70 Beyond PD-L1: novel PD-1 biomarkers identified by driving T cell dysfunction in vitro

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A76-A76
Author(s):  
Simarjot Pabla ◽  
Tenzing Khendu ◽  
Dhan Chand ◽  
Bulent Aksoy ◽  
Benjamin Duckless ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cancer Cells ◽  
Tumor Cells ◽  
Clinical Outcomes ◽  
Tumor Escape ◽  
Cell Dysfunction ◽  
Wide Range ◽  
T Cell Dysfunction

BackgroundAnti-PD-1 therapies have achieved durable clinical responses in a wide range of malignancies, but responses are limited to a small subset of patients. Expression of PD-L1 on tumor cells by immunohistochemistry (IHC) has been applied as a companion diagnostic for anti-PD-1 therapy. However, recent studies have called in to question the reliability of this method to predict response.MethodsHere we developed a novel platform that integrates in vitro pharmacogenomic and functional data with clinical pharmacodynamic responses to immunotherapy using proprietary in silico approaches. The data originate from a long-term co-culture of primary antigen-specific T cells and cancer cells which drives T cells to a terminally dysfunctional, PD-1 refractory state. T cell effector functions and gene expression changes were monitored in the presence or absence of anti-PD-1 antibody or genetic knockouts. RNA expression signatures were refined with a randomized sliding window approach to generate a deep learning neural network for PD-1 response prediction.ResultsWe defined five T cell states associated with distinct phenotypic and molecular features - naïve, active, effector, transition and dysfunction. Among the genes that were selectively expressed in the dysfunction state, we identified a 96-gene signature that is closely associated with clinical outcomes to anti-PD-1 therapy. In PD-1 treated patients across multiple solid tumor indications, this signature correlates with objective response rate and outperforms traditional metrics such as tumor mutation burden or PD-L1 IHC signal. Moreover, this signature combines with tumor sequencing data to generate a powerful machine-learning model that predicts anti-PD-1 responses in metastatic melanoma patients with significantly higher accuracy than PD-L1 IHC. Having established that the T cell states in our co-culture relate to clinical outcomes, we leveraged the system to investigate the molecular basis for PD-1 responses. Single cell mapping of transition state T cells in the presence of anti-PD-1 revealed an expanded population of T cells that co-expresses PD-1, TIGIT and activation markers. Likewise, PD-L1 knockout on cancer cells identified the TIGIT ligand, CD155, as a potential tumor escape mechanism to anti-PD-1 therapy. Consistent with this, the combination of PD-1 and TIGIT blockade enhanced T cell cytotoxicity of tumor cells relative to monotherapies.ConclusionsAgenus’ T cell dysfunction platform combines deep in vitro profiling and AI-based approaches to predict clinical outcomes. Here, we defined a predictive biomarker signature that outperforms standard PD-L1 IHC. Further, we identified known (TIGIT) and potentially novel combination partners predicted to enhance the durability of anti-PD-1 responses.Ethics ApprovalNot ApplicableConsentNot Applicable

TIGIT Induces (CD3+) T Cell Dysfunction by Inhibiting Glucose Metabolism and its Reversal by TIGIT and/or PD-1 Blockade in Colorectal Cancer

2021 ◽  
Author(s):  
qi shao ◽  
Lei Wang ◽  
maoling yuan ◽  
Xiaohong Jin ◽  
changping wu
Keyword(s):  
Colorectal Cancer ◽  
T Cells ◽  
T Cell ◽  
Glucose Metabolism ◽  
Tumor Growth ◽  
Tumor Cells ◽  
Peripheral Blood ◽  
Cancer Tissue ◽  
Cell Dysfunction ◽  
T Cell Dysfunction

Abstract Background: T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT) is an immunosuppressive receptor expressed on the surface of immune cells, suppressing immune responses by activating the intracellular negative regulatory signals. TIGIT plays an important role in the pathogenesis of various tumors, but its immune escape in colorectal cancer remains unclear.Methods: In this study, TIGIT expression in the peripheral blood and tissue microarrays was detected flow cytometry and immunofluorescence and its relationship with prognosis was evaluated. The proliferation and cytokines of TIGIT+ T cells were measured. Glucose metabolism and key enzymes were detected by qPCR or western blot. After establishing the co-cultured system and xenotransplant models, TIGIT antibody alone or combined with PD-1 antibody was blocked to observe the tumor growth.Results: We found that the proportion of CD3+TIGIT+ T cells was increased in peripheral blood and cancer tissue in colorectal cancer patients when compared with the healthy donors. These cells exhibited functional defects, low proliferative activity, impaired cytokine production and reduced glucose metabolism. A strong association was also observed between the elevated TIGIT expression and poor prognosis. In the in vitro co-culture assays of T cells and tumor cells, the suppressed glucose metabolic activity of T cells was reversed by TIGIT blockade. In addition, this blockade induced the apoptosis and reduced G2/M transit in tumor cells. The antitumor efficacy of TIGIT Ab therapy was further demonstrated in a human colorectal xenograft mice model while co-blockers of TIGIT and PD-1 exhibited synergistic suppressing effects on tumor growth.Conclusions: It is suggest that while TIGIT induces CD3+ T cell dysfunction in colorectal cancer, co-targeting TIGIT and PD-1 can lead to an effective antitumor response and may serve as a novel therapeutic strategy for colorectal patients.

scholarly journals 663 Media based on the metabolic composition of tumor interstitial fluid reveals persistent T cell dysfunction induced through arginine deprivation and exposure to the oncometabolite phosphoethanolamine

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A691-A691
Author(s):  
Yupeng Wang ◽  
Chufan Cai ◽  
Dayana Rivadeneira ◽  
Alexander Muir ◽  
Greg Delgoffe
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Interstitial Fluid ◽  
Cytokine Production ◽  
Cell Dysfunction ◽  
Kennedy Pathway ◽  
T Cell Dysfunction ◽  
Tumor Interstitial Fluid

BackgroundWhile CD8 T cells are crucial for anti-tumor immunity, tumor infiltrating CD8 T cells encounter stressors which deviate their differentiation to a dysfunctional, exhausted phenotype. T cell functions are closely regulated by T cell metabolism, and the dysfunctional vasculature in tumor tissues and the deregulated metabolism of tumor cells lead to depletion of nutrients and accumulation of metabolic wastes in the tumor microenvironment (TME). Thus, the unbalanced levels of the nutrients and the metabolic wastes might skew the metabolism of T cells thus contributing to T cell dysfunction.MethodsOvalbumin-specific OT-I cells were activated with SIINFEKL/IL2 and cultured with IL2. The tumor interstitial fluid media (TIFM) was formulated based on the concentrations of the metabolites measured in the tumor interstitial fluid of pancreatic ductal adenocarcinoma.1 Purified arginine and phosphoethanolamine (PEtn) were used to change their levels in TIFM/RPMI1640 culture. Expression level of cytokines and PD-1 was measured by flow cytometry.ResultsWe sought to determine how T cells would differentiate, in vitro, if they were exposed only to the metabolites present in the TME. Using media formulated to model the metabolic composition of tumor interstitial fluid (TIFM),1 we show that CD8 T cells develop features of exhausted T cells in the TIFM culture: reduced proliferation, increased expression of PD-1 and decreased cytokine production. Using 'dropout' and 'add-back' approaches, we found arginine levels as a major contributor to the proliferation defect observed in TIFM-cultured T cells. Arginine was sufficient to restore proliferative capacity to T cells cultured in TIFM, but had no effect on the inhibited cytokine production. We then asked which metabolites were enriched in the TIFM, finding that PEtn, an intermediate in the ethanolamine branch of the Kennedy pathway and an oncometabolite enriched in the interstitial of many solid tumors, up-regulates PD-1 expression and compromises the cytokine production of the cells in culture. Depletion of Pcyt2, the metabolizing enzyme of PEtn and the rate limiting enzyme in the Kennedy pathway, makes CD8 T cells resistant to the effects of PEtn.ConclusionsOur data shows that the metabolic environment in the TME can be recapitulated in vitro and is sufficient to drive T cell dysfunction. Arginine depletion acts as a major inhibitor of T cell proliferation in the TME, but the oncometabolite PEtn drives a hypofunctional effector fate of T cells. Targeting PEtn metabolism via Pcyt2 depletion or inhibition is a potential target to reinvigorate T cells and enhance anti-tumor immunity.ReferenceSullivan MR, Danai LV, Lewis CA, Chan SH, Gui DY, Kunchok T, Dennstedt EA, Vander Heiden MG, Muir A. Quantification of microenvironmental metabolites in murine cancers reveals determinants of tumor nutrient availability. Elife 2019;;8:e44235. doi: 10.7554/eLife.44235. PMID: 30990168; PMCID: PMC6510537.

A CD19/CD3 Bispecific Tandab, AFM11, Recruits T Cells To Potently and Safely Kill CD19+ Tumor Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4405-4405
Author(s):  
Eugene Zhukovsky ◽  
Uwe Reusch ◽  
Carmen Burkhardt ◽  
Stefan Knackmuss ◽  
Ivica Fucek ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Cancer Cells ◽  
Tumor Cells ◽  
Cd8 T Cells ◽  
Cross Reactivity ◽  
Equity Ownership

Abstract To harness the potent tumor-killing capacity of T cells for the treatment of CD19+ malignancies, we developed a humanized bispecific tetravalent antibody, with two binding sites for CD3 and CD19, the CD19/CD3 RECRUIT-TandAb AFM11. CD19 is expressed from early B cell development through differentiation into plasma cells, and is an attractive alternative to CD20 as a target for the development of therapeutic antibodies to treat B cell malignancies such as Non Hodgkin Lymphoma. Since native antibodies cannot recruit T cells, we engineered a bispecific anti-CD19/anti-CD3 TandAb. The tumor-specific CD19 antigen module targets the TandAb to cancer cells, while simultaneously, the CD3 effector module recruits and activates T cells, leading to cancer cell lysis. The advantages of the TandAb technology, relative to other bi-functional fragment antibody scaffolds, include: improved pharmacokinetics (PK) enabling intravenous dosing, more drug-like properties, and avidity-enhanced efficacy for the targeting and killing of tumor cells. We evaluated in vitro efficacy and safety using CD19+ cell lines, and in vivo efficacy in a murine NOD/scid xenograft model reconstituted with human PBMC. Further, we used standard preclinical IND enabling assays to evaluate tissue cross reactivity, PK, and toxicological profile (local tolerance, hematocompatibility, effects on hematopoesis, etc). In vitro assays demonstrated the higher potency and efficacy of target cell lysis by AFM11 relative to a bispecific tandem scFv (that is currently in clinical evaluation). CD8+ T cells dominate early AFM11-mediated cytotoxicity (4 hrs) while after 24 hrs both CD4+ and CD8+ T cells equally contribute to tumor lysis with EC50 between 0.5 – 5 pM; cytotoxicity was independent of CD19 cell-surface density. AFM11 exhibited similar cytotoxicity over effector:target ratios ranging from 5:1 to 1:5, and facilitated serial T cell-killing of its targets. The advantage of AFM11 over the bispecific tandem scFv was most pronounced at lower effector:target ratios. AFM11 activated T cells only in the presence of CD19+ cells. In PBMC cultures, AFM11 induced CD69 and CD25 expression, T cell proliferation, and production of IFN-γ, TNF-α, IL-2, IL-6, and IL-10. Depletion of CD19+ cells from PBMC abrogated these effects, demonstrating that the T cell activation is strictly CD19+ target-dependent. Thus, AFM11 should not elicit the devastating cytokine release observed when full-length antibodies bind CD3. Up to one week co-incubation with AFM11 did not inhibit T cell cytotoxicity, suggesting that the TandAb does not induce anergy. In vivo, AFM11 induced dose-dependent growth inhibition of Raji tumors; a single 0.5 mg/kg dose exhibited efficacy similar to 5 daily injections. In the tissue cross reactivity study, only tissues containing CD19+ and CD3+ cells were stained by AFM11; all other tissues, including vital organs, displayed no cross reactivity. Similarly, no local intolerance was observed in rabbits, and no effect on myeloid and erythroid progenitors was observed in a colony-forming assay. Strong accumulation of 125I-labeled AFM11 was observed in the tumors of mice engrafted with CD19+ cancer cells, and no unspecific organ accumulation was observed. Finally, evaluated on the basis of Cmax and the area under the curve (AUC), AFM11 exhibited dose linearity (20 – 500 mg AFM11 dose range) upon single i.v. bolus administration in mice; half-life (T1/2) ranged from 18.4 to 22.9 hr. In summary, AFM11 is a highly efficacious novel drug candidate for the treatment of CD19+ malignancies with an advantageous safety profile and anticipated dosing regimen. Disclosures: Zhukovsky: Affimed Therapeutics AG: Employment, Equity Ownership. Reusch:Affimed Therapeutics AG: Employment. Burkhardt:Affimed Therapeutics AG: Employment. Knackmuss:Affimed Therapeutics AG: Employment. Fucek:Affimed Therapeutics AG: Employment. Eser:Affimed Therapeutics AG: Employment. McAleese:Affimed Therapeutics AG: Employment. Ellwanger:Affimed Therapeutics AG: Employment. Little:Affimed Therapeutics AG: Consultancy, Equity Ownership.

scholarly journals P11.10 The IFNγ pathway mediates brain metastasis formation of breast cancer

2019 ◽  
Vol 21 (Supplement_3) ◽  
pp. iii44-iii44
Author(s):  
R Pedrosa ◽  
J M Kros ◽  
B Schrijver ◽  
R Marques ◽  
P Leenen ◽  
...  
Keyword(s):  
Breast Cancer ◽  
T Cells ◽  
T Cell ◽  
T Lymphocytes ◽  
Cancer Cells ◽  
Tumor Cells ◽  
Breast Cancer Cells ◽  
T Cell Response ◽  
Activated T Cells

Abstract BACKGROUND In previous work we showed the prominence of the T-cell response in the formation of brain metastases of primary ER negative breast cancers (Mustafa et al, Acta Neuropathol 2018). We also showed that breast cancer cells co-cultured with stimulated T lymphocytes overexpress Guanylate-binding protein 1 (GBP1) accompanying increased trespassing ability through an in vitro blood-brain barrier (BBB) model. In addition, we demonstrated a predilection for metastasizing to brain of breast cancer cells that were co-cultured with activated T cells in a mouse model. We now scrutinize the importance of the IFNγ pathway for tresspassing of the tumor cells through the BBB following T cell contact. MATERIAL AND METHODS Anti-hIFN-γ-IgA antibodies were used to neutralize the IFNγ effects on the tumor cells. The effects on the tumor cells is only due to native IFNγ produced by activated T cells, not by recombinant IFNγ. Since the IFNγ expression itself enhances its expression by the T cells, we blocked IFNγ receptors prior to adding CD3+ T cell conditioned media to the breast cancer cells. The receptor blocking was achieved by antibodies to the IFNγα and IFNγβ subunits. Activation of the STAT1 pathway was monitored by GBP1 expression. For functional read-out the in vitro BBB model was used. RESULTS The presence of T-lymphocyte-secreted IFNγ in the primary breast cancer microenvironment activates the STAT1-dependent IFNγ pathway in breast cancer cells, endowing them with an increased ability to trespass the in vitro BBB. Moreover, direct inhibition of soluble IFNγ, or blocking of the IFNγ-specific receptor in breast cancer cells significantly decreases their ability to cross the BBB. CONCLUSION The results illustrate the specific action of T lymphocytes in the formation of cerebral metastasis involves the IFNγ signaling pathway as one of the crucial entangled pathways Subsequent studies should aim at the interference with the IFNγ pathway to develop preventive strategies against the formation of cerebral metastases of breast cancer.

scholarly journals TIGIT Induces (CD3+) T Cell Dysfunction in Colorectal Cancer by Inhibiting Glucose Metabolism

2021 ◽  
Vol 12 ◽  
Author(s):  
Qi Shao ◽  
Lei Wang ◽  
Maoling Yuan ◽  
Xiaohong Jin ◽  
Zhiming Chen ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
T Cells ◽  
T Cell ◽  
Glucose Metabolism ◽  
Tumor Cells ◽  
Immune Escape ◽  
Strong Association ◽  
Cancer Tissue ◽  
Cell Dysfunction ◽  
T Cell Dysfunction

T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT) is an immunosuppressive receptor expressed on the surface of immune cells, suppressing immune responses by activating the intracellular negative regulatory signals. TIGIT plays an important role in the pathogenesis of various tumors, but its immune escape in colorectal cancer remains unclear. We found that the proportion of CD3+TIGIT+ T cells was increased in peripheral blood and cancer tissue in colorectal cancer patients when compared with the healthy donors. These cells exhibited functional defects, low proliferative activity, impaired cytokine production and reduced glucose metabolism. A strong association was also observed between the elevated TIGIT expression and poor prognosis in this cohort. In the in vitro co-culture assays of T cells and tumor cells, the suppressed glucose metabolic activity of T cells was reversed by TIGIT blockade. In addition, this blockade induced the apoptosis and reduced G2/M transit in tumor cells. The antitumor efficacy of TIGIT Ab therapy was further demonstrated in a human colorectal xenograft mice model while co-blockers of TIGIT and PD-1 exhibited synergistic suppressing effects on tumor growth. These results suggest that while TIGIT induces CD3+ T cell dysfunction in colorectal cancer, co-targeting TIGIT and PD-1 can lead to an effective antitumor response and may serve as a novel therapeutic strategy for colorectal patients.

scholarly journals 687 Enhancement of anti-tumor immunity by ICT01: a novel g9d2 T cell-activating antibody targeting butyrophilin-3A (BTN3A)

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A726-A726
Author(s):  
Aude de Gassart ◽  
Patrick Brune ◽  
LE Suong ◽  
Sophie Agaugue ◽  
Emmanuel Valentin ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
T Cell Activation ◽  
Cell Subset ◽  
List Type ◽  
Human Cancers ◽  
Wide Range

BackgroundgdT-cells are innate-like lymphocytes described as potent killer of cancer cells whose infiltration into tumors is associated with a positive prognosis.1 2 This supports gd T-cells use in cancer immunotherapy. BTN3A, which belongs to the B7-subfamily of Ig proteins, is required for the recognition of malignant or infected cells by human g9d2 T-cells by sensing intracellular accumulation of phosphoantigens.3 ImCheck Therapeutics is developing ICT01, a humanized anti-BTN3A (IgG1, Fc-silenced), g9d2 T-cell-activating antibody for the treatment of patients with solid or hematologic tumors.MethodsA complete IND-enabling program was conducted to characterize the preclinical activity and safety of ICT01. ICT01 effects on human and cynomolgus PBMCs were characterized in vitro using flow cytometry. ICT01-mediated killing activity of g9d2 T-cells was assessed using in vitro co-cultures with tumor and non-tumor cells. Immunocompromised mice bearing human tumors and adoptively transferred with human g9d2 T cells were used to assess ICT01 anti-tumor activity in vivo. The PK, PD and safety of intravenous ICT01 (0.1 to 100 mg/kg single- and repeated-dose) were evaluated in Cynomolgus monkeys.ResultsICT01 selectively binds to all three BTN3A isoforms with high affinity (<10nM). When assayed in human and cynomolgus PBMCs in vitro, ICT01 promoted a robust and specific activation of g9d2 T-cells as shown by concentration dependent increase in cell surface CD69 and CD25 and cytokines secretion (IFNγ, TNFα). In co-culture experiments, ~20% of target occupancy on tumor cells is sufficient for maximal g9d2 T-cell degranulation (e.g. CD107a/b expression). ICT01-activated g9d2 T-cells continuously and serially kill a wide range of tumor cells in multi-day co-culture conditions. In contrast, non-tumoral BTN3A-expressing B cells, HUVEC and fibroblasts were unaffected. In mouse AML and ovarian cancer models, repeated injections of ICT01 delayed tumor growth and significantly prolonged animal survival. In primates, ICT01 exposure and target engagement was dose-dependent, with all tested doses producing a specific g9d2 T cell activation and trafficking out of the circulation within 1 hour. ICT01 administration was well tolerated with no safety signals observed at doses up to 25 mg/kg/week based on clinical, laboratory, and anatomic pathology parameters.ConclusionsThe combined in vitro and in vivo pharmacology data provide evidence that ICT01 is an attractive and novel therapeutic approach for enhancing the innate anti-tumor potential of g9d2 T-cells by activating BTN3A. Importantly, ICT01 did not affect healthy BTN3A-expressing cells, and NHP studies confirmed ICT01 safety with a wide therapeutic index. Therefore, ICT01 is being tested in the ongoing EVICTION trial (NCT04243499).Ethics ApprovalPseudonymized samples isolated from healthy volunteers’ whole blood by ImCheck Therapeutics under the agreement n° 7173 between ImCheck Therapeutic SAS and EFS PACA (Etablissement Français du Sang Provence-Alpes-cote d’Azur)ReferencesGentles AJ, Newman AM, Liu CL, et al. The prognostic landscape of genes and infiltrating immune cells across human cancers. Nature Medicine 2015;21(8):938–945.Tosolini M, Pont F, Poupot M, et al. Assessment of tumor-infiltrating TCRVγ9Vδ2 γδ lymphocyte abundance by deconvolution of human cancers microarrays. OncoImmunology. 2017;6(3):e1284723.Harly C, Guillaume Y, Nedellec S, et al. Key implication of CD277/butyrophilin-3 (BTN3A) in cellular stress sensing by a major human γδ T-cell subset. Blood 2012;120(11):2269–2279.

scholarly journals PD-1 does not mark tumor-infiltrating CD8+ T cell dysfunction in human gastric cancer

2020 ◽  
Vol 8 (2) ◽  
pp. e000422
Author(s):  
Yang Shen ◽  
Yongsheng Teng ◽  
Yipin Lv ◽  
Yongliang Zhao ◽  
Yuan Qiu ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd8 T Cell ◽  
Human Gastric Cancer ◽  
Cell Dysfunction ◽  
T Cell Dysfunction ◽  
Tgf Β1

BackgroundOverexpression of programmed cell death protein 1 (PD-1) is linked to CD8+ T cell dysfunction and contributes to tumor immune escape. However, the prevalence and functional regulations of PD-1 expression on CD8+ T cells in human gastric cancer (GC) remain largely unknown.MethodsFlow cytometry was performed to analyze the level, phenotype, functional and clinical relevance of PD-1+CD8+ T cells in GC patients. Peripheral blood CD8+ T cells were purified and subsequently exposed to culture supernatants from digested primary GC tumor tissues (TSN) in vitro for PD-1 expression and functional assays. Tumor responses to adoptively transferred TSN-stimulated CD8+ T cells or to the TSN-stimulated CD8+ T cell transfer combined with an anti-PD-1 antibody injection were measured in an in vivo xenograft mouse model.ResultsGC patients’ tumors showed a significantly increased PD-1+CD8+ T cell infiltration. However, these GC-infiltrating PD-1+CD8+ T cells showed equivalent function to their PD-1−CD8+ counterparts and they did not predict tumor progression. High level of transforming growth factor-β1 (TGF-β1) in tumors was positively correlated with PD-1+CD8+ T cell infiltration, and in vitro GC-derived TGF-β1 induced PD-1 expression on CD8+ T cells via Smad3 signaling, whereas Smad2 signaling was involved in GC-derived TGF-β1-mediated CD8+ T cell dysfunction. Furthermore, GC-derived TGF-β1-mediated CD8+ T cell dysfunction contributed to tumor growth in vivo that could not be attenuated by PD-1 blockade.ConclusionsOur data highlight that GC-derived TGF-β1 promotes PD-1 independent CD8+ T cell dysfunction. Therefore, restoring CD8+ T cell function by a combinational PD-1 and TGF-β1 blockade might benefit future GC immunotherapy.

scholarly journals 670 Oxidative stress originating in the mitochondria damages telomeres sufficient to drive certain features of T cell dysfunction

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A698-A698
Author(s):  
Dayana Rivadeneira ◽  
Jess Yana ◽  
Sanjana Thosar ◽  
Marcel Bruchez ◽  
Patricia Lynn ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
T Cells ◽  
T Cell ◽  
Oxidative Damage ◽  
Telomeric Dna ◽  
Cell Dysfunction ◽  
Ros Accumulation ◽  
T Cell Dysfunction ◽  
Infiltrating Lymphocytes

BackgroundThe functional state of infiltrating lymphocytes is a critical determinant of antitumor immunity and immunotherapy response. Key factors responsible for driving T cell dysfunction are metabolic barriers such as nutrient competition, low oxygen tension and damaging byproducts, like reactive oxygen species (ROS). ROS are critical contributors to T cell dysfunction observed during aging as well as in the tumor microenvironment. While we have shown ROS accumulation drives T cell exhaustion in part by altering signaling, ROS can affect other cellular functions further contributing to T cell dysfunction. One of the main downstream consequences observed with ROS accumulation is DNA damage, in particular telomeric DNA. However, little is known on whether telomeric shortening vs damage and the associated response affects T cell fate and function.MethodsWe performed telomeric analyses of endogenous tumor-infiltrating T cells. In vitro, direct induction of oxidative stress in mitochondria or telomeres was performed using a photosensitizer approach employing fluorogen-activating peptide (FAP)1 targeted to the mitochondria (COXVIII-FAP) or to the telomeric shelterin protein TRF1, which produces singlet oxygen and 8-oxoguanine specifically in the mitochondria or at the telomeres.2 Our lab generated mouse models to express this telomeric-FAP (Rosa26-LSL-TRF1-FAP) or mitochondrial-FAP (Rosa26-LSL-COXVIII-FAP) specifically in T cells (Cd4Cre).ResultsTelomere analysis of tumor-infiltrating exhausted (PD1hiTim3+) vs non-exhausted (PD1int) cells revealed exhausted T cells do not have shorter telomeres (like senescent T cells), but rather damaged telomeres. Using a photosensitizer strategy to specifically induce oxidative damage to the mitochondria, we recapitulated our previous work in vitro, resulting in ROS cascades, severe T cell dysfunction, and damage to telomeres. We next directly assessed the role of telomere oxidative damage using a telomeric-targeted photosensitizer, showing oxidative damage to telomeres promotes a persistent T cell dysfunction, resulting in sustained Tim3 and PD1 expression and severely decreased functions. However, oxidative damage to telomeric DNA alone does not induce all of the features of T cell exhaustion, suggesting that telomere damage is a crucial piece, but not fully sufficient to drive T cell dysfunction.ConclusionsOur data support a model where oxidative stress originating in the mitochondria alters cellular biology in part by damaging telomeric DNA. While this activity does not shorten telomeres, it induces stress responses sufficient to deviate differentiation into a dysfunctional phenotype. While telomeric damage alone does not fully recapitulate exhaustion, it nevertheless remains a crucial part of the dysfunctional phenotype in tumor infiltrating lymphocytes.ReferencesHe J, Wang Y, Missinato MA, Onuoha E, Perkins LA, Watkins SC, St Croix CM, Tsang M, Bruchez MP. A genetically targetable near-infrared photosensitizer Nat Methods 2016;13:263–268.Fouquerel E, Barnes RP, Uttam S, Watkins SC, Bruchez MP, Opresko PL. Targeted and persistent 8-Oxoguanine base damage at telomeres promotes telomere loss and crisis. Mol Cell 2019;75(1):117–130.e6.

scholarly journals IL-15 Enhances the Persistence and Function of BCMA-targeting CAR-T Cells Compared to IL-2 or IL-15/IL-7 by Limiting CAR-T Cell Dysfunction and Differentiation

Cancers ◽  
2021 ◽  
Vol 13 (14) ◽  
pp. 3534
Author(s):  
Anthony M. Battram ◽  
Mireia Bachiller ◽  
Victor Lopez ◽  
Carlos Fernández de Larrea ◽  
Alvaro Urbano-Ispizua ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Dysfunction ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
T Cell Dysfunction ◽  
And Function

Chimeric antigen receptor (CAR)-T cell immunotherapy has revolutionized the treatment of B-lymphoid malignancies. For multiple myeloma (MM), B-cell maturation antigen (BCMA)-targeted CAR-T cells have achieved outstanding complete response rates, but unfortunately, patients often relapse within a year of receiving the therapy. Increased persistence and reduced dysfunction are crucial features that enhance the durability of CAR-T cell responses. One of the factors that influence CAR-T cell in vivo longevity and loss of function, but which has not yet been extensively studied for BCMA-directed CAR-T cells, are the cytokines used during their production. We here compared the impact of IL-2, IL-15 and a combination of IL-15/IL-7 on the phenotype and function of ARI2h, an academic BCMA-directed CAR-T cell that is currently being administered to MM patients. For this study, flow cytometry, in vitro cytotoxicity assays and analysis of cytokine release were performed. In addition, ARI2h cells expanded with IL-2, IL-15, or IL-15/IL-7 were injected into MM tumor-bearing mice to assess their in vivo efficacy. We demonstrated that each of the cytokine conditions was suitable for the expansion of ARI2h cells, with clear in vitro activity. Strikingly, however, IL-15-produced ARI2h cells had improved in vivo efficacy and persistence. When explored further, it was found that IL-15 drove a less-differentiated ARI2h phenotype, ameliorated parameters related to CAR-T cell dysfunction, and lowered the release of cytokines potentially involved in cytokine release syndrome and MM progression. Moreover, we observed that IL-15 was less potent in inducing T cell senescence and DNA damage accumulation, both of which may contribute to an unfavorable CAR-T cell phenotype. These findings show the superiority of IL-15 to IL-2 and IL-15/IL-7 in the quality of anti-BCMA CAR-T cells, particularly their efficacy and persistence, and as such, could improve the duration of responses if applied to the clinical production of CAR-T cells for patients.

scholarly journals Tim-3 adaptor protein Bat3 is a molecular checkpoint of T cell terminal differentiation and exhaustion

2021 ◽  
Vol 7 (18) ◽  
pp. eabd2710
Author(s):  
Chen Zhu ◽  
Karen O. Dixon ◽  
Kathleen Newcomer ◽  
Guangxiang Gu ◽  
Sheng Xiao ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Adaptor Protein ◽  
Transcriptional Analysis ◽  
T Cell Exhaustion ◽  
Cell Exhaustion ◽  
Cell Dysfunction ◽  
Autoreactive T Cell ◽  
Autoimmune Conditions ◽  
T Cell Dysfunction

T cell exhaustion has been associated with poor prognosis in persistent viral infection and cancer. Conversely, in the context of autoimmunity, T cell exhaustion has been favorably correlated with long-term clinical outcome. Understanding the development of exhaustion in autoimmune settings may provide underlying principles that can be exploited to quell autoreactive T cells. Here, we demonstrate that the adaptor molecule Bat3 acts as a molecular checkpoint of T cell exhaustion, with deficiency of Bat3 promoting a profound exhaustion phenotype, suppressing autoreactive T cell–mediated neuroinflammation. Mechanistically, Bat3 acts as a critical mTORC2 inhibitor to suppress Akt function. As a result, Bat3 deficiency leads to increased Akt activity and FoxO1 phosphorylation, indirectly promoting Prdm1 expression. Transcriptional analysis of Bat3−/− T cells revealed up-regulation of dysfunction-associated genes, concomitant with down-regulation of genes associated with T cell effector function, suggesting that absence of Bat3 can trigger T cell dysfunction even under highly proinflammatory autoimmune conditions.

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