Search

Immune checkpoint inhibition using monoclonal antibodies targeting cytotoxic T-lymphocyte antigen-4 (CTLA-4) and programmed cell death receptor-1 (PD-1) to enhance cytotoxic T-lymphocyte response against tumours has been used in the management of several malignancies including metastatic melanoma. Although preliminary studies in patients with untreated metastatic melanoma demonstrated significantly increased progression-free survival, there is an unmet need for clinical decision-making tools that can be used for monitoring the efficacy of these costly agents, their potential side effects and the identification of patients with the most suitable immunological profile for this treatment approach. The development of non-invasive imaging methods and imaging biomarkers of response to immune checkpoint inhibitors could therefore provide important diagnostic and prognostic information. This project builds on existing technologies in Cambridge to develop structural and functional imaging approaches for monitoring tumour response to immune checkpoint inhibitors. Magnetic resonance imaging (MRI), radiolabelling of neutrophils for single-photon emission computed tomography (SPECT) imaging and intravital imaging are explored as part of a co-clinical study on metastatic melanoma patients and syngeneic mouse models of cancer immunotherapy.

CD4+ effector (Teff) and regulatory (Treg) T cells play different roles in the immune system. Teffs promote innate and adaptive immune responses, while Tregs suppress immune responses, maintain immunological tolerance and prevent autoimmune diseases. Naïve CD4+ T cell can be induced in vitro to become Teffs or inducible Tregs (iTregs). This work focused on comparing the spatiotemporal organisation of T cell signalling intermediates upon activation of Teffs and iTregs, using the Tg4 murine model of experimental autoimmune encephalomyelitis. Live cell imaging data of Tg4 Teffs were previously acquired. Compared to Teffs, iTregs were found to show impaired spatiotemporal patterning of LAT, TCR-ζ and F-actin, suggesting their activation to be defective. T cell-APC couples formed by iTregs were found to be less stable. iTregs show a reduced ability to secrete IFN-γ and tend to secrete more IL-10 than Teffs. To investigate whether iTreg function can be controlled by proximal T cell signalling, CTLA-4 and PD-1 coinhibitory pathways, the spatiotemporal distribution of LAT during iTreg activation were manipulated. Results show that blocking CTLA-4 and PD-1 significantly inhibited iTreg generation. Dual blockade of CTLA-4 and PD-1 was found to improve cell coupling and partially enhance LAT accumulation at the T cell-APC interface. Previous work has shown that fusing LAT with the PKC-θ V3 or Vav1 SH3SH2SH3 domain enhances LAT central accumulation at the interface under attenuated T cell stimulation. Here we show that in iTreg activation, fusion with PKC-θ V3 excessively enhanced LAT overall accumulation at the interface, but slightly reduced their IFN-γ and IL-10 secretion, while fusion with Vav1 SH3SH2SH3 fully restored LAT overall accumulation at the interface, and slightly enhanced IL-10 secretion. Both conditions improved cell coupling. These data indicate that blocking coinhibitory pathways and enhancing LAT spatiotemporal organisation improve iTreg activation, but regulate their immunosuppressive function to a small extent.

The Unfolded Protein Response In T Cell Thymic Development And Peripheral DifferentiationAbstractbyBRENDAN M. BARTONEffector T cell differentiation involves coordination of transcription factor networks reinforced by signals integrated from their microenvironment. Herein, I demonstrate that the tripartite unfolded protein response (UPR) integrated signals in vitro downstream of T cell receptor signaling and co-stimulation and in vivo in response to viral infection. While mice with T cell specific deficiency of individual UPR enzymes mounted normal anti-viral responses, mice containing T cells devoid of all three UPR enzymes markedly failed to generate anti-viral T cell responses and exhibited early exhaustion. Transcriptome and epigenetic analysis revealed UPR-deficient CD4+ and CD8+ antiviral T cells failed to upregulate the Ets1-Id2 transcription factor network to promote chromatin accessibility at multiple effector gene loci. Re-expression of the transcription factor XBP1s downstream of UPR sensor IRE1α was sufficient to rescue these defects. Thus, I demonstrate the underlying compensation of UPR enzymes and an interdependent role of UPR sensors in T cell effector differentiation.

The administration of soluble antigenic peptides is known to be effective at inducing tolerance in naïve antigen-reactive CD4+ T cells. This observation forms the basis of antigen-based therapy, which offers the potential to specifically target the auto-reactive CD4+ T cells involved in driving autoimmune disease pathogenesis, whilst leaving the rest of the immune system intact. The prophylactic administration of soluble autoantigen-derived peptides has proven to be effective at inhibiting disease induction in various experimental models of autoimmune disease. However, the clinical requirement is to switch off the activated antigen-experienced CD4+ T cells that are present during an ongoing immune response. The effect of soluble peptide administration of antigenexperienced CD4+ T cells is poorly understood, and several clinical trials using peptides in multiple sclerosis patients had to be halted due to the exacerbation of disease. This thesis characterises the effect of soluble peptide administration on pathogenic antigen-experienced CD4+ T cells, using experimental autoimmune encephalomyelitis (EAE) as a model of autoimmune disease of the central nervous system. Using traceable myelin-reactive T cells from Tg4 mice, it was determined that soluble peptide administration induces substantial expansion of antigen-experienced CD4+ T cells. Despite the increase in number, these cells were no longer able to induce EAE. Production of effector cytokine was significantly decreased in peptide treated antigen-reactive CD4+ T cells, and this correlated with high level expression of the co-inhibitory molecule PD-1. The induction of tolerance in both naïve and antigen-experienced CD4+ T cells was found to be dependent upon PD-1 expression, whereby peptide treatment of naïve and antigen-experienced CD4+ T cells that were deficient in PD-1, did not inhibit disease induction. This thesis identifies a novel mechanism of peptide-induced tolerance in CD4+ T cells, and demonstrates that soluble peptide administration can induce tolerance in antigen-experienced T cells.

Melanoma is a highly malignant neoplasia with high rates of metastasis in humans and dogs. Regardless of being considered a highly immunogenic neoplasm, the function of the immune system is hampered by the expression of immune checkpoint molecules by the cancer cells. In contrast, soft tissue sarcomas are poorly immunogenic, as Tumor infiltrating Lymphocytes are lacking, or when present they are usually at the periphery of the tumor. Still, soft tissue sarcomas are considered immunosuppressed. Checkpoint molecules from the PD-axis are overexpressed in numerous human malignant neoplasia and have recently gained attention with a few reports in canine tumors. Immunotherapies against these checkpoint molecules have shown great efficacy in humans, but in order to determine translational approaches into canine patients, more research is needed. Here we determined the gene expression of Programed Death receptor-1, and its ligands PD-L1 and PD-L2 in canine tumors with two distinct immune profiles. Our results show that regardless of their immune profiles, melanoma versus soft tissue sarcoma, checkpoint molecules expression was higher in malignant tumors with a higher grade. Additionally, we evaluated the expression of these molecules in a set of patients that received histotripsy, which is a non-invasive and non-thermal ultrasound focused therapy that induces mechanical stress to the cells, leading to liquefactive necrosis. Here we reported a focal decrease of the expression of these checkpoint molecules in tissue sections obtain at the treatment interface, compared to those taken from untreated areas of the tumor. In addition, a positive relationship was noticed between the infiltration of CD3+ T lymphocytes and the expression of these checkpoint molecules in both canine melanoma, and soft tissue sarcoma. Our findings demonstrate that immunotherapies targeting these checkpoint molecules have a great potential for efficacy in canine neoplasia, along or combined with tumor ablation therapies that increased immune cell infiltration in poorly immunogenic neoplasia.

Immune-related adverse events (irAEs) are a potentially life-threatening complication of PD-1 cancer immunotherapy in patients. A deeper understanding of irAEs is essential to inform strategies to treat and monitor irAEs. This knowledge may also uncover information about the pathogenesis of spontaneously arising autoimmune diseases. In this thesis, I investigated how anti-PD1 affects autoimmunity by undertaking two independent studies. First, I studied how the cellular states of diabetogenic T cells in anti-PD-1-induced T1D differ from those in spontaneous T1D in non-obese diabetic (NOD) mice. Using a combination of scRNA-seq, TCR-seq, and flow cytometry studies, I identified significant differences in the phenotype of diabetogenic T cells mediating anti-PD-1-induced T1D compared to spontaneous T1D. Anti-PD-1-induced T1D showed a significant increase in proliferative and terminally exhausted/effector-like CD8+ T cells, while spontaneous T1D is largely dominated by memory T cell subsets. Moreover, I determined that anti-PD-1 increases TCR sharing between the pancreas and periphery, which suggests that analysis of blood T cells may be an effective strategy to monitor irAEs rather than relying on the autoimmune target organ. Secondly, I examined the effect of interactions of PD-1 and TIGIT on T cells in autoimmunity. Given the critical roles of conventional CD4+ T cells (Tcon) and Treg in promoting and suppressing EAE, respectively, I analyzed the transcriptional profiles of CD4+ T cells in the spleen and CNS of mice with EAE treated with anti-PD-1 at peak of disease and used the TCR as a molecular barcode to identify individual clonotypes. I found that anti-PD-1 exacerbated disease by promoting clonotype expansion of Th1 in the central nervous system (CNS) and reduced the proportion of Treg in the CNS that have shared TCRs in the spleen. These findings suggest that anti-PD-1 reduces Treg in the CNS which allows for clonotype expansion of encephalitogenic Tcon. Thus, using a combination of in vivo mouse models, transcriptomic, and phenotypic analyses, I have revealed the blood as a potential site for monitoring irAEs, identified differences in diabetogenic T cells in anti-PD-1-induced and spontaneous T1D; and determined differences in Treg and Tcon clonotypes in EAE in mice treated with anti-PD-1 versus controls.

Immunotherapy has emerged as one of the four “standard” cancer therapies, alongside surgery, chemotherapy, and radiotherapy. Immune checkpoint inhibitor (ICI) therapy is an immunotherapy that blocks inhibitory immune checkpoint interactions, allowing T cells and other immune cells to kill tumor cells. In the tumor microenvironment, there is often overexpression of immune checkpoint proteins, whose binding interaction with cytotoxic T cells and other immune cells results in the dampening of the antitumor response. Programmed cell death protein 1 (PD-1) and T-lymphocyte-associated protein 4 (CTLA-4) are the two most targeted immune checkpoint proteins. Antibodies against PD-1 and CTLA-4, as well as other checkpoint proteins, are approved for clinical use as well as in clinical trials. While ICIs have changed the treatment landscape for many cancers, particularly those with significant immunogenicity, only 20-40% of patients respond to ICI therapy. Many factors are behind the lack of response and resistance, and significant efforts are aimed at improving the response to ICI therapy. One major area is modulating the gut microbiome, as it is well-established that microbial dysbiosis is associated with various human diseases. The concept is that by modulating the microbiome, we might be able to return it to a composition more similar to that seen in healthy individuals or provide microorganisms beneficial to clinical response. In the case of ICI therapy, it is proposed that there is a connection between certain microbial species and the immune system via metabolites and other signaling effects. The microbiome can be manipulated through many methods, including fecal microbiota transplantation (FMT), transferring bacterial isolates or consortia, probiotics, antibiotics, and soluble dietary fiber. For clinical insights, it is important to consider how the pre-treatment microbiome of patients may affect their response to ICI therapy, as well as how their microbiomes can be manipulated to enhance their response. Initial clinical trials have been promising, but this is an emerging field with additional work to be done. Particularly, a better understanding of the microorganisms involved in the response to ICI therapy and the mechanism by which they communicate with the immune system is essential. Future studies will need to be much larger to reduce noise between studies and to allow for emerging computational techniques to be applied.

Immune checkpoint-inhibitor combinations are the best therapeutic option for advanced hepatocellular car-cinoma (HCC) patients, but improvements in efficacy are needed to improve response rates. We develop a multifocal HCC model to test immunotherapies by introducing c-myc using hydrodynamic gene transfer along with CRISPR-Cas9-mediated disruption of p53 in mouse hepatocytes. Additionally, induced co -expression of luciferase, EGFP, and the melanosomal antigen gp100 facilitates studies on the underlying immunological mechanisms. We show that treatment of the mice with a combination of anti-CTLA-4 + anti-PD1 mAbs results in partial clearance of the tumor with an improvement in survival. However, the addi-tion of either recombinant IL-2 or an anti-CD137 mAb markedly improves both outcomes in these mice. Combining tumor-specific adoptive T cell therapy to the aCTLA-4/aPD1/rIL2 or aCTLA-4/aPD1/aCD137 reg-imens enhances efficacy in a synergistic manner. As shown by multiplex tissue immunofluorescence and intravital microscopy, combined immunotherapy treatments enhance T cell infiltration and the intratumoral performance of T lymphocytes.

Aortic aneurysms, a permanent enlargement in the aorta, is a devastating disease with a high mortality rate after rupture. Aortic aneurysms exhibit strong sex dimorphisms: men are at higher risk than women while women experience worse outcomes if diagnosed. Currently, the only intervention is by open surgery or endovascular repair, and no drugs are approved for blunting the disease progression. This highlights the needs for mechanistic studies for sex-specific therapeutic targets. Our lab established the mouse model in which mineralocorticoid receptor (MR) agonist aldosterone induces aortic aneurysms in the presence of high salt in male mice age-dependently, mimicking the aging pattern in clinical observation. However, whether the Aldo-salt model exhibits sex dimorphisms like humans is unknown. The current study first demonstrated a strong sex-dependent phenotype in Aldo-salt-induced aortic aneurysms in 10-month-old male and female mice. Next, we performed gonadectomy on male and female mice to study the effects of male and female sex hormones on Aldo-salt-induced aortic aneurysms respectively, and we identified two distinct hormone-specific mechanisms. In male mice, we showed that orchiectomy provided strong protection against aneurysm development mediated by the androgen-androgen receptor axis. Next, by unbiased RNA-seq, we revealed that androgen deprivation is associated with upregulated T cell receptor signaling and PD-1 signaling pathway. Moreover, we indicated that increased PD-1+ T cells in the aorta were associated with splenectomy-attenuated aortic aneurysms in response to Aldo-salt. To gain mechanistic insights into the regulation between androgen and PD-1, we performed a series of experiments and demonstrated that PD-1 was upregulated in the spleen of orchiectomized mice than that of sham mice at both protein and mRNA levels. Moreover, we showed that AR can bind to the PD-1 promoter and exert suppression on PD-1 transcription. The PD-1 changes in the spleen were also mirrored in the blood and partially in the aorta, suggesting possible effects on vascular inflammation via circulating T cells. Most importantly, by injecting an anti-PD-1 antibody and adoptive transfer of PD-1-/- T cells, we successfully reinstated the aortic aneurysms formation in orchiectomized mice. Furthermore, using Ang II and HFD in intact male mice, we showed that PD-1-/- exacerbated the aortic aneurysms compared to wildtype mice. Collectively, we demonstrated the important role of PD-1 in the pathogenesis of aortic aneurysms, and its transcription was suppressed by the androgen receptor. In female mice, we found that ovariectomy abolished the female protection against Aldo-salt-induced aortic aneurysms in 11-12-month-old mice. Such effects were not observed in 3-month-old mice, suggesting the age-dependent fashion as in humans. Next, we used multiple approaches to study the underlying mechanisms involved both aging and estrogen, and we found that MR showed a strong aging pattern, and it was upregulated, activated, and translocated into the nucleus upon aldosterone only with the absence of estrogen. Interestingly, such changes were more sensitive in vascular endothelial cells. Moreover, specific deletion of MR from endothelium mitigated the Aldo-salt-induced aortic aneurysms in aged ovariectomized mice, possibly via the downstream cellular senescence. In summary, our findings demonstrate the sex dimorphisms in Aldo-salt-induced aortic aneurysms. The promoting role of androgen and the protective role of estrogen are by two distinct mechanisms: 1) warranting the caution for cancer patients on immune checkpoint inhibitors of the potential risk of developing aortic aneurysms; and 2) giving rise to MR as a potential therapeutic target specifically for aged menopausal women.

Programmed Death Ligand 1 (PD-L1) overexpressed on cancer cells can help tumors evade the immune system by triggering the coinhibitory receptor Programmed Death 1 (PD-1) on T-cells, subsequently inhibiting T-cell activity. PD-L1 binding to PD-1 is reciprocally regulated by two proteins, CD80 and CKLF-like MARVEL-containing Transmembrane 6 (CMTM6). CD80 is a key immunostimulatory ligand that binds PD-L1 in cis on the same membrane, and blocks PD-L1:PD-1 inhibitory signaling. Conversely, CMTM6 is a multi-pass transmembrane protein that prevents the degradation of PD-L1, and thereby promotes PD-L1:PD-1 inhibitory signaling. However, the biochemical nature of CMTM6:PD-L1 interaction, and how it may be influenced by CD80, is poorly defined. Here, I established an intact cell-based, quantitative, and functional assay of CMTM6 to determine the molecular requirements for PD-L1 and CMTM6 interaction, and its potential regulation by CD80. Using domain-swapping and cellular reconstitution, I found that PD-L1 chimeras with the transmembrane (TM), extracellular (EC), or intracellular (IC) domains of PD-L1 swapped with that of the CMTM6 non-client PD-L2, were all upregulated by CMTM6, but to a lesser extent than wild-type PD-L1. The TM replacement showed the greatest impairment in CMTM6-mediated upregulation, suggesting that CMTM6 primarily interacts with the TM of PD-L1. Finally, co-expression of CD80 did not affect the magnitude of the CMTM6 upregulation of PD-L1, suggesting that cis-CD80:PD-L1 interaction does not perturb CMTM6:PD-L1 interaction. These data provide a better understanding of the trimolecular relationship between PD-L1, CMTM6, and CD80.

Background The Spanish Melanoma Group (GEM) developed a national registry of patients with melanoma infected by SARS-CoV-2 ( GRAVID ).Methods The main objective was to describe the COVID-19 fatality rate in patients with melanoma throughout the pandemic, as well as to explore the effect of melanoma treatment and tumor stage on the risk of COVID-19 complications. These are the final data of the register, including cases from February 2020 to September 2021.Results One hundred-fifty cases were registered. Median age was 68 years (range 6-95), 61 (40%) patients were females, and 63 (42%) patients had stage IV. Thirty-nine (26%) were on treatment with immunotherapy, and 17 (11%) with BRAF-MEK inhibitors. COVID-19 was resolved in 119 cases, including 85 (57%) patients cured, 15 (10%) that died due to melanoma, and 20 (13%) that died due to COVID-19. Only age over 60 years, cardiovascular disorders, and diabetes mellitus increased the risk of death due to COVID-19, but not advanced melanoma stage nor melanoma systemic therapies. Three waves have been covered by the register: February-May 2020, August-November 2020, and December 2020-April 2021. The first wave had the highest number of registered cases and COVID-19 mortality.Conclusion Tumor stage or melanoma treatments are non-significant prognostic factors for COVID-19 mortality. During the pandemic in Spain there was a downward trend in the number of patients registered across the waves, as well as in the severity of the infection.

Coinhibitory immunoreceptor programmed cell death-1 (PD-1, CD279) plays a critical role in mediating T cell exhaustion and blockade of this pathway can promote antitumor immunity. While PD-1 pathway inhibitors are revolutionizing cancer therapy, only a subset of patients respond and show durable remission, highlighting the need to better understand the basic mechanisms by which the PD-1 pathway inhibits T cell functions. Studies assessing PD-1 signaling mechanisms have the potential to identify novel targets for therapeutic intervention in cancer to improve patient responses. To identify novel mechanisms of PD-1 function, we utilized proximity labeling as an unbiased approach to survey proteins proximal to PD-1 following ligation and identified the mechanosensitive cation channel PIEZO1 as a primary target of PD-1-mediated inhibition. Stimulation of CD8+ T cells through the T cell receptor (TCR) and costimulatory receptor CD28 triggered the activation of PIEZO1 at the immune synapse, while simultaneous ligation of PD-1 countered this activation. We found that mice lacking PIEZO1 selectively on CD8+ T cells exhibited significantly increased tumor growth marked by impaired CD8+ T cell function and this could not be rescued by PD-1 blockade. Conversely, CD8+ tumor infiltrating lymphocytes (TILs) from wild-type (WT) mice treated with PIEZO1 agonist showed greater functionality compared to controls. Coadministration of PIEZO1 agonist and PD-1 blockade significantly reduced tumor burden and improved survival in a PD-1 blockade-unresponsive tumor model. These findings identify PIEZO1 inhibition as an important mechanism by which PD-1 signaling regulates CD8+ T cell functions and suggest that modulating PIEZO1 activity on CD8+ T cells may be a novel approach to augment cancer immunotherapy. To determine how PD-1 tyrosine-phosphorylation motifs differentially impact downstream signaling, we relied on PD-1 mutant mouse models and proteomic methodology to uncouple the functions of PD-1 immune tyrosine-based inhibition motif (ITIM) and immune tyrosine-based switch motif (ITSM), as well as other potential signaling motifs. We found that ITSM-mutant mice controlled tumor growth similar to PD-1 knockout (KO) mice, but did not full recapitulate PD-1 KO phenotypes. Furthermore, ITIM-mutant mice showed no significant improvement in tumor control, similar to WT mice. To further characterize these differences, we performed proximity phospho-proteomic analysis and PD-1 phospho-peptide pulldowns to assess PD-1 binding interactions and phosphorylation alterations. We confirmed that the ITSM recruits inhibitory phosphatases and kinases to dampen TCR-mediated activation and discovered that the ITIM associates with proteins involved in ubiquitin-mediated degradation, suggesting that the ITIM regulates the degradation of TCR effector proteins. Novel phospho-serine sites identified on the cytoplasmic tail of PD-1 also exhibited unique protein binding behavior, implying that PD-1 retains residual signaling function beyond the ITIM and ITSM. While additional experimentation is necessary to confirm these findings, our results significantly advance our mechanistic understanding of PD-1-mediated signaling. Together, our studies have discovered a novel regulatory mechanism of PD-1-mediated inhibition and have substantially built upon our understanding of the function of PD-1 signaling motifs. Specifically, our findings demonstrate that PD-1 inhibition of PIEZO1 is therapeutically relevant in cancer and that novel PD-1 phosphorylation sites and binding partners may participate in uncharacterized functions of PD-1.

T-cells are an essential component to the immune system, but they do not act alone and are instead a component in the body’s immune system. PD-1 and its ligands PD-L1 and PD-L2 play an important role in the regulation of T-cells which are incredibly important to the treatment of cancer. Tumors have been able to hijack the PD-1 inhibitory pathway to evade our body’s immune response. PD-1 pathway blockade, therefore, can serve as an important approach for cancer immunotherapy. However, we do not fully understand the mechanism by which PD-1 regulates anti-tumor immunity. With the datasets derived from experiments by the Sharpe Lab, we hope to answer two important questions. First, what does PD-1 regulate in a cell intrinsic manner compared to bystander effects on other cells in the tumor micro-environment? Second, which gene expression changes predict response as opposed to resistance to tumor clearance when PD-1 is present or deleted? We hypothesize that a cell intrinsic loss of PD-1 is necessary for improved T-cell fitness and effector functions. This project aims to help answer these questions in three ways. First, the development of an RNA-sequencing pipeline allows the researchers in the lab to analyze the results of datasets that are generated. Second, conducting pathway analyses provides a broader picture of the gene expression landscape, including provide a more complete picture of the tumor micro-environment by indicating which pathways and cellular processes are enriched for the genes affected by the deletion of PD-1. Third, the development of a statistical model which makes predictions on which gene expression changes predict response as opposed to resistance. The results of the statistical model indicate which genes are more closely related to PD-1 when it is deleted versus when it is present. This will help us better understand immune response as opposed to resistance to PD-1 cancer immunotherapy, and its effects on tumor growth. This model therefore provides a valuable tool to the community that would allow researchers to probe the gene expression landscape around PD-1.

T cells constitute one of the main agents in the adaptive immune system. When in contact with an antigen presenting cell (APC), the T cell receptor (TCR), located in the plasma membrane of T cells, recognises and binds cognate antigen presented in major histocompatibility complexes (pMHC) located in the APC. This interaction triggers a signalling cascade via the TCR associated CD3 complex, that induces T cell activation and proliferation. TCR mediated activation is modified by a number of co-stimulatory and co-inhibitory receptors including Cluster of Differentiation 28 (CD28) which enhances T cell activation and Programmed death- 1 (PD-1), which inhibits T-cell activation. When PD-1 is bound to its ligands PD-L1 or PD-L2, it transduces an inhibitory signal marked by SHP2 phosphatase recruitment, that blocks the signalling cascade of TCR or CD28. Under normal conditions PD-1 plays a role in maintaining peripheral tolerance, however some viruses and cancer can manipulate this system to evade immunological surveillance. We hypothesise that PD-1 and TCR or CD28 need to colocalise on a scale that allows SHP2 bound to the cytoplasmic tail of PD-1 to reach and dephosphorylate phosphotyrosines on the target receptor (tens of nm). In order to study this, we used super-resolution microscopy techniques, specifically direct stochastic optical reconstruction microscopy (dSTORM) and point accumulation for imaging nanoscale topography (PAINT). We also assessed the segregation of membrane-recruited SHP-2 with ZAP70 and PI3K to investigate the mechanisms of PD-1 inhibition. Our data suggest that the TCR and PD1 do not significantly colocalize on the nanoscale when both receptors are engaged. However, SHP2 and ZAP70 do seem to be segregated, indicating that SHP2 does dephosphorylate the tails of the TCRs that are found in close proximity. In addition, PD-1 and CD28 colocalize on the nanoscale upon ligand engagement. Nonetheless, SHP2 and PI3K appear to be colocalized as well instead of segregated, denoting lack of interaction between the enzymes. This research sheds significant light on the regulatory mechanism behind adaptive immune system control, and it may provide the tools to overcome T-cell exhaustion and dysfunction in chronic diseases, such as cancer.

Lung cancer is the leading cause of cancer related mortality in the United States, accounting for more than 142,000 estimated deaths in 2019. The major subtype of lung cancer is non-small cell lung cancer (NSCLC), which represents 85% of all cases. Despite the advancement in understanding the molecular basis of NSCLC, the 5-year survival rate is less than 20%. The current treatment strategies for advanced stage patients rely on molecularly-targeted therapies, cytotoxic chemotherapy and immunotherapeutic agents. Because of the complexity and heterogeneity of lung tumors, intrinsic and acquired resistance mechanisms ultimately result in failure to respond to these therapies and early relapse. Combinatorial strategy that involves targeting multiple aspects of tumorigenesis may represent a new avenue for therapeutic intervention in lung cancer management. Estrogen signaling has been frequently shown to be an important mediator of lung cancer progression and metastasis. In a non-genomic fashion, ER mutually interacts with human epidermal growth factor receptors (HERs) to promote lung cancer proliferation and growth. Targeting ER signaling with the antiestrogen fulvestrant has shown moderate activities in preclinical models of lung cancer. The reciprocal interaction between ER and EGFR could limit the activity of using anti-ER agent alone. In a phase II clinical trial, combining fulvestrant with the selective EGFR tyrosine kinase inhibitor erlotinib showed an enhanced activity over erlotinib alone and improved the survival outcomes in NSCLC patients. However, the improvement in overall median survival was modest. Recent retrospective clinical analysis demonstrated that genes contained in the prediction analysis of microarray 50 (PAM50) provide prognostic information in high ERβ+ lung cancer patients. The 7-gene model includes c-Myc, MIA, CXXC5, FGFR4, Grb-7, FOXC1, and PgR. In high-risk patients, who tend to have a poor prognosis and short median survival, c-Myc, MIA, CXXC5, FGFR4, Grb-7, FOXC1 are overexpressed and PgR is downregulated. Importantly, the 7-gene model described one interacting network that includes ER and HER2/HER3 as the top upstream regulators for the 7-gene panel. In fact, a significant association between ERβ and HER2 expression was found, in which 70.2 % of ERβ-positive cases were positive for HER2 compared to 34.5% of ERβ-negative cases. HER3, when analyzed with HER2, showed also positive association with ERβ. These observations suggest that ERβ and HER2/HER3 pathways define lung tumors with very aggressive biology, indicating that blocking both pathways could be more efficacious than either one of them alone. These observations also could explain why the magnitude of response was modest when fulvestrant was combined with erlotinib, as erlotinib has weak activities against HER2 and HER3. Combining a pan-HER inhibitor such as dacomitinib (inhibits EGFR, HER2 and HER4) with fulvestrant could be more efficacious than either agent alone and could produce a gene signature that predicts better clinical outcomes in NSCLC. Immune checkpoint inhibitors have changed the treatment paradigm for several solid tumors, including lung cancer. Antibodies that target programmed death receptor 1(PD1) or its ligand (PD-L1) have proven great efficacy for certain patients with lung cancer. Patients who respond to these agents tend to have durable effects and longer survival outcomes; however, only 20-29% of patients are predicted to respond. Several combination strategies are being evaluated clinically to maximize the efficacy of these agents and improve the response rate. Preclinical evidence found that the ER blocker fulvestrant effectively sensitizes lung cancer cells to T cell and natural killer (NK) cell mediated cytotoxicity effects. In addition, estrogen signaling is a critical mediator for myeloid derived suppressor cells (MDSCs), which are largely known for their immunosuppressive effects. Evidence also demonstrated that EGFR inhibitors possess dual immunomodulatory effects that include upregulation of major histocompatibility complex I and II (MHC I and II), inducing the recruitment of immune cells, and inhibiting other tyrosine kinases essential for immune cells function. Synergy was observed when EGFR TKI and PD1 inhibitor was combined in an- EGFR-mutant model. Altogether, these previous observations encouragingly support the hypothesis that use of triple therapy (fulvestrant, pan-HER TKI and an immune checkpoint blocker) will be a promising treatment approach for lung cancer. Testing this hypothesis is the subject of this dissertation. To evaluate the therapeutic potential of combining ER blocker with pan-HER in NSCLC, we chose fulvestrant, as an antiestrogen, and dacomitinib, as a tyrosine kinase inhibitor that targets EGFR, HER2, and HER4. We first evaluated the efficacy of this combination in three different human NSCLC cell lines and assessed the ability of this treatment approach to produce a gene signature that predicts better clinical outcomes. We utilized three different cell lines that represent three different categories of NSCLC population; EGFR-mutant, KRAS-mutant and EGFR and KRAS wild-type. Next, we investigated the immunomodulatory effects of this combination treatment on macrophages and CD8+ T cells in vitro. We used a novel syngeneic lung cancer model (FVBW-17/FVB-N) to evaluate the therapeutic effectiveness of fulvestrant-dacomitinib in combination with a mouse anti-PD1. Major results The combination of fulvestrant and dacomitinib significantly suppressed NSCLC cell growth in vitro and produced a combination index < 0.5, indicating strong synergy. The combination also showed potent downregulation of HER activity and marked decrease in amphiregulin (AREG) and neuregulin (NRG1-β1) expression. Importantly, the combination, but not single agents, completely reversed the gene signature associated with poor prognosis. C-Myc, MIA, CXXC5, FGFR4, FOXC1 and Grb7 were downregulated and PgR was upregulated following the combination treatment. The combination significantly reduced c-Fos, JunB and pCREB DNA-binding activities. The c-Fos/Ap-1 inhibitor t-5224, but not CBP-CREB inhibitor, was able to partially mimic the effects of the combination in reversing the gene signature. In vivo, the combination treatment demonstrated a robust synergistic antitumor effect in NSCLC cell lines that were engrafted subcutaneously in immunodeficient mice. Tumor regression was observed in the majority of tumors following the combination treatment. A drastic decrease in HER activity and downstream signaling were observed with the combination, along with a significant decrease in AREG and NRG1-β1 expression. In situ proximity ligation assay revealed a significant decrease in the active dimerization of both p-HER2/p-HER3 and p-HER2/p-EGFR dimers following the combination treatment. Additionally, the gene signature was also completely reversed by the combination but not with single agents. In the EGFR mutant model, the survival of mice was improved after treatment discontinuation, tumors that recurred were less aggressive, and two mechanisms of resistance commonly associated with HER TKIs were blocked. To evaluate the immunomodulatory effects of the combination, bone marrow-derived macrophages and CD8+ T cells were treated with the combination. Surprisingly, macrophages lost their phagocytic function and behave more like M2-macrophages by expressing high IL-10, CD206 and PD1 following the combination treatment. Mechanistically, dacomitinib induced downregulation of phospho-Syk, and fulvestrant was not able to overcome this effect. In CD8+ T cells, the combination impaired the function of T cells, induced high PD1 expression, and severally reduced IFN-ϒ and TNF-α production. These debilitating effects were mostly attributed to the downregulation of Src Family kinases activities, as less phospho-SFK Y416 was detected following the combination treatment. In vivo, adding anti-PD1 antibody to the combination treatment enhanced the immune function and improved the antitumor effects. In a sequential approach, where anti-PD1 was administrated after the combination treatment, the average tumor volume was 4-fold less than placebo and this effect was synergistic. In comparison, the triple therapy given concomitantly showed a 2-fold decrease compared to placebo. Sequential triple therapy was also significantly better than concomitant triple therapy. None of the drugs alone show any sign of activities. Interestingly, after one week of administrating fulvestrant and dacomitinib, tumors showed high immune cell infiltration (inflamed tumor microenvironment), with relatively high PD1 expression on CD8+ T cells. In contrast, the concomitant triple therapy showed a significant increase in CD8+ T cells infiltration but with a decrease in PD1 expression, and fewer M2 macrophages. Conclusion and Significance Retrospective analysis of NSCLC patients revealed that the interaction between HER2/HER3 and ERβ contributes to poor outcomes in NSCLC patients with high ERβ expression. Here, we report that targeting ERβ with the antiestrogen fulvestrant, along with targeting multiple HER pathways with the pan-HER TKI dacomitinib produced synergistic antitumor effects in preclinical models of human ERβ+ NSCLC. The robust antitumor effect seen was accompanied by the ability of this combination treatment to produce a gene signature that predicts better clinical outcomes. The 7-gene model could serve as a predictive tool for identifying patients who will more likely respond to the treatment. These data strongly support its clinical use, giving the fact that both drugs are clinically used for cancer patients. Convincing evidence also suggest an immunomodulatory effect of ER signaling and HER pathways. The combination of fulvestrant and dacomitinib suppressed phagocytic macrophages and CD8+ T cell functions and upregulated PD1 expression. These effects were largely contributed by dacomitinib for its inhibitory effects on Syk and Src kinases on immune cells and might limit the clinical utility of the fulvestrant/dacomitinib combination. However, the ability of the combination to turn the TME into an inflamed microenvironment with upregulated PD1 could potentiate the tumor-mediated killing effects by immunotherapy. The synergistic antitumor effect observed with adding anti-PD1 in a sequential manner after the administration of fulvestrant and dacomitinib is highly encouraging. The significance of these observations is that the triple therapy has shown excellent antitumor effects in a highly aggressive tumor model that is unresponsive to immune checkpoint inhibitor alone or fulvestrant-dacomitinib combination. The clinical usefulness of this approach can have therapeutic applications for other solid tumor models, particularly in tumors that are less inflamed and are unlikely to respond to immune checkpoint inhibitors.

Immunotherapy has changed the paradigm of cancer treatments. In this way, several combinatorial strategies based on monoclonal antibodies (mAb) such as anti (a)-PD-1 or anti (a)-PD-L1 are often reported to yield promising clinical benefits. However, the pharmacokinetic (PK) behavior of these mAbs is a critical issue that requires selective analytical techniques. Indeed, few publications report data on a-PD1/a-PD-L1 exposure and its relationship with therapeutic or toxic effects. In this regard, preclinical assays allow the time profiles of antibody plasma concentrations to be characterized rapidly and easily, which may help to increase PK knowledge. In this study, we have developed and validated two in-house ELISAs to quantify a-PD-1 and a-PD-L1 in plasma collected from tumor-bearing mice. The linear range for the a-PD-1 assay was 2.5–125 ng/mL and 0.11–3.125 ng/mL for the a-PD-L1 assay, whereas the intra-and inter-day precision was lower than 20% for both analytes. The PK characterization revealed a significant decrease in drug exposure after administration of multiple doses. Plasma half-life for a-PD-1 was slightly shorter (22.3 h) than for a-PD-L1 (46.7 h). To our knowledge, this is the first reported preclinical ELISA for these immune checkpoint inhibitors, which is sufficiently robust to be used in different preclinical models. These methods can help to understand the PK behavior of these antibodies under different scenarios and the relationship with response, thus guiding the choice of optimal doses in clinical settings.

Immune checkpoint blockade using monoclonal antibodies (mAbs) able to block programmed death-1 (PD-1)/PD-L1 axis represents a promising treatment for cancer. However, it requires repetitive systemic administration of high mAbs doses, often leading to adverse effects. We generated a novel nanobody against PD-1 (Nb11) able to block PD-1/PD-L1 interaction for both mouse and human molecules. Nb11 was cloned into an adeno-associated virus (AAV) vector downstream of four different promoters (CMV, CAG, EF1α, and SFFV) and its expression was analyzed in cells from rodent (BHK) and human origin (Huh-7). Nb11 was expressed at high levels in vitro reaching 2–20 micrograms/mL with all promoters, except SFFV, which showed lower levels. Nb11 in vivo expression was evaluated in C57BL/6 mice after intravenous administration of AAV8 vectors. Nb11 serum levels increased steadily along time, reaching 1–3 microgram/mL two months post-treatment with the vector having the CAG promoter (AAV-CAG-Nb11), without evidence of toxicity. To test the antitumor potential of this vector, mice that received AAV-CAG-Nb11, or saline as control, were challenged with colon adenocarcinoma cells (MC38). AAV-CAG-Nb11 treatment prevented tumor formation in 30% of mice, significantly increasing survival. These data suggest that continuous expression of immunomodulatory nanobodies from long-term expression vectors could have antitumor effects with low toxicity.

Abstract: T cells are a vital part of the adaptive immune system that function to eliminate foreign threats while remaining tolerant to healthy-self. During T cell development in the thymus, T cells are generated with various T cell receptor (TCR) specificities, such that they can respond to a near endless variety of threats. However, some T cells deleteriously generate a TCR specific for self and thus, have the potential to induce autoimmune disease. To prevent this, self-reactive thymocytes undergo negative selection after receiving a high affinity signal in the thymus. T cells are negatively selected against ubiquitous antigen (UbA) in the thymic cortex or tissue restricted antigen (TRA) in the thymic medulla. Various molecular factors are differentially important for ensuring self-tolerance to either UbA or TRA; one such protein is programmed cell death protein 1 (PD-1). Given that our group has previously investigated the role of PD-1 in T cell tolerance to UbA, we used the OT-I TCR transgenic mouse model to investigate the role of PD-1 in T cell tolerance to TRA. We determined that CD8+ T cells from OT-I Bim-/- > RIP-mOVA bone marrow chimeras were intrinsically impaired but were not functionally rescued by blocking PD-1 signalling in vitro. Subsequently, we generated OT-I PD-1-/- > RIP-mOVA chimeras and found that 100% of OT-I PD-1-/- > RIP-mOVA chimeras develop autoimmunity, compared to 0% of control OT-I WT > RIP-mOVA controls. We then isolated OT-I PD-1-/- T cells from the thymus of OT-I PD-1-/- > RIP-mOVA chimeras and found that clonal deletion was intact, suggesting that PD-1 signals impact T cell tolerance outside of deletional mechanisms. In the periphery, we found a greater number of CD8+ T cells from OT-I PD-1-/- > RIP-mOVA chimeras compared to OT-I WT > RIP-mOVA controls, and these cells exhibited differences in proliferation capacity, trafficking potential and cytokine production. Collectively, this data suggests that PD-1 plays a significant role in establishing T cell tolerance to TRA via non-deletional tolerance mechanisms.

Seminal research conducted over the last two decades has confirmed that the immune system plays a crucial role in the processes that regulate tumor growth and development. So-called immune surveillance is an idea that has persisted for decades, and evidence supporting the hypothesis that the immune system detects and destroys neoplastic growth has accumulated over time. More recently the understanding of the role of immune checkpoints in cancer has led to breakthrough immunotherapeutic drugs such as ipilimumab (anti-CTLA-4 antibody), and pembrolizumab and nivolumab (anti-PD-1 antibodies). These drugs, also known as checkpoint inhibitors, act by blocking inhibitory immune receptors on T cells thereby enhancing the ability of the immune system to mount an anti-tumoral response. While immunotherapies can lead to durable, robust responses, the rate of response is quite variable across and within tumor types (Topalian, Taube, Anders, & Pardoll, 2016). There is currently an intense effort to characterize what differentiates responders from non-responders that will lead to the development of predictive criteria capable of identifying these populations prior to the initiation of treatment. For instance, the FDA has approved companion diagnostics that quantify the amount of programmed cell death ligand 1 (PD-L1) there is within a tumor for prediction of response to nivolumab and pembrolizumab (Topalian, Taube, Anders, & Pardoll, 2016). However, a recent analysis found that the response rate to anti-PD-1 antibody in PD-L1+ tumors was 48% while PD-L1- tumors still maintained a 15% response rate (Sunshine & Taube, 2015), demonstrating the difficulty that exists in predicting absolute response to these antibodies. Ongoing work at Merck Research Laboratories (MRL) in Boston is focused on developing next generation immuno-oncology therapeutics. Understanding the molecular mechanisms that regulate response and non-response is central to this work. Traditional mouse tumor models relied on implantation of cells derived from human tumors, called xenografts, that required immunodeficient hosts. These models were suitable for the study of cytotoxic drugs designed to kill tumor cells directly. However, they would not be suitable for the study of tumor-immune interaction. The preclinical research at Merck currently relies heavily on mouse syngeneic tumor models that, crucially, use mice with an intact immune system capable of mounting immune responses directed towards the tumor. Here, tumor bearing mice are generated by exogenous implantation of cultured tumor cell lines derived from primary mouse tumors obtained from either naturally occurring (spontaneous) or carcinogen-induced tumors raised in mice of the same genetic background. Upon subcutaneous inoculation implanted tumor cells grow into solid tumors that can be easily measured for response to therapeutic intervention. The most commonly used murine syngeneic model at MRL uses the MC38 colon adenocarcinoma cell line. Tumors generated from these cells demonstrate robust responsiveness to murine anti-PD-1 antibody (mDX400). Typical response rates in mice bearing 80-120mm3 tumors are between 60-80% complete regressions (with additional partial responses) following 5 milligram/kilogram (mg/kg) dosing of mDX400, administered intraperitoneally (IP) every 5 days (Q5D) for approximately 15-20 days (data not shown). As MC38 tumors grow larger than this 100mm3 average size they have markedly lower response rates. It is possible that immune cell populations contribute to this observation. Phenotyping of explanted dissociated 80-120mm3 tumors via mass cytometry time of flight (Helios, Fluidigm) revealed that as much as 50% of cells within these tumors are CD45+ immune cell infiltrate. Furthermore, the PD-1/PD-L1 axis is abundant: 60+% of T cells express PD-1, ~90% of myeloid cells express PD-L1, and among CD45- cells, which consist of tumor and stromal cells, PD-L1 expression is nearly 50% (data not shown). Moreover, in tumors of this size the CD4+ T cell compartment is typically 10-12% CD25+Foxp3+ regulatory T cells (Treg). Opportunistic phenotyping of large tumors (600mm3 and 1000mm3) revealed drastic increases in Treg cells within the tumor infiltrating lymphocytes (TIL): ~30% and ~60% of total CD4+, respectively. Several lines of research have demonstrated the importance of IL-10 in the activation and anti-tumor activity of tumor resident CD8+ cytotoxic T cells, which is type I IFN dependent (Emmerich, et al., 2012) (Mumm, et al., 2011) (Stewart, et al., 2013). Additionally, it has been shown that the majority of intratumoral IL-10 is generated by Treg, which plays an important role in restraining tumorigenic Th17 inflammation (Stewart, et al., 2013). Therefore, one possible explanation for the loss of responsiveness to anti-PD-1 treatment as tumors progress is that while Treg numbers increase they become dysfunctional and lose the ability to both suppress Th17 inflammation and activate intratumoral CD8+ T cells. Elucidating the role of the type I IFN/IL-10/Treg axis in this phenomenon could provide valuable insight into strategies for treating patients that respond poorly to checkpoint inhibitor therapy. The current study interrogates whether changes in the type I IFN/IL-10/Treg axis within the tumor microenvironment contributes to poor therapeutic response in syngeneic tumors across tumor progression through a comprehensive profiling of surface markers and cytokine production.

Reactivation of cytotoxic CD8+ T-cell responses has set a new direction for cancer immunotherapy. Neutralizing antibodies targeting immune checkpoint programmed cell death protein 1 (PD-1) or its ligand (PD-L1) have been particularly successful for tumor types with limited therapeutic options such as melanoma and lung cancer. However, reactivation of T cells is only one step toward tumor elimination, and a substantial fraction of patients fails to respond to these therapies. In this context, combination therapies targeting more than one of the steps of the cancer-immune cycle may provide significant benefits. To find the best combinations, it is of upmost importance to understand the interplay between cancer cells and all the components of the immune response. This review focuses on the elements of the complement system that come into play in the cancer-immunity cycle. The complement system, an essential part of innate immunity, has emerged as a major regulator of cancer immunity. Complement effectors such as C1q, anaphylatoxins C3a and C5a, and their receptors C3aR and C5aR1, have been associated with tolerogenic cell death and inhibition of antitumor T-cell responses through the recruitment and/or activation of immunosuppressive cell subpopulations such as myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs), or M2 tumor-associated macrophages (TAMs). Evidence is provided to support the idea that complement blocks many of the effector routes associated with the cancer-immunity cycle, providing the rationale for new therapeutic combinations aimed to enhance the antitumor efficacy of anti-PD-1/PD-L1 checkpoint inhibitors.

The TNF blockade therapy is currently a well-established treatment option for a variety of autoimmune diseases such as rheumatoid arthritis (RA), psoriasis or Crohn's disease, given the proinflammatory role of TNF in the course of these diseases. Importantly, TNF neutralization is also used for the treatment of corticosteroid-refractory immune-related adverse events (irAEs) induced by the combined anti-PD-1 and anti-CTLA-4 immunotherapy. The manifestation of these toxicities is an important limiting factor for the successful implementation of the inhibitory checkpoint blockade therapy (ICB), restraining its anti-tumor efficacy. In our recent study (Perez-Ruiz et al., Nature 569(7756): 428-432.), we analyzed the potential impact of prophylactic TNF neutralization therapy in the anti-PD1/CTLA-4 efficacy. Through several mouse models, we demonstrated that TNF neutralization ameliorated ICB-exacerbated colitis in addition to improving ICB-dependent anti-tumor efficacy. Similar results were obtained after prophylactic TNF blockade in graft vs host xenografted mouse models with human immune cells, which showed a reduction in colitis and hepatitis. Importantly, there was a preservation of the immunotherapeutic control of xenografted tumors after ICB treatment. Moreover, TNF and TNF-dependent gene expression is upregulated in the colon mucosa from patients affected by colitis as a side effect of ipilimumab and nivolumab. Our results, thus, provide evidence of the successful combination of prophylactic TNF neutralization with ICB therapy strategy to ameliorate toxicities, while keeping or even ameliorating anti-tumor efficacy. The prophylactic TNF blockade strategy is clinically feasible since excellent TNF inhibitors have been approved for the treatment of autoimmunity and are used for the immune-related serious adverse events in immunotherapy.

The treatment of cancer, especially of various types of solid tumors, has been revolutionized by the blockade of the PD-1/PD-L1 pathway by immune checkpoint inhibitors. Their success amongst hematologic malignancies, however, has been limited so far to the treatment of classic Hodgkin’s lymphoma, which portrays a typical overexpression of PD-1 ligands (PD-L1, PD-L2) as a consequence of changes in chromosome 9p24.1. Their current application in multiple myeloma (MM) is rather uncertain, as discordant results have been reported by distinct research groups concerning especially the expression of PD-1/PD-L1 molecules on malignant plasma cells or on the responsible immune effector cell populations, respectively. In MM it seems that an approach based on combination treatment might be appropriate as unsatisfactory results have been yielded by monotherapy with PD-1/PD-L1 inhibitors. Immunomodulatory drugs, which are the current cornerstone of MM treatment, are the most logical partners as they possess many possibly synergistic effects. Nevertheless, the initially optimistic results have become disappointing due to the excessive and unpredictable toxicity of the combination of pembrolizumab with lenalidomide or pomalidomide. The FDA has suspended or put on hold several phase 3 trials in relapsed as well as in newly diagnosed myeloma patients. There are also other potentially synergistic and promising combinations, such as the anti-CD38 monoclonal antibody daratumumab, irradiation, etc. Not only the effective partner but also the correct timing of the initiation of the PD-1/PD-L1 inhibitors treatment seems to be of utmost importance. These strategies are currently being examined in various stages of myeloma such as during consolidation post autologous stem cell transplantation, targeting minimal residual disease or even in high risk smoldering myeloma.

Background: Programmed death-ligand 1 (PD-L1) may be induced by oncogenic signals or can be upregulated via interferon gamma (IFN-y). We have explored whether the expression of IFNG, the gene encoding IFN-y, is associated with clinical response to the immune checkpoint blockade in non-small cell lung cancer (NSCLC) and melanoma patients. The role of inflammation-associated transcription factors STAT3, IKBKE, STAT1 and other associated genes has also been examined.

Despite numerous advances in targeted therapy and immunotherapy in the last decade, lung cancer continues to present the highest mortality rate of all cancers. Targeted therapy based on specific genomic alterations, together with PD-1 and CTLA-4 axis blocking-based immunotherapy, have significantly improved survival in advanced non-small cell lung cancer (NSCLC) and both therapies are now well-established in this clinical setting. However, it is time for immunotherapy to be applied in patients with early-stage disease, which would be an important qualitative leap in the treatment of lung cancer patients with curative intent. Preliminary data from a multitude of studies are highly promising, but therapeutic decision-making should be guided by an understanding of the molecular features of the tumour and host. In the present review, we discuss the most recently published studies and ongoing clinical trials, controversies, future challenges and the role of biomarkers in the selection of best therapeutic options.

Radiotherapy (RT) is currently used in more than 50% of cancer patients during the course of their disease in the curative, adjuvant or palliative setting. RT achieves good local control of tumor growth, conferring DNA damage and impacting tumor vasculature and the immune system. Formerly regarded as a merely immunosuppressive treatment, pre- and clinical observations indicate that the therapeutic effect of RT is partially immune mediated. In some instances, RT synergizes with immunotherapy (IT), through different mechanisms promoting an effective antitumor immune response. Cell death induced by RT is thought to be immunogenic and results in modulation of lymphocyte effector function in the tumor microenvironment promoting local control. Moreover, a systemic immune response can be elicited or modulated to exert effects outside the irradiation field (so called abscopal effects). In this review, we discuss the body of evidence related to RT and its immunogenic potential for the future design of novel combination therapies.

Cancer is the second most common cause of death in the United States behind heart disease. While current treatments such as surgery, chemotherapy, and radiation therapy are effective and widely used, medicine is moving towards more targeted and personalized therapies. Immunotherapy is one such treatment that utilizes the patient’s own immune system to target and eliminate tumor cells. It allows for the patient’s adaptive immune system to bypass the self-tolerance mechanisms used by the cancerous cells and be activated against the cancer. Two such self-tolerant mechanisms that are co-opted by tumor cells are the interactions between CTLA-4 and T lymphocytes and the interactions between PD-1 and PD-L1. Blocking these interactions allows for the recruitment of CTLs to the site of the tumor and subsequent attack. CTLA-4 and PD-1 are inhibitory costimulators that play a role in the suppression of the adaptive immune system. The interaction of these receptors with their respective ligands leads to self-tolerance, and is a common mechanism used as a protective measure against autoimmune reactions. Monoclonal antibodies against these two receptors and ligand have been tested in clinical trials and have shown efficacy against ovarian cancers, non-small cell lung carcinomas, colon cancers, and melanomas. By targeting the inhibitory signals, these monoclonal antibodies expose cancer cells as being “non-self” thus prompting the immune system to attack. Now, studies are focusing on combination therapies, which combine chemotherapeutics or other monoclonal antibodies with PD-1 and CTLA-4 inhibitors to enhance the effectiveness of the drug. However, drawbacks and side effects to the therapy range from fatigue and nausea to development of autoimmune diseases. It brings forward that future studies will need a panel of predictive biomarkers to identify the best candidates for the immunotherapy. While there are many obstacles, such as a lower than expected efficacy of the immunotherapy, the progress made has important implications in the development of personalized medicine.

Checkpoint blockade immunotherapy takes advantage of an endogenous anti-cancer immune response and promotes tumor killing by altering the balance of signals that control T-cell activity. Dramatic and durable responses have led to FDA approvals for anti-programmed- death-1 (anti-PD1) therapy across a broad range of cancer types, however only a fraction of patients benefit and some with an initial response will later progress. The specific mechanisms of tumor-immune evasion or resistance in this setting are not well established, but understanding them will be critical for accurate prognosis, proper therapy selection, and identification of next- generation drug targets.To investigate mechanistic correlates of anti-PD1 response and resistance in melanoma, we used high-throughput sequencing to analyze genomic, transcriptomic, and T-cell data in whole-tumor biopsies and early passage cell lines from 1) longitudinally sampled lesions from patients with an initial response followed by late progression (acquired resistance) and 2) pre-treatment lesions in cohorts of patients with or without response (primary resistance).Whole exome sequencing from four cases of late acquired resistance revealed evidence of in-situ clonal selection for new homozygous loss-of-function mutations in the interferon-receptor associated kinases JAK1 and JAK2 and in beta-2-microglobulin (B2M, a co-factor required for class I antigen presentation). These JAK mutations abolished sensitivity to interferon-gamma (IFNγ), including IFNγ-induced PD-L1 expression and growth arrest. In complementary work, we evaluated the frequency of interferon insensitivity (or lack of PD-L1 inducibility) and interferon pathway mutations in PD1-therapy na�ve cohorts. Among the handful of JAK homozygous loss-of-function mutations we identified from patients with response data, all were found in non-responders.Two other retrospective cohort studies revealed additional response-correlated insights. Transcriptomic data from pre-treatment biopsies showed enrichment for a coordinated set of genes associated with epithelial-to-mesenchymal transition (EMT), wound-healing, angiogenesis, and hypoxia in PD1 non-responders. By contrast, we found the rare desmoplastic melanoma subtype (DM) was unusually sensitive to anti-PD1 therapy, with a 70% response rate double that of unselected cutaneous melanoma. While often a histological diagnosis, we observed our DM cohort was characterized by a high mutational load and NF1 driver subtype.Finally, we worked with colleagues at Caltech on enabling studies with a new technology for sensitive identification of neo-antigen specific T-cell responses that is optimized for downstream T-cell capture and receptor cloning. Here, the goal was to aid in assessment of the diversity and dynamics of defined tumor antigen-specific T-cells and to allow for their ex-vivo production.

There is a growing interest in metabolomic profiling of immune cells, as there is accumulating evidence that metabolism influences immune cell function and fate decisions. The non-adherent nature of these cells presents certain challenges not factored into extraction protocols developed for adherent cells. Here, we present a method developed for metabolite extraction from human T-cells that emphasizes simplicity and economy. We apply this method to profile the metabolic changes effected by in-vitro programmed cell death 1 receptor (PDCD1/PD-1) immune checkpoint engagement by liquid chromatography and mass spectrometry. We demonstrate that we can efficiently confirm the known reduction in aerobic glycolysis and glutaminolysis that accompanies PD-1 signaling and extend this by showing a block in de novo nucleoside phosphate biosynthesis.

Abstract: T lymphocytes (T cells) are powerful directors and effectors of immunity. The system of pseudo-random rearrangements of the T cell receptor (TCR) loci that underlie their ability to recognize a vast universe of molecular patterns is at once useful and dangerous, because many T cells develop TCR that can recognize self, and while most developing T cells with high affinity for self are removed from the repertoire during thymic development, the process is not perfect and some dangerous clones escape. These newly generated T cells have only passed through one “filtration” process in the thymus, and therefore peripheral tolerance mechanisms are critical in order to avoid autoimmunity. Homeostasis of T cells in the periphery is tightly regulated by competition for a finite resource pool, including homeostatic cytokines and relevant peptide:MHC (pMHC) complexes with which a T cell can interact and receive at least a “tonic” or greater signal. In conditions of lymphopenia, which can arise as a consequence of viral infections, clinical interventions, and other stimuli, resources are in excess and T cells will undergo a process of lymphopenia-induced proliferation (LIP) to fill their available niche. Importantly, LIP is strongly associated with inflammatory disease. The co-inhibitory receptor programmed death-1 (PD-1) is expressed on T cells where it provides inhibitory signals that help prevent inappropriate T cell activation or keep T cells in an unresponsive state. PD-1-/- mice are predisposed to infrequent and mild autoimmunity. In contrast, reconstitution of the lymphoid compartment of lymphopenic adult Rag-/- mice with PD-1-/- hematopoietic stem cells (HSC) gives rise to a rapid and severe systemic autoimmune disease shortly after the first newly generated T cells emerge from the thymus. Thus a “three strikes” combination of newly generated T cells, deficiency of PD-1, and LIP synergizes to promote autoimmunity. Our goal herein was to explore the mechanisms and cells underlying the disease in this model. One of PD-1’s ligands, PD-L1, has been associated with the generation of peripheral FoxP3+ regulatory T cells (pTreg), which are critical mediators of immune tolerance. Also, while PD-1 is known to be up-regulated following conventional T cell activation, we hypothesized that PD-1 might control the comparatively weak “tonic” signals T cells receive in response to self pMHC, and in the setting of LIP of newly generated T cells, this lack of restraint on normally tonic signals led to a widespread self pMHC-directed polyclonal response. We examined the role of PD-1 in the control of pTreg generation and found that deficiency of PD-1 does not preclude pTreg generation or regulatory T cell function. Instead, PD-1 is important for restraining the expansion of both conventional and regulatory cells in a lymphopenic host, suggesting that clinical approaches to tumour immunotherapy mediated by PD-1 blockade are unlikely to work by inhibiting tumour-associated pTreg generation and may actually expand them. We also found that PD-1 indeed controls LIP of T cells in response to tonic pMHC signals, and thus PD-1-/- T cells out-expanded WT cells in competitive in vivo assay in an MHC-dependent manner but independent of signals from the IL-7 receptor. Using a model system where we could purify newly generated T cells from the periphery of an adult, lymphoreplete mouse, we found that these cells were not rapidly tolerized upon contact with the periphery but instead maintained their autoimmune-generating potential for a period of time. Mice lacking MHC Class II expression were spared from autoimmunity, suggesting that CD4+ T cells are the main effectors of disease in the PD-1-/- HSC model. Furthermore, disease was not dependent on either of two canonical T cell killing mechanisms, the Perforin-Granzyme B pathway or the Fas/FasL pathway. Together these data may suggest that disease in our model is driven by a cytokine storm, and beyond advancing our basic understanding of how co-inhibitors control homeostasis of T cells, this model may be useful for developing approaches to treat diseases associated with cytokinemias, such as graft-versus-host disease or immune reconstitution inflammatory syndrome in human immunodeficiency virus patients.

Chlamydia trachomatis infection is the most common bacterial sexually transmitted disease in the United States. Irregular screening to identify infected individuals and a lack of sterilizing immunity to C. trachomatis has led to a dramatic increase in the number of reported C. trachomatis infections over the last twenty years. Repeated infections with C. trachomatis lead to serious sequelae such as pelvic inflammatory disease and ectopic pregnancy, which can result in infertility.

A variety of mechanisms act to prevent the inappropriate targeting of host tissues by the immune system, but these mechanisms can fail, leading to development of autoimmunity. Type 1 Diabetes is an autoimmune disease caused by T cell-mediated destruction of the insulin-producing beta cells in the pancreatic islets of Langerhans. This work focused on understanding islet-reactive CD4 T cells during Type 1 Diabetes progression by comparing T cell responses in diabetes-susceptible non-obese diabetic (NOD) mice to diabetes-resistant (B6.g7) mice. Our knowledge of how these cells are normally regulated and how that regulation breaks down leading to diabetes is limited due to a lack of reagents to track these cells in mice with normal T cell repertoires. The goal of this work was to develop models to study physiological numbers of islet-reactive CD4 T cells in mice in order to gain a better understanding of how these cells are regulated with an emphasis on one critical inhibitory pathway involving Programmed-Death 1 (PD-1). Using these models, we showed that in diabetes-susceptible NOD mice, the majority of islet-reactive CD4 T cells become activated, but the pathogenic subset capable of contributing to disease was relatively small. Rather, the majority of the population was adequately controlled by the host through induction of T cell anergy. In diabetes-resistant mice, islet-reactive CD4 T cells were present, but failed to become activated and subsequently did not infiltrate the pancreas. The inhibitory receptor PD-1 was important for suppressing CD4 T cell effector functions in NOD mice, including proliferation, trafficking to the pancreas, and localization within the pancreas. However, blockade of PD-1 did not promote CD4 T cell trafficking to the pancreas in diabetes-resistant mice. Unexpectedly, in NOD mice, PD-1 was predominantly involved in suppressing the functions of an activated effector population, not maintaining the anergic population. These findings provide new insight into the regulation of islet-reactive CD4 T cells, and show that PD-1 differentially contributes to the suppression of these cells.

Abstract: The immune system must be able to mount a response against pathogens and transformed cells while remaining tolerant to healthy host tissue. A key process for ensuring this self-tolerance is the negative selection of self-reactive thymocytes. Expression of Programmed Death-1 (PD-1), a co-inhibitory member of the CD28 family associated with dampened peripheral immune responses,was found to be upregulated in 20-40% of thymocytes undergoing negative selection in the HYcd4model of thymic development. Although analysis of gene and protein expression directly ex vivo indicates that PD-1- and PD-1+ thymocytes are equally apoptotic, PD-1+ thymocytes appear to be protected from apoptosis in an in vitro stimulation assay. Analysis of HYcd4PD-1-/- mice indicates that thymocytes receive a higher intensity signal in the absence of PD-1. Future work utilizing HYcd4PD-1-/- mice will increase our understanding of the role of PD-1 in thymic negative selection.