Enhanced recruitment of genetically modified CX3CR1-positive human T cells into Fractalkine/CX3CL1 expressing tumors: importance of the chemokine gradient.

BACKGROUND: Adoptive T-cell based immunotherapies constitute a promising approach to treat cancer, however, a major problem is to obtain effective and long-lasting anti-tumor responses. Lack of response may be due to insufficient trafficking of specific T cells to tumors. A key requirement for efficient migration of cytotoxic T cells is that they express chemokine receptors that match the chemokines produced by tumor or tumor-associated cells. METHODS: In this study, we investigated whether the in vivo tumor trafficking of activated T cells could be enhanced by the expression of the chemokine receptor CX3CR1. Two human colorectal cancer cell lines were used to set up a xenograft tumor model in immunodeficient mice; the NCI-H630, constitutively expressing the chemokine ligand CX3CL1 (Fractalkine), and the RKO cell line, transduced to express CX3CL1. RESULTS: Human primary T cells were transduced with the receptor CX3CR1-eGFP. Upon in vivo adoptive transfer of genetically modified CX3CR1-T cells in mice bearing NCI-H630 tumors, enhanced lymphocyte migration and tumor trafficking were observed, compared to mice receiving Mock-T cells, indicating improved homing ability towards ligand-expressing tumor cells. Furthermore, significant inhibition of tumor growth was found in mice receiving modified CX3CR1-T cells. In contrast, tumors formed by RKO cells transduced with the ligand (RKO-CX3CL1) were not affected, nor more infiltrated upon transfer of CX3CR1-T lymphocytes, likely because high levels of the chemokine were shed by tumor cells in the systemic circulation, thus nullifying the blood-tissue chemokine gradient. CONCLUSIONS: This study demonstrates that ectopic expression of CX3CR1 enhanced the homing of adoptively transferred T cells towards CX3CL1-producing tumors, resulting in increased T cell infiltration in tumor tissues and decreased tumor growth. Our results also establish that a correct chemokine gradient between the systemic circulation and the tumor is an essential requirement in adoptive T-cell based immunotherapy to efficiently recruit T cell effectors at the correct sites

T cell retargeting with MHC class I-restricted antibodies: the CD28 costimulatory domain enhances antigen-specific cytotoxicity and cytokine production

T cells require both primary and costimulatory signals for optimal activation. The primary Ag-specific signal is delivered by engagement of the TCR. The second Ag-independent costimulatory signal is mediated by engagement of the T cell surface costimulatory molecule CD28 with its target cell ligand B7. However, many tumor cells do not express these costimulatory molecules. We previously constructed phage display derived F(AB), G8, and Hyb3, Ab-based receptors with identical specificity but distinct affinities for HLA-A1/MAGE-A1, i.e., "TCR-like" specificity. These chimeric receptors comprised the FcepsilonRI-gamma signaling element. We analyzed whether linking the CD28 costimulation structure to it (gamma + CD28) could affect the levels of MHC-restricted cytolysis and/or cytokine production. Human scFv-G8(POS) T lymphocytes comprising the gamma + CD28 vs the gamma signaling element alone produced substantially more IL-2, TNF-alpha, and IFN-gamma in response to HLA-A1/MAGE-A1(POS) melanoma cells. Also a drastic increase in cytolytic capacity of scFv-G8(POS) T cells, equipped with gamma + CD28 vs the gamma-chain alone was observed

Enhanced recruitment of genetically modified CX3CR1-positive human T cells into Fractalkine/CX3CL1 expressing tumors: Importance of the chemokine gradient

Background: Adoptive T-cell based immunotherapies constitute a promising approach to treat cancer, however, a major problem is to obtain effective and long-lasting anti-tumor responses. Lack of response may be due to insufficient trafficking of specific T cells to tumors. A key requirement for efficient migration of cytotoxic T cells is that they express chemokine receptors that match the chemokines produced by tumor or tumor-associated cells. Methods: In this study, we investigated whether the in vivo tumor trafficking of activated T cells could be enhanced by the expression of the chemokine receptor CX3CR1. Two human colorectal cancer cell lines were used to set up a xenograft tumor model in immunodeficient mice; the NCI-H630, constitutively expressing the chemokine ligand CX3CL1 (Fractalkine), and the RKO cell line, transduced to express CX3CL1. Results: Human primary T cells were transduced with the receptor CX3CR1-eGFP. Upon in vivo adoptive transfer of genetically modified CX3CR1-T cells in mice bearing NCI-H630 tumors, enhanced lymphocyte migration and tumor trafficking were observed, compared to mice receiving Mock-T cells, indicating improved homing ability towards ligand-expressing tumor cells. Furthermore, significant inhibition of tumor growth was found in mice receiving modified CX3CR1-T cells. In contrast, tumors formed by RKO cells transduced with the ligand (RKO-CX3CL1) were not affected, nor more infiltrated upon transfer of CX3CR1-T lymphocytes, likely because high levels of the chemokine were shed by tumor cells in the systemic circulation, thus nullifying the blood-tissue chemokine gradient. Conclusions: This study demonstrates that ectopic express

Consumption of β-glucans to spice up T cell treatment of tumors: a review

Introduction: Adoptive T-cell treatments of solid cancers have evolved into a robust therapy with objective response rates surpassing those of standardized treatments. Unfortunately, only a limited fraction of patients shows durable responses, which is considered to be due to a T cell-suppressive tumor microenvironment (TME). Here we argue that naturally

Immunotherapy in Glioblastoma: Current Shortcomings and Future Perspectives

Glioblastomas are aggressive, fast-growing primary brain tumors. After standard-of-care treatment with radiation in combination with temozolomide, the overall prognosis of newly diagnosed patients remains poor, with a 2-year survival rate of less than 20%. The remarkable survival benefit gained with immunotherapy in several extracranial tumor types spurred a variety of experimental intervention studies in glioblastoma patients. These ranged from immune checkpoint inhibition to vaccinations and adoptive T cell therapies. Unfortunately, almost all clinical outcomes were universally disappointing. In this perspective, we provide an overview of immune interventions performed to date in glioblastoma patients and re-evaluate their performance. We argue that shortcomings of current immune therapies in glioblastoma are related to three major determinants of resistance, namely: low immunogenicity; immune privilege of the central nervous system; and immunosuppressive micro-environment. In this perspective, we propose strategies that are guided by exact shortcomings to sensitize glioblastoma prior to treatment with therapies that enhance numbers and/or activation state of CD8 T cells

Engineering Orthogonal Polypeptide GalNAc-Transferase and UDP-Sugar Pairs

O-Linked α-N-acetylgalactosamine (O-GalNAc) glycans constitute a major part of the human glycome. They are difficult to study because of the complex interplay of 20 distinct glycosyltransferase isoenzymes that initiate this form of glycosylation, the polypeptide N-acetylgalactosaminyltransferases (GalNAc-Ts). Despite proven disease relevance, correlating the activity of individual GalNAc-Ts with biological function remains challenging due to a lack of tools to probe their substrate specificity in a complex biological environment. Here, we develop a “bump–hole” chemical reporter system for studying GalNAc-T activity in vitro. Individual GalNAc-Ts were rationally engineered to contain an enlarged active site (hole) and probed with a newly synthesized collection of 20 (bumped) uridine diphosphate N-acetylgalactosamine (UDP-GalNAc) analogs to identify enzyme–substrate pairs that retain peptide specificities but are otherwise completely orthogonal to native enzyme–substrate pairs. The approach was applicable to multiple GalNAc-T isoenzymes, including GalNAc-T1 and -T2 that prefer nonglycosylated peptide substrates and GalNAcT-10 that prefers a preglycosylated peptide substrate. A detailed investigation of enzyme kinetics and specificities revealed the robustness of the approach to faithfully report on GalNAc-T activity and paves the way for studying substrate specificities in living systems

Lack of B and T cell reactivity towards IDH1(R132H) in blood and tumor tissue from LGG patients

Purpose Mutations in the isocitrate dehydrogenase-1 gene (IDH1) occur at high frequency in grade II–III gliomas (LGGs). IDH1 mutations are somatic, missense and heterozygous afecting codon 132 in the catalytic pocket of the enzyme. In LGG, most mutations (90%) result in an arginine to histidine substitution (IDH1R132H) providing a neo-epitope that is expressed in all tumor cells. To assess the immunogenic nature of this epitope, and its potential use to develop T cell treatments, we measured IDH1R132H-specifc B and T cell reactivity in blood and tumor tissue of LGG patients. Methods Sera from IDH1R132H-mutated LGG patients (n=27) were assayed for the presence of a neo-specifc antibody response using ELISA. In addition, PBMCs (n=36) and tumor-infltrating lymphocytes (TILs, n=10) were measured for T cell activation markers and IFN-γ production by fow cytometry and ELISA. In some assays, frequencies of CD4 T cells specifc for mutated peptide presented by HLA-DR were enriched prior to T cell monitoring assays. Results Despite high sensitivity of our assay, we failed to detect IDH1R132H-specifc IgG in sera of LGG patients. Similarly, we did not observe CD4 T cell reactivity towards IDH1R132H in blood, neither did we observe such reactivity following preenrichment of frequencies of IDH1R132H-specifc CD4 T cells. Finally, we did not detect IDH1R132H-specifc CD4 T cells among TILs. Conclusions The absence of both humoral and cellular responses in blood and tumors of LGG patients indicates that IDH1R132H is not sufciently immunogenic and devaluates its further therapeutic exploitation, at least in the majority of LGG patients

Structural equation modelling analysis on relationships of job demands and resources with work engagement, burnout and work ability: an observational study among physicians in Dutch hospitals

Objective: To investigate to what extent work engagement mediates the relationships of job resources with work ability, and to what extent burnout mediates the relationships of job demands and resources with work ability. Design: Multicentre observational study. Setting: Academic and non-academic hospitals in the Netherlands. Participants: Physicians (n=385) participated in this study. Primary and secondary outcome measures: We measured work ability with selected items from the validated Questionnaire of Experience and Evaluation of Work 2.0 (QEEW V.2.0), work engagement with the Utrecht Work Engagement Scale and burnout with the exhaustion subscale of the Oldenburg Burnout Inventory. The job demand ‘workload’ and job resources ‘development opportunities’, ‘participation in decision-making’, ‘inspirational leadership’ and ‘relationships with colleagues’ were measured using the QEEW V.2.0. The job demand ‘bureaucratic burden’ was measured with the Three Item Red Tape scale. A structural equation model was built to answer our research question. Results: Work engagement mediated relationships of job resources with physicians’ work ability, and burnout mediated relationships of job resources and demands with work ability. Development opportunities (β=0.39, SE=0.12, p<0.001), participation in decision-making (β=0.18, SE=0.08, p=0.028) and relationships with colleagues (β=0.19, SE=0.19, p=0.002) were positively related to work engagement. Development opportunities (β=−0.20, SE=0.08, p=0.004) were negatively related and workload (β=0.51, SE=0.19, p<0.001) was positively related to burnout. Work engagement (β=0.22, SE=0.04, p<0.001) was positively related and burnout (β=−0.56, SE=0.06, p<0.001) was negatively related to work ability. Conclusions: Physicians’ work engagement and burnout mediated the relationships of various job demands and resources with their work ability. More work-engaged and less burned-out physicians reported better work ability. Hospitals may attenuate excessive workloads and facilitate development opportunities, participation in decision-making and good collegial relationships to enhance physicians’ occupational well-being and performance