Citations

Abstract Cytotoxic T lymphocytes (CTL) are indispensable in anti-tumor immunity. Although CTLs are prone to exhaustion in patients with advanced cancer, T cell resiliency explains the presence of tumor-reactive CTLs that are less exhausted, capable of cytolytic function, expansion, and rebound in response to immunotherapy to reject metastatic malignancies. However, the features of resilient T cells have not been clearly defined. In this report, we demonstrate that peripheral CX3CR1+ CD8+ T cells with low mitochondrial membrane potential rebounded CTL function quickly after radiation therapy in patients with large tumor burden portraying their functional resiliency. Furthermore, CX3CR1+ CD8+ T cell with low, but not high, mitochondrial membrane potential are highly cytotoxic, accumulate less reactive oxygen species (ROS), and express more Malic enzyme 1 (ME1). ME1 overexpression increases ATP production in a glycolysis-independent manner while concurrently curtailing excessive ROS in activated CD8+ T cells; and expands CX3CR1+NKG7+ effector CD8+ T cells with enhanced cytotoxicity. Importantly, transfection of ME1 mRNA promotes tumoricidal activity in CD8+ T cells from patients with advanced cancers. Our study reveals a mechanism used by CTLs to balance excessive ROS via ME1 to maintain a metabolic and functional resiliency. Modification of ME1 expression in CTLs may be a novel method to improve the efficacy of cancer immunotherapy by preventing T cell exhaustion. Supported by grants from Mayo Clinic Foundation and NIH (R01CA 256927-2)

Abstract PD-1- and PD-L1-targeted immune checkpoint blockade therapy has shown great clinical potential. Unfortunately, this therapy has been ineffective in a majority of patients bearing solid tumors due to the highly immunosuppressive nature of the tumor microenvironment. As macrophages compose the most abundant myeloid cell subset within the tumor, current macrophage-targeted treatments aim to deplete macrophages within the tumor to reduce immune suppression. However, our goal is to determine whether αPD-1 and αPD-L1 therapy can harness macrophage potential by shifting tumor associated macrophages (TAMs) from a suppressive M2 to a pro-inflammatory M1 phenotype. Our preliminary data has shown that αPD-1/αPD-L1 treatment of mice bearing B16 murine melanoma leads to controlled tumor growth and this was accompanied by a higher M1:M2 ratio and CD8 T-cell abundance. Therefore, we hypothesized that αPD-1/αPD-L1 therapy may drive TAMs towards a pro-inflammatory phenotype and to become better activators of CD8 T-cells. By analyzing antigen presentation machinery on TAMs, we show that in the presence of tumor cells, αPD-L1 blockade upregulates expression of MHC1 and MHC2 on TAMs. Unexpectedly, direct αPD-L1 blockade on TAMs leads to reduced CD80 surface due to mechanisms still to be elucidated. Further, we analyzed co-cultures of αPD-L-treated TAMs loaded with ovalbumin and naïve OT1 cells, which led to greater proliferation of naïve CD8 T-cells compared to their isotype control counterparts. Our observations thus far support the ability of αPD-1/αPD-L1 therapy to induce an M1-phenotype shift among macrophages and also αPD-L1 blockade alone can drive TAMs to become better CD8 T-cell activators. Mayo Clinic David and Margaret Grohne Cancer Immunotherapy and Mayo Clinic Immune Monitoring Core Pilot Awards

Abstract Although immune checkpoint blockade that targets the PD-L1 has revolutionized the treatment of human cancers, only a small proportion of patients positively respond to this form of treatment. A limitation of these therapies is that they only target and inhibit PD-L1’s extracellular function, which is to bind its receptor on PD-1 on T cells and inhibit T cell cytotoxicity. Recent discoveries have shown that PD-L1 expressed on tumor cells can signal intrinsically and independently of PD-1 ligation to promote tumor cell survival. Besides tumor cells, host myeloid cells have been shown to have high expression of PD-L1, therefore contributing to the immunosuppressive nature of the tumor. However, PD-L1 intrinsic signaling in human myeloid cells has not been studied. Therefore, the purpose of our work is to determine the intrinsic functions of PD-L1 in myeloid cells and how this impacts anti-tumor immunity. To study this, we used a novel PD-L1 antibody (H1A) that does not block PD-L1’s binding with PD-1, but directs PD-L1 for degradation, thereby decreasing total PD-L1 expression. Additionally, we developed a chimeric mouse model in which the murine PD-1/PD-L1 has been replaced with the human version. This allows us to evaluate PD-L1’s intrinsic function in vivo. In our preliminary studies, the degradation of PD-L1 in human monocytes led to increased chemokine release as well as increased conversion of effector T cells. Additionally, H1A treatment of tumor-bearing mice led to lower tumor burden in a CD8 T cell dependent manner. Based on these results, PD-L1 intrinsic signaling has the potential to regulate the immune-stimulatory response of monocytes, thereby limiting effector T cell responses against cancer. Supported by grants from NIH (R0192389) and Mayo Clinic graduate school of biomedical sciences

Abstract Only a small cohort of patients show durable responses to immune checkpoint inhibitor (ICI) therapy and the mechanisms regulating CD8 T cell responses to ICIs are yet to be elucidated. To that end, we explored T cell intrinsic factors that may be attributed to low ICI therapy response rates. We have identified Dual Specificity Phosphatase 2 (DUSP2) as a potential negative regulator of ICI response within CD8 T cells. We demonstrated that DUSP2 expression in human CD8 T cells is transient, peaking at two-hours post-T cell stimulation with rapid waning thereafter. Downregulation of DUSP2, via siRNA transfection, increased cytotoxicity of human CD8 T cells activated for 48-hours prior to inclusion in a four-hour tumor killing co-culture assay, despite the return of DUSP2 expression to baseline levels at this time-point. Additionally, DUSP2 overexpression, via mRNA transfection, decreased release of effector molecules (Granzyme B and Interferon-gamma) from human CD8 T cells at 24-hours post-activation when compared to control mRNA. When we utilized RNA-sequencing to analyze and compare differentially expressed genes between responders and non-responders of ICI therapy, we found DUSP2 expression in CX3CR1 positive CD8 T cells to be consistently decreased in responders but not in non-responders. Since CX3CR1 positive CD8 T cells identify a highly resilient and cytotoxic subset in response to ICI therapy, we hypothesize that DUSP2 negatively regulates ICI-responsive effector CD8 T cells. Thus, uncovering the role of DUSP2 in CD8 T cells may provide a target to improve the efficacy of ICIs in patients with limited therapeutic alternatives. Mayo Clinic Department of Immunology Pilot Award Mayo Clinic Center for Individualized Medicine

Abstract Though anti-PD-1/PD-L1 immune checkpoint blockade therapy has demonstrated robust anti-tumor immunity in human patients with advanced cancers such as melanoma, only a fraction of patients achieved such durable anti-tumor responses. Among all the non-responders, some patients responded initially but developed resistance to the therapy in the later treatments. This reflects an urgent need to understand the role of PD-1 signals in CD8+ T cells at priming and at effector stages during tumor development for the benefit of immunotherapy. The controversy of rapid tumor progression in PD-1 conditional knockout in regulatory T cells and tumor regression in PD-1 targeted deletion in regulatory T cells indicated germline PD-1 knockout mice might not be sophisticated enough to study the role of PD-1 in CD8+ T cells during tumor development. Thus, we produced a CD8 T cell-specific PD-1 knockout mouse model (CD8 E8ICre-PD-1fl/fl, also called CD8-PD-1 cKO mice) in which PD-1 is deleted in CD8+ T cells in the peripheral tissues. When CD8-PD-1 cKO mice were challenged with B16-OVA mouse melanoma cells, we found a delayed tumor growth and prolonged survival of CD8-PD-1 cKO compared to control mice. However, after 20 days of tumor challenge, the delayed tumor growth rebound in CD8-PD-1 cKO mice. In an OVA antigen immunization model, we found increased effector cells and tissue resident cells in the spleen of CD8-PD-1 cKO mice compared with control mice on day 7 after immunization. Taken together, our results suggest that PD-1 may restrain the expansion of effector T cells that contribute to the early phase of tumor control, but the augmented effector T cell response may cause an attrition of memory T cell pool that will be needed for long term protection. R01CA256927

Abstract PD-L1/PD-1 receptor pathway represents an important way of cancer cells to escape immune surveillance, yet only small portions of patients can benefit from PD-1/PD-L1 inhibitors. This is at least partially due to incomplete understanding of PD-1/PD-L1 biology in cancer. Although the interaction of extracellular domains between PD-L1 and PD-1 on cancer cells and immune cells is well studied, the function of PD-L1 inside cells, however, has not been well established. In our previous data, we found that intracellular PD-L1 acts as an RNA binding protein and interacts with NBS1 and BRCA1 mRNAs. Furthermore, intracellular PD-L1 protects targeted RNAs from degradation by RNA exosome, therefore increasing cellular resistance to DNA damage. Since Clinical approved PD-1/PD-L1 inhibitors have little effect on intracellular PD-L1, we sought to find new drugs to target intracellular PD-L1. Here we developed a new PD-L1 antibody, H1A, which has a different effect on PD-L1. Instead of blocking of PD-1/PD-L1 interaction, we found that this antibody disrupts the interaction of PD-L1 and CMTM6, which leads to degradation of PD-L1 by the lysosome. Therefore, H1A can unleash host antitumor activity and enhance the effects of DNA damaging therapy in cancer cells at the same time. Indeed, applying H1A in vivo significantly sensitized tumors to radiation and chemotherapy in a PDX models. Moreover, in a humanized immune competent TNBC PDX model, H1A monotherapy exhibited better therapeutic efficacy compared with clinically available PD-L1 inhibitor, suggesting that H1A activates anti-tumor immunity through PD-L1 degradation. Therefore, H1A may be a potential new drug to target both intracellular and extracellular PD-L1 to improve cancer therapy.

Abstract Aging is considered to be a major risk factor for host morbidity and mortality upon acute respiratory virus infections. To better elucidate the immune determinants of viral pathogenesis during aging, we chronological characterized lung cellular and molecular profiles following influenza virus (PR8) infection in young or aged mice till 60 days post infection. Time-course scRNAseq coupled with high dimensional flow cytometry enabled the description of changes in both immune and stromal compartment between young and aged hosts. We further zoomed into the myeloid compartment and focused on the dynamic responses of lung macrophages given their complex roles in viral pathogenesis, tissue repair and lung fibrosis. Enlarged Monocyte-derived Macrophage (MoM) population as well as decreased resident-Alveolar Macrophage (AM) population were observed in the aged mice when compared to those of the young mice. Despite the self-sustain nature of AMs during homeostasis, it has been reported that Ly6Chi monocytes can gain AM identity following severe lung damage like influenza virus infection. We found that the gain of AM identity from monocytes was abrogated in the aged hosts, which likely contributed to the age-associated impairment in lung recovery after influenzas virus infection. Additionally, trajectory analysis with scRNAseq allowed us to identify IFNα/γ signaling to be potential pathways contributing to such defect. This notion is also supported by elevated IFNγ level in BAL from the aged host during lung recovery. Our results thus have improved our understanding on the cellular and molecular mechanisms by which aged hosts exhibit severe diseases and delayed recovery after respiratory viral infection.

27 Background: Cholangiocarcinoma (CCA) is a devastating cancer with few effective treatment options. We have previously discovered LCK can activate AXL, a TAM receptor tyrosine kinase (RTK), by phosphorylation of AXL-Y866 ( J Hepatol 2022). AXL is known to confer therapeutic resistance to solid cancers. However, the role of AXL in CCA, and the impact of AXL-Y866 activation by LCK still remains to be elucidated. Here, we investigated the significance of AXL expression, signaling, and activation by LCK on Y866. Methods: We first evaluated the expression levels of AXL in CCA and its association with patient outcome using The Cancer Genome Atlas (TCGA) dataset. Next, to evaluate whether AXL inactivation sensitizes CCA cells to gemcitabine and cisplatin (GC), AXL downregulation was achieved via siRNA approach and the selective AXL inhibitor bemcentinib (Bem) using human CCA cell lines, HuCCT1 and RBE, and murine SB-1. We examined 50% inhibitory concentration (IC50) value on GC with or without AXL knockdown (KD) using cell viability assay. Then we assessed the efficacy of the combination of GC and Bem utilizing Calcusyn software and apoptosis by Annexin V assay. I n vivo efficacy was assessed using both a patient derived CCA xenograft and SB-1 a syngeneic model of CCA treated with vehicle, GC, Bem, or the combination. Next, we defined the interactome of wildtype AXL- Y866 compared to AXL-Y866F using biotin proximity labelling methods (BioID), validation of downstream target proteins using Western blot (WB), and a functional study with AXL-/- cells reconstituted with either AXL WT, or AXL Y866F. Results: In TCGA cohorts, AXL transcripts are more abundant than in normal adjacent liver, and higher levels are correlated with worse clinical outcome. In in vitro study, IC50 values of GC decreased after AXL KD. Synergistic effects were observed in the combination. The combination caused increased apoptosis compared to other treatments. In the in vivo studies, the combination suppressed tumor growth and the Ki67 levels, and the phosphorylation levels of PEAK1 decreased in the combination group compared to other groups. In BioID experiments, among the 222 proteins which were detected in both AXL WT and AXL Y866F interactomes, PEAK1 kinase, which localizes to actin cytoskeleton and focal adhesions, was most altered when Y866 was inactivated by the Y866F mutation. In gene ontology analysis, cell-cell adhesion and focal adhesion related proteins were enriched. In WB, we validated that the phosphorylation of PEAK1 decreased after AXL or LCK knockout (KO) and after Bem treatment in above in vivo study. In cell viability assay, reconstitution of AXL KO cells with WT AXL increased the IC50 to GC while reconstitution with AXL-Y866F did not. Conclusions: AXL inhibition sensitizes CCA cells to cytotoxic chemotherapy in preclinical model. LCK-AXL Y866-PEAK1 signaling axis is a potential target for the treatment of CCA.

Objective: This study aims at establishing benchmark values for best achievable outcomes following open major anatomic hepatectomy for liver tumors of all dignities. Background: Outcomes after open major hepatectomies vary widely lacking reference values for comparisons among centers, indications, types of resections, and minimally invasive procedures. Methods: A standard benchmark methodology was used covering consecutive patients, who underwent open major anatomic hepatectomy from 44 high-volume liver centers from 5 continents over a 5-year period (2016–2020). Benchmark cases were low-risk non-cirrhotic patients without significant comorbidities treated in high-volume centers (≥30 major liver resections/year). Benchmark values were set at the 75th percentile of median values of all centers. Minimum follow-up period was 1 year in each patient. Results: Of 8044 patients, 2908 (36%) qualified as benchmark (low-risk) cases. Benchmark cutoffs for all indications include R0 resection ≥78%; liver failure (grade B/C) ≤10%; bile leak (grade B/C) ≤18%; complications ≥grade 3 and CCI ® ≤46% and ≤9 at 3 months, respectively. Benchmark values differed significantly between malignant and benign conditions so that reference values must be adjusted accordingly. Extended right hepatectomy (H1, 4-8 or H4-8) disclosed a higher cutoff for liver failure, while extended left (H1-5,8 or H2-5,8) were associated with higher cutoffs for bile leaks, but had superior oncologic outcomes, when compared to formal left hepatectomy (H1-4 or H2-4). The minimal follow-up for a conclusive outcome evaluation following open anatomic major resection must be 3 months. Conclusion: These new benchmark cutoffs for open major hepatectomy provide a powerful tool to convincingly evaluate other approaches including parenchymal-sparing procedures, laparoscopic/robotic approaches, and alternative treatments, such as ablation therapy, irradiation, or novel chemotherapy regimens.