scholarly journals The Impact of the Bone Marrow Microenvironment on T Cell Redirection Therapeutics

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2752-2752
Author(s):  
Priyanka Nair-Gupta ◽  
Stephen Rudnick ◽  
Leopoldo Luistro ◽  
Diana Chin ◽  
Melissa Smith ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Stromal Cells ◽  
Cell Activation ◽  
Leukemic Cells ◽  
Anti Cancer ◽  
The Impact

Abstract Redirecting T cells to specifically target and kill malignant cells has been validated as an effective anti-cancer strategy in the clinic with the approval of CD19xCD3 BiTE (Blincyto) for acute lymphoblastic leukemia. However, the immunosuppressive nature of the tumor microenvironment potentially poses a significant hurdle to T cell therapies. For instance, the bone marrow (BM) niche is appreciated to be a site of immune privilege at steady state to allow for normal hematopoiesis and immune cell generation. Additionally, in hematological malignancies, the BM niche is protective to leukemic stem cells, a phenomenon that has minimized the efficacy of several anti-cancer drugs including chemotherapy, targeted small molecule inhibitors and antibody based therapies. In this study, we investigated the impact of the BM microenvironment on T cell redirection. Using antibodies made with the Genmab DuoBody® technology targeting specific tumor antigens (CD123 and BCMA) and CD3, we observed that co-culture of acute myeloid leukemia (AML; KG-1, MOLM-13 and OCI-AML5) or multiple myeloma (MM; H929 and RPMI8226) cell lines with bone marrow stromal cell lines (HS-5 and HS-27a) significantly protected leukemic cells from DuoBody®-T cell mediated lysis in vitro. Specifically, co-culture of bone marrow stromal cells in killing assays led to a 20-50% decrease in the maximum observed cytotoxicity and a 3-10 fold weaker EC50, reflecting an impact on both the efficacy and potency of bispecific DuoBody® antibodies. Similar results were also observed with primary stromal cells obtained from healthy donors. Furthermore, presence of stromal cells in a humanized xenograft AML model attenuated tumor growth inhibition (TGI) observed with DuoBody® treatment (78% TGI with MOLM-13 vs 15% TGI with MOLM-13+HS-5). Impaired TGI correlated with reduced T cell activation (7 fold decrease in CD25 upregulation) and production of granzyme B (8 fold reduction), providing one potential mechanism to explain loss of activity of the DuoBody® antibody. In vitro trans-well redirection assays revealed that cell-cell contact with stromal cells was crucial for reduced T cell activation and target cell survival relative to controls. Additionally, leukemic cells not killed by T cells were observed to preferentially cluster around stromal cells. We propose that target cells can evade T cell death by a stromal cell dependent mechanism involving activation of multiple pro-survival and anti-apoptotic pathways in leukemic cells in addition to suppressed activation of T cells. We are currently studying pathways mediating the cross-talk between cancer, immune and stromal cells. A better understanding of the mechanisms underlying the protective and immunosuppressive nature of the BM microenvironment will be instrumental to the design of more effective CD3 redirected therapeutics or novel combinatorial regimens for robust anti-cancer responses. Disclosures Nair-Gupta: Janssen: Employment. Rudnick:Janssen Pharmaceuticals R&D: Employment. Luistro:Janssen: Employment. Chin:Janssen: Employment. Smith:Janssen Research & Development, LLC: Employment, Equity Ownership. McDaid:Janssen Pharmaceuticals Research and Development: Employment. Li:Janssen: Employment. Pillarisetti:Janssen Research and Development, LLC: Employment. Baldwin:Janssen: Employment. Packman:Janssen: Employment. Elsayed:Janssen: Employment. Attar:Janssen: Employment. Gaudet:Janssen Pharmaceuticals R&D: Employment, Other: Stock options, Patents & Royalties: pending, not yet issued.

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.

The Acquired CD40L Deficiency in B-CLL Is Reversible and Due To Contact Dependant and Independent Factors.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2813-2813
Author(s):  
Andrea G.S. Buggins ◽  
Julie Richards ◽  
Piers E.M. Patten ◽  
Ghulam J. Mufti ◽  
Stephen Devereux
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Cell Contact ◽  
Normal Donor ◽  
The Impact

Abstract Interactions between CD40L (CD154) and its receptor CD40, are of central importance in T-cell mediated B-cell activation, proliferation and isotype switching and the regulation of antigen presentation by dendritic cells. Peripheral blood T lymphocytes from patients with B cell chronic lymphocytic leukemia (B-CLL) have an acquired defect of activation induced CD40L expression that might contribute to the immunodeficiency characteristic of the disease. In view of recent reports that T-cells within bone marrow and lymph node pseudofollicles express CD40L we have re-examined the mechanism of the deficiency in PB cells. Activation of normal resting naïve and memory (CD45RA+ and −) T-cells with PMA and ionomycin increased the number expressing surface CD40L from a mean of 2 and 0% to 81.2 and 66.5% respectively. In line with previous reports, up-regulation of CD40L by CD4+45RA+ and CD4+45RA− T-cells from patients with B-CLL was reduced to a mean of 11.8% and 2.6%. This defect is reversible since removal of T-cells from the malignant clone by CD3 selection increased the number of cells able to up-regulate CD40L to 62.2% and 61.8% for naïve and memory CD4 subsets. To investigate whether this phenomenon is due to cell contact or soluble mediators, washed leukemic cells or supernatant (SN) harvested from the same cell number were incubated with normal donor T-cells for 48 hours then stimulated for 4 hours with PMA and Ionomycin. B-CLL SN reduced CD40L up-regulation by a mean of 51% (range 14–86, n=17, p<0.0001) but co-culture reduced this further to a mean of 17% (range 7–33%, n=8, p<0.0001). Other markers of T-cell activation were similarly affected, for example T-cell IL-2 production was reduced to 40.1% ( 6.2% SEM, p < 0.0001) of the level seen in the absence of B-CLL SN. Although SN and cell contact both prevented CD40L up-regulation, only cell contact caused its down-regulation in pre-activated T-cells (reduced to 94% of normal with SN, [range 93–96%, n=3, p=0.5] and 14% of normal with co-culture [range 4.3–21.7%, n=6, p p<0.0001]). The acquired CD40L deficiency observed in patients with B-CLL is thus reversible and mediated by contact with leukemic cells and soluble mediator(s). B-CLL cells are known to secrete a number of factors that might produce this effect. Studies using blocking monoclonal antibodies and immuno-adsorption excluded the most likely candidates including TGF-beta, soluble CD40 and soluble IL-2R. These findings indicate that regulation of the CD40/CD40L system in B-CLL is more complex than previously reported however the impact of this on disease pathogenesis needs to be determined.

Secondary Lymphoid Tissues Are Not Required for the Induction of GVHD Following Allogeneic BMT: A Shifting Paradigm for T Cell Allo-Activation.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3170-3170
Author(s):  
Ines A. Silva ◽  
Krystyna Olkiewicz ◽  
Jacquelyn M. Fisher ◽  
Meghana N. Chaudhary ◽  
Kevin Vannella Vannella ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Major Complication ◽  
Scoring Systems ◽  
Lymphoid Tissues ◽  
Target Tissue

Abstract Allogeneic (allo) bone marrow transplantation (BMT) is the only curative option for many patients with malignant and non-malignant diseases. Acute graft versus host disease (GVHD) is the major complication of allo-BMT and limits the utility of this treatment strategy. The induction of GVHD fundamentally depends upon the activation of donor T cells by host antigen presenting cells (APCs), and the prevailing hypothesis is that these critical interactions occur in secondary lymphoid organs, such as lymph nodes (LN) Peyer’s patches (PP), and spleen (SP). We tested this hypothesis by using a well established, MHC disparate, murine SCT system (Balb/c → B6) and homozygous aly/aly (alymphoplasia) mice that are deficient in all LN and PP and heterozygous aly/+ littermate controls. Lethally irradiated, splenectomized, aly/aly mice (LN/PP/SP −/ −) and aly/+ sham mice (LN/PP/SP +/+) received BMT either from syngeneic (aly/aly) or allo (Balb/c) donors. In some experiments, wild-type B6 recipients of B6 or Balb/c BMT served as additional negative and positive GVHD controls respectively. The severity of GVHD was assessed by survival and well-described scoring systems of both clinical and target organ disease. As expected, greater than 95% of syngeneic (syn) BMT recipients survived and were indistinguishable from naïve, un-transplanted controls, whereas LN/PP/SP +/+ mice receiving allo-BMT showed significant signs of GVHD with ~40% mortality by day 49. All LN/PP/SP −/ − allo-BMT recipients also survived, but surprisingly, examination demonstrated that they too developed significant clinical GVHD compared to syn controls (score: 3.2 vs. 0.85) that was comparable in severity to LN/PP/SP +/+ mice (3.1). Moreover, histopathologic analysis demonstrated that LN/PP/SP −/ − allo-BMT recipients developed significantly greater GVHD target tissue damage in the liver, intestinal tract and skin compared to syn controls. In fact, LN/PP/SP −/ − allo-BMT recipients developed more severe hepatic GVHD compared to allo littermate (LN/PP/SP +/+) controls (30.8±1.9 vs. 20.7±2.2; p < 0.01). Similar differences in liver GVHD was also seen between allo groups as early as day 7 (16.0±2.2 vs. 7.3±0.9; p < 0.01). We next tested the ability of host aly/aly and aly/+ APCs to stimulate donor Balb/c T cells in vitro. No differences in proliferation, IFN γ production or CTL generation were detected, thus showing that the allo-stimulatory capacity of host APCs was not different between groups. In order to ascertain what extra-lymphoid host tissues might serve as initial sites for allo-antigen exposure, we examined donor T cell expansion (CD3+), activation (CD69+) and proliferation (CFSE) in the bone marrow compartment 3 days after BMT. We found that in each case, LN/PP/SP −/ − allo-BMT recipients had significantly higher numbers / divisions compared to allo, littermate, (LN/PP/SP +/+) controls. Collectively, these data challenge the paradigm that secondary lymphoid tissues are required for GVHD induction, and suggest that the bone marrow may represent an alternative site for allo-antigen recognition and donor T cell activation.

scholarly journals Development of a CD123xCD3 Bispecific Antibody (JNJ-63709178) for the Treatment of Acute Myeloid Leukemia (AML)

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2824-2824 ◽  
Author(s):  
François Gaudet ◽  
Jennifer F Nemeth ◽  
Ronan McDaid ◽  
Yingzhe Li ◽  
Benjamin Harman ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
T Cell Activation ◽  
Stock Options ◽  
Cell Activation ◽  
Bispecific Antibody ◽  
Tumor Regression

Abstract AML is a cancer of the myeloid lineage that is characterized by the accumulation of abnormal white blood cells in the bone marrow and blood. Existing therapies do not lead to cures, partially due to their inability to eliminate residual leukemic stem cells (LSCs) in the bone marrow. T-cell redirection has been shown to be an effective method of treatment for hematologic malignancies (eg, blinatumomab) and represents an attractive approach to treat AML. CD123 (α-chain of the interleukin-3 receptor) has been shown to be expressed on the surface of AML blasts and LSCs. To eradicate CD123+ cells, we developed a bispecific antibody (JNJ-63709178) using the Genmab DuoBody® technology that can bind both CD123 on tumor cells and CD3 on T cells. JNJ-63709178 is a humanized IgG4 bispecific antibody with silenced Fc function. This antibody is able to recruit T cells to CD123-expressing tumor cells and induce the killing of these tumor cells in vitro (MOLM-13, OCI-AML5 and KG-1; EC50 = 0.51-0.91 nM). In contrast, this antibody does not kill CD123- cell lines, demonstrating the specificity of cytotoxicity. Consistently, the degree of cell killing correlated with the level of T-cell activation (CD69 and CD25) and cytokine release (TGF-β and TNF-α). Control bispecific antibodies containing a null arm (viral epitope) paired with a CD123 arm (CD123xnull) or a CD3 arm (nullxCD3) did not induce cytotoxicity or T-cell activation in the assays tested. JNJ-63709178 had no effect on T-cell activation when incubated with T cells alone. In AML murine xenograft models, JNJ-63709178 was able to suppress tumor growth and induce tumor regression (MOLM-13 and KG-1, respectively) in the presence of human peripheral blood mononuclear cells (PBMCs) or T cells. Tumor regression correlated with the infiltration of T cells in the tumor and the expression of T-cell activation markers such as CD25, PD1 and TIM3. Furthermore, this antibody was able to induce the killing of primary CD123+ cancer cells from the blood of patients with AML without the need to supplement with fresh T cells (EC50 = 0.83 nM). These results indicate that JNJ-63709178 can potently and specifically kill CD123+ cancer cells in vitro, in vivo and ex vivo. Pharmacokinetic studies in cynomolgus monkeys support twice weekly dosing for human studies. JNJ-63709178 is currently being investigated in a Phase 1 clinical trial in relapsed and refractory AML (ClinicalTrials.gov ID: NCT02715011). Disclosures Gaudet: Janssen Pharmaceuticals R&D: Employment, Other: Stock options, Patents & Royalties: pending, not yet issued. Nemeth:Janssen Pharmaceuticals R&D: Employment, Other: stock, Patents & Royalties: patent pending. McDaid:Janssen Pharmaceuticals Research and Development: Employment. Li:Janssen: Employment. Harman:Janssen Pharmaceuticals R&D: Employment. Millar:Janssen Pharmaceuticals R&D: Employment, Other: stock options. Teplyakov:Janssen Pharmaceuticals R&D: Employment. Wheeler:Janssen Pharmaceuticals R&D: Employment. Luo:Janssen Pharmaceuticals R&D: Employment. Tam:Janssen Pharmaceuticals R&D: Employment, Other: stocks, Research Funding. Wu:Janssen Pharmaceuticals R&D: Employment. Chen:Janssen Pharmaceuticals R&D: Employment. Rudnick:Janssen Pharmaceuticals R&D: Employment. Chu:Janssen Pharmaceuticals R&D: Employment. Hughes:Janssen Pharmaceuticals R&D: Employment. Luistro:Janssen: Employment. Chin:Janssen: Employment. Babich:Janssen: Employment. Kalota:Janssen Pharmaceuticals R&D: Employment, Other: stock. Singh:Janssen Pharmaceuticals R&D: Employment, Other: stock options. Salvati:Janssen Pharmaceuticals R&D: Employment, Other: stock options, Patents & Royalties: patent. Elsayed:Janssen: Employment, Other: stock options. Attar:Janssen: Employment.

scholarly journals Exploiting an Anti-CD3/CD33 Bispecific Antibody to Redirect Donor T Cells Against HLA Loss Leukemia Relapses

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 513-513
Author(s):  
Pier Edoardo Rovatti ◽  
Laura Zito ◽  
Eleonora Draghi ◽  
Monika Herrmann ◽  
Anetta Marcinek ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Research Funding ◽  
Cell Activation ◽  
Bispecific Antibody ◽  
Leukemic Cells ◽  
Donor T Cells ◽  
Hla Loss

Background Genomic loss of mismatched HLAs ("HLA loss") represents a frequent modality by which acute myeloid leukemia (AML) evades immune recognition from donor T cells after partially HLA-incompatible allogeneic hematopoietic cell transplantation (allo-HCT). One important consequence of this post-transplantation relapse mechanism is that infusions of lymphocytes from the original donor become ineffectual, prompting the search for alternative therapeutic options. Here, to circumvent the loss of physiological T cell receptor-HLA interactions in these patients, we tested the ability of an anti-CD3/CD33 bispecific antibody (BsAb) to re-target donor T cells towards HLA loss relapses. Methods For short-term in vitro experiments, T cells were co-cultured with the MOLM-13 AML cell line or with primary patient blasts for 96 hours in presence or absence of an anti-CD3/CD33 BsAb. As readouts, we measured T cell activation (as surface expression of CD25 and CD69) and the absolute counts and relative proportion of effectors and targets. For long-term in vitro experiments, we established mixed lymphocyte cultures (MLCs) of T cells purified from two patients after haploidentical HCT and primary AML blasts obtained from the same patients at the time of diagnosis. After sequential stimulations, the co-cultures were tested against targets of interest, with or without addition of the BsAb. Functional readouts were T cell degranulation (measured as CD107a expression), antigen-specific activation (as CD137/41-BB expression) and target-specific cytotoxicity (measured by time-lapse live cell imaging over a 48 hour time span). For in vivo experiments, human leukemic cells were infused intravenously into non-irradiated NSG mice, followed by intraperitoneal infusion of T cells and daily administration of the BiTE compound. Results First, we retrospectively analyzed immunophenotypic data of 36 AML patients who experienced HLA loss relapses at our Institution, documenting robust expression of CD33 on the surface of the relapsed leukemia in 35 of them (97%; Figure 1A). By short-term co-culture experiments we titrated the BsAb concentration to be used for subsequent in vitro assays to 100 ng/ml, and the most informative effector:target ratio to 1:3. Then, we established MLCs by stimulating T cells collected from two patients after partially HLA-incompatible allo-HCT with AML blasts collected from the same patients at the time of diagnosis. In both cases, donor-derived T cells robustly responded against the patient blasts both in term of degranulation (Figure 1B) and of antigen-specific activation (Figure 1C). As expected, when we tested the same T cells against the patient leukemia at time of HLA loss relapse, we detected no T cell-mediated responses. Noticeably, when the BsAb was added, in both cases we detected a strong response not only against the diagnosis but also against the HLA loss variants, indicating that T cells were effectively re-targeted towards leukemic cells. Similar results were obtained also by live cell imaging, measuring target cell apoptosis over 48 hours of recording: also in this assay, in fact, donor T cells recognized and killed leukemia at diagnosis (45% of detection area positive for apoptosis dye) and failed to recognize its HLA loss relapse counterpart (32% of area positive for apoptosis dye). Addition of the BsAb to the co-cultures had a minor effect on recognition of the original disease (45% of area positive for apoptosis dye) but drove dramatic cell death of HLA loss blasts (80% of area positive for apoptosis dye), demonstrating that the BsAb induced not only T cell activation but also and most importantly target cell killing (Figure 1D). Finally, we modeled the BsAb activity in vivo, showing that, whereas the sole infusion of human T cells is not able to prevent the outgrowth of leukemia in the bone marrow of NSG mice, addition of the bispecific antibody leads to effective disease clearance (Figure 1E). Conclusions Our results demonstrate that anti-CD3/CD33 BsAbs can effectively redirect donor T cells against HLA loss leukemia variants, resulting in their rapid and effective killing. Taken together, these promising findings strongly support translation of this approach to ad hoc designed early-phase clinical trials, to provide a rational therapy for this increasingly recognized but still treatment-orphan modality of post-transplantation relapse. Figure 1 Disclosures Subklewe: Janssen: Consultancy; Miltenyi: Research Funding; Pfizer: Consultancy, Honoraria; Oxford Biotherapeutics: Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria; Morphosys: Research Funding; Roche: Consultancy, Research Funding; AMGEN: Consultancy, Honoraria, Research Funding. Vago:Moderna Therapeutics: Research Funding; GenDx: Research Funding.

scholarly journals Augmentation of T-cell activation by oscillatory forces and engineered antigen-presenting cells

2019 ◽  
Author(s):  
Fatemeh S. Majedi ◽  
Mohammad Mahdi Hasani-Sadrabadi ◽  
Timothy J. Thauland ◽  
Song Li ◽  
Louis-S. Bouchard ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Signal Strength ◽  
Antigen Presenting Cells ◽  
Oscillatory Movement ◽  
Antigen Presenting ◽  
The Impact

AbstractActivation of T cells by antigen presenting cells allows them to proliferate, produce cytokines, and kill infected or cancerous cells. We and others have shown that T cell receptors receive and in fact require mechanical forces from their own movements and the movements of antigen presenting cells. Emulation of T cell activation in vitro allows for the massive expansion of T cells necessary for clinical applications. In this paper, we studied the impact of augmenting novel artificial antigen presenting cells of various sizes and antigenic signal strength with mechanical, oscillatory movement. We showed that dynamic culture roughly doubles signal strength as compared to conventional, static culture. We demonstrated that tuning the strength of signal to a “sweet spot” allows for robust expansion of induced regulatory T cells, which is impeded by approaches that simply maximize activation.

Bone marrow microenvironment controls the in vivo differentiation of murine dendritic cells into osteoclasts

Blood ◽  
2008 ◽  
Vol 112 (13) ◽  
pp. 5074-5083 ◽  
Author(s):  
Abdelilah Wakkach ◽  
Anna Mansour ◽  
Romain Dacquin ◽  
Emmanuel Coste ◽  
Pierre Jurdic ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Bone Cells ◽  
Activated T Cells

Abstract Finding that activated T cells control osteoclast (OCL) differentiation has revealed the importance of the interactions between immune and bone cells. Dendritic cells (DCs) are responsible for T-cell activation and share common precursors with OCLs. Here we show that DCs participate in bone resorption more directly than simply through T-cell activation. We show that, among the splenic DC subsets, the conventional DCs have the higher osteoclastogenic potential in vitro. We demonstrate that conventional DCs differentiate into functional OCLs in vivo when injected into osteopetrotic oc/oc mice defective in OCL resorptive function. Moreover, this differentiation involves the presence of activated CD4+ T cells controlling a high RANK-L expression by bone marrow stromal cells. Our results open new insights in the differentiation of OCLs and DCs and offer new basis for analyzing the relations between bone and immune systems.

T Cell Pathway Deficiencies in Chronic Lymphocityc Leukemia: Partial Restoration with OKT3/IL-2 Activation.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4692-4692
Author(s):  
Mauro Di Ianni ◽  
Lorenzo Moretti ◽  
Beatrice Del Papa ◽  
Maria De Ioanni ◽  
Adelmo Terenzi ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Activated T Cells ◽  
Mutational Status ◽  
The Impact

Abstract As Chronic Lymphocytic Leukemia (CLL) is associated with several defects in the T cell compartment, the impact of tumour burden on the autologous immune system was studied. Gene expression profiles (using Applied Biosystem Human Genome Microarray) identified 237 genes with significantly increased expression and 221 genes with significantly decreased expression (p<0.05) in CD3+ cells from CLL patients compared with healthy donors. Panther software analysis identified 34/237 upregulated genes and 26/221 downregulated genes that were involved in specific pathways, mainly cell differentiation and proliferation, survival, apoptosis, cytoskeleton formation, vesicle trafficking and T cell activation. The 26 dowregulated genes included Zap70, a member of the syk family protein tyrosine kinase, which is involved in T-cell activation. Zap-70 results were validated by mRNA quantification by RT-PCR (−1.77 fold in comparison with healthy controls) and by flow-cytometric analysis (Mean Intensity Fluorescence=33±12 vs 80±23.62 in controls, p<0.05). To test the hypothesis that activation with OKT3 /IL-2 could bypass these T cell deficiencies, activated T cells from 20 patients with CLL were tested in vitro for cytotoxicity (using the 51chromium release assay) against mutated and unmutated (according to IgVH mutational status) autologous B cells, DAUDI, K562 and P815 cell lines. After 10 days’ culture, the T cell count remained unchanged; CD8 cells expanded more than CD4; TCR spectratyping analysis indicated no differences in TCR repertoires. Activation restored the ZAP-70 mRNA (+1.67 fold). The 51chromium release cytotoxicity assay showed an index > 30% in 5/20 patients. The other 15 were partially cytotoxic against P815, K562 and Daudi. Cell line analysis in all 20 confirmed prevalently T cell-mediated cytotoxicity and poor NK/LAK activity. Cytotoxicity did not correlate with B cell mutational status. We tested the cytotoxic activity of autologous activated T cells in NOD/SCID mice co-transplanted with leukaemic B cells. Only activated T cells exerting cytotoxicity vs autologous B-cell CLL prevent CLL in human-mouse chimera, as confirmed by PCR and FACS analysis which visualised only CD3+ cells. In conclusion, in patients with CLL, activating autologous T cells with OKT3 /IL-2 bypasses, at least in part, the T cell immunological deficiencies. These in vitro and in vivo findings might serve to throw light on new mechanisms that could be exploited in immunotherapy designed to exert disease control.

scholarly journals Ex vivo modelling of PD-1/PD-L1 immune checkpoint blockade under acute, chronic, and exhaustion-like conditions of T-cell stimulation

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Alexander Roberts ◽  
Lindsay Bentley ◽  
Tina Tang ◽  
Fay Stewart ◽  
Chiara Pallini ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Immune Checkpoint ◽  
Inflammatory Cytokine ◽  
Cell Activation ◽  
Ex Vivo ◽  
In Vitro Assays ◽  
The Impact

AbstractBlockade of PD-1/PD-L1 interactions is proving an exciting, durable therapeutic modality in a range of cancers whereby T cells are released from checkpoint inhibition to revive their inherent anti-tumour activity. Here we have studied various ways to model ex vivo T cell function in order to compare the impact of the clinically utilised anti-PD-1 antibody, pembrolizumab (Keytruda) on the activation of human T cells: focussing on the release of pro-inflammatory IFNγ and anti-inflammatory IL-10 to assess functionality. Firstly, we investigated the actions of pembrolizumab in an acute model of T-cell activation with either immature or mature allogeneic dendritic cells (DCs); pembrolizumab enhanced IFNγ and IL-10 release from purified CD4+ T-cells in the majority of donors with a bias towards pro-inflammatory cytokine release. Next, we modelled the impact of pembrolizumab in settings of more chronic T-cell activation. In a 7-day antigen-specific response to EBV peptides, the presence of pembrolizumab resulted in a relatively modest increase in both IFNγ and IL-10 release. Where pembrolizumab was assessed against long-term stimulated CD4+ cells that had up-regulated the exhaustion markers TIM-3 and PD-1, there was a highly effective enhancement of the otherwise exhausted response to allogeneic DCs with respect to IFNγ production. By contrast, the restoration of IL-10 production was considerably more limited. Finally, to assess a direct clinical relevance we investigated the consequence of PD-1/PD-L1 blockade in the disease setting of dissociated cells from lung and colon carcinomas responding to allogeneic DCs: here, pembrolizumab once more enhanced IFNγ production from the majority of tumour preparations whereas, again, the increase in IL-10 release was modest at best. In conclusion, we have shown that the contribution of PD-1—revealed by using a canonical blocking antibody to interrupt its interaction with PD-L1—to the production of an exemplar pro- and anti-inflammatory cytokine, respectively, depends in magnitude and ratio on the particular stimulation setting and activation status of the target T cell. We have identified a number of in vitro assays with response profiles that mimic features of dissociated cell populations from primary tumours thereby indicating these represent disease-relevant functional assays for the screening of immune checkpoint inhibitors in current and future development. Such in vitro assays may also support patient stratification of those likely to respond to immuno-oncology therapies in the wider population.

ATG5 Dependent Autophagy Uncouples T Cell Functions and Modulates Experimental Graft-Versus-Host Disease

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 149-149
Author(s):  
Katherine Oravecz-Wilson ◽  
Corinne Rossi ◽  
Chen Liu ◽  
Tomomi Toubai ◽  
Hiroya Tamaki ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
T Cell Proliferation ◽  
Apoptotic Proteins

Abstract ATG5 is a key protein that regulates autophagy, a vital cellular process whose role in various immune cells is poorly understood. A recent report showed that the deficiency of autophagy gene Atg16l1 in host DCs increased graft-vs-host disease (GVHD). Nevertheless, the direct role of autophagy in regulating T cell alloreactivity after bone marrow transplant (BMT) is unknown. In order to investigate the role of autophagy in T cells, we first analyzed the changes in autophagosome marker LC3 upon WT T cell activation. TCR stimulation with anti-CD3/CD28, increased cytosolic LC3-I and its membrane-bound LC3-II form. Interestingly, we found that the upregulation of LC3 was predominant in dividing cells, which lead us to hypothesize that autophagy is essential for T cell proliferation. Therefore we next explored if the deficiency in autophagy impaired T cell proliferation utilizing B6 T cell-specific ATG5 knockout (ATG5 KO T cell) mouse and hydroxychloroquine (CQ, a known inhibitor of autophagy). As hypothesized, when compared with WT controls, both CQ treated WT T cells and the ATG5 KO T cells, in vitro, demonstrated a significant decrease in proliferation as demonstrated by 3H-thymidine incorporation and CFSE staining (p<0.0001) and were associated with a failure to upregulate LC3. These effects were observed after anti-CD3/CD28 TCR stimulation as well as following allogenic stimulation in a mixed lymphocyte reaction (MLR) with bone marrow-derived dendritic cells (DCs) from BALB/c mice. The reduction in T cell proliferation was accompanied by a significant increase in apoptosis (p<0.0001). However it was not associated with a decrease in T-helper (TH) signature cytokines (IFNγ, IL-2, IL-17, IL-4) suggesting no impact on T cell differentiation. Furthermore ATG5 deficiency also did not alter T cell activation as determined by upregulation of NFAT and ZAP70. Thus lack of autophagy lead to the decrease survival of T cell along with decreased proliferation after TCR stimulation but did not affect TH differentiation and T cell activation. Given the in vitro observations, we hypothesized that ATG5 KO T cells would also induce less GVHD following allogenic bone marrow transplantation (BMT). Utilizing clinically relevant MHC-mismatched B6 → BALB/c BMT model, we lethally irradiated (800cGy) WT-BALB/c mice and transplanted 5x106 T cell-depleted bone marrow from WT-B6 mice along with 0.5x106 splenic T cells purified from ATG5 KO or WT- B6 mice. WT-BALB/c TCD BM and T cells were used for syngeneic controls. Consistent with in vitro results, ATG5 KO T cells showed decreased proliferation in vivo but showed no difference in Th1/Th17 differentiation. Allogenic animals transplanted with ATG5 KO T cells also showed a significantly improved survival (p=0.001) and reduced GVHD severity (p=0.03). Phenotypic analyses prior to BMT showed that ATG5 KO T cells show decreased CD62L and an increased expression of CD44. Because naïve (CD62L+CD44-) T cells are critical for GVHD, we next explored if the observed improvement in GVHD could be due to a decreased population of these naïve T cells in the transplant inoculum of ATG5 KO animals. Therefore, using the same BMT model and design, we transplanted WT-BALB/c mice 0.5x106 isolated splenic CD62L+ T cells only from either ATG5 KO or WT-B6 animals and observed that GVHD mortality was reduced in the allo-recipients of ATG5 KO T cells compared with WT T cells (p=0.005). To determine the potential molecular mechanism, we next hypothesized that upon activation ATG KO T cells may show alterations in pro and anti-apoptotic proteins. We observed that ATG5 KO failed to increase Bcl-2 level and showed a decrease in Bcl-XL level upon TCR stimulation compared to WT T cells. In contrast to the relative lack of anti-apoptotic proteins, they displayed similar levels of the pro-apoptotic proteins BIM, Bak and Bax. These results suggest that an imbalance between pro- and anti-apoptotic factors is likely a cause for the reduced T cell expansion by ATG5 KO T cells. Our results collectively demonstrate that inhibition of autophagy decreases T cell expansion but not its differentiation in vitro and in vivo. Furthermore contrary to the aggravation of GVHD when autophagy is targeted in host DCs, it mitigated GVHD when targeted in donor T cells. Thus the net impact of manipulating autophagy after allogeneic BMT on GVHD is dependent on which immune cell subsets are being targeted. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures No relevant conflicts of interest to declare.

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