Exhausted CD8 T (Tex) cells in chronic viral infection and cancer have sustained co-expression of inhibitory receptors (IRs). Tex cells can be reinvigorated by blocking IRs, such as PD-1, but synergistic reinvigoration and enhanced disease control can be achieved by co-targeting multiple IRs including PD-1 and LAG-3. To dissect the molecular changes intrinsic when these IR pathways are disrupted, we investigated the impact of loss of PD-1 and/or LAG-3 on Tex cells during chronic infection. These analyses revealed distinct roles of PD-1 and LAG-3 in regulating Tex cell proliferation and effector functions, respectively. Moreover, these studies identified an essential role for LAG-3 in sustaining TOX and Tex cell durability as well as a LAG-3-dependent circuit that generated a CD94/NKG2+ subset of Tex cells with enhanced cytotoxicity mediated by recognition of the stress ligand Qa-1b, with similar observations in humans. These analyses disentangle the non-redundant mechanisms of PD-1 and LAG-3 and their synergy in regulating Tex cells.

NK-cell reactivity against cancer is conceivably suppressed in the tumor microenvironment by the interaction of the inhibitory receptor NKG2A with the non-classical MHC-I molecules HLA-E in humans or Qa-1b in mice. We found that intratumoral delivery of NK cells attains significant therapeutic effects only if co-injected with anti-NKG2A and anti-Qa-1b blocking monoclonal antibodies against solid mouse tumor models. Such therapeutic activity was contingent on endogenous CD8 T cells and type-1 conventional dendritic cells (cDC1). Moreover, the anti-tumor effects were enhanced upon combination with systemic anti-PD-1 mAb treatment and achieved partial abscopal efficacy against distant non-injected tumors. In xenografted mice bearing HLA-E-expressing human cancer cells, intratumoral co-injection of activated allogeneic human NK cells and clinical-grade anti-NKG2A mAb (monalizumab) synergistically achieved therapeutic effects. In conclusion, these studies provide evidence for the clinical potential of intratumoral NK cell-based immunotherapies that exert their anti-tumor efficacy as a result of eliciting endogenous T-cell responses.

BACKGROUND: Malignant melanoma is one kind of rare cancer in human and animals, which occurs not only in skin but also in the mucous membranes of the nose, mouth, anus, digestive tract, and in the uvea (choroid) of the eye. In order to develop therapies, a better understanding of the genetic landscape and signal pathways are needed. Numerous studies highlighted the importance of NKG2A-HLA-E in tumor immunotherapy, but the function and mechanism of HLA-E in tumor cells have seldom been studied alone. The statistical analyses from publicly available database Oncomine and The Cancer Genome Atlas (TCGA) showed that HLA-E is highly expressed in melanoma compared to non-transformed counterparts. In addition, melanoma patients with HLA-E high expression had a worse OS rate than the patients with low expression. These data indicate the importance of HLA-E in human melanoma. Qa-1b is the homolog of HLA-E in mouse.
OBJECTIVE: To investigate the function and mechanism of Qa-1b in mouse melanoma.
METHODS: Mouse melanoma cell line B16-F10 and allogenic melanoma model were used to investigate the function and mechanism of Qa-1b in melanoma.
RESULTS: Qa-1b was highly expressed in B16-F10 compared with normal mouse epidermal cells. Qa-1b knockdown inhibited B16-F10 cell proliferation and migration in vitro and allogenic melanoma process in vivo. Furthermore, Qa-1b knockdown promoted cell cycle arrest at G0/G1 phase and cell apoptosis through Ras-Raf-MAPK signal pathway.
CONCLUSIONS: Qa-1b functions as an oncogenic factor and it can be used as a new therapeutic target in melanoma.

The immune co-receptor CD8 molecule (CD8) has two subunits, CD8α and CD8β, which can assemble into homo or heterodimers. Nonclassical (class-Ib) major histocompatibility complex (MHC) molecules (MHC-Ibs) have recently been identified as ligands for the CD8αα homodimer. This was demonstrated by the observation that histocompatibility 2, Q region locus 10 (H2-Q10) is a high-affinity ligand for CD8αα which also binds the MHC-Ib molecule H2-TL. This suggests that MHC-Ib proteins may be an extended source of CD8αα ligands. Expression of H2-T3/TL and H2-Q10 is restricted to the small intestine and liver, respectively, yet CD8αα-containing lymphocytes are present more broadly. Therefore, here we sought to determine whether murine CD8αα binds only to tissue-specific MHC-Ib molecules or also to ubiquitously expressed MHC-Ib molecules. Using recombinant proteins and surface plasmon resonance-based binding assays, we show that the MHC-Ib family furnishes multiple binding partners for murine CD8αα, including H2-T22 and the CD94/NKG2-A/B-activating NK receptor (NKG2) ligand Qa-1b We also demonstrate a hierarchy among MHC-Ib proteins with respect to CD8αα binding, in which Qa-1b > H2-Q10 > TL. Finally, we provide evidence that Qa-1b is a functional ligand for CD8αα, distinguishing it from its human homologue MHC class I antigen E (HLA-E). These findings provide additional clues as to how CD8αα-expressing cells are controlled in different tissues. They also highlight an unexpected immunological divergence of Qa-1b/HLA-E function, indicating the need for more robust studies of murine MHC-Ib proteins as models for human disease.

The HLA-E homolog in the mouse (Qa-1b) is a conserved MHC class Ib molecule presenting monomorphic peptides to germline-encoded natural killer receptor CD94/NKG2A. Previously, we demonstrated the replacement of this canonical peptide by a diverse peptidome upon deficiency of the TAP peptide transporter. Analysis of this Qa-1b-restricted T cell repertoire against these non-mutated neoantigens revealed characteristics of conventional hypervariable CD8+ T cells, but also of invariant T cell receptor (TCR)αβ T cells. A shared TCR Vα chain was used by this subset in combination with a variety of Vβ chains. The TCRs target peptide ligands that are conserved between mouse and man, like the identified peptide derived from the transcriptional cofactor Med15. The thymus selection was studied in a TCR-transgenic mouse and emerging naïve CD8+ T cells displayed a slightly activated phenotype, as witnessed by higher CD122 and Ly6C expression. Moreover, the Qa-1b protein was dispensable for thymus selection. Importantly, no self-reactivity was observed as reported for other MHC class Ib-restricted subsets. Naïve Qa-1b restricted T cells expanded, contracted, and formed memory cells in vivo upon peptide vaccination in a similar manner as conventional CD8+ T cells. Based on these data, the Qa-1b restricted T cell subset might be positioned closest to conventional CD8+ T cells of all MHC class Ib populations.

NK cells can reduce anti-viral T cell immunity during chronic viral infections, including infection with the lymphocytic choriomeningitis virus (LCMV). However, regulating factors that maintain the equilibrium between productive T cell and NK cell immunity are poorly understood. Here, we show that a large viral load resulted in inhibition of NK cell activation, which correlated with increased expression of Qa-1b, a ligand for inhibitory NK cell receptors. Qa-1b was predominantly upregulated on B cells following LCMV infection, and this upregulation was dependent on type I interferons. Absence of Qa-1b resulted in increased NK cell-mediated regulation of anti-viral T cells following viral infection. Consequently, anti-viral T cell immunity was reduced in Qa-1b- and NKG2A-deficient mice, resulting in increased viral replication and immunopathology. NK cell depletion restored anti-viral immunity and virus control in the absence of Qa-1b. Taken together, our findings indicate that lymphocytes limit NK cell activity during viral infection in order to promote anti-viral T cell immunity.