T-cell immunoglobulin domain and mucin domain-3 (TIM-3, also known as HAVCR2) is an activation-induced inhibitory molecule involved in tolerance and shown to induce T-cell exhaustion in chronic viral infection and cancers. Under some conditions, TIM-3 expression has also been shown to be stimulatory. Considering that TIM-3, like cytotoxic T lymphocyte antigen 4 (CTLA-4) and programmed death 1 (PD-1), is being targeted for cancer immunotherapy, it is important to identify the circumstances under which TIM-3 can inhibit and activate T-cell responses. Here we show that TIM-3 is co-expressed and forms a heterodimer with carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1), another well-known molecule expressed on activated T cells and involved in T-cell inhibition. Biochemical, biophysical and X-ray crystallography studies show that the membrane-distal immunoglobulin-variable (IgV)-like amino-terminal domain of each is crucial to these interactions. The presence of CEACAM1 endows TIM-3 with inhibitory function. CEACAM1 facilitates the maturation and cell surface expression of TIM-3 by forming a heterodimeric interaction in cis through the highly related membrane-distal N-terminal domains of each molecule. CEACAM1 and TIM-3 also bind in trans through their N-terminal domains. Both cis and trans interactions between CEACAM1 and TIM-3 determine the tolerance-inducing function of TIM-3. In a mouse adoptive transfer colitis model, CEACAM1-deficient T cells are hyper-inflammatory with reduced cell surface expression of TIM-3 and regulatory cytokines, and this is restored by T-cell-specific CEACAM1 expression. During chronic viral infection and in a tumour environment, CEACAM1 and TIM-3 mark exhausted T cells. Co-blockade of CEACAM1 and TIM-3 leads to enhancement of anti-tumour immune responses with improved elimination of tumours in mouse colorectal cancer models. Thus, CEACAM1 serves as a heterophilic ligand for TIM-3 that is required for its ability to mediate T-cell inhibition, and this interaction has a crucial role in regulating autoimmunity and anti-tumour immunity.

Tumour-specific CD8 T cell dysfunction is a differentiation state that is distinct from the functional effector or memory T cell states^1-6. Here we identify the nuclear factor TOX as a crucial regulator of the differentiation of tumour-specific T (TST) cells. We show that TOX is highly expressed in dysfunctional TST cells from tumours and in exhausted T cells during chronic viral infection. Expression of TOX is driven by chronic T cell receptor stimulation and NFAT activation. Ectopic expression of TOX in effector T cells in vitro induced a transcriptional program associated with T cell exhaustion. Conversely, deletion of Tox in TST cells in tumours abrogated the exhaustion program: Tox-deleted TST cells did not upregulate genes for inhibitory receptors (such as Pdcd1, Entpd1, Havcr2, Cd244 and Tigit), the chromatin of which remained largely inaccessible, and retained high expression of transcription factors such as TCF-1. Despite their normal, 'non-exhausted' immunophenotype, Tox-deleted TST cells remained dysfunctional, which suggests that the regulation of expression of inhibitory receptors is uncoupled from the loss of effector function. Notably, although Tox-deleted CD8 T cells differentiated normally to effector and memory states in response to acute infection, Tox-deleted TST cells failed to persist in tumours. We hypothesize that the TOX-induced exhaustion program serves to prevent the overstimulation of T cells and activation-induced cell death in settings of chronic antigen stimulation such as cancer.

T-cell exhaustion, or an impaired capacity to secrete cytokines and proliferate with overexpression of immune checkpoint receptors, occurs during chronic viral infections but has also been observed in tumors, including hepatocellular carcinomas (HCCs). We investigated features of exhaustion in CD8+ T cells isolated from HCC specimens.

We obtained HCC specimens, along with adjacent nontumor tissues and blood samples, from 90 patients who underwent surgical resection at Asan Medical Center (Seoul, Korea) from April 2016 through April 2018. Intrahepatic lymphocytes and tumor-infiltrating T cells were analyzed by flow cytometry. Tumor-infiltrating CD8+ T cells were sorted by flow cytometry into populations based on expression level of programmed cell death 1 (PDCD1 or PD1): PD1-high, PD1-intermediate, and PD1-negative. Sorted cells were analyzed by RNA sequencing. Proliferation and production of interferon gamma (IFNG) and tumor necrosis factor (TNF) by CD8+ T cells were measured in response to anti-CD3 and antibodies against immune checkpoint receptors including PD1, hepatitis A virus cellular receptor 2 (HAVCR2 or TIM3), lymphocyte activating 3 (LAG3), or isotype control. Tumor-associated antigen-specific CD8+ T cells were identified using HLA-A*0201 dextramers. PDL1 expression on tumor tissue was assessed by immunohistochemistry.

PD1-high, PD1-intermediate, and PD1-negative CD8+ T cells from HCCs had distinct gene expression profiles. PD1-high cells expressed higher levels of genes that regulate T-cell exhaustion than PD1-intermediate cells. PD1-high cells expressed TIM3 and LAG3, and low proportions of TCF1+, TBEThigh/eomesoderminlow, and CD127+. PD1-high cells produced the lowest amounts of IFNG and TNF upon anti-CD3 stimulation. Differences in the PD1 expression patterns of CD8+ T cells led to the identification of 2 subgroups of HCCs: HCCs with a discrete population of PD1-high cells were more aggressive than HCCs without a discrete population of PD1-high cells. HCCs with a discrete population of PD1-high cells had higher levels of predictive biomarkers of response to anti-PD1 therapy. Incubation of CD8+ T cells from HCCs with a discrete population of PD1-high cells with antibodies against PD1 and TIM3 or LAG3 further restored proliferation and production of IFNG and TNF in response to anti-CD3.

We found HCC specimens to contain CD8+ T cells that express different levels of PD1. HCCs with a discrete population of PD1-high CD8+ T cells express TIM3 and/or LAG3 and produce low levels of IFNG and TNF in response to anti-CD3. Incubation of these cells with antibodies against PD1 and TIM3 or LAG3 further restore proliferation and production of cytokines; HCCs with a discrete population of PD1-high CD8+ T cells might be more susceptible to combined immune checkpoint blockade-based therapies.

Innate immunity is an indispensable arm of tumor immune surveillance, and the liver is an organ with a predominance of innate immunity, where mucosal-associated invariant T (MAIT) cells are enriched. However, little is known about the phenotype, functions, and immunomodulatory role of MAIT cells in hepatocellular carcinoma (HCC).Experimental Design: The distribution, phenotype, and function of MAIT cells in patients with HCC were evaluated by both flow cytometry (FCM) and in vitro bioassays. Transcriptomic analysis of MAIT cells was also performed. Prognostic significance of tumor-infiltrating MAIT cells was validated in four independent cohorts of patients with HCC.

Despite their fewer densities in HCC tumor than normal liver, MAIT cells were significantly enriched in the HCC microenvironment compared with other mucosa-associated organs. Tumor-derived MAIT cells displayed a typical CCR7^-CD45RA^-CD45RO⁺CD95⁺ effector memory phenotype with lower costimulatory and effector capabilities. Tumor-educated MAIT cells significantly upregulated inhibitory molecules like PD-1, CTLA-4, TIM-3, secreted significantly less IFNγ and IL17, and produced minimal granzyme B and perforin while shifting to produce tumor-promoting cytokines like IL8. Transcriptome sequencing confirmed that tumor-derived MAIT cells were reprogrammed toward a tumor-promoting direction by downregulating genes enriched in pathways of cytokine secretion and cytolysis effector function like NFKB1 and STAT5B and by upregulating genes like IL8, CXCL12, and HAVCR2 (TIM-3). High infiltration of MAIT cells in HCC significantly correlated with an unfavorable clinical outcome, revealed by FCM, qRT-PCR, and multiplex IHC analyses, respectively.

HCC-infiltrating MAIT cells were functionally impaired and even reprogrammed to shift away from antitumor immunity and toward a tumor-promoting direction.See related commentary by Carbone, p. 3199.

Natural killer (NK) cells are critical to both innate and adaptive immunity. However, the development and heterogeneity of human NK cells are yet to be fully defined. Using single-cell RNA-sequencing technology, here we identify distinct NK populations in human bone marrow and blood, including one population expressing higher levels of immediate early genes indicative of a homeostatic activation. Functionally matured NK cells with high expression of CX3CR1, HAVCR2 (TIM-3), and ZEB2 represents terminally differentiated status with the unique transcriptional profile. Transcriptomic and pseudotime analyses identify a transitional population between CD56^bright and CD56^dim NK cells. Finally, a donor with GATA2^T354M mutation exhibits reduced percentage of CD56^bright NK cells with altered transcriptome and elevated cell death. These data expand our understanding of the heterogeneity and development of human NK cells.