Ex vivo modulation of intact tumor fragments with anti-PD-1 and anti-CTLA-4 influences the expansion and specificity of tumor-infiltrating lymphocytes.

Checkpoint inhibition (CPI) therapy and adoptive cell therapy with autologous tumor-infiltrating lymphocytes (TIL-based ACT) are the two most effective immunotherapies for the treatment of metastatic melanoma. While CPI has been the dominating therapy in the past decade, TIL-based ACT is beneficial for individuals even after progression on previous immunotherapies. Given that notable differences in response have been made when used as a subsequent treatment, we investigated how the qualities of TILs changed when the ex vivo microenvironment of intact tumor fragments were modulated with checkpoint inhibitors targeting programmed death receptor 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). Initially, we show that unmodified TILs from CPI-resistant individuals can be produced, are overwhelmingly terminally differentiated, and are capable of responding to tumor. We then investigate these properties in ex vivo checkpoint modulated TILs finding that that they retain these qualities. Lastly, we confirmed the specificity of the TILs to the highest responding tumor antigens, and identified this reactivity resides largely in CD39+CD69+ terminally differentiated populations. Overall, we found that anti-PD-1 will alter the proliferative capacity while anti-CTLA4 will influence breadth of antigen specificity.

Neutropenia-related aspergillosis in non-transplant haematological patients hospitalised under ambient air versus purified air conditions.

BACKGROUND: To reduce the risk of invasive aspergillosis (IA), air purification by high-efficiency particulate air filtration and laminar air flow (HEPA/LAF) is standard of care in allogeneic blood stem cell transplantation. Its use in non-transplant haematological patients is inconsistent. OBJECTIVES: We sought to assess the incidence and outcome of pulmonary IA in non-transplant patients with life-threatening neutropenia by comparing an ambient air hospitalisation period (2008-2011) with a subsequent HEPA/LAF hospitalisation period (2012-2014). PATIENTS AND METHODS: We compared 204 consecutive patients with acute myeloid leukaemia, acute lymphoblastic leukaemia or aplastic anaemia completing 534 neutropenia-related hospitalisations under ambient air conditions with 126 such patients completing 437 neutropenia-related hospitalisations under HEPA/LAF conditions. IA was defined using the 2008 EORTC/MSG criteria. RESULTS: Within a 7-year study period, we observed one 'proven', three 'probable' and 73 'possible' IAs, most often during acute leukaemia remission induction. Their frequency rose with increasing duration of life-threatening neutropenia (1-10 days, 1.8%; >40 days, 35.2%) and concomitant severe anaemia (0 days, 3.2%; >20 days, 31.0%). Multiple logistic regression revealed a strong correlation between IA incidence and hospitalisation under HEPA/LAF conditions (odds ratio [OR], 0.368 [95% confidence interval, 0.207-0.654]; p < .001) and duration of neutropenia (OR, 1.043 [1.023-1.062] per day; p < .001) and anaemia (OR, 1.044 [1.008-1.081] per day; p = .016). IA-associated fatal outcomes were non-significantly reduced under HEPA/LAF (OR, 0.077 [0.005-1.151]; p = .063). The protective effect of HEPA/LAF was not seen under posaconazole prophylaxis (OR, 0.856 [0.376-1.950]; p = .711). CONCLUSIONS: Implementation of HEPA/LAF was associated with a significant reduction in neutropenia-related IA in non-transplant haematological patients.

Peptide vaccination activating Galectin-3-specific T cells offers a novel means to target Galectin-3-expressing cells in the tumor microenvironment.

Galectin-3 (Gal3) can be expressed by many cells in the tumor microenvironment (TME), including cancer cells, cancer-associated fibroblasts, tumor-associated macrophages, and regulatory T cells (Tregs). In addition to immunosuppression, Gal3 expression has been connected to malignant cell transformation, tumor progression, and metastasis. In the present study, we found spontaneous T-cell responses against Gal3-derived peptides in PBMCs from both healthy donors and cancer patients. We isolated and expanded these Gal3-specific T cells in vitro and showed that they could directly recognize target cells that expressed Gal3. Finally, therapeutic vaccination with a long Gal3-derived peptide epitope, which induced the expansion of Gal3-specific CD8+ T cells in vivo, showed a significant tumor-growth delay in mice inoculated with EO771.LMB metastatic mammary tumor cells. This was associated with a significantly lower percentage of both Tregs and tumor-infiltrating Gal3+ cells in the non-myeloid CD45+CD11b- compartment and with an alteration of the T-cell memory populations in the spleens of Gal3-vaccinated mice. These results suggest that by activating Gal3-specific T cells by an immune-modulatory vaccination, we can target Gal3-producing cells in the TME, and thereby induce a more immune permissive TME. This indicates that Gal3 could be a novel target for therapeutic cancer vaccines.

CTLA-4 blockade boosts the expansion of tumor-reactive CD8+ tumor-infiltrating lymphocytes in ovarian cancer.

Adoptive cell therapy (ACT) with autologous tumor-infiltrating lymphocytes (TILs) can induce durable complete tumor regression in patients with advanced melanoma. Efforts are currently underway to expand this treatment modality to other cancer types. In the microenvironment of ovarian cancer, the engagement of co-inhibitory immune checkpoint molecules such as CTLA-4 can lead to the inactivation of TILs. Thus, approaches that directly manipulate co-inhibitory pathways within the tumor microenvironment might improve the expansion of tumor-reactive TILs. The initial expansion of TILs for ACT from tumor fragments provides a window of opportunity to manipulate an intact tumor microenvironment and improve CD8+ T-cell output and TIL tumor reactivity. To exploit this, we used a CTLA-4-blocking antibody, added during the initial TIL culture, and found that the blockade of CTLA-4 favored the propagation of CD8+ TILs from ovarian tumor fragments. Interestingly, adding the CTLA-4 blocking antibody in the initial phase of the TIL culture resulted in more potent anti-tumor TILs in comparison to standard TIL cultures. This phenotype was preserved during the rapid expansion phase. Thus, targeting CTLA-4 within the intact tumor microenvironment of tumor fragments enriches tumor-reactive TILs and may improve clinical outcome of TIL-based ACT in ovarian cancer.

Inflammation induced PD-L1-specific T cells.

PD-L1-specific T cells are a natural part of the T-cell repertoire in humans. Hence, we have previously described spontaneous CD8+ and CD4+ T-cell reactivity against PD-L1 in the peripheral blood of patients with various cancers as well as in healthy donors. It is well described that the expression of the PD-L1 protein is introduced in cells by pro-inflammatory cytokines, e.g. IFN-γ. In the current study, we were able to directly link inflammation with PD-L1-specific T cells by showing that inflammatory mediators such as IFN-γ generate measurable numbers of PD-L1-specific T cells in human PBMCs as well as in in vivo models. These PD-L1-specific T cells can vigorously modulate the cell compartments of the local environment. PD-L1-specific T cells may be important for immune homeostasis by sustaining the ongoing inflammatory response by the suppression of regulatory cell function both directly and indirectly.

Correction: Tumour-reactive T cell subsets in the microenvironment of ovarian cancer.

Since the publication of this paper, the authors noticed that the funding information was not complete. The correct funding information is now shown in the Acknowledgements section. Acknowledgements The studies were supported by grants from the OvaCure Foundation, the Danish Cancer Society Research Foundation, the Anticancer Fund, Aase og Ejnar Danielsens Foundation and the Independent Research Fund Denmark (grant number DFF-4183-00597).

Tumour-reactive T cell subsets in the microenvironment of ovarian cancer.

BACKGROUND: Solid malignancies are frequently infiltrated with T cells. The success of adoptive cell transfer (ACT) with expanded tumour-infiltrating lymphocytes (TILs) in melanoma warrants its testing in other cancer types. In this preclinical study, we investigated whether clinical-grade TILs could be manufactured from ovarian cancer (OC) tumour specimens. METHODS: Thirty-four tumour specimens were obtained from 33 individual patients with OC. TILs were analysed for phenotype, antigen specificity and functionality. RESULTS: Minimally expanded TILs (Young TILs) were successfully established from all patients. Young TILs contained a high frequency of CD3+ cells with a variable CD4/CD8 ratio. TILs could be expanded to clinical numbers. Importantly, recognition of autologous tumour cells was demonstrated in TILs in >50% of the patients. We confirmed with mass spectrometry the presentation of multiple tumour antigens, including peptides derived from the cancer-testis antigen GAGE, which could be recognised by antigen-specific TILs. Antigen-specific TILs could be isolated and further expanded in vitro. CONCLUSION: These findings support the hypothesis that patients with OC can benefit from ACT with TILs and led to the initiation of a pilot clinical trial at our institution . TRIAL REGISTRATION: clinicaltrials.gov: NCT02482090.

SATB1 in Malignant T Cells.

Deficient expression of SATB1 hampers thymocyte development and results in inept T-cell lineages. Recent data implicate dysregulated SATB1 expression in the pathogenesis of mycosis fungoides, the most frequent variant of cutaneous T-cell lymphoma. Here, we report on a disease stage-associated decrease of SATB1 expression and an inverse expression of STAT5 and SATB1 in situ. STAT5 inhibited SATB1 expression through induction of microRNA-155. Decreased SATB1 expression triggered enhanced expression of IL-5 and IL-9 (but not IL-6 and IL-32), whereas increased SATB1 expression had the opposite effect, indicating that the microRNA-155 target SATB1 is a repressor of IL-5 and IL-9 in malignant T cells. In accordance, inhibition of STAT5 and its upstream activator JAK3 triggered increased SATB1 expression and a concomitant suppression of IL-5 and IL-9 expression in malignant T cells. In conclusion, we provide a mechanistic link between the proto-oncogenic JAK3/STAT5/microRNA-155 pathway, SATB1, and cytokines linked to CTCL severity and progression, indicating that SATB1 dysregulation is involved in cutaneous T-cell lymphoma pathogenesis.