and thereby overcome negative costimulation of T cells. With CTLA-4 Nb16 stimulation, dendritic cell/hepatocellular carcinoma fusion cells (DC/HepG2-FCs) enhanced autologous CD8+ T cell proliferation and production of IFN-γ in vitro, thereby leading to enhanced killing of tumor cells. Using this approach in the context of adoptive CD8+ immunotherapy led to a marked suppression of tumor growth in murine NOD/SCID hepatocarcinoma or breast cancer xenograft models. We also observed significantly increased tumor cell apoptosis, and corresponding increases in murine survival. These findings thus demonstrate that in response to nanobody stimulation, DC/tumor cells-FC-induced specific CTLs exhibit superior anti-tumor efficacy, making this a potentially valuable means of achieving better adoptive immunotherapy outcomes in cancer patients.

scholarly journals Allergen-Induced C5a/C5aR1 Axis Activation in Pulmonary CD11b+ cDCs Promotes Pulmonary Tolerance through Downregulation of CD40

Cells ◽  
2020 ◽  
Vol 9 (2) ◽  
pp. 300 ◽  
Author(s):  
Konstantina Antoniou ◽  
Fanny Ender ◽  
Tillman Vollbrandt ◽  
Yves Laumonnier ◽  
Franziska Rathmann ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cd4 T Cells ◽  
Cell Activation ◽  
T Cell Proliferation ◽  
C5a Receptor ◽  
The Impact

Activation of the C5/C5a/C5a receptor 1 (C5aR1) axis during allergen sensitization protects from maladaptive T cell activation. To explore the underlying regulatory mechanisms, we analyzed the impact of C5aR1 activation on pulmonary CD11b+ conventional dendritic cells (cDCs) in the context of house-dust-mite (HDM) exposure. BALB/c mice were intratracheally immunized with an HDM/ovalbumin (OVA) mixture. After 24 h, we detected two CD11b+ cDC populations that could be distinguished on the basis of C5aR1 expression. C5aR1− but not C5aR1+ cDCs strongly induced T cell proliferation of OVA-reactive transgenic CD4+ T cells after re-exposure to antigen in vitro. C5aR1− cDCs expressed higher levels of MHC-II and CD40 than their C5aR1+ counterparts, which correlated directly with a higher frequency of interactions with cognate CD4+ T cells. Priming of OVA-specific T cells by C5aR1+ cDCs could be markedly increased by in vitro blockade of C5aR1 and this was associated with increased CD40 expression. Simultaneous blockade of C5aR1 and CD40L on C5aR1+ cDCs decreased T cell proliferation. Finally, pulsing with OVA-induced C5 production and its cleavage into C5a by both populations of CD11b+ cDCs. Thus, we propose a model in which allergen-induced autocrine C5a generation and subsequent C5aR1 activation in pulmonary CD11b+ cDCs promotes tolerance towards aeroallergens through downregulation of CD40.

Donor Requirements For CD4+CD25+FoxP3+ Regulatory T Cells Capable Of Suppressing CD4+ and CD8+ Conventional T Cell Proliferation and Graft Versus Host Disease

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4484-4484 ◽  
Author(s):  
Antonio Pierini ◽  
Lucrezia Colonna ◽  
Maite Alvarez ◽  
Dominik Schneidawind ◽  
Byung-Su Kim ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Animal Models ◽  
Graft Versus Host Disease ◽  
Third Party ◽  
T Cell Proliferation ◽  
Graft Versus Host

Adoptive transfer of CD4+CD25+FoxP3+ regulatory T cells (Tregs) prevents graft versus host disease (GvHD) in several animal models and following allogeneic hematopoietic cell transplantation (HCT) in clinical trials. In these models donor derived Tregs have been mainly used as they share the same major histocompatibility complex (MHC) with conventional CD4+ and CD8+ T cells (Tcons) that are primarily responsible for GvHD onset and persistence. Third-party derived Tregs are a promising alternative tool for cellular therapy as they can be prepared in advance, screened for pathogens and activity and banked. In this study we explored MHC disparities between Tregs and Tcons in HCT to evaluate the impact of these different cell populations in GvHD prevention and survival after transplant. Methods and Results We evaluated the ability of highly purified Treg to suppress proliferation of C57BL/6 (H-2b) Tcons following exposure to irradiated splenocytes from BALB/C (H-2d) mice in vitro in a mixed lymphocyte reaction (MLR). Either donor derived C57BL/6 (H-2b) or third party FVB (H-2q) Tregs suppressed Tcon proliferation at the Treg/Tcon ratios of 1:2 and 1:4. The same Treg population effectively suppressed different MHC derived Tcons where BALB/C (H-2d) or FVB (H-2q, third-party) Tcons were incubated with irradiated splenocytes from C57BL/6 (H-2b) mice and were effectively suppressed with BALB/C (H-2d) Tregs. In the MLR, third-party Tregs present the same activation molecule expression patterns as MHC matched Tregs: CTLA4 and LAG3 expression is enhanced after stimulation with interleukin-2 (IL-2) and anti-CD3/CD28 beads, while MHC class II molecule expression is increased after 3-4 days of culture with Tcons and irradiated splenocytes. Furthermore third-party and MHC matched Tregs express the same levels of interleukin-10 (IL-10). We translated these results to in vivo studies in animal models. In these studies T cell depleted bone marrow (TCD BM) from C57BL/6 (H-2b) mice was injected into lethally irradiated (total body irradiation, 8 Gy) BALB/C (H-2d) recipient mice. 2 days later GvHD was induced by injecting luc+ donor derived Tcons (1x106/mouse). Using this model GvHD was evaluated following the adoptive transfer of freshly isolated CD4+CD25+FoxP3+ Tregs derived from BALB/C (H-2d, host type), C57BL/6 (H-2b, donor type), FVB (H-2q, third-party) or BALB/B (H-2b, minor mismatched with the donor, major mismatched with the host) mice at the different Treg/Tcon ratios of 1:1, 1:2 and 1:4. As expected, donor Tregs exerted the strongest dose dependent GvHD protection (p = 0.028), while host Tregs did not improve mouse survival (p = 0.58). Third-party and minor mismatched with the donor Tregs improved mouse survival (third-party and minor mismatched with the donor respectively, p = 0.028 and p = 0.17) but mice had worse GvHD score profiles (both p< 0.001) and could not recover their weight as well as mice treated with donor Tregs (both p< 0.001). In vivoTcon bioluminescent imaging confirmed these results showing a reduced Tcon proliferation in mice treated with donor, third-party and minor mismatched with the donor Tregs, the first exerting the strongest effect (after 6 weeks of observation, p< 0.001). Conclusions Our studies indicate that MHC disparities between Tregs and Tcons do not represent an insurmountable barrier for Treg function. In vitro and in vivo data strongly suggest that Tregs can suppress Tcon proliferation without requiring MHC matching. In vivo GvHD prevention efficiency was affected by MHC disparities with donor derived Treg being the most effective, however, third party Treg also resulted in GvHD attenuation. These studies indicate that both donor and third party Treg could be effective in clinical application raising the possibility of screening and banking Treg for use. Further, these studies highlight the need for activation of the Treg on host tissues to effectively suppress conventional T cell proliferation and GvHD induction. Disclosures: No relevant conflicts of interest to declare.

Hydroxyurea Is Most Suitable for Cytoreduction of AML Prior to CD33/CD3 Bispecific BiTE® Antibody (AMG 330) Therapy: Uncompromised T-Cell Proliferation Ex-Vivo and CD33 Upregulation on AML Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 986-986 ◽  
Author(s):  
Christina Krupka ◽  
Franziska Brauneck ◽  
Felix S Lichtenegger ◽  
Peter Kufer ◽  
Roman Kischel ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Cell Function ◽  
Expression Level ◽  
T Cell Proliferation ◽  
Hl60 Cells ◽  
T Cell Function ◽  
Equity Ownership

Abstract Bispecific T-cell engager (BiTE®) antibodies represent a promising tool for anti-leukemic immunotherapy. The CD19/CD3-bispecific antibody blinatumomab was shown to be active in refractory and relapse patients with B-precursor acute lymphoblastic leukemia (Topp et al, ASCO 2014). Transient, blinatumomab-mediated cytokine release syndrome has been linked to target cell numbers as this phenomenon is predominantly observed within the first treatment cycle. In our previous work, we demonstrated that the bispecific CD33/CD3 BiTE® antibody AMG 330 is able to induce activation and proliferation of residual autologous T-cells and effectively mediates lysis of primary acute myeloid leukemia (AML) cells (Krupka et al, Blood 2014; 123(3):356-65). We hypothesize that in AML patients with high initial leukocyte counts (WBC > 30.000/μl) a cytoreductive phase prior to AMG 330 therapy might be beneficial to reduce the incidence and severity of cytokine mediated toxicity. Ideally, the cytoreductive drug does not impair T-cell function or reduce target antigen expression level. In the current study, we evaluated the effect of cytarabine (20 µM), decitabine (5 µM), azacitidine (1 µM and 5 µM) and hydroxyurea (10 µM and 100 µM) on T-cell proliferation and function in close analogy to potential treatment algorithms for AML. Healthy donor (HD) T-cells were pre-incubated with the cytoreductive drugs for 72 hours. T-cells were CFSE-labeled and co-cultured with either HL60 or MV4-11 cells (effector cell:target (E:T) ratio 1:1) in the presence or absence of AMG 330 (5 ng/ml). After 3 days of co-culture, lysis of HL60 cells and T-cell proliferation was assessed by flow cytometry. Pretreatment of T-cells with cytarabine completely abrogated T-cell function (lysis of HL60 cells: untreated (UT): 96.9% vs 20 µM: 4.2%) and significantly impaired T-cell proliferation (UT: 31.2% vs 20 µM: 4.6%). These findings correlated to data using primary AML samples collected 3 and 6 days after discontinuation of cytarabine treatment. After a 3-day chemotherapy-free interval, we observed no relevant T-cell proliferation and lysis of AML cells upon the addition of AMG 330 to the ex-vivo long-term culture system (lysis of AML cells on day 12: 30%; fold change T-cell expansion 0.9). After a 6-day treatment-free interval, high T-cell proliferation and cytotoxicity against primary AML cells were observed (lysis of AML cells on day 12: 61%; fold change T-cell expansion: 3.1). In contrast to cytarabine, decitabine treatment only marginally impaired T-cell function. Similarly, pre-incubation with azacitidine did not convey a negative effect on T-cell function (lysis of HL60 cells: UT: 100% vs 1 µM: 94.9% vs 5µM: 86.8%; proliferation: UT: 90.9% vs 1 µM: 80% vs 5 µM: 66.8%). Pretreatment with hydroxyurea had the least impact on T-cell performance. It did not impair T-cell function (lysis of HL60 cells: UT: 100% vs 10 µM: 100% vs 100 µM: 100%) and proliferation compared to untreated controls (UT: 92.9% vs 100 µM 90.8% vs 10 µM 92.9%). As we have previously shown that the level of CD33 expression correlates to kinetics of AMG 330-mediated lysis (Krupka et.al, EHA 2014), we analyzed the effect of the cytoreductive agents on CD33 expression level in AML cell lines and primary AML cells. Five AML cell lines (HL60, MV4-11, PL21, OCI-AML3, KG1a) and a primary AML patient sample were cultured in the presence or absence of decitabine (5 µM and 50 µM), azacitidine (1 µM and 5 µM) or hydroxyurea (10 µM and 100 µM) for 72 hours. The change of CD33 expression level was evaluated by flow cytometry (median fluorescence intensity, MFI). No significant changes in CD33 expression level were observed after culture of AML cell lines and primary AML cells with decitabine or azacitidine. In contrast, hydroxyurea upregulated surface expression of CD33 on 2/5 cell lines (HL60 and PL21) in a dose dependent manner (HL 60 MFI Ratio: UT 134.9 vs 10 µM 171.3 vs 100 µM 210; PL21 MFI Ratio: UT 166.9 vs 10 µM 177.9 vs 100 µM 191.8). In summary, we could show that pretreatment with hydroxyurea did not impair T-cell function and proliferation. In addition, we observed an upregulation of CD33 expression on AML cell lines. As the BiTE® technology relies on T-cell function and target antigen expression level, sequential and combinatorial immuno-chemotherapeutic approaches need to address both issues. Our data support the use of hydroxyurea in AML patients that require cytoreduction prior to AMG 330 treatment. Disclosures Krupka: AMGEN Inc.: Research Funding. Kufer:AMGEN Research (Munich): Employment; AMGEN Inc.: Equity Ownership. Kischel:AMGEN Research (Munich): Employment; AMGEN Inc.: Equity Ownership. Zugmaier:AMGEN Inc.: Equity Ownership; AMGEN Research (Munich): Employment. Sinclair:AMGEN Inc.: Employment, Equity Ownership. Newhall:AMGEN Inc.: Employment, Equity Ownership. Frankel:AMGEN Inc.: Employment, Equity Ownership. Baeuerle:AMGEN Research (Munich): Employment; AMGEN Inc.: Equity Ownership. Riethmüller:AMGEN Inc.: Equity Ownership. Subklewe:AMGEN Inc.: Research Funding.

scholarly journals Targeting Antigen Presenting   Cells to Treat Autoimmune  Inflammation

2021 ◽  
Author(s):  
◽  
Aras Toker
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Mhc Class Ii ◽  
T Cell Proliferation ◽  
Target Cells ◽  
Antigen Presenting

<p>Glatiramer acetate (GA) is approved for the treatment of relapsing-remitting multiple sclerosis (MS), and can suppress experimental autoimmune encephalomyelitis (EAE), a murine model of human MS. GA treatment is associated with the induction of anti-inflammatory TH2 responses and with the antigen specific expansion of regulatory T cells that counteract or inhibit pathogenic events in MS and EAE. These T cell mediated mechanisms of protection are considered to be a result of modulation of antigen presenting cells (APCs) by GA, rather than direct effects on T cells. However, it is unknown if GA preferentially targets a specific APC subset or can act through multiple APCs in vivo. In addition, GA-modulated innate cells may also exhibit direct antigen non-specific suppression of autoreactive cells. One objective of this study was to identify the in vivo target cell population of GA and to assess the potential of the target cells to antigen non-specifically suppress immune responses. Fluorophor-labelled GA bound to monocytes after intravenous injections, suggesting that monocytes may be the primary target of GA in vivo. In addition, intravenous GA treatment enhanced the intrinsic ability of monocytes to suppress T cell proliferation, both in vitro and in vivo. The findings of this study therefore suggest that GA-induced monocytes may contribute to GA therapy through direct mechanisms of antigen non-specific T cell immunosuppression. A further objective of this work was to investigate the potential of an in vivo drug targeting approach. This approach was hypothesised to increase the uptake of GA by the target cells and substantially improve GA treatment through antigen specific mechanisms such as induction of TH2 or regulatory T cells. Targeting antigens to professional APCs with an anti-MHC class II antibody resulted in significantly enhanced T cell proliferation in vitro. However, no EAE suppression occurred when GA was targeted to MHC class II in vivo. In addition, targeting GA specifically to monocytes also failed to suppress EAE. These findings suggest that GA treatment may selectively modulate monocytes to enhance their ability to inhibit autoreactive T cells, which could be part of the mechanism by which GA ameliorates MS. Targeting GA to a specific cell type may not be a powerful approach to improve treatment, because increased proliferation of GA specific T cells is not sufficient for disease suppression, and conjugation to antibodies may functionally reduce GA to a mere antigen devoid of immunomodulatory capacity.</p>

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