The CXCR4/STAT3/IL-10 Pathway Controls the Immunoregulatory Function of Chronic Lymphocytic Leukemia (CLL) and Can be Modulated By Lenalidomide

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1709-1709 ◽  
Author(s):  
Hila Shaim ◽  
Zeev Estrov ◽  
Takuya Sekine ◽  
Kayo Kondo ◽  
Peter P. Ruvolo ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Research Funding ◽  
Cell Function ◽  
Blocking Antibody ◽  
Cell Dysfunction ◽  
T Cell Suppression ◽  
T Cell Function ◽  
Stat3 Phosphorylation ◽  
T Cell Dysfunction

Abstract Patients with CLL experience generalized immune suppression, susceptibility to infections and secondary malignancies that likely involve complex bi-directional interactions between leukemic cells, components of the tumor microenvironment and immune effectors. CLL cells are capable of secreting IL-10 and exhibit regulatory functions comparable to that of normal B10 cells, a regulatory B cell subset that suppresses effector T-cell function through STAT3-mediated production of IL-10. However, the underlying mechanisms governing IL-10 production by CLL cells are not fully understood. The chemokine CXCL12 is constitutively secreted by bone marrow stroma in CLL, and binds CXCR4 to direct chemotaxis, support tumor survival and activate various signaling pathways, including STAT3. Thus, we investigated if CXCL12 can enhance IL-10 production by activating the STAT3 pathway in CLL. Using peripheral blood mononuclear cells (PBMC) from 24 CLL patients who had not received therapy for ≥2 years, we showed that CXCL12 can enhance IL-10 production by CLL cells by activating S727-STAT3. This effect was CXCR4-mediated since blocking the CXCR4-CXCL12 interaction with a blocking antibody abolished CXCL12-induced IL-10 production. Addition of the STAT3 inhibitor curcubitacin to the culture also abrogated CXCL12-induced IL-10 production, confirming an important role for S727-STAT3 as a mediator of CXCL12-CXCR4-induced IL-10 production by CLL cells. We next determined if activation of the CXCR4-CXCL12-STAT3-IL10 pathway in CLL is important in mediating their immunoregulatory function. Culture of primary CLL withCXCL12 induced significantly more suppression of CD3+ T cell function, including TNF-α, IFN-γ and IL-2 production, and CD107a degranulation, compared to CD3+ T cells cultured with untreated CLL cells or with CXCL12 alone. The addition of IL-10 blocking antibody to the co-culture completely reversed T cell dysfunction, supporting an important for IL-10 in CLL-mediated T-cell suppression. IL-10 has been reported to induce T cell suppression through phosphorylation of Y705-STAT3.. Blocking IL-10 also prevented CLL-induced phosphorylation of Y705-STAT3 in T cells, confirming an important role for CLL-derived IL-10 in activation of Y705-STAT3 and induction of T cell dysfunction. Lenalidomide is an immune-modulatory drug with clinical efficacy in CLL and was recently reported to inhibit STAT3 phosphorylation. To investigate if lenalidomide can inhibit CXCL12-induced STAT3 phosphorylation, we treated CLL cells with lenalidomide and measured p-S727-STAT3 levels. Exposure of CLL cells to 10µM/ml lenalidomide prevented CXCL12-induced increase in p-S727-STAT3 and resulted in significant reduction in the IL-10 response by CLL cells. Lenalidomide also suppressed IL-10-induced Y705-STAT3 phosphorylation in healthy T cells, thus reversing CLL-induced T cell dysfunction. We next confirmed the in vivo relevance of our findings using PBMC cryopreserved from patients treated with lenalidomide monotherapy (NCT00535873). When compared to pretreatment samples, CLL cells from on-treatment samples produced less IL-10 and showed significantly improved T cell function. We thus conclude that the capacity of CLL to produce IL-10 is mediated by the CXCL12-CXCR4-STAT3 pathway and may contribute to immunodeficiency in patients. Lenalidomide can reverse CLL-induced immunosuppression through multiple mechanisms that involve abrogation of the CXCL12-CXCR4-S727STAT3-mediated IL-10 response by CLL B cells and prevention of IL-10-induced phosphorylation of Y705-STAT3 in T cells. Disclosures Estrov: incyte: Consultancy, Research Funding. Wierda:Glaxo-Smith-Kline Inc.: Research Funding; Celgene Corp.: Consultancy. Rezvani:Pharmacyclics: Research Funding.

scholarly journals Chronic Lymphocytic Leukemia Actively Induces T-Cell Dysfunction By Contact-Dependent Signaling Via CD24 and CD52

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3714-3714
Author(s):  
Jaco A. C. Van Bruggen ◽  
Fleur Peters ◽  
Gaspard Cretenet ◽  
J. Joseph Melenhorst ◽  
Eric Eldering ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Research Funding ◽  
Cell Activation ◽  
Cell Function ◽  
Cell Dysfunction ◽  
T Cell Function ◽  
Car T ◽  
T Cell Dysfunction

Abstract Introduction Success rates of autologous T cell-based therapies, such as CAR-T cell therapy, in chronic lymphocytic leukemia (CLL) have been suboptimal and correlate with failure of activation and proliferation of T cells in vitro and in vivo. Previous data showing that impaired CD8 T-cell activation, proliferation and metabolic reprogramming could be restored by purifying CLL T cells via cell-sorting (van Bruggen et al., Blood, 2019) indicating that an as yet unknown, CLL-derived factor is responsible for acquired T-cell dysfunction. In this study we aim to elucidate the mechanistic basis of CLL-mediated T-cell dysfunction. Results Dynamic analysis of αCD3/CD28 stimulated autologous T cells in presence of CLL cells over a period of 9 days revealed that T-cell activation (CD25, CD71, CD95 and PD-1) in CLL is in fact not impaired but occurs in a delayed fashion. CLL T cells reached peak activation after 5-6 days in contrast to 2-3 days for age-matched healthy donors. (Fig. 1A). This delayed T cell receptor-induced T cell activation was largely normalized with tumor cell depletion by flow-sorting prior to activation. Accordingly, in absence versus presence of autologous CLL cells, CAR-T cells derived from CLL patients showed enhanced proliferation, cytokine production and cytotoxicity, indicating potential clinical relevance. These findings show that T cells in CLL are not (terminally) exhausted but that a CLL-derived factor interferes with proper T-cell activation, leading to a delay in activation and impaired proliferation and cytotoxicity. We attempted to identify the mechanism of action in which CLL cells induce T cell dysfunction and whether these suppressive effects are mediated through a soluble factor secreted by CLL cells or by contact-dependent mechanisms. Previous studies have shown that CD40 activation of CLL cells results in increased expression of key surface-expressed adhesion and costimulatory molecules, but also in alterations of immune-modulatory cytokines secretion. This model was therefore used to decipher mechanisms of CLL-mediated T cell dysfunction. CD40-activation of CLL cells resulted in improved T-cell activation and proliferation upon αCD3/CD28 stimulation in a contact-dependent manner (based on trans-well experiments; Fig. 1B ). Several clinically approved kinase inhibitors were tested to identify signaling cascades involved in CD40-mediated alleviation of T-cell dysfunction. Only pre-treatment of CLL cells with the SRC-inhibitor dasatinib (100nM) abrogated the enhanced T-cell activation induced by CD40-activated CLL cells. Additional control experiments excluded direct effects of dasatinib on T cell function. Dasatinib did not reduce expression of co-stimulatory markers on CD40-activated CLL cells, indicating that lack of co-stimulation was not the sole explanation for CLL-mediated T cell dysfunction. RNA sequencing of CD40-stimulated CLL cells treated with or without dasatinib and filtered for membrane-bound factors revealed the Sialic acid-binding Ig-like lectin 10 (Siglec-10) ligands CD24 and CD52 as potential candidates responsible for inhibiting T-cell function in CLL, which we confirmed at the protein level. We also found increased expression of Siglec-10 on CLL T cells, suggesting a role for Siglec-10 ligation in inhibition of the TCR signaling cascade. Indeed, inhibition of Siglec-10 ligation by blocking CD24, and CD52 antibodies subsequently improved T-cell activation despite presence of CLL cells (Fig. 1C). Conclusion These results demonstrate that T cells derived from CLL patients are not terminally dysfunctional and can be revived. Our observations indicate that CLL cells actively suppress (CAR) T-cell function in a contact-dependent fashion through CD24- and CD52-mediated Siglec-10 ligation. These proteins might represent targets for therapeutic intervention aimed at enhancing T-cell function in CLL. Figure 1 Figure 1. Disclosures Kater: Genmab, LAVA: Other: Ad Board, Steering Committee; Abbvie: Honoraria, Other: Ad Board, Research Funding; Janssen, AstraZeneca: Other: Ad Board, steering committee, Research Funding; BMS, Roche/Genentech: Other: Ad Board, , Research Funding.

scholarly journals Expansion of Monocytic Myeloid-Derived Suppressor Cells Dampens T Cell Function in HIV-1-Seropositive Individuals

2012 ◽  
Vol 87 (3) ◽  
pp. 1477-1490 ◽  
Author(s):  
Aiping Qin ◽  
Weiping Cai ◽  
Ting Pan ◽  
Kang Wu ◽  
Qiong Yang ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Function ◽  
Suppressor Cells ◽  
Cell Contact ◽  
Myeloid Derived Suppressor Cells ◽  
Cell Dysfunction ◽  
Arginase 1 ◽  
T Cell Function ◽  
T Cell Dysfunction ◽  
Hiv 1

ABSTRACTT lymphocyte dysfunction contributes to human immunodeficiency virus type 1 (HIV-1) disease progression by impairing antivirus cellular immunity. However, the mechanisms of HIV-1 infection-mediated T cell dysfunction are not completely understood. Here, we provide evidence that expansion of monocytic myeloid-derived suppressor cells (M-MDSCs) suppressed T cell function in HIV-1-infected individuals. We observed a dramatic elevation of M-MDSCs (HLA-DR−/lowCD11b+CD33+/highCD14+CD15−cells) in the peripheral blood of HIV-1-seropositive subjects (n= 61) compared with healthy controls (n= 51), despite efficacious antiretroviral therapy for nearly 2 years. The elevated M-MDSC frequency in HIV-1+subjects correlated with prognostic HIV-1 disease markers, including the HIV-1 load (r= 0.5957;P< 0.0001), CD4+T cell loss (r= −0.5312;P< 0.0001), and activated T cells (r= 0.4421;P= 0.0004). Functional studies showed that M-MDSCs from HIV-1+subjects suppressed T cell responses in both HIV-1-specific and antigen-nonspecific manners; this effect was dependent on the induction of arginase 1 and required direct cell-cell contact. Further investigations revealed that direct HIV-1 infection or culture with HIV-1-derived Tat protein significantly enhanced human MDSC generationin vitro, and MDSCs from healthy donors could be directly infected by HIV-1 to facilitate HIV-1 replication and transmission, indicating that a positive-feedback loop between HIV-1 infection and MDSC expansion existed. In summary, our studies revealed a novel mechanism of T cell dysfunction in HIV-1-infected individuals and suggested that targeting MDSCs may be a promising strategy for HIV-1 immunotherapy.

scholarly journals Treatment with Acalibrutinib, Ibrutinib and CD19 CAR T Cells Restore the Number of Granulocytic Myeloid Derived Supressor Cells in CLL-Bearing Mice

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3032-3032
Author(s):  
Arantxa Romero-Toledo ◽  
Robin Sanderson ◽  
John G. Gribben
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Population ◽  
Research Funding ◽  
Cell Function ◽  
Car T Cells ◽  
Car T Cell ◽  
T Cell Function ◽  
Car T ◽  
Secondary Lymphoid Organs

The complex crosstalk between malignant chronic lymphocytic leukemia (CLL) cells and the tumor microenvironment (TME) is not fully understood. CLL is associated with an inflammatory TME and T cells exhibit exhaustion and multiple functional defects, fully recapitulated in Eµ-TCL1 (TCL1) mice and induced in healthy mice by adoptive transfer (AT) of murine CLL cells, making it an ideal model to test novel immunotherapies for this disease. Myeloid-derived suppressor cells (MDSCs), a non-leukemic cell type within the TME, are immature myeloid cells with the ability to suppress T cell function and promote Treg expansion. In humans, CLL cells can induce conversion of monocytes to MDSCs provoking their accumulation in peripheral blood (PB). MDSCs include two major subsets granulocytic (Gr) and monocytic (M)-MDSC. In mice, Gr-MDSCs are defined as CD11b+Ly6G+Ly6Clo and M-MDSC as CD11b+Ly6G-Ly6Chi. Both murine and human MDSCs express BTK. We observed that in CLL-bearing mice, MDSCs cells are lost in PB as disease progresses. Treatment with both BTK inhibitors (BTKi), ibrutinib (Ibr) and acalabrutinib (Acala), result in shift of T cell function from Th2 towards Th1 polarity and increase MDSC populations in vivo. We aimed to determine whether combination treatment with BTKi and chimeric antigen receptor (CAR) T cells renders recovery of the MDSC population in CLL-bearing mice. To address this question we designed a two-part experiment, aiming to mimic the clinically relevant scenario of pre-treatment of CLL with BTKi to improve CAR T cell function. Part 1 of our experiment consisted of 4 groups (n=12) of 2.5 month old C57/Bl6 mice. Three groups had AT with 30x106 TCL1 splenocytes. A fourth group of WT mice remained CLL-free as a positive control and donors for WT T cells. When PB CLL load reached >10% (day 14) animals were randomized to either Ibr or Acala at 0.15 mg/l in 2% HPBC or no treatment for 21 days. All animals from part 1 were culled at day 35 post-AT and splenic cells were isolated, analyzed and used to manufacture CAR T cells. WT, CLL, Ibr and Acala treated T cells were activated and transduced with a CD19-CD28 CAR to treat mice in part 2. Here, 50 WT mice were given AT with 20x106 TCL1 splenocytes for CLL engraftment. All mice were injected with lymphodepleting cyclophosphamide (100mg/kg IP) one day prior to IV CAR injection. At day 21 post-AT, mice were treated with WT CAR, CLL CAR, IbrCAR, AcalaCAR or untransduced T cells. MDSC sub-populations were monitored weekly in PB and SP were analysed by flow cytometry. As malignant CD19+CD5+ cells expands in PB, the overall myeloid (CD19-CD11b+) cell population was not affected, but MDSCs significantly decreased (p<0.0001). Treatment with Acala, but not Ibr restores total MDSCs. However, MDSC impairment occurs in the Gr- but not M- MDSC population and both Acala and Ibr restores this population (Figure 1a). When we examined the spleen, treatment with both Ibr (p<0.001) and Acala (p<0.001) reduced CD5+CD19+ cells, whereas neither BTKi affected the overall myeloid (CD19-CD11b+) cell population. Gr-MDSCs were restored by both treatments whilst M-MDSCs were only restored after Ibr treatment (p<0.001 in each case). In part 2 of this experiment we observed that treatment with all CAR-T cell groups provokes the clearance of all CD19+CD5+ cells. The overall CD19-CD11b+ population stays the same across all mice groups 35 days after treatment in PB with any group of CAR and untransduced T cells. Overall MDSC population is maintained following all CAR T cells compared to CLL-bearing mice (p<0.0001) and it is the Gr- but not the M- MDSC population which is recovered in PB (Figure 1b). These parts of the experiments can of course be influenced by treatment with cyclophosphamide. We conclude that novel therapies for CLL treatment have an effect not only in CLL cells but also in non-malignant cell components of the TME. In this animal model of CLL, the rapid expansion of CLL cells in PB and secondary lymphoid organs provokes loss of MDSC, particularly the Gr-MDSC subpopulation is affected. Treatment with BTKi and CAR T cells provokes clearance of CLL cells in PB and spleen allowing MDSC recovery; suggesting this may be BTK and ITK independent. We continue to explore secondary lymphoid organs to further characterize the shift of the CLL microenvironment from an immunosuppressive to an immune effective one and its impact on immune function in this model. Disclosures Sanderson: Kite/Gilead: Honoraria. Gribben:Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria, Research Funding; Acerta/Astra Zeneca: Consultancy, Honoraria, Research Funding.

A Failure to Start: Aborted Activation of CAR T Cells in Chronic Lymphocytic Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 681-681
Author(s):  
McKensie Collins ◽  
Weimin Kong ◽  
Inyoung Jung ◽  
Meng Wang ◽  
Stefan M Lundh ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Research Funding ◽  
Cell Dysfunction ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
T Cell Dysfunction ◽  
Low Levels

Introduction: Chronic Lymphocytic Leukemia (CLL) is a CD19+ B-cell malignancy that accounts for approximately 25% of adult leukemia diagnoses in the developed world. While conventional therapies have some efficacy, there are few curative therapeutic options and many patients ultimately progress to relapsed or refractory disease. CD19-targeting chimeric antigen receptor (CAR) T cell therapy has provided some hope, but induces complete remission in only 26% of patients. This suboptimal response rate is believed to be due to T cell dysfunction and immune-suppression by CLL cells, the mechanisms of which are poorly understood. Results: To understand the causes of CAR T cell dysfunction in CLL we investigated the defects that CLL cells induced in normal donor CD19-targeting CAR T cells. CAR T cells were repeatedly stimulated at 5-day intervals with either primary CLL cells from patients or a CD19-expressing control cell line (aAPC). Repeat stimulation of CAR T cells with aAPCs resulted in 5.36 ± .94 population doublings after three stimulations, whereas CLL cells only evoked 2.39 ± .92 population doublings. We performed phenotyping, proliferation analysis, and cytokine analysis of stimulated CAR T cells. CLL-stimulated T cells appeared un-activated, with low levels of PD-1, LAG3, and TIM3, low levels of cytokine production, and a high proportion of non-cycling cells as measured by Ki67 staining. We first hypothesized that CLL cells induce an altered epigenetic program that prevents effector function and is stabilized by successive stimulations. To test this, we stimulated CAR T cells with CLL cells or aAPCs as indicated in Fig. 1A. CLL-stimulated CAR T cells failed to proliferate or produce cytokines, but subsequent stimulation with aAPCs rescued these functions (Fig. 1B). Further, CLL-stimulated CAR T cells did not differentiate, suggesting that CLL cells do not induce stable defects but rather insufficiently activate CAR T cells (Fig. 1C). These cells also appeared un-activated as indicated by low levels of PD-1 and Ki67. We then used flow cytometry to assess expression of costimulatory and inhibitory molecules on the primary CLL samples. We found that the levels of co-stimulatory and adhesion molecules, namely CD80/CD86 and CD54/CD58 respectively were found at low frequencies, and where present were expressed at low levels. This suggested that one mechanism behind the lack of CAR T cell effector responses may be that a lack of co-stimulation prevents proper CAR T cell targeting of these cells. Towards this, we incubated CLL cells with a murine fibroblast line expressing CD40 ligand for 24 hours with IL-4 to activate the CLL cells. We found that this activation significantly increased expression of CD80, CD86, CD54, and CD58 on the CLL cells. We then used these cells to stimulate CAR T cells in our re-stimulation assay and found that CAR T cells were able to proliferate in response to these activated CLLs (Fig. 1D). In addition, CAR T cells stimulated with activated CLL cells formed more cell-to-cell conjugates than those stimulated with un-activated CLL cells. These data suggest not only that insufficient activation of CAR T cells may be responsible for the poor response rates to CAR T cell therapy in CLL, but also implicate a need for additional co-stimulation in this CAR T cell setting. Another contributing factor may be immune suppression by CLL cells. To determine if CLL cells are immune-suppressive, we used a co-culture assay to stimulate CAR T cells with aAPCs and CLL cells mixed at known ratios. Interestingly, all mixed cultures proliferated similarly, suggesting that CLL cells did not prevent T cell activation in the presence of a strong activation signal. We also found that CLL cells are responsive to IL-2, as addition of this cytokine to culture media prolongs survival of CLL cells out to 10 days depending on the dose. This suggests that CLL cells express a functional IL-2 receptor and may be taking up IL-2 from the culture media, further impairing T cell activation. In support of this, supplementing IL-2 into our CLL/CAR T cell co-cultures rescued T cell proliferative capacity. Taken together, these data suggest that T cell dysfunction in CLL is the result of insufficient activation rather than true functional defects. Disclosures June: Novartis: Research Funding; Tmunity: Other: scientific founder, for which he has founders stock but no income, Patents & Royalties. Melenhorst:National Institutes of Health: Research Funding; Parker Institute for Cancer Immunotherapy: Research Funding; Novartis: Research Funding, Speakers Bureau; IASO Biotherapeutics, Co: Consultancy; Simcere of America, Inc: Consultancy; Shanghai Unicar Therapy, Co: Consultancy; Colorado Clinical and Translational Sciences Institute: Membership on an entity's Board of Directors or advisory committees; Genentech: Speakers Bureau; Stand Up to Cancer: Research Funding; Incyte: Research Funding.

Multiple Inhibitory Receptors Are Expressed on Central Memory and Memory Stem T Cells Infiltrating the Bone Marrow of AML Patients Relapsing after Allo-HSCT

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4564-4564 ◽  
Author(s):  
Maddalena Noviello ◽  
Francesco Manfredi ◽  
Tommaso Perini ◽  
Giacomo Oliveira ◽  
Filippo Cortesi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Research Funding ◽  
Central Memory ◽  
Cd8 Cells ◽  
Effector Functions ◽  
Cell Dysfunction ◽  
T Cell Dysfunction ◽  
Cell Effector

Abstract Background:Allogeneic Hematopoietic Stem Cell Transplantation (HSCT) is the only cure for high-risk acute myeloid leukemia (AML). Unfortunately, relapse still remains the major cause of death after HSCT. We investigated if T-cell dysfunction is associated to post-transplant relapse. Patients and Methods: To this,we longitudinally analyzed the T-cell dynamics in bone marrow (BM) and peripheral blood (PB) of 32 AML patients receiving HSCT from HLA identical (HLAid, 20 pts) or HLA haploidentical (haplo, 12 pts) donors. Samples were analysed by multi-parametric flow cytometry to investigate the expression of inhibitory receptors (IRs) on CD4 and CD8 T-cell subsets defined by CD45RA, CD62L and CD95 expression, and to assess the proportion of regulatory T cells (Tregs; CD4+CD25+FoxP3+). Results were also analyzed with the BH-SNE algorithm, an unbiased computational method for the analysis of FACS data. To evaluate T-cell effector functions, the CD107a degranulation assay was performed and the production of cytokines (IL-2, IFNg and TNFa) was measured by intracellular staining. BM and PB were collected 60 days after HSCT and at relapse (median 237 days; 16 pts) or, when complete remission was maintained (CR; 16 pts), at 1 year. Samples from 8 healthy donors (HD) were used as controls. Results:After transplant, BM and PB T cells showed a lower CD4/CD8 ratio (p<0.01) and a preferential late differentiation profile (p<0.05) when compared to HD. A higher proportion of BM Tregs was documented at relapse (p<0.01), independently from the donor source. We next investigated the expression of several IRs as T-cell exhaustion markers. After haplo-HSCT, PD-1, CTLA-4, 2B4 and Tim-3 were significantly upregulated in BM and PB T cells at all time-points, compared to HD and independently from the clinical outcome. Conversely, after HLAid-HSCT, at the late time-point, patients who relapsed, displayed a higher frequency of BM infiltrating T cells expressing PD-1, CTLA-4 and Tim-3 than CR pts (p<0.05) and HD (p<0.01). We then investigated the profile of each T-cell subset in our cohort. In the BM of HD the IR expression was confined to effector memory and effectors. While a similar IR distribution was observed in CR, at relapse, PD-1, 2B4 and Tim-3 were also upregulated in BM infiltrating central memory (p<0.01) and memory stem T cells (p<0.05). Interestingly, at relapse, leukemia expressed PD-L1 (9/9 cases) and Galectin-9 (6/9). The levels of Tim-3 on BM CD8 cells associates with that of Galectin-9 on autologous blasts (p<0.05), suggesting a preferential role for this immunomodulatory axis after HSCT. Based on phenotype similarities, the BH-SNE algorithm positioned HD samples separately from transplanted pts in bi-dimensional maps. 93 significant clusters were identified. Clusters associated with relapse after HLA-id (5) and after haplo (15) were composed of T cells expressing multiple IRs, while CR-specific clusters were diminished in IR fluorescence. To verify whether the T-cell exhaustion phenotypic profile at relapse associates with functional impairment, we evaluated T-cell effector functions upon polyclonal stimulation. Strikingly, we observed a lower degranulation ability of CD8 cells at relapse when compared to CR (p<0.05). In two patients, selected based on samples availability, we isolated and expanded by rapid expansion protocol (REP) T cells expressing one or more IRs (IR+) or no IR (IR-). Expansion rates were high and similar in IR+ and IR- T cells (mean fold increase 624 and 781, respectively at day 21). The degranulation ability measured ex-vivo in those patients (mean 4.4% on CD8 cells) was dramatically increased upon REP expansion (95% and 88.9% for IR+ and IR-, respectively). Similarly, the frequency of IFN-g producing CD8 cells increased in IR+ and IR- cells upon REP, indicating that the T-cell dysfunction observed at relapse can be efficiently reversed. We next challenged IR+ and IR- T cells against autologous blasts. Preliminary results suggest that IR+ T cells are enriched in leukemia specificity (elimination index of 66% and 44% in IR+ and IR- cells respectively at an E/T ratio of 100:1). Conclusions: After HSCT, the molecular signature of exhausted CD8 cells in relapsing pts includes PD-1, CTLA-4, 2B4 and Tim-3. The expression of IRs on early differentiated central memory and memory stem T cells at relapse suggests a wide, though reversible, immunological dysfunction mediated by AML relapsing blasts. Disclosures Bondanza: TxCell: Research Funding; MolMed SpA: Research Funding; Formula Pharmaceuticals: Honoraria. Ciceri:MolMed SpA: Consultancy. Bonini:TxCell: Membership on an entity's Board of Directors or advisory committees; Molmed SpA: Consultancy.

scholarly journals Dynamic Expressions of TIGIT on Splenic T Cells and TIGIT-Mediated Splenic T Cell Dysfunction of Mice With Chronic Toxoplasma gondii Infection

2021 ◽  
Vol 12 ◽  
Author(s):  
Haoran Li ◽  
Jing Zhang ◽  
Changwei Su ◽  
Xiaowei Tian ◽  
Xuefang Mei ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Toxoplasma Gondii ◽  
Cell Function ◽  
Immune Cell ◽  
Rt Pcr ◽  
Cell Dysfunction ◽  
T Cell Dysfunction ◽  
Toxoplasma Gondii Infection ◽  
Cellular Immune

As an immunosuppressive receptor, T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT) play a critical part in cellular immune regulation mediated by pathogen infection. Whereas, TIGIT expression on splenic T cells in hosts infected with Toxoplasma gondii cysts has not been studied. In this study, we detected TIGIT expression and the changes of immune function in the spleen by flow cytometry and real-time PCR (RT-PCR). We found that TIGIT expression on splenic T cells increased significantly post infection. At the same time, splenic TIGIT+TCM cells were activated and transformed into TIGIT+TEM cells during the infection, and the cytotoxicity of TIGIT+ T cells was reduced in the later stage of infection. This study shows that chronic T. gondii infection can upregulate TIGIT expression on the surface of T cells and affect immune cell function.

scholarly journals Optimizing T Cell-Based Therapy for Glioblastoma

2021 ◽  
Vol 12 ◽  
Author(s):  
Aida Karachi ◽  
Farhad Dastmalchi ◽  
Saina Nazarian ◽  
Jianping Huang ◽  
Elias J. Sayour ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Function ◽  
Treatment Strategies ◽  
Cell Dysfunction ◽  
Cell Based Therapy ◽  
Car T ◽  
Transfer Strategies ◽  
T Cell Dysfunction ◽  
T Cell Transfer

Evading T cell surveillance is a hallmark of cancer. Patients with solid tissue malignancy, such as glioblastoma (GBM), have multiple forms of immune dysfunction, including defective T cell function. T cell dysfunction is exacerbated by standard treatment strategies such as steroids, chemotherapy, and radiation. Reinvigoration of T cell responses can be achieved by utilizing adoptively transferred T cells, including CAR T cells. However, these cells are at risk for depletion and dysfunction as well. This review will discuss adoptive T cell transfer strategies and methods to avoid T cell dysfunction for the treatment of brain cancer.

scholarly journals Treatment-Free Intervals Mitigate T-Cell Exhaustion Induced By Continuous CD19xCD3-BiTE® Construct Stimulation in Vitro

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 44-45
Author(s):  
Nora Zieger ◽  
Alyssa Nicholls ◽  
Jan Wulf ◽  
Gerulf Hänel ◽  
Maryam Kazerani Pasikhani ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Research Funding ◽  
Cell Function ◽  
Granzyme B ◽  
T Cell Exhaustion ◽  
Specific Lysis ◽  
Cell Exhaustion ◽  
T Cell Function

The bispecific T-cell engager (BiTE®) blinatumomab is approved for treatment of relapsed/refractory B-cell precursor acute lymphoblastic leukemia and applied as continuous infusion over 28 days. The overall response rate to blinatumomab reported in clinical trials was 43 % and correlated to T-cell expansion (Zugmaier et al. 2015). In chronic viral infections, continuous antigen stimulation induces T-cell exhaustion, defined by phenotypic changes and functional impairment (Wherry 2011). Thus, we hypothesized that continuous BiTE® construct stimulation leads to T-cell exhaustion and that a treatment-free interval (TFI) reverses progressive T-cell dysfunction. To simulate continuous application of a BiTE® construct in vitro, T-cell long-term co-cultures were set up. Healthy donor T cells were stimulated in the presence of CD19+ OCI-Ly1 cells for 28 days with AMG 562, a half-life extended CD19 and CD3 specific BiTE® construct. T cells were harvested from the co-culture every 3-4 days between day 7 and 28 and assessed for markers of T-cell exhaustion: (1) AMG 562-mediated cytotoxicity of T cells was evaluated as specific lysis of CD19+ Ba/F3 target cells after 3 days, (2) T-cell expansion during the cytotoxicity assay was calculated as fold change (FC) of CD2+ counts, (3) Cytokine secretion of AMG 562-stimulated T cells was evaluated in co-culture supernatants by cytometric bead array (CBA) or after PMA/Ionomycine stimulation via intracellular cytokine staining (ICCS), (4) T-cell metabolic fitness was determined by Mito- and Glycolytic Stress Test using a Seahorse Analyzer, and (5) expression of the exhaustion-related transcription factor TOX was assessed by multiparameter flow cytometry. In order to assess the effect of a TFI on T-cell function, we cultured T cells and CD19+ OCI-Ly1 cells in the absence of AMG 562 from day 7-14 and 21-28 and compared their activity to T cells stimulated continuously with AMG 562. On day 7 of continuous (CONT) AMG 562 stimulation, we observed high cytotoxic and proliferative potential (% specific lysis=93±0.2, FC=2.9±0.2) as well as high IFN-g and TNF-a secretion analyzed by ICCS (% CD8+IFN-g+TNF-a+=23±6.7). However, cytotoxicity and proliferation decreased gradually until day 28 (% specific lysis=28±8.9; FC=0.6±0.1). CBA analysis confirmed decreasing secretion of IFN-g (day 3: 61113±12482, day 24: 3085±1351 pg/ml) and TNF-a (day 3: 1160±567, day 24: 43±7.6 pg/ml) as well as decreased IL-2 and granzyme B levels in culture supernatants. We furthermore observed highest mitochondrial fitness and basal glycolysis in T cells on day 7 of stimulation (basal OCR=2.2±0.6, maximal OCR=3.7±1.0, SRC=1.5±1.1 pmol/min/1000 cells, basal ECAR=2.0±0.4 mpH/min/1000 cells) which decreased until day 28 (basal OCR=0.4±0.2, maximal OCR=1.5±0.5, SRC=1.0±0.2 pmol/min/1000 cells, basal ECAR=0.5±0.2 mpH/min/1000 cells). In concordance, TOX increased during continuous stimulation (MFI ratio CD8+ day 7=6±0.8 to 12±0.8 on day 28). Strikingly, implementation of a TFI of 7 days led to superior cytotoxicity in T cells compared to continuously stimulated T cells (% specific lysis on day 14 CONT=34±4.2, TFI=99±2.2) and granzyme B production (CD8+; MFI ratio on day 14 CONT=124±11, TFI=303±34). Furthermore, increased proliferation during the cytotoxicity assay was observed in previously rested T cells (FC CONT=0.2±0.0, TFI=1.6±0.6). Although T cell function also decreased over time in TFI T cells, they maintained a strikingly higher cytotoxic potential (CONT=6±4.4, TFI=52±9.9) as well as higher granzyme B production (CONT=25±2, TFI=170±11) on day 28 compared to continuously stimulated T cells. In addition, TFI T cells showed increased IFN-g and TNF-a secretion after PMA/Ionomycine stimulation on day 28 (% CD8+IFN-g+TNF-a+ CONT=21±3.8, TFI=38±11.6). Our in vitro results demonstrate that continuous AMG 562 exposure negatively impacts T-cell function. Comprehensive analysis of T-cell activity in an array of functional assays suggests that continuous BiTE® construct exposure leads to T-cell exhaustion which can be mitigated through TFI. Currently, T cells from patients receiving blinatumomab are being analyzed to confirm the clinical relevance of our findings. Furthermore, RNA-Seq of continuously vs. intermittently AMG 562-exposed T cells will help us to understand underlying transcriptional mechanisms of BiTE® construct induced T-cell exhaustion. Disclosures Zieger: AMGEN Research Munich: Research Funding. Buecklein:Pfizer: Consultancy; Novartis: Research Funding; Celgene: Research Funding; Amgen: Consultancy; Gilead: Consultancy, Research Funding. Brauchle:AMGEN Inc.: Research Funding. Marcinek:AMGEN Research Munich: Research Funding. Kischel:AMGEN: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Subklewe:Gilead Sciences: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Morphosys: Research Funding; Seattle Genetics: Research Funding; AMGEN: Consultancy, Honoraria, Research Funding; Janssen: Consultancy; Roche AG: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Celgene: Consultancy, Honoraria.

scholarly journals A Signature of T Cell Exhaustion Is Enriched in the Bone Marrow (BM) of AML Patients and Shared with Immune Exhaustion Signatures of Solid Tumors

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2711-2711
Author(s):  
Hanna A. Knaus ◽  
Hubert Hackl ◽  
Amanda Blackford ◽  
Sofia Berglund ◽  
Raul Montiel-Esparza ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Induction Chemotherapy ◽  
Cd8 T Cells ◽  
Research Funding ◽  
Post Treatment ◽  
Cell Dysfunction ◽  
Immune Exhaustion ◽  
T Cell Dysfunction

Background: T cells are key players in immunotherapy but our understanding of their role and function in AML is limited. We have previously shown that following induction chemotherapy, the phenotypic and transcriptional profiles of peripheral blood (PB) CD8+ T cells in AML diverge between complete responders (CR) and non-responders (NR) to treatment (Knaus et al, JCI insight 2018). Further defining AML-induced T cell dysfunction is critical for immune monitoring and developing novel immunotherapeutic strategies. Aims: 1. To characterize transcriptional differences between PB vs BM T cells of AML patients, similar to concept of circulating vs tumor infiltrating lymphocytes (TILs), and compare them to established immunologic signatures of solid tumors. 2. To examine the cumulative expression of multiple inhibitory receptors (IRs) on AML CD8+ T cells and their dynamics in PB vs BM at diagnosis (PRE-treatment) and POST-induction chemotherapy. Methods: To study transcriptional signatures, we FACS-purified CD8+ T cells from BM of 6 AML patients (3 CR and 3 NR) PRE- and POST-induction chemotherapy whose PB T cells we analyzed and published previously (Knaus et al, JCI insight 2018). Samples were hybridized to the Human Prime View Gene Expression Array (Affymetrix). Expression fold change (FC), p-values and FDR were calculated. We compared our data set to those of dysfunctional T-cell signatures from non-small cell lung cancer (NSCLC: Guo et al, Nature 2018), colorectal cancer (CRC; Zhang et al, Nature 2018) and hepatocellular carcinoma (HCC; Zheng et al, Cell 2017) using gene set enrichment analyses (GSEA) and considered a normalized enrichment score NES>2 and FDR<0.1 as enriched. Flow cytometry data on paired PB and BM samples from AML patients (n=32) PRE- and POST-treatment and healthy controls (HC; n=21) were used to calculate the IR-score (Thommen et al, Cancer Immunol Res 2015). The IR-score summarizes the cumulative relative amount of expression of 7 different IRs on CD8+ T cells. To determine the predictive ability of the IR score to differentiate CR from NR, Receiver Operating Characteristic (ROC) analysis was conducted using logistic regression, and the area under the ROC curve (AUC) was calculated. Results: Immune signatures of AML CD8+ T cells at diagnosis, particularly in the BM, overlapped with transcriptomic exhaustion signatures of TILs from other malignancies (Figure 1-Overlap CRC, HCC, NSCLC), in particular that of CRC. Further, AML BM but not PB T cells showed a high enrichment for a common immune exhaustion signature shared by all 3 solid tumor entities and included IRs like HAVCR2, PDCD1, CTLA4, LAG3 and TIGIT, and other genes (e.g. TNFRSF9, CD27, IFNG, FASLG, CD39/ENTPD1) (Figure 1). Interestingly, this signature seemed to discriminate CR from NR patients only in the BM compartment, and at both PRE- and POST-treatment, thus providing a rationale for future exploration as a predictive biomarker of response. We found that the flow-cytometry-based IR-score strongly discriminated between CR and NR in both the PB and the BM compartment POST-treatment with an AUC of 0.70 and 0.84, respectively. While IR-score including all 7 markers provided the best discriminatory value in the PB, the combination of only two senescence markers, CD57 and KLRG1, discriminated CR from NR patients in both PB and BM with an AUC of 0.69 and 0.79, respectively. The combination of 5 exhaustion markers (2B4, BTLA, Tim3, PD-1, and CD160) discriminated CR from NR patients only in the BM (AUC 0.79). Conclusion: We found that in AML, T cells display a signature of immune exhaustion which is more prominent in the tumor milieu (e.g BM), similar to exhaustion signatures of solid cancers, can discriminate CR from NR, and persists in CR. Multiple genes, such as TNFRSF9, CD27, IFNG, FASLG, and CD39/ENTPD1, were higher expressed in CR patients both PRE and POST-treatment, suggesting that this signature also includes genes that could be useful for discriminating less dysfunctional T cell states. The phenotypic IR score was increased only in NR POST-treatment, indicating that assessment of a limited number of phenotypic markers may not be sufficient to address overall changes within T cells. Further exploration of AML-imposed T cell dysfunction is ongoing to inform better integration of immune-based therapies to augment anti-leukemia immunity. Disclosures Zeidner: Celgene: Consultancy, Honoraria, Research Funding; Daiichi Sankyo: Honoraria; Tolero: Honoraria, Research Funding; Pfizer: Honoraria; AsystBio Laboratories: Consultancy; Merck: Research Funding; Takeda: Research Funding; AbbVie: Honoraria; Agios: Honoraria. Gojo:Jazz: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Merck: Research Funding; Juno: Research Funding; Amgen Inc: Consultancy, Honoraria, Research Funding; Amphivena: Research Funding. Luznik:Merck: Research Funding, Speakers Bureau; Genentech: Research Funding; AbbVie: Consultancy; WindMiL Therapeutics: Patents & Royalties: Patent holder.

scholarly journals CD3xCD19 Dart Treatment Is Efficient in Venetoclax Resistant CLL and Reverses T Cell Dysfunction

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3043-3043
Author(s):  
Anne W. J. Martens ◽  
Susanne R. Janssen ◽  
Ingrid A.M. Derks ◽  
Sanne Tonino ◽  
Eric Eldering ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Death ◽  
T Cell ◽  
Board Of Directors ◽  
Cd8 T Cells ◽  
Research Funding ◽  
Cell Killing ◽  
Advisory Committees ◽  
Cell Dysfunction ◽  
T Cell Dysfunction

Intro - Agents targeting the apoptosis pathway, like the Bcl-2 inhibitor venetoclax, are highly effective in chronic lymphocytic leukemia (CLL). However, not all patients experience deep responses and acquired resistance has already been described. T cell mediated lysis is another tool currently exploited in hematologic malignancies. In contrast to acute lymphoblastic leukemia (ALL) however, efficacy of autologous based T cell therapy, such as CAR T cells, in CLL has been low. This is linked to a CLL mediated acquired T cell dysfunction. Bispecific T cell engagers targeting CD19 are successfully applied in ALL, but whether it overcomes the acquired T cell dysfunction in CLL is unknown. We therefore tested efficacy of a CD3xCD19 Dual Affinity Re-Targeting molecule (DART) in CLL. Since it has been observed that bispecific antibodies can overcome deficient synapse formation in CLL (Robinson et al, 2018) and based on our assumption that T cell mediated lysis differs from venetoclax-mediated killing, we hypothesized that usage of a CD3xCD19 DART in CLL overcomes T cell dysfunction and will be effective against venetoclax resistant CLL. Methods - Co-culture of CLL derived or aged-matched healthy donor (HD) CD4+ and/or CD8+ T cells with (CD40 activated) primary CLL or CD19+ cell lines JeKo-1 or Ramos in presence of CD3xCD19 (JNJ-64052781), CD3xFITC, anti-CD3/28 antibodies was performed. R esults - JeKo-1 cells were highly sensitive to CD3xCD19 mediated HD T cell killing with close to 70% of lysis in a concentration of 10ng/mL using an E:T ratio of 4:1. In the same conditions, primary CLL cells proved sensitive for CD3xCD19 mediated HD T cell killing with 50% of lysis. Killing was observed irrespective of IGHV mutation or chemorefractory status. We next compared HD with CLL-derived T cells by measuring activation levels between direct TCR (anti-CD3/CD28) and CD3xCD19 stimulation. As described, TCR stimulation resulted in impaired CLL CD4+ and CD8+ T cell activation and proliferation when compared to HD. In contrast, treatment of CLL derived PBMCs with CD3xCD19 did not resulted in dysfunctional CLL-derived T cell responses (Fig 1A-C). Consistently, co-culture of CLL derived CD4+, CD8+ or a combination with either JeKo-1 or allogeneic CLL cells in the presence of CD3xCD19 resulted in significant cytotoxicity (Fig. 1D). In the allogeneic setting, cytotoxic capacity of CD4+ T cells was similar to their CD8+ counterparts. When targeting autologous CLL, a benefit was observed when both CD4+ and CD8+ T cells were present (Fig. 1D). We then studied whether venetoclax resistant CLL cells could be targeted by CD3xCD19 mediated T cell killing. Bcl-2 overexpressing Ramos were equally lysed in presence of the CD3xCD19 DART as their wildtype counterpart, indicating that Bcl-2 expression does not inhibit CD3xCD19 mediated cell death. Following CLL cell stimulation by CD40 ligation, anti-apoptotic Bcl-XL, Bfl-1 and Mcl-1 are highly induced (Thijssen et al., 2015) resulting in venetoclax resistance (Fig 1E). Nevertheless, CD40L stimulated CLL cells were as efficiently lysed upon CD3xCD19 treatment as unstimulated CLL. (Fig 1F). This indicates that an augmented apoptotic threshold does not impact efficacy of CD3xCD19. Further examination of the mechanism of CD3xCD19 mediated killing showed that lysis depended on granzymes, as blocking granule exocytosis prevented cell death. Independence of the mitochondrial apoptotic pathway was shown by equal sensitivity to CD3xCD19 mediated T cell lysis comparing BAX/BAK knockout Jeko-1 cells to the parental cell line. Also, caspase blockage did not inhibit cell death, pointing to apoptosis independent killing. In concordance, PARP cleavage could only be detected when caspase activity was not blocked. Conclusion - This is the first report describing reversal of CLL mediated T cell dysfunction by applying a CD3xCD19 DART. Furthermore, it shows that venetoclax resistant CLL can still be efficiently targeted by T cells, in a non-apoptotic fashion. These results imply that T cell mediated therapy could be used alongside venetoclax. Figure 1 Disclosures Eldering: Celgene: Research Funding; Roche: Research Funding; Janssen Pharmaceutical Companies: Research Funding. van der Windt:Genmab: Employment. Kater:Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta: Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche/Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding.

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