Regulation of the immune tolerance system determines the susceptibility to HLA-mediated abacavir-induced skin toxicity

Idiosyncratic drug toxicity (IDT) associated with specific human leukocyte antigen (HLA) allotype is a rare and unpredictable life-threatening adverse drug reaction for which prospective mechanistic studies in humans are difficult. Here, we show the importance of immune tolerance for IDT onset and determine whether it is susceptible to a common IDT, HLA-B*57:01-mediated abacavir (ABC)-induced hypersensitivity (AHS), using CD4+ T cell-depleted programmed death-1 receptor (PD-1)-deficient HLA-B*57:01 transgenic mice (B*57:01-Tg/PD-1−/−). Although AHS is not observed in B*57:01-Tg mice, ABC treatment increases the proportion of cytokine- and cytolytic granule-secreting effector memory CD8+ T cells in CD4+ T cell-depleted B*57:01-Tg/PD-1−/− mice, thereby inducing skin toxicity with CD8+ T cell infiltration, mimicking AHS. Our results demonstrate that individual differences in the immune tolerance system, including PD-1highCD8+ T cells and regulatory CD4+ T cells, may affect the susceptibility of humans to HLA-mediated IDT in humans.

A biomimetic assay platform for the interrogation of antigen-dependent anti-tumor T-cell function

Overcoming tumor-mediated immunosuppression and enhancing cytotoxic T-cell activity within the tumor microenvironment are two central goals of immuno-oncology (IO) drug discovery initiatives. However, exploratory assays involving immune components are often plagued by low-throughput and poor clinical relevance. Here we present an innovative ultra-high-content assay platform for interrogating T-cell-mediated killing of 3D multicellular tumor spheroids. Employing this assay platform in a chemical genomics screen of 1800 annotated compounds enabled identification of small molecule perturbagens capable of enhancing cytotoxic CD8+ T-cell activity in an antigen-dependent manner. Specifically, cyclin-dependent kinase (CDK) and bromodomain (BRD) protein inhibitors were shown to significantly augment anti-tumor T-cell function by increasing cytolytic granule and type II interferon secretion in T-cells in addition to upregulating major histocompatibility complex (MHC) expression and antigen presentation in tumor cells. The described biotechnology screening platform yields multi-parametric, clinically-relevant data and can be employed kinetically for the discovery of first-in-class IO therapeutic agents.

Immune phenotype of patients with stage IV metastatic inflammatory breast cancer

Background Inflammatory breast cancer (IBC) is a rare but aggressive carcinoma characterized by severe erythema and edema of the breast, with many patients presenting in advanced metastatic disease. The “inflammatory” nature is not due to classic immune-mediated inflammation, but instead results from tumor-mediated blockage of dermal lymphatic ducts. Previous work has shown that expression of PD-L1 on tumor cells can suppress T cell activation in triple-negative (TN) non-IBC breast cancer. In the present work, we investigated immune parameters in peripheral blood of metastatic IBC patients to determine whether cellular components of the immune system are altered, thereby contributing to pathogenesis of the disease. These immune parameters were also compared to PD-1 and PD-L1 expression in IBC tumor biopsies. Methods Flow cytometry-based immune phenotyping was performed using fresh peripheral blood from 14 stage IV IBC patients and compared to 11 healthy age-similar control women. Immunohistochemistry for CD20, CD3, PD-1, and PD-L1 was performed on tumor biopsies of these metastatic IBC patients. Results IBC patients with Stage IV disease had lymphopenia with significant reductions in circulating T, B, and NK cells. Reductions were observed in all subsets of CD4+ T cells, whereas reductions in CD8+ T cells were more concentrated in memory subsets. Immature cytokine-producing CD56bright NK cells expressed higher levels of FcγRIIIa and cytolytic granule components, suggesting accelerated maturation to cytolytic CD56dim cells. Immunohistochemical analysis of tumor biopsies demonstrated moderate to high expression of PD-1 in 18.2% of patients and of PD-L1 in 36.4% of patients. Interestingly, a positive correlation was observed between co-expression levels of PD-L1 and PD-1 in tumor biopsies, and higher expression of PD-L1 in tumor biopsies correlated with higher expression of cytolytic granule components in blood CD4+ T cells and CD56dim NK cells, and higher numbers of CD8+ effector memory T cells in peripheral blood. PD-1 expression in tumor also correlated with increased infiltration of CD20+ B cells in the tumor. Conclusions Our results suggest that while lymphocyte populations are severely compromised in stage IV IBC patients, an immune response toward the tumor had occurred in some patients, providing biological rationale to evaluate PD-1/PD-L1 immunotherapies for IBC.

PBRM1 and the glycosylphosphatidylinositol biosynthetic pathway promote tumor killing mediated by MHC-unrestricted cytotoxic lymphocytes

Major histocompatibility complex (MHC)–unrestricted cytotoxic lymphocytes (CLs) such as natural killer (NK) cells can detect and destroy tumor and virus-infected cells resistant to T cell–mediated killing. Here, we performed genome-wide genetic screens to identify tumor-intrinsic genes regulating killing by MHC-unrestricted CLs. A group of genes identified in our screens encode enzymes for the biosynthesis of the glycosylphosphatidylinositol (GPI) anchor, which is not involved in tumor response to T cell–mediated cytotoxicity. Another gene identified in the screens was PBRM1, which encodes a subunit of the PBAF form of the SWI/SNF chromatin-remodeling complex. PBRM1 mutations in tumor cells cause resistance to MHC-unrestricted killing, in contrast to their sensitizing effects on T cell–mediated killing. PBRM1 and the GPI biosynthetic pathway regulate the ligands of NK cell receptors in tumor cells and promote cytolytic granule secretion in CLs. The regulators identified in this work represent potential targets for cancer immunotherapy.

SerpinB1 controls encephalitogenic T helper cells in neuroinflammation

SerpinB1, a protease inhibitor and neutrophil survival factor, was recently linked with IL-17–expressing T cells. Here, we show that serpinB1 (Sb1) is dramatically induced in a subset of effector CD4 cells in experimental autoimmune encephalomyelitis (EAE). Despite normal T cell priming, Sb1−/− mice are resistant to EAE with a paucity of T helper (TH) cells that produce two or more of the cytokines, IFNγ, GM-CSF, and IL-17. These multiple cytokine-producing CD4 cells proliferate extremely rapidly; highly express the cytolytic granule proteins perforin-A, granzyme C (GzmC), and GzmA and surface receptors IL-23R, IL-7Rα, and IL-1R1; and can be identified by the surface marker CXCR6. In Sb1−/− mice, CXCR6+ TH cells are generated but fail to expand due to enhanced granule protease-mediated mitochondrial damage leading to suicidal cell death. Finally, anti-CXCR6 antibody treatment, like Sb1 deletion, dramatically reverts EAE, strongly indicating that the CXCR6+ T cells are the drivers of encephalitis.

SM protein Munc18-2 facilitates transition of Syntaxin 11-mediated lipid mixing to complete fusion for T-lymphocyte cytotoxicity

The atypical lipid-anchored Syntaxin 11 (STX11) and its binding partner, the Sec/Munc (SM) protein Munc18-2, facilitate cytolytic granule release by cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. Patients carrying mutations in these genes develop familial hemophagocytic lymphohistiocytosis, a primary immunodeficiency characterized by impaired lytic granule exocytosis. However, whether a SNARE such as STX11, which lacks a transmembrane domain, can support membrane fusion in vivo is uncertain, as is the precise role of Munc18-2 during lytic granule exocytosis. Here, using a reconstituted “flipped” cell–cell fusion assay, we show that lipid-anchored STX11 and its cognate SNARE proteins mainly support exchange of lipids but not cytoplasmic content between cells, resembling hemifusion. Strikingly, complete fusion is stimulated by addition of wild-type Munc18-2 to the assay, but not of Munc18-2 mutants with abnormal STX11 binding. Our data reveal that Munc18-2 is not just a chaperone of STX11 but also directly contributes to complete membrane merging by promoting SNARE complex assembly. These results further support the concept that SM proteins in general are part of the core fusion machinery. This fusion mechanism likely contributes to other cell-type–specific exocytic processes such as platelet secretion.

CD8 T cell-mediated killing of orexinergic neurons induces a narcolepsy-like phenotype in mice

Narcolepsy with cataplexy is a rare and severe sleep disorder caused by the destruction of orexinergic neurons in the lateral hypothalamus. The genetic and environmental factors associated with narcolepsy, together with serologic data, collectively point to an autoimmune origin. The current animal models of narcolepsy, based on either disruption of the orexinergic neurotransmission or neurons, do not allow study of the potential autoimmune etiology. Here, we sought to generate a mouse model that allows deciphering of the immune mechanisms leading to orexin+ neuron loss and narcolepsy development. We generated mice expressing the hemagglutinin (HA) as a “neo-self-antigen” specifically in hypothalamic orexin+ neurons (called Orex-HA), which were transferred with effector neo-self-antigen–specific T cells to assess whether an autoimmune process could be at play in narcolepsy. Given the tight association of narcolepsy with the human leukocyte antigen (HLA) HLA-DQB1*06:02 allele, we first tested the pathogenic contribution of CD4 Th1 cells. Although these T cells readily infiltrated the hypothalamus and triggered local inflammation, they did not elicit the loss of orexin+ neurons or clinical manifestations of narcolepsy. In contrast, the transfer of cytotoxic CD8 T cells (CTLs) led to both T-cell infiltration and specific destruction of orexin+ neurons. This phenotype was further aggravated upon repeated injections of CTLs. In situ, CTLs interacted directly with MHC class I-expressing orexin+ neurons, resulting in cytolytic granule polarization toward neurons. Finally, drastic neuronal loss caused manifestations mimicking human narcolepsy, such as cataplexy and sleep attacks. This work demonstrates the potential role of CTLs as final effectors of the immunopathological process in narcolepsy.