Adoptive transfer of ex vivo expanded tumor infiltrating lymphocytes (TILs) has led to clinical benefit in some patients with melanoma but has not demonstrated convincing efficacy in other solid ...cancers. Whilst the presence of TILs in many types of cancer is often associated with better clinical prognosis, their function has not been systematically evaluated across cancer types. Responses to immunological checkpoint inhibitors in a wide range of cancers, including those for which adoptive transfer of expanded TILs has not shown clinical benefit, has clearly delineated a number of tumor type associated with tumor-reactive lymphocytes capable of effecting tumor remissions. Neuroblastoma is an aggressive childhood solid cancer in which immunotherapy with GD2-directed antibodies confers a proven survival advantage through incompletely understood mechanisms. We therefore evaluated the feasibility of ex vivo expansion of TILs from freshly resected neuroblastoma tumors and the potential therapeutic utility of TIL expansions. TILs were successfully expanded from both tumor biopsies or resections. Significant numbers of NKT and γδT cells were identified alongside the mixed population of cytotoxic (CD8+) and helper (CD4+) T cells of both effector and central memory phenotypes. Isolated TILs were broadly non-reactive against autologous tumor and neuroblastoma cell lines, so enhancement of neuroblastoma killing was attained by transducing TILs with a second-generation chimeric antigen receptor (CAR) targeting GD2. CAR-TILs demonstrated antigen-specific cytotoxicity against tumor cell lines. This study is the first to show reproducible expansion of TILs from pediatric neuroblastoma, the high proportion of innate-like lymphocytes, and the feasibility to use CAR-TILs therapeutically.
Abstract Background Neuroblastoma is the commonest cancer in infants. Survival in high-risk groups is low (40–50%). Newer treatments are needed to improve survival and morbidity. Cytomegalovirus ...(CMV) is a common viral infection, which increases γδ T cells. We investigated the use of CMV-reactive γδ T cells as a potential new immunotherapy. Methods Peripheral blood mononuclear cells from 30 paediatric haemato-oncology patients with or without CMV infection were analysed by flow cytometry. γδ T cells were expanded, and then co-cultured with CMV targets or neuroblastoma cells. T-cell activation, cytokine secretion, killing activity, and mechanisms were determined by ELISA, MTT colorimetric assay, and blocking assays. Novel γδ T cell receptors (TCR) were identified by sequencing. Findings In paediatric haemato-oncology patients, acute CMV led to higher proportions of total γδ T cells (p=0·0002) with the dominant subtype Vδ1 (p=0·0035). CMV-reactive γδ T cells were of the effector memory phenotype and expanded to clinically significant numbers for adoptive transfer. When compared with γδ T cells from CMV-negative patients, CMV-reactive γδ T cells had statistically significantly higher interferon γ release (p<0·001) in co-cultures with CMV-infected fibroblasts and showed cytolytic activity against CMV-infected fibroblasts and neuroblastoma cells which was mediated by γδ TCR and the NKG2D receptor. Sequencing showed that the dominant γδ T-cell chains in CMV-infected patients were Vδ1 Vγ2. Within the TCRs, CDR3 sequences had minimal diversity, γ chains had wide variations, and we identified a novel subpopulation in some patients. Interpretation We show that acute CMV infection in paediatric haemato-oncology patients leads to an increase in the Vδ1 Vγ2 subtype of γδ T cells. They can be expanded for adoptive transfer and are likely to retain killing ability post expansion. They recognise and kill CMV-infected targets and neuroblastoma cells via the γδ TCR and the NKG2D receptor. We have identified a novel TCR in a subpopulation of CMV-positive patients. Funding Great Ormond Street Charity, National Institute for Health Research Biomedical Research Centre, Olivia Hodson Cancer Fund.
Gamma delta T (γδT) lymphocytes are primed for rapid function, including cytotoxicity toward cancer cells, and are a component of the immediate stress response. Following activation, they can ...function as professional antigen-presenting cells. Chimeric antigen receptors (CARs) work by focusing T cell function on defined cell surface tumor antigens and provide essential costimulation for robust activation. Given the natural tropism of γδT cells for the tumor microenvironment, we hypothesized that their transduction with CARs might enhance cytotoxicity while retaining their ability to migrate to tumor and act as antigen-presenting cells to prolong the intratumoral immune response. Using a GD2-targeting CAR as a model system, we showed that γδT cells of both Vδ1 and Vδ2 subsets could be expanded and transduced to sufficient numbers for clinical studies. The CAR added to the cells’ innate cytotoxicity by enhancing GD2-specific killing of GD2-expressing cancer cell lines. Migration toward tumor cells in vitro was not impaired by the presence of the CAR. Expanded CAR-transduced Vδ2 cells retained the ability to take up tumor antigens and cross presented the processed peptide to responder alpha beta T (αβT) lymphocytes. γδ CAR-T cell products show promise for evaluation in clinical studies of solid tumors.
Chimeric antigen receptors (CARs) have been successfully expressed in human T cells to redirect their cytotoxic effector function against cancer antigens. A rare subset of T cells called gamma delta T cells is shown to be amenable to genetic modification by CARs and to retain the additional functional characteristic of antigen cross presentation.
Background Neuroblastoma is the commonest cancer in infants. Survival in high-risk groups is low (40-50%). Newer treatments are needed to improve survival and morbidity. Cytomegalovirus (CMV) is a ...common viral infection, which increases γδ T cells. We investigated the use of CMV-reactive γδ T cells as a potential new immunotherapy. Methods Peripheral blood mononuclear cells from 30 paediatric haemato-oncology patients with or without CMV infection were analysed by flow cytometry. γδ T cells were expanded, and then co-cultured with CMV targets or neuroblastoma cells. T-cell activation, cytokine secretion, killing activity, and mechanisms were determined by ELISA, MTT colorimetric assay, and blocking assays. Novel γδ T cell receptors (TCR) were identified by sequencing. Findings In paediatric haemato-oncology patients, acute CMV led to higher proportions of total γδ T cells (p=0·0002) with the dominant subtype Vδ1 (p=0·0035). CMV-reactive γδ T cells were of the effector memory phenotype and expanded to clinically significant numbers for adoptive transfer. When compared with γδ T cells from CMV-negative patients, CMV-reactive γδ T cells had statistically significantly higher interferon γ release (p<0·001) in co-cultures with CMV-infected fibroblasts and showed cytolytic activity against CMV-infected fibroblasts and neuroblastoma cells which was mediated by γδ TCR and the NKG2D receptor. Sequencing showed that the dominant γδ T-cell chains in CMV-infected patients were Vδ1 Vγ2. Within the TCRs, CDR3 sequences had minimal diversity, γ chains had wide variations, and we identified a novel subpopulation in some patients. Interpretation We show that acute CMV infection in paediatric haemato-oncology patients leads to an increase in the Vδ1 Vγ2 subtype of γδ T cells. They can be expanded for adoptive transfer and are likely to retain killing ability post expansion. They recognise and kill CMV-infected targets and neuroblastoma cells via the γδ TCR and the NKG2D receptor. We have identified a novel TCR in a subpopulation of CMV-positive patients. Funding Great Ormond Street Charity, National Institute for Health Research Biomedical Research Centre, Olivia Hodson Cancer Fund.
Background Neuroblastoma is the commonest cancer in infants. Survival in high-risk groups is low (40-50%). Newer treatments are needed to improve survival and morbidity. Cytomegalovirus (CMV) is a ...common viral infection, which increases gamma delta T cells. We investigated the use of CMV-reactive gamma delta T cells as a potential new immunotherapy. Methods Peripheral blood mononuclear cells from 30 paediatric haemato-oncology patients with or without CMV infection were analysed by flow cytometry. gamma delta T cells were expanded, and then co-cultured with CMV targets or neuroblastoma cells. T-cell activation, cytokine secretion, killing activity, and mechanisms were determined by ELISA, MTT colorimetric assay, and blocking assays. Novel gamma delta T cell receptors (TCR) were identified by sequencing. Findings In paediatric haemato-oncology patients, acute CMV led to higher proportions of total gamma delta T cells (p=0.0002) with the dominant subtype V delta 1 (p=0.0035). CMV-reactive gamma delta T cells were of the effector memory phenotype and expanded to clinically significant numbers for adoptive transfer. When compared with gamma delta T cells from CMV-negative patients, CMV-reactive gamma delta T cells had statistically significantly higher interferon gamma release (p<0.001) in co-cultures with CMV-infected fibroblasts and showed cytolytic activity against CMV-infected fibroblasts and neuroblastoma cells which was mediated by gamma delta TCR and the NKG2D receptor. Sequencing showed that the dominant gamma delta T-cell chains in CMV-infected patients were V delta 1 V gamma 2. Within the TCRs, CDR3 sequences had minimal diversity, gamma chains had wide variations, and we identified a novel subpopulation in some patients. Interpretation We show that acute CMV infection in paediatric haemato-oncology patients leads to an increase in the V delta 1 V gamma 2 subtype of gamma delta T cells. They can be expanded for adoptive transfer and are likely to retain killing ability post expansion. They recognise and kill CMV-infected targets and neuroblastoma cells via the gamma delta TCR and the NKG2D receptor. We have identified a novel TCR in a subpopulation of CMV-positive patients. Funding Great Ormond Street Charity, National Institute for Health Research Biomedical Research Centre, Olivia Hodson Cancer Fund.
ObjectivesOver 80% of patients diagnosed with childhood cancer in the UK will survive five or more years from diagnosis; as such, there is an increasing population of adults living with long-term ...effects of treatment. Cancer treatment can have negative effects on future fertility and reproductive function, and this is a recognised source of anxiety for patients and families.1,2 As such, it is recommended that fertility should be discussed with all patients diagnosed with childhood cancer, with fertility preservation offered if appropriate.3,4 At present, there are funding discrepancies across the U.K. for fertility preservation in cancer patients.5 The Children’s Cancer and Leukaemia Group (CCLG) Reproductive and Sexual Health Group are undertaking a U.K.-wide audit to assess whether fertility is being discussed and whether fertility preservation is being offered/accepted. This audit is ongoing; here we present interim analysis from initial results.MethodsAll 20 U.K. CCLG principle treatment centres were invited to participate, collecting six months of data on all male and female patients, under 18, with a new diagnosis of cancer. Data were collected on the following: diagnosis; age and pubertal status; risk of proposed treatment to fertility; details of fertility discussion; and whether fertility preservation was offered/accepted. Anonymised data was analysed centrally.ResultsTo date, six centres have submitted data on 273 patients diagnosed with childhood cancer, at a median age of 6.5 years (range 0–17). Treatment intention was curative in 260 (95%) patients. Fertility was discussed in 180 (65.9%) of cases; in 129 cases (71.7%), only the parents/carers were present for this discussion, with the patient also present in 51 cases. Referral for fertility preservation was offered to 56 (20.5%) patients; of those offered fertility preservation, 38 (67.9%) patients accepted.ConclusionData collection is ongoing for this national audit. However, this interim analysis suggests that, despite guidance that fertility should be discussed with all patients diagnosed with cancer, this standard is not being met. Where fertility is discussed, the patient is only included in the discussion 28% of the time. In order to optimise access to fertility preservation in eligible patients, as well as address the concerns of patients/families, discussion of fertility risk must be incorporated into consultations with newly diagnosed childhood cancer patients.ReferencesChow EJ, Stratton KL, Leisenring WM, Oeffinger KC, Sklar CA, Donaldson SS, et al. Pregnancy after chemotherapy in male and female survivors of childhood cancer treated between 1970 and 1999: A report from the Childhood Cancer Survivor Study cohort. Lancet Oncol. 2016 May 1;17(5):567–76.Langeveld NE, Grootenhuis MA, Voûte PA, de Haan RJ, van den Bos C. Quality of life, self-esteem and worries in young adult survivors of childhood cancer. Psychooncology. 2004;13(12):867–81.Mulder RL, Font-Gonzalez A, Green DM, Loeffen EAH, Hudson MM, Loonen J, et al. Fertility preservation for male patients with childhood, adolescent, and young adult cancer: recommendations from the PanCareLIFE Consortium and the International Late Effects of Childhood Cancer Guideline Harmonization Group. Lancet Oncol. 2021;22(2):e57–67.Mulder RL, Font-Gonzalez A, Hudson MM, van Santen HM, Loeffen EAH, Burns KC, et al. Fertility preservation for female patients with childhood, adolescent, and young adult cancer: recommendations from the PanCareLIFE Consortium and the International Late Effects of Childhood Cancer Guideline Harmonization Group. Lancet Oncol. 2021;22(2):e45–56.Latif S, Martins Da Silva S, Davies M, Mavrelos D, Foo X, Sangster P, et al. Fertility preservation provision in the NHS: a national assessment of care policies. Hum Fertil. 2022;0(0):1–6.
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