Mechanisms of response and resistance to anti-PD-1 immunotherapy

Immune checkpoint inhibition with anti-PD-1 and anti-CTLA-4 monoclonal antibodies has revolutionised the treatment of metastatic melanoma. However, most patients fail to respond or develop acquired resistance. A greater understanding of the mechanisms of response and resistance to checkpoint-based immunotherapies is required. Key to this is the need to identify the exact immune cell phenotypes responsible for anti-PD-1 and/or anti-CTLA-4 response, including the factors responsible for their recruitment and retention in tumours. Currently clinical trials are exploring novel strategies for overcoming resistance to anti-PD-1 immunotherapy, specifically through the targeting of alternative checkpoint receptors. In this thesis, we investigate the cells underlying response to anti-PD-1 checkpoint immunotherapy and show that CD103+ tumor resident CD8 T cells are strongly associated with patient overall survival in melanoma and expand significantly early during treatment with anti-PD-1. We show that IL-15 expression, but not tumor mutation burden, is associated with a higher density of these cells in patient tumors. Next, we investigate the expression profile of alternative checkpoint markers in primary, regional, and metastatic melanoma disease. Specifically, we show that only a small subset of tumor-infiltrating leukocytes expresses alternative co-stimulatory and co-inhibitory markers at any stage of disease, with PD-1 negative tumors lacking alternative targets compared to PD-1 positive tumors. Lastly, we investigate predictive biomarkers of response to second-line combination ant-PD-1+ anti-CTLA-4 after failure to first-line anti-PD-1 therapy. We characterize these patient tumors and identify the proportion of TCF7+ CD8 T cells and CD4 T cells as immune phenotypes associated with second-line response to combination treatment. Collectively, this thesis represents a significant step forward in our understanding of the mechanisms of response and resistance to immunotherapy

LNK suppresses interferon signaling in melanoma.

LNK (SH2B3) is a key negative regulator of JAK-STAT signaling which has been extensively studied in malignant hematopoietic diseases. We found that LNK is significantly elevated in cutaneous melanoma; this elevation is correlated with hyperactive signaling of the RAS-RAF-MEK pathway. Elevated LNK enhances cell growth and survival in adverse conditions. Forced expression of LNK inhibits signaling by interferon-STAT1 and suppresses interferon (IFN) induced cell cycle arrest and cell apoptosis. In contrast, silencing LNK expression by either shRNA or CRISPR-Cas9 potentiates the killing effect of IFN. The IFN-LNK signaling is tightly regulated by a negative feedback mechanism; melanoma cells exposed to IFN upregulate expression of LNK to prevent overactivation of this signaling pathway. Our study reveals an unappreciated function of LNK in melanoma and highlights the critical role of the IFN-STAT1-LNK signaling axis in this potentially devastating disease. LNK may be further explored as a potential therapeutic target for melanoma immunotherapy

Compartmentalization of total and virus-specific tissue-resident memory CD8+ T Cells in human lymphoid organs

Disruption of T cell memory during severe immune suppression results in reactivation of chronic viral infections, such as Epstein Barr virus (EBV) and Cytomegalovirus (CMV). How different subsets of memory T cells contribute to the protective immunity against these viruses remains poorly defined. In this study we examined the compartmentalization of virus-specific, tissue resident memory CD8+ T cells in human lymphoid organs. This revealed two distinct populations of memory CD8+ T cells, that were CD69+CD103+ and CD69+CD103-, and were retained within the spleen and tonsils in the absence of recent T cell stimulation. These two types of memory cells were distinct not only in their phenotype and transcriptional profile, but also in their anatomical localization within tonsils and spleen. The EBV-specific, but not CMV-specific, CD8+ memory T cells preferentially accumulated in the tonsils and acquired a phenotype that ensured their retention at the epithelial sites where EBV replicates. In vitro studies revealed that the cytokine IL-15 can potentiate the retention of circulating effector memory CD8+ T cells by down-regulating the expression of sphingosine-1-phosphate receptor, required for T cell exit from tissues, and its transcriptional activator, Kruppel-like factor 2 (KLF2). Within the tonsils the expression of IL-15 was detected in regions where CD8+ T cells localized, further supporting a role for this cytokine in T cell retention. Together this study provides evidence for the compartmentalization of distinct types of resident memory T cells that could contribute to the long-term protection against persisting viral infections

Spatial mapping reveals granuloma diversity and histopathological superstructure in human tuberculosis.

The hallmark of tuberculosis (TB) is the formation of immune cell-enriched aggregates called granulomas. While granulomas are pathologically diverse, their tissue-wide heterogeneity has not been spatially resolved at the single-cell level in human tissues. By spatially mapping individual immune cells in every lesion across entire tissue sections, we report that in addition to necrotizing granulomas, the human TB lung contains abundant non-necrotizing leukocyte aggregates surrounding areas of necrotizing tissue. These cellular lesions were more diverse in composition than necrotizing lesions and could be stratified into four general classes based on cellular composition and spatial distribution of B cells and macrophages. The cellular composition of non-necrotizing structures also correlates with their proximity to necrotizing lesions, indicating these are foci of distinct immune reactions adjacent to necrotizing granulomas. Together, we show that during TB, diseased lung tissue develops a histopathological superstructure comprising at least four different types of non-necrotizing cellular aggregates organized as satellites of necrotizing granulomas

Melanoma protective anti-tumor immunity activated by catalytic DNA

Melanoma incidence is increasing worldwide, and although drugs such as BRAF/MEK small-molecule inhibitors and immune checkpoint antibodies improve patient outcomes, most patients ultimately fail these therapies and alternative treatment strategies are urgently needed. DNAzymes have recently undergone clinical trials with signs of efficacy and no serious adverse events attributable to the DNAzyme. Here we investigated c-Jun expression in human primary and metastatic melanoma. We also explored the role of T cell immunity in DNAzyme inhibition of primary melanoma growth and the prevention of growth in non-treated tumors after the cessation of treatment in a mouse model. c-Jun was expressed in 80% of melanoma cells in human primary melanomas (n = 17) and in 83% of metastatic melanoma cells (n = 38). In contrast, c-Jun was expressed in only 11% of melanocytes in benign nevi (n = 24). Dz13, a DNAzyme targeting c-Jun/AP-1, suppressed both Dz13-injected and untreated B16F10 melanoma growth in the same mice, an abscopal effect relieved in each case by administration of anti-CD4/anti-CD8 antibodies. Dz13 increased levels of cleaved caspase-3 within the tumors. New, untreated melanomas grew poorly in mice previously treated with Dz13. Administration of anti-CD4/anti-CD8 antibodies ablated this inhibitory effect and the tumors grew rapidly. Dz13 inhibited c-Jun expression, reduced intratumoral vascularity (vascular lumina area defined by CD31 staining), and increased CD4+ cells within the tumors. This study provides the first demonstration of an abscopal effect of a DNAzyme on tumor growth and shows that Dz13 treatment prevents growth of subsequent new tumors in the same animal. Dz13 may be useful clinically as a therapeutic antitumor agent by preventing tumor relapse through adaptive immunity

Combination nivolumab and ipilimumab or nivolumab alone in melanoma brain metastases: a multicentre randomised phase 2 study

Nivolumab monotherapy and combination nivolumab plus ipilimumab increase proportions of patients achieving a response and survival versus ipilimumab in patients with metastatic melanoma; however, efficacy in active brain metastases is unknown. We aimed to establish the efficacy and safety of nivolumab alone or in combination with ipilimumab in patients with active melanoma brain metastases.This multicentre open-label randomised phase 2 trial was done at four sites in Australia, in three cohorts of immunotherapy-naive patients aged 18 years or older with melanoma brain metastases. Patients with asymptomatic brain metastases with no previous local brain therapy were randomly assigned using the biased coin minimisation method, stratified by site, in a 30:24 ratio (after a safety run-in of six patients) to cohort A (nivolumab plus ipilimumab) or cohort B (nivolumab). Patients with brain metastases in whom local therapy had failed, or who had neurological symptoms, or leptomeningeal disease were enrolled in non-randomised cohort C (nivolumab). Patients in cohort A received intravenous nivolumab 1 mg/kg combined with ipilimumab 3 mg/kg every 3 weeks for four doses, then nivolumab 3 mg/kg every 2 weeks; patients in cohort B or cohort C received intravenous nivolumab 3 mg/kg every 2 weeks. The primary endpoint was intracranial response from week 12. Primary and safety analyses were done on an intention-to-treat basis in all patients who received at least one dose of the study drug. This trial is registered with ClinicalTrials.gov, number NCT02374242, and is ongoing for the final survival analysis.Between Nov 4, 2014, and April 21, 2017, 79 patients were enrolled; 36 in cohort A, 27 in cohort B, and 16 in cohort C. One patient in cohort A and two in cohort B were found to be ineligible and excluded from the study before receiving the study drug. At the data cutoff (Aug 28, 2017), with a median follow up of 17 months (IQR 8-25), intracranial responses were achieved by 16 (46%; 95% CI 29-63) of 35 patients in cohort A, five (20%; 7-41) of 25 in cohort B, and one (6%; 0-30) of 16 in cohort C. Intracranial complete responses occurred in six (17%) patients in cohort A, three (12%) in cohort B, and none in cohort C. Treatment-related adverse events occurred in 34 (97%) of 35 patients in cohort A, 17 (68%) of 25 in cohort B, and eight (50%) of 16 in cohort C. Grade 3 or 4 treatment-related adverse events occurred in 19 (54%) patients in cohort A, four (16%) in cohort B, and two (13%) in cohort C. No treatment-related deaths occurred.Nivolumab combined with ipilimumab and nivolumab monotherapy are active in melanoma brain metastases. A high proportion of patients achieved an intracranial response with the combination. Thus, nivolumab combined with ipilimumab should be considered as a first-line therapy for patients with asymptomatic untreated brain metastases.Melanoma Institute Australia and Bristol-Myers Squibb

LNK suppresses interferon signaling in melanoma.

LNK (SH2B3) is a key negative regulator of JAK-STAT signaling which has been extensively studied in malignant hematopoietic diseases. We found that LNK is significantly elevated in cutaneous melanoma; this elevation is correlated with hyperactive signaling of the RAS-RAF-MEK pathway. Elevated LNK enhances cell growth and survival in adverse conditions. Forced expression of LNK inhibits signaling by interferon-STAT1 and suppresses interferon (IFN) induced cell cycle arrest and cell apoptosis. In contrast, silencing LNK expression by either shRNA or CRISPR-Cas9 potentiates the killing effect of IFN. The IFN-LNK signaling is tightly regulated by a negative feedback mechanism; melanoma cells exposed to IFN upregulate expression of LNK to prevent overactivation of this signaling pathway. Our study reveals an unappreciated function of LNK in melanoma and highlights the critical role of the IFN-STAT1-LNK signaling axis in this potentially devastating disease. LNK may be further explored as a potential therapeutic target for melanoma immunotherapy

CD69⁺CD103⁺CD8⁺ T cells localize near the epithelial barrier in tonsils.

<p>The locations of CD69⁺CD8⁺ T cell subsets were determined by immunohistochemistry. (A) Immunofluorescence microscopy images of human tonsils show the localization of CD8 (green), CD69 (blue) and CD103 (red). Scale bar represents 100 μm. (B) Higher magnification of areas 1 & 2 show the localization of CD103⁺CD69⁺CD8⁺ and CD103CD69⁺CD8⁺ T cells within the tonsils. (C) White arrowheads point to CD103⁺CD69⁺CD8⁺ T cells in areas 1 and CD103^—CD69⁺CD8⁺ T cells in area 2. (C) Quantitative analysis of the distance of CD103⁺CD3⁺CD8⁺ T cells from the epithelium shows majority localizing near the epithelial surface (P = 0.0022 by two-tailed Mann Whitney U-test). (D) Immunofluorescence microscopy of human spleen sections shows the localization of CD8 (green), CD69 (blue) and CD103 (red). Scale bar represents 100 μm. Higher magnification of areas 1 and 2 show the distribution of CD103⁺CD69⁺CD8⁺ and CD103^—CD69⁺CD8⁺ T cells. White arrowheads in area 1 show the CD103⁺CD69⁺CD8⁺ T cells and in area 2 show the CD103^—CD69⁺CD8⁺ T cells.</p

Constitutive expression of IL-15 within tonsils.

<p>Immunofluorescence microscopy of frozen section of the tonsils show the presence of IL-15 expressing cells in the T cell areas and the epithelial lining of the tissue. Sections were stained with anti-IL-15 (red), anti-CD8 (green) and anti-IgM (blue). Yellow dashed line marks the epithelial barrier surface and the white dashed-lines show B cell follicles (B). ‘T” indicates the extra-follicular regions where T cells localize.</p

IL-15 and TGF-β co-operate to extinguish expression of <i>KLF2</i> and <i>S1PR1</i>.

<p>(A) Representative flow cytometry plots show the expression of CD69 and CD103 by CD8⁺ T cells following 7 day culture under different conditions: unstimulated (US), IL-15 or IL-15 + TGF-β and polyclonal stimulation (TAE). (B) Plot shows the proportion of CD69⁺CD103^- (left panel) and CD69⁺CD103⁺ (right panel) CD8⁺ T cells following 7 day culture with different cytokines. Data are represented as the mean and SEM of 5–11 donors. (C) Representative flow cytometry plots show the expression of CD69, CD137 and dilution of cell trace violet (CTV) dye following stimulation of circulating CD8⁺ T cells for 7 days with TAE beads (upper panels) or IL-15 (lower panels). (D) Representative flow cytometry plot and graph show the expression of CD69 and the dilution of cell trace violet dye following stimulation of purified circulating naïve (n = 4), TCM (n = 2), TEM (n = 8) and TEMRA (n = 5) CD8⁺ T cells for 7 days with IL-15. (E-F) Plots show the relative expression of <i>S1PR1</i> (E) and <i>KLF2</i> (F) in CD69⁺ or CD69^—CD8⁺ T cells following culture for 7 days with no stimulation or stimulation with IL-15 with and without TGF-β. Purified circulating CD8⁺ T cells were cultured for 7 days and the resulting CD69⁺ and CD69^—populations were purified by cell sorting. The expression levels of KLF2 and S1pr1 were quantified by RT-PCR. Individual dots represent different samples and the data is represented as the mean ± SEM. Statistical analysis was performed using one-way ANOVA and Tukey test. P<0.05 is noted with * and P<0.005 is noted with **. (G-H) The ability of IL-15 induced CD69⁺CD8⁺ T cells to migrate to S1P and CCL5 (20 nM) was tested in trans-well migration assays. Cultured cells were sorted as stated above (for F) and their ability to migrate towards different concentrations of S1P (G) or CCL5 (20 nM) (H) was determined. Data represent the mean and SEM of three independent experiments using three different donors. Statistical analysis was performed using two-way ANOVA and the p value was < 0.05.</p