Evolutionary Pressure against MHC Class II Binding Cancer Mutations.

The anti-cancer immune response against mutated peptides of potential immunological relevance (neoantigens) is primarily attributed to MHC-I-restricted cytotoxic CD8+ T cell responses. MHC-II-restricted CD4+ T cells also drive anti-tumor responses, but their relation to neoantigen selection and tumor evolution has not been systematically studied. Modeling the potential of an individual's MHC-II genotype to present 1,018 driver mutations in 5,942 tumors, we demonstrate that the MHC-II genotype constrains the mutational landscape during tumorigenesis in a manner complementary to MHC-I. Mutations poorly bound to MHC-II are positively selected during tumorigenesis, even more than mutations poorly bound to MHC-I. This emphasizes the importance of CD4+ T cells in anti-tumor immunity. In addition, we observed less inter-patient variation in mutation presentation for MHC-II than for MHC-I. These differences were reflected by age at diagnosis, which was correlated with presentation by MHC-I only. Collectively, our results emphasize the central role of MHC-II presentation in tumor evolution.

Disulfiram's journey from rubber vulcanization to T-cell activation.

Disulfiram, a drug prescribed for the treatment of alcohol use disorders for more than 60 years, has recently been repurposed for cancer treatment. New work in The EMBO Journal now describes a disulfiram role in immunotherapy of cancer, involving direct binding to Lck to mediate activation of tumor-infiltrating T cells.

Subcellular location of source proteins improves prediction of neoantigens for immunotherapy.

Antigen presentation via the major histocompatibility complex (MHC) is essential for anti-tumor immunity. However, the rules that determine which tumor-derived peptides will be immunogenic are still incompletely understood. Here, we investigated whether constraints on peptide accessibility to the MHC due to protein subcellular location are associated with peptide immunogenicity potential. Analyzing over 380,000 peptides from studies of MHC presentation and peptide immunogenicity, we find clear spatial biases in both eluted and immunogenic peptides. We find that including parent protein location improves the prediction of peptide immunogenicity in multiple datasets. In human immunotherapy cohorts, the location was associated with a neoantigen vaccination response, and immune checkpoint blockade responders generally had a higher burden of neopeptides from accessible locations. We conclude that protein subcellular location adds important information for optimizing cancer immunotherapies.

Autoimmune alleles at the major histocompatibility locus modify melanoma susceptibility.

Autoimmunity and cancer represent two different aspects of immune dysfunction. Autoimmunity is characterized by breakdowns in immune self-tolerance, while impaired immune surveillance can allow for tumorigenesis. The class I major histocompatibility complex (MHC-I), which displays derivatives of the cellular peptidome for immune surveillance by CD8+ T cells, serves as a common genetic link between these conditions. As melanoma-specific CD8+ T cells have been shown to target melanocyte-specific peptide antigens more often than melanoma-specific antigens, we investigated whether vitiligo- and psoriasis-predisposing MHC-I alleles conferred a melanoma-protective effect. In individuals with cutaneous melanoma from both The Cancer Genome Atlas (n = 451) and an independent validation set (n = 586), MHC-I autoimmune-allele carrier status was significantly associated with a later age of melanoma diagnosis. Furthermore, MHC-I autoimmune-allele carriers were significantly associated with decreased risk of developing melanoma in the Million Veteran Program (OR = 0.962, p = 0.024). Existing melanoma polygenic risk scores (PRSs) did not predict autoimmune-allele carrier status, suggesting these alleles provide orthogonal risk-relevant information. Mechanisms of autoimmune protection were neither associated with improved melanoma-driver mutation association nor improved gene-level conserved antigen presentation relative to common alleles. However, autoimmune alleles showed higher affinity relative to common alleles for particular windows of melanocyte-conserved antigens and loss of heterozygosity of autoimmune alleles caused the greatest reduction in presentation for several conserved antigens across individuals with loss of HLA alleles. Overall, this study presents evidence that MHC-I autoimmune-risk alleles modulate melanoma risk unaccounted for by current PRSs.

Structure-selected RBM immunogens prime polyclonal memory responses that neutralize SARS-CoV-2 variants of concern.

Successful control of the COVID-19 pandemic depends on vaccines that prevent transmission. The full-length Spike protein is highly immunogenic but the majority of antibodies do not target the virus: ACE2 interface. In an effort to affect the quality of the antibody response focusing it to the receptor-binding motif (RBM) we generated a series of conformationally-constrained immunogens by inserting solvent-exposed RBM amino acid residues into hypervariable loops of an immunoglobulin molecule. Priming C57BL/6 mice with plasmid (p)DNA encoding these constructs yielded a rapid memory response to booster immunization with recombinant Spike protein. Immune sera antibodies bound strongly to the purified receptor-binding domain (RBD) and Spike proteins. pDNA primed for a consistent response with antibodies efficient at neutralizing authentic WA1 virus and three variants of concern (VOC), B.1.351, B.1.617.2, and BA.1. We demonstrate that immunogens built on structure selection can be used to influence the quality of the antibody response by focusing it to a conserved site of vulnerability shared between wildtype virus and VOCs, resulting in neutralizing antibodies across variants.

IRE1α regulates macrophage polarization, PD-L1 expression, and tumor survival.

In the tumor microenvironment, local immune dysregulation is driven in part by macrophages and dendritic cells that are polarized to a mixed proinflammatory/immune-suppressive phenotype. The unfolded protein response (UPR) is emerging as the possible origin of these events. Here we report that the inositol-requiring enzyme 1 (IRE1α) branch of the UPR is directly involved in the polarization of macrophages in vitro and in vivo, including the up-regulation of interleukin 6 (IL-6), IL-23, Arginase1, as well as surface expression of CD86 and programmed death ligand 1 (PD-L1). Macrophages in which the IRE1α/X-box binding protein 1 (Xbp1) axis is blocked pharmacologically or deleted genetically have significantly reduced polarization and CD86 and PD-L1 expression, which was induced independent of IFNγ signaling, suggesting a novel mechanism in PD-L1 regulation in macrophages. Mice with IRE1α- but not Xbp1-deficient macrophages showed greater survival than controls when implanted with B16.F10 melanoma cells. Remarkably, we found a significant association between the IRE1α gene signature and CD274 gene expression in tumor-infiltrating macrophages in humans. RNA sequencing (RNASeq) analysis showed that bone marrow-derived macrophages with IRE1α deletion lose the integrity of the gene connectivity characteristic of regulated IRE1α-dependent decay (RIDD) and the ability to activate CD274 gene expression. Thus, the IRE1α/Xbp1 axis drives the polarization of macrophages in the tumor microenvironment initiating a complex immune dysregulation leading to failure of local immune surveillance.

The unfolded protein response links tumor aneuploidy to local immune dysregulation.

Aneuploidy is a chromosomal abnormality associated with poor prognosis in many cancer types. Here, we tested the hypothesis that the unfolded protein response (UPR) mechanistically links aneuploidy and local immune dysregulation. Using a single somatic copy number alteration (SCNA) score inclusive of whole-chromosome, chromosome arm, and focal alterations in a pan-cancer analysis of 9,375 samples in The Cancer Genome Atlas (TCGA) database, we found an inverse correlation with a cytotoxicity (CYT) score across disease stages. Co-expression patterns of UPR genes changed substantially between SCNAlow and SCNAhigh groups. Pathway activity scores showed increased activity of multiple branches of the UPR in response to aneuploidy. The PERK branch showed the strongest association with a reduced CYT score. The conditioned medium of aneuploid cells transmitted XBP1 splicing and caused IL-6 and arginase 1 transcription in receiver bone marrow-derived macrophages and markedly diminished the production of IFN-γ and granzyme B in activated human T cells. We propose the UPR as a mechanistic link between aneuploidy and immune dysregulation in the tumor microenvironment.

PERK-mediated induction of microRNA-483 disrupts cellular ATP homeostasis during the unfolded protein response.

Endoplasmic reticulum (ER) stress activates the unfolded protein response (UPR), which reduces levels of misfolded proteins. However, if ER homeostasis is not restored and the UPR remains chronically activated, cells undergo apoptosis. The UPR regulator, PKR-like endoplasmic reticulum kinase (PERK), plays an important role in promoting cell death when persistently activated; however, the underlying mechanisms are poorly understood. Here, we profiled the microRNA (miRNA) transcriptome in human cells exposed to ER stress and identified miRNAs that are selectively induced by PERK signaling. We found that expression of a PERK-induced miRNA, miR-483, promotes apoptosis in human cells. miR-483 induction was mediated by a transcription factor downstream of PERK, activating transcription factor 4 (ATF4), but not by the CHOP transcription factor. We identified the creatine kinase brain-type (CKB) gene, encoding an enzyme that maintains cellular ATP reserves through phosphocreatine production, as being repressed during the UPR and targeted by miR-483. We found that ER stress, selective PERK activation, and CKB knockdown all decrease cellular ATP levels, leading to increased vulnerability to ER stress-induced cell death. Our findings identify miR-483 as a downstream target of the PERK branch of the UPR. We propose that disruption of cellular ATP homeostasis through miR-483-mediated CKB silencing promotes ER stress-induced apoptosis.

Ubi Maior Minor Cessat.

This article is a tribute to Eli Sercarz and draws on his proposal of a hierarchical order of T-cell determinants in antigen presentation and T-cell activation. Here I revisit the concept of dominance and crypticity in the context of lymphocyte cooperation in the generation of the adaptive immune response, with emphasis on Th-Th cooperation. The remarks made in this article serve as a basis for a reassessment of the unresponsiveness of self-tumor antigens and how to leverage cryptic T-cell determinants operationally to generate antitumor T-cell immunity.

Tapping CD4 T cells for cancer immunotherapy: the choice of personalized genomics.

Cellular immune responses that protect against tumors typically have been attributed to CD8 T cells. However, CD4 T cells also play a central role. It was shown recently that, in a patient with metastatic cholangiocarcinoma, CD4 T cells specific for a peptide from a mutated region of ERBB2IP could arrest tumor progression. This and other recent findings highlight new opportunities for CD4 T cells in cancer immunotherapy. In this article, I discuss the role and regulation of CD4 T cells in response to tumor Ags. Emphasis is placed on the types of Ags and mechanisms that elicit tumor-protective responses. I discuss the advantages and drawbacks of cancer immunotherapy through personalized genomics. These considerations should help to guide the design of next-generation therapeutic cancer vaccines.

Synthesis and delivery of short, noncoding RNA by B lymphocytes.

Evolutionarily conserved short (20-30 nucleotides) noncoding RNAs (microRNAs) are powerful regulators of gene expression in a variety of physiological and pathological processes. As such, means to efficiently modulate microRNA function constitute an important therapeutic opportunity. Here we demonstrate that primary B lymphocytes can be genetically programmed with nonviral plasmid DNA for the biogenesis and delivery of antisense sequences (anti-microRNA) against microRNA-150 (miR-150). Within 18 h of transfection with an anti-miR-150 construct, primary B lymphocytes secrete ∼3,000 copies of anti-miR-150 molecules per cell. Anti-miR-150 molecules released by B lymphocytes were internalized by CD8 T lymphocytes during cross-priming in vitro and in vivo, resulting in marked down-regulation of endogenous miR-150. However, such internalization was not observed in the absence of cross-priming. These results suggest that shuttling anti-miR-150 molecules from B lymphocytes to T cells requires the activation of receiver T cells via the antigen receptor. Finally, anti-miR-150 synthesized in B cells were secreted both as free and extracellular vesicle-associated fractions, but only extracellular vesicle-associated anti-miR-150 were apparently taken up by CD8 T cells. Collectively, these data indicate that primary B lymphocytes represent an efficient platform for the synthesis and delivery of short, noncoding RNA, paving the way for an approach to immunogenomic therapies.

Transmission of endoplasmic reticulum stress and pro-inflammation from tumor cells to myeloid cells.

Metabolic, infectious, and tumor cell-intrinsic noxae can all evoke the endoplasmic reticulum (ER) stress response in tumor cells, which is critical for tumor cell growth and cancer progression. Evidence exists that the ER stress response can drive a proinflammatory program in tumor cells and macrophages but, to our knowledge, a role for the tumor ER stress response in influencing macrophages and inflammation in the tumor microenvironment has not been suggested. Here we show that macrophages cultured in conditioned medium from ER-stressed tumor cells become activated, and themselves undergo ER stress with the up-regulation of Grp78, Gadd34, Chop, and Xbp-1 splicing, suggesting a general activation of the ER stress-signaling pathways. Furthermore, these macrophages recapitulate, amplify and expand the proinflammatory response of tumor cells. We term this phenomenon "transmissible" ER stress. Although neither Toll-like receptor (TLR)2 nor interleukin 6 receptor (IL6R) signaling is involved, a reduction was observed in the transmission of ER stress to TLR4 KO macrophages, consistent with the fact that a second signal through TLR4 combined with exposure to tumor ER stress-conditioned medium results in a faster ER stress response and an enhancement of proinflammatory cytokine production in macrophages. The injection of tumor ER stress-conditioned medium into WT mice elicited a generalized ER stress response in the liver. We suggest that transmissible ER stress is a mechanism through which tumor cells can control myeloid cells by directing them toward a proinflammatory phenotype, thus facilitating tumor progression.

Integrated germline and somatic features reveal divergent immune pathways driving ICB response.

Immune Checkpoint Blockade (ICB) has revolutionized cancer treatment, however mechanisms determining patient response remain poorly understood. Here we used machine learning to predict ICB response from germline and somatic biomarkers and interpreted the learned model to uncover putative mechanisms driving superior outcomes. Patients with higher T follicular helper infiltrates were robust to defects in the class-I Major Histocompatibility Complex (MHC-I). Further investigation uncovered different ICB responses in MHC-I versus MHC-II neoantigen reliant tumors across patients. Despite similar response rates, MHC-II reliant responses were associated with significantly longer durable clinical benefit (Discovery: Median OS=63.6 vs. 34.5 months P=0.0074; Validation: Median OS=37.5 vs. 33.1 months, P=0.040). Characteristics of the tumor immune microenvironment reflected MHC neoantigen reliance, and analysis of immune checkpoints revealed LAG3 as a potential target in MHC-II but not MHC-I reliant responses. This study highlights the value of interpretable machine learning models in elucidating the biological basis of therapy responses.

Inhalable dry powders of microRNA-laden extracellular vesicles prepared by thin-film freeze-drying.

Extracellular vesicles (EVs) are endogenous vesicles that comprise a variety of submicron vesicular structures. Among these, exosomes have been widely investigated as delivery systems for small and large molecules. Herein, the thin-film freeze-drying technology was utilized to engineer aerosolizable dry powders of miR-335-laden induced EVs (iEV-335) generated in B cells for potential delivery into the lung to treat primary lung cancer and/or pulmonary metastases. The size distribution, structure, and morphology of iEV-335 were preserved after they were subjected to thin-film freeze-drying with the proper excipients. Importantly, iEV-335, in liquid or reconstituted from thin-film freeze-dried powders, were equally effective in downregulating SOX4 gene expression in LM2 human triple-negative mammary cancer cells. The iEV-335 dry powder compositions showed mass median aerodynamic diameters (MMAD) of around 1.2 µm with > 60 % of the emitted doses had an MMAD of ≤ 3 µm, indicating that the powders can potentially achieve efficient deposition within the alveolar region following oral inhalation, which is desirable for treatment of primary lung cancer and pulmonary metastases. Overall, it is concluded that it is feasible to apply thin-film freeze-drying to prepare aerosolizable dry powders of iEVs for pulmonary delivery.

Tumor stress inside out: cell-extrinsic effects of the unfolded protein response in tumor cells modulate the immunological landscape of the tumor microenvironment.

The unfolded protein response (UPR) is a eukaryotic cellular adaptive mechanism that functions to cope with stress of the endoplasmic reticulum (ER). Accumulating evidence demonstrates that the tumor microenvironment contains stressors that elicit a UPR, which has been demonstrated to be a cell-intrinsic mechanism crucial for tumorigenesis. In addition, the UPR is a source of proinflammatory signaling whose downstream mediators may hamper antitumor immunity. We discuss how the UPR may impair Ag presentation, which could result in defective T cell priming, also leading to tumor escape and growth. Further, we discuss the recent finding that ER stress and attendant proinflammation can be transmitted from ER-stressed tumor cells to myeloid cells. The ideas presented suggest that, in addition to being a cell-intrinsic mechanism of tumor survival, the tumor UPR can serve as a cell-extrinsic regulator of tumorigenesis by remodeling the immune response in the tumor microenvironment.

Transcriptional analysis links B cells and TERT expression to favorable prognosis in head and neck cancer.

Telomerase reverse transcriptase (TERT) is a conserved self-tumor antigen overexpressed in ∼85% of tumor cells and is immunogenic in cancer patients. The effect of TERT expression on the regulation of intratumor adaptive immunity has not yet been investigated. We used RNA sequencing data from The Cancer Genome Atlas (TCGA) in 11 solid tumor types to investigate potential interactions between TERT expression, and B and T cell infiltrate in the tumor microenvironment. We found a positive correlation between TERT expression, B and T cells in four cancer types with the strongest association in head and neck squamous cell carcinoma (HSNCC). In HNSCC a Bhigh/TERThigh signature was associated with improved progression-free survival (PFS) (P = 0.0048). This effect was independent of HPV status and not shared in comparable analysis by other conserved tumor antigens (NYESO1, MUC1, MAGE, and CEA). Bhigh/TERThigh HNSCC tumors also harbored evidence of tertiary lymphoid structure (TLS) such as signatures for germinal center (GC) and switched memory B cells, central memory CD4 and effector memory CD8 T cells. Bhigh/TERThigh HNSCC tumors also showed an up-regulation of genes and pathways related to B and T cell activation, proliferation, migration, and cytotoxicity, while factors associated with immunosuppression and cancer cell invasiveness were down-regulated. In summary, our study uncovers a new association between high TERT expression and high B cell infiltrate in HNSCC, suggesting a potential benefit from therapeutic strategies that invigorate intratumor TERT-mediated T-B cooperation.

Germline modifiers of the tumor immune microenvironment implicate drivers of cancer risk and immunotherapy response.

With the continued promise of immunotherapy for treating cancer, understanding how host genetics contributes to the tumor immune microenvironment (TIME) is essential to tailoring cancer screening and treatment strategies. Here, we study 1084 eQTLs affecting the TIME found through analysis of The Cancer Genome Atlas and literature curation. These TIME eQTLs are enriched in areas of active transcription, and associate with gene expression in specific immune cell subsets, such as macrophages and dendritic cells. Polygenic score models built with TIME eQTLs reproducibly stratify cancer risk, survival and immune checkpoint blockade (ICB) response across independent cohorts. To assess whether an eQTL-informed approach could reveal potential cancer immunotherapy targets, we inhibit CTSS, a gene implicated by cancer risk and ICB response-associated polygenic models; CTSS inhibition results in slowed tumor growth and extended survival in vivo. These results validate the potential of integrating germline variation and TIME characteristics for uncovering potential targets for immunotherapy.

The Unfolded Protein Response at the Tumor-Immune Interface.

The tumor-immune interface has surged to primary relevance in an effort to understand the hurdles facing immune surveillance and cancer immunotherapy. Reports over the past decades have indicated a role for the unfolded protein response (UPR) in modulating not only tumor cell fitness and drug resistance, but also local immunity, with emphasis on the phenotype and altered function of immune cells such as myeloid cells and T cells. Emerging evidence also suggests that aneuploidy correlates with local immune dysregulation. Recently, we reported that the UPR serves as a link between aneuploidy and immune cell dysregulation in a cell nonautonomous way. These new findings add considerable complexity to the organization of the tumor microenvironment (TME) and the origin of its altered function. In this review, we summarize these data and also discuss the role of aneuploidy as a negative regulator of local immunity.