William Nicholas Haining

Background: Resistance to immune checkpoint inhibitors represents a major therapeutic challenge, as less than 50% of patients with melanoma achieve long-term response to immune checkpoint inhibitor therapy. One mechanism of acquired resistance involves somatic mutations, such as loss of beta-2 microglobulin ( B2m ), that enable tumor cells to evade T cell-mediated killing. Methods: This study used single-cell RNA-seq, flow cytometry, and ex vivo functional assays to characterize tumor-infiltrating immune cells in antigen presentation-deficient tumors. Tumor-bearing mice were treated with anti-PD-1 or CD40 agonist antibodies and cell depletion or cytokine blocking antibodies to define mechanisms of action. Analysis of published human RNA-seq datasets was performed to dissect the contributions of inflammatory monocytes to patient outcomes. Results: We found an increase in immunosuppressive macrophages in B2m -null tumors. We hypothesized that repolarizing myeloid cells may restore control of tumor growth. Treatment with CD40 agonist antibody, which promotes differentiation of monocytes and macrophages towards a proinflammatory phenotype, reduced tumor growth and improved survival in B2m -null melanoma and colorectal cancer models. Unexpectedly, both CD8 ⁺ T cells and NK cells, but not CD4 ⁺ T cells, were required for the efficacy of CD40 agonist, even though CD8 ⁺ T cells cannot directly recognize antigen presentation-deficient tumor cells. Instead, these lymphocytes control tumor growth via secretion of IFNγ, as depletion of IFNγ inhibited the therapeutic effect of CD40 agonist. IFNγ receptor ( Ifngr1 ) expression was required on host cells, not tumor cells, for CD40 agonist-mediated tumor control. Single-cell analysis identified a distinct population of inflammatory monocytes that were enriched for an IFNγ response signature in CD40 agonist-treated tumors, suggesting that these cells may be important for tumor control. Analysis of human bulk and single-cell RNA-seq datasets demonstrated that an inflammatory monocyte signature derived from our data was associated with improved patient outcomes and response to immune checkpoint inhibitors. Conclusions: These data demonstrate that CD8 ⁺ T cells contribute to tumor control even in the absence of direct antigen presentation by tumor cells. More broadly, our work suggests that strategies to activate the effector functions of inflammatory monocytes may limit tumor growth and overcome acquired resistance to immune checkpoint inhibitors.

Predicting cellular responses to therapeutics is a promising approach for novel target discovery. However, state-of-the-art computational models designed to predict perturbation effects struggle to generalize and outperform simple baselines. We present DINOcell, a weakly supervised framework that adapts self-distillation to single-cell transcriptomics for predicting perturbation effects. We demonstrate that DINOcell outperforms baselines in predicting the effects of single gene perturbations. Furthermore, DINOcell accurately predicts non-additive effects of combination perturbations, indicating its capacity to model complex genetic interactions. Finally, we show that DINOcell learns representations that capture biological signals and is a promising, generalizable approach for in silico perturbation modeling, providing a valuable tool for accelerating therapeutic target discovery.

PD-1 is a key negative regulator of CD8⁺ T cell activation and is highly expressed by exhausted T cells in cancer and chronic viral infection. Although PD-1 blockade can improve viral and tumor control, physiological PD-1 expression prevents immunopathology and improves memory formation. The mechanisms driving high PD-1 expression in exhaustion are not well understood and could be critical to disentangling its beneficial and detrimental effects. Here, we functionally interrogated the epigenetic regulation of PD-1 using a mouse model with deletion of an exhaustion-specific PD-1 enhancer. Enhancer deletion exclusively alters PD-1 expression in CD8⁺ T cells in chronic infection, creating a ‘sweet spot’ of intermediate expression where T cell function is optimized compared to wild-type and Pdcd1-knockout cells. This permits improved control of chronic infection without additional immunopathology. Together, these results demonstrate that tuning PD-1 via epigenetic editing can reduce CD8⁺ T cell dysfunction while avoiding excess immunopathology.

A systems analysis was conducted to determine the potential molecular mechanisms underlying differential immunogenicity and protective efficacy results of a clinical trial of the radiation-attenuated whole-sporozoite PfSPZ vaccine in African infants. Innate immune activation and myeloid signatures at prevaccination baseline correlated with protection from P. falciparum parasitemia in placebo controls. These same signatures were associated with susceptibility to parasitemia among infants who received the highest and most protective PfSPZ vaccine dose. Machine learning identified spliceosome, proteosome, and resting DC signatures as prevaccination features predictive of protection after highest-dose PfSPZ vaccination, whereas baseline circumsporozoite protein–specific (CSP-specific) IgG predicted nonprotection. Prevaccination innate inflammatory and myeloid signatures were associated with higher sporozoite-specific IgG Ab response but undetectable PfSPZ-specific CD8 ⁺ T cell responses after vaccination. Consistent with these human data, innate stimulation in vivo conferred protection against infection by sporozoite injection in malaria-naive mice while diminishing the CD8 ⁺ T cell response to radiation-attenuated sporozoites. These data suggest a dichotomous role of innate stimulation for malaria protection and induction of protective immunity by whole-sporozoite malaria vaccines. The uncoupling of vaccine-induced protective immunity achieved by Abs from more protective CD8 ⁺ T cell responses suggests that PfSPZ vaccine efficacy in malaria-endemic settings may be constrained by opposing antigen presentation pathways.

Summary: Tumor-specific cytotoxic T cells unleashed by the blockade of immune checkpoints have to overcome a hostile tumor microenvironment (TME). They start from very small numbers of T cells with tumor antigen specificity and, despite expansion, likely remain at a numerical disadvantage to the tumor cells they target. To overcome these obstacles, we propose that T cells need to change the TME to make it permissive for their antitumor effects by altering the phenotype of cells beyond the cancer cells they are in physical contact with. In this process, IFNγ secreted by tumor-specific T cells plays a critical role, as it changes the expression of hundreds of genes in cancer cells and other immune cells in the TME up to 40 layers of cells away from their location, effectively turning these cells into enablers of the antitumor immune response. In this perspective, we postulate that the clinical activity of cancer immunotherapy with immune-checkpoint blocking antibodies and adoptively transferred T cells requires that cancer cells facilitate the antitumor immune response. IFNγ effectively changes the balance of power in the TME to enable the antitumor activity of tumor antigen–specific cytotoxic T cells.

The immune system can eliminate tumors, but checkpoints enable immune escape. Here, we identify immune evasion mechanisms using genome-scale in vivo CRISPR screens across cancer models treated with immune checkpoint blockade (ICB). We identify immune evasion genes and important immune inhibitory checkpoints conserved across cancers, including the non-classical major histocompatibility complex class I (MHC class I) molecule Qa-1^b/HLA-E. Surprisingly, loss of tumor interferon-γ (IFNγ) signaling sensitizes many models to immunity. The immune inhibitory effects of tumor IFN sensing are mediated through two mechanisms. First, tumor upregulation of classical MHC class I inhibits natural killer cells. Second, IFN-induced expression of Qa-1^b inhibits CD8⁺ T cells via the NKG2A/CD94 receptor, which is induced by ICB. Finally, we show that strong IFN signatures are associated with poor response to ICB in individuals with renal cell carcinoma or melanoma. This study reveals that IFN-mediated upregulation of classical and non-classical MHC class I inhibitory checkpoints can facilitate immune escape.

T cell therapies for cancer have shown striking clinical efficacy in different tumor settings. Integration of a transgene cassette expressing an antigen receptor into the genome enables T cells to recognize specific tumor antigens. Viral vectors and transposons can introduce transgenes into primary human T cells. However these approaches cannot control genomic location and have limited control over the copy number of cassette insertions. Here, we used epigenetic analysis, transcriptional profiling, high-throughput gene-editing and T cell functional experiments to identify the optimal genomic locus amenable to non-viral genome targeting. First we analyzed the epigenetic landscape of primary human T cells to identify regions (“gene deserts”) that are accessible but devoid of annotated regulatory elements or coding sequences. Candidate loci were validated using CRISPR/Cas9 genome editing to directly cut and a marker gene cassette was inserted into these sites. Knock-in efficiency and transgene expression stability in primary human T cells were evaluated for all loci. Evaluation of 40 loci demonstrated substantial variability (10 fold) in transgene expression and confirmed that not all sites were equivalent in their ability to sustain transgene expression. Next, we evaluated 8 most promising candidate loci for compatibility with complex T cell programs embodied by integrated circuits, containing a priming receptor (PrimeR) that triggers expression of a CAR in response to a priming antigen. We identified one integration site (termed “GS94”) that supported: 1) stable and high PrimeR expression; 2) high and inducible CAR expression; and 3) a superior T cell cytotoxic and cytokine secretion profile. Finally, we assessed off-target cutting mediated by the CRISPR sgRNA targeting GS94. The sgRNA is the most specific out of the candidates evaluated with iGUIDE and targeted PCR. We were unable to detect any measurable off-target activity by targeted PCR on predicted sites. The GS94 site is a component of the integrated circuit T cell (ICT) therapy, AB-1015, which is now under development for the treatment of high-grade serous carcinoma ovarian tumors. AB-1015 includes an inserted cassette that contains a logic gate and two shRNAs that enhance the potency and solid tumor microenvironment resistance of the AB-1015 ICT cells. These computational and experimental approaches can be generalized to identify new safe harbor sites for different cell types, expanding the genome engineering toolkit for diverse cell therapy applications. Citation Format: Grace X.Y. Zheng, Somya Khare, Brendan Galvin, Robby Moot, Aaron Cooper, Michelle Nguyen, Michelle Tan, Shan Sabri, Audre May, Jun Fung, Anzhi Yao, Andrea Liu, Matt Drever, Steve Santoro, W Nicholas Haining, Tarjei Mikkelsen. Identification of a safe harbor CRISPR-Cas9 integration site for improved cell therapy safety and potency [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2840.

The response of naive CD8 ⁺ T cells to their cognate antigen involves rapid and broad changes to gene expression that are coupled with extensive chromatin remodeling, but the mechanisms governing these changes are not fully understood. Here, we investigated how these changes depend on the basic leucine zipper ATF-like transcription factor Batf, which is essential for the early phases of the process. Through genome scale profiling, we characterized the role of Batf in chromatin organization at several levels, including the accessibility of key regulatory regions, the expression of their nearby genes, and the interactions that these regions form with each other and with key transcription factors. We identified a core network of transcription factors that cooperated with Batf, including Irf4, Runx3, and T-bet, as indicated by their colocalization with Batf and their binding in regions whose accessibility, interactions, and expression of nearby genes depend on Batf. We demonstrated the synergistic activity of this network by overexpressing the different combinations of these genes in fibroblasts. Batf and Irf4, but not Batf alone, were sufficient to increase accessibility and transcription of key loci, normally associated with T cell function. Addition of Runx3 and T-bet further contributed to fine-tuning of these changes and was essential for establishing chromatin loops characteristic of T cells. These data provide a resource for studying the epigenomic and transcriptomic landscape of effector differentiation of cytotoxic T cells and for investigating the interdependency between transcription factors and its effects on the epigenome and transcriptome of primary cells.

T cell exhaustion is associated with failure to clear chronic infections and malignant cells. Defining the molecular mechanisms of T cell exhaustion and reinvigoration is essential to improving immunotherapeutic modalities. Here we confirmed pervasive phenotypic, functional and transcriptional differences between memory and exhausted antigen-specific CD8⁺ T cells in human hepatitis C virus (HCV) infection before and after treatment. After viral cure, phenotypic changes in clonally stable exhausted T cell populations suggested differentiation toward a memory-like profile. However, functionally, the cells showed little improvement, and critical transcriptional regulators remained in the exhaustion state. Notably, T cells from chronic HCV infection that were exposed to antigen for less time because of viral escape mutations were functionally and transcriptionally more similar to memory T cells from spontaneously resolved HCV infection. Thus, the duration of T cell stimulation impacts exhaustion recovery, with antigen removal after long-term exhaustion being insufficient for the development of functional T cell memory.

T cell exhaustion is an induced state of dysfunction that arises in response to chronic infection and cancer. Exhausted CD8⁺ T cells acquire a distinct epigenetic state, but it is not known whether that chromatin landscape is fixed or plastic following the resolution of a chronic infection. Here we show that the epigenetic state of exhaustion is largely irreversible, even after curative therapy. Analysis of chromatin accessibility in HCV- and HIV-specific responses identifies a core epigenetic program of exhaustion in CD8⁺ T cells, which undergoes only limited remodeling before and after resolution of infection. Moreover, canonical features of exhaustion, including super-enhancers near the genes TOX and HIF1A, remain ‘epigenetically scarred.’ T cell exhaustion is therefore a conserved epigenetic state that becomes fixed and persists independent of chronic antigen stimulation and inflammation. Therapeutic efforts to reverse T cell exhaustion may require new approaches that increase the epigenetic plasticity of exhausted T cells.