No monkey business: why studying NK cells in non-human primates pays off

Human NK (hNK) cells play a key role in mediating host immune responses against various infectious diseases. For practical reasons, the majority of the data on hNK cells has been generated using peripheral blood lymphocytes. In contrast, our knowledge of NK cells in human tissues is limited, and not much is known about developmental pathways of hNK cell subpopulations in vivo. Although research in mice has elucidated a number of fundamental features of NK cell biology, mouse, and hNK cells significantly differ in their subpopulations, functions, and receptor repertoires. Thus, there is a need for a model that is more closely related to humans and yet allows experimental manipulations. Non-human primate models offer numerous opportunities for the study of NK cells, including the study of the role of NK cells after solid organ and stem cell transplantation, as well as in acute viral infection. Macaque NK cells can be depleted in vivo or adoptively transferred in an autologous system. All of these studies are either difficult or unethical to carry out in humans. Here we highlight recent advances in rhesus NK cell research and their parallels in humans. Using high-throughput transcriptional profiling, we demonstrate that the human CD56bright and CD56dim NK cell subsets have phenotypically and functionally analogous counterparts in rhesus macaques. Thus, the use of non-human primate models offers the potential to substantially advance hNK cell research

Loss of CCR7 Expression on CD56brightCD56^{bright} NK Cells Is Associated with a CD56dimCD16+CD56^{dim}CD16^{+} NK Cell-Like Phenotype and Correlates with HIV Viral Load

NK cells are pivotal sentinels of the innate immune system and distinct subpopulations in peripheral blood have been described. A number of studies addressed HIV-induced alterations of NK cell phenotype and functionality mainly focusing on $CD56^{dim}CD16^{+}$ and $CD56^{-} CD16^{+}$ NK cells. However, the impact of HIV-infection on $CD56^{bright}$ NK cells is less well understood. Here we report a rise of $CD56^{bright}$ NK cells in HIV-infected individuals, which lack CCR7-expression and strongly correlate with HIV viral load. $CCR7^{−} CD56^{bright}$ NK cells were characterized by increased cytolytic potential, higher activation states and a more differentiated phenotype. These cells thus acquired a number of features of $CD56^{dim}CD16^{+}$ NK cells. Furthermore, $CD56^{bright}$ NK cells from HIV patients exhibited higher degranulation levels compared to uninfected individuals. Thus, chronic HIV-infection is associated with a phenotypic and functional shift of $CD56^{bright}$ NK cells, which provides a novel aspect of HIV-associated pathogenesis within the NK cell compartment

Distinct transcriptional changes in non-small cell lung cancer patients associated with multi-antigenic RNActive® CV9201 immunotherapy

ABSTRACT We recently completed a phase I/IIa trial of RNActive® CV9201, a novel mRNA-based therapeutic vaccine targeting five tumor-associated antigens in non-small cell lung cancer (NSCLC) patients. The aim of the study presented here was to comprehensively analyze changes in peripheral blood during the vaccination period and to generate hypotheses facilitating the identification of potential biomarkers correlating with differential clinical outcomes post RNActive® immunotherapy. We performed whole-genome expression profiling in a subgroup of 22 stage IV NSCLC patients before and after initiation of treatment with CV9201. Utilizing an analytic approach based on blood transcriptional modules (BTMs), a previously described, sensitive tool for blood transcriptome data analysis, patients segregated into two major clusters based on transcriptional changes post RNActive® treatment. The first group of patients was characterized by the upregulation of an expression signature associated with myeloid cells and inflammation, whereas the other group exhibited an expression signature associated with T and NK cells. Patients with an enrichment of T and NK cell modules after treatment compared to baseline exhibited significantly longer progression-free and overall survival compared to patients with an upregulation of myeloid cell and inflammatory modules. Notably, these gene expression signatures were mutually exclusive and inversely correlated. Furthermore, our findings correlated with phenotypic data derived by flow cytometry as well as the neutrophil-to-lymphocyte ratio. Our study thus demonstrates non-overlapping, distinct transcriptional profiles correlating with survival warranting further validation for the development of biomarker candidates for mRNA-based immunotherapy

Phase Ib evaluation of a self-adjuvanted protamine formulated mRNA-based active cancer immunotherapy, BI1361849 (CV9202), combined with local radiation treatment in patients with stage IV non-small cell lung cancer

Background: Preclinical studies demonstrate synergism between cancer immunotherapy and local radiation, enhancing anti-tumor effects and promoting immune responses. BI1361849 (CV9202) is an active cancer immunotherapeutic comprising protamine-formulated, sequence-optimized mRNA encoding six non-small cell lung cancer (NSCLC)-associated antigens (NY-ESO-1, MAGE-C1, MAGE-C2, survivin, 5T4, and MUC-1), intended to induce targeted immune responses. Methods: We describe a phase Ib clinical trial evaluating treatment with BI1361849 combined with local radiation in 26 stage IV NSCLC patients with partial response (PR)/stable disease (SD) after standard first-line therapy. Patients were stratified into three strata (1: non-squamous NSCLC, no epidermal growth factor receptor (EGFR) mutation, PR/SD after ≥4 cycles of platinum- and pemetrexed-based treatment [n = 16]; 2: squamous NSCLC, PR/SD after ≥4 cycles of platinum-based and non-platinum compound treatment [n = 8]; 3: non-squamous NSCLC, EGFR mutation, PR/SD after ≥3 and ≤ 6 months EGFR-tyrosine kinase inhibitor (TKI) treatment [n = 2]). Patients received intradermal BI1361849, local radiation (4 × 5 Gy), then BI1361849 until disease progression. Strata 1 and 3 also had maintenance pemetrexed or continued EGFR-TKI therapy, respectively. The primary endpoint was evaluation of safety; secondary objectives included assessment of clinical efficacy (every 6 weeks during treatment) and of immune response (on Days 1 [baseline], 19 and 61). Results: Study treatment was well tolerated; injection site reactions and flu-like symptoms were the most common BI1361849-related adverse events. Three patients had grade 3 BI1361849-related adverse events (fatigue, pyrexia); there was one grade 3 radiation-related event (dysphagia). In comparison to baseline, immunomonitoring revealed increased BI1361849 antigen-specific immune responses in the majority of patients (84%), whereby antigen-specific antibody levels were increased in 80% and functional T cells in 40% of patients, and involvement of multiple antigen specificities was evident in 52% of patients. One patient had a partial response in combination with pemetrexed maintenance, and 46.2% achieved stable disease as best overall response. Best overall response was SD in 57.7% for target lesions. Conclusion: The results support further investigation of mRNA-based immunotherapy in NSCLC including combinations with immune checkpoint inhibitors. Trial registration: ClinicalTrials.gov, Identifier: NCT01915524

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

97TiP Identification of therapeutic targets in patients with squamous cell carcinoma of the head and neck who progress on or after anti-PD-(L)1 therapy: An IMMUcan sub-project

Background: Nivolumab and pembrolizumab are approved for the treatment of recurrent/metastatic Squamous Cell Carcinoma of the Head and Neck (SCCHN). However, a significant number of patients are not responding to PD-(L)1 inhibitors and, ultimately, the majority of the patients will finally progress. No standard of care exists for patients who progress after platinum-based chemotherapy or PD-1 inhibitors. Median overall survival of these patients is dismal (5-6 months). Tumor molecular mechanisms and the role of the immune micro-environment implicated in disease progression on or after anti-PD1 therapy need to be better characterized to identify new therapeutic targets. This will guide the rational development of therapeutic combinations. Trial design: IMMUcan is a European translational research study aiming at generating broad molecular and cellular profiling data of the tumor and its microenvironment from up to 3000 patients derived from 5 different cancer types: head and neck, colorectal, lung, renal and breast cancers. Collaborations with clinical trials are also possible, such as EORTC-HNCG-1559 study (UPSTREAM trial). This biomarker-driven umbrella trial enrolls patients with recurrent/metastatic SCCHN, progressing after platinum-based chemotherapy. The majority of the enrolled patients were pre-treated with PD-(L)1 inhibitors (anti-PD-(L)1 exposed patients), while a control population is also available (anti-PD-(L)1 naïve patients). Biological materials of these two distinct populations will be analyzed in IMMUcan: Whole Exome Sequencing, RNA sequencing, multiplex immunofluorescence and Imaging Mass Cytometry. The biological characteristics together with the clinical data will be analyzed to characterize the immune molecular profile of SCCHN that progress on or after anti-PD-(L)1 therapy with the final aim to identify therapeutic targets and to guide the development of rational therapeutic combinations. Those results will be completed with patients prospectively enrolled in the Head and Neck cohort of IMMUcan, via the EORTC-SPECTA infrastructure. For more information on IMMUcan, please visit https://immucan.eu/

HIV Infection Is Associated with a Preferential Decline in Less-Differentiated CD56dim CD16+ NK Cells▿

HIV-1 infection is characterized by loss of CD56dim CD16+ NK cells and increased terminal differentiation on various lymphocyte subsets. We identified a decrease of CD57− and CD57dim cells but not of CD57bright cells on CD56dim CD16+ NK cells in chronic HIV infection. Increasing CD57 expression was strongly associated with increasing frequencies of killer immunoglobulin-like receptors (KIRs) and granzyme B-expressing cells but decreasing percentages of cells expressing CD27+, HLA-DR+, Ki-67+, and CD107a. Our data indicate that HIV leads to a decline of less-differentiated cells and suggest that CD57 is a useful marker for terminal differentiation on NK cells

CCR7⁻CD56^bright NK cells exhibit phenotypic features of CD56^dimCD16⁺ cells.

<p>Representative expression data of CD62L, NKG2A, CD27, KIR3DL1, CD69 and CD16 on gated CCR7⁺ or CCR7⁻ CD56^bright cells and respective summary data including CD56^dimCD16⁺ NK cells, from untreated HIV-seropositive individuals. Numbers represent percentages of gated events and horizontal bars in dot plots indicate mean values. *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001.</p