HLA-E–restricted HIV-specific T cells offer exciting possibilities for immunotherapy. However, HLA-E binding peptides are rare. A recent study showed that in HLA-B*57:01–positive people with HIV, the peptide that dominates the T cell response, KAFSPEVIPMF (KF11), also stimulates HLA-E–restricted T cells, even though direct binding of this peptide to HLA-E could not be demonstrated. We therefore changed position 2 alanine for methionine in the peptide (referred to as KMF11), which greatly enhanced binding to HLA-E. This enabled the generation of stabilized HLA-E-KMF11 tetramers, which were used to select and then grow specific T cell clones from T cells of HLA-B*57:01–negative blood donors primed with this peptide in vitro. Approximately 20% of these T cell clones reacted with HLA-E–positive cells presenting the native KF11 peptide. Furthermore, these T cells inhibited replication of HIV-1 NL4-3 in CD4 + T cells in vitro. Therefore, this native peptide can be presented by HLA-E to CD8 + T cells, although priming in vivo may depend on cross-reactivities to classical MHC-Ia types. Nevertheless, such T cells could be exploitable for immunotherapy given the conservation of this HIV-1 peptide epitope and the non-polymorphism in HLA-E.

A high-affinity antibody response to both infection and vaccination critically relies on the ability of B cells to capture and process antigen for presentation to CD4+ T cells. The cellular processes from antigen recognition to full B cell activation require a finely orchestrated series of events, involving signalling and intracellular trafficking mechanisms. Here, we describe a novel regulator of B cell receptor (BCR) endocytosis and intracellular trafficking. Multidomain trafficking protein sorting-related receptor with A-type repeats (SorLA) associates with the BCR and regulates uptake of both soluble and substrate-bound antigens. SorLA deletion results in altered BCR-antigen intracellular trafficking to degradative compartments, modulating eventual antigen presentation. Crucially, this change in antigen trafficking results in a significant reduction in plasma cells and humoral responses in vivo. Given the critical importance of antigen presentation in immunity, as well as autoimmune disease and malignancy, these results identify a new cellular pathway in B cell biology with potential implications for immune regulation.

Natural killer (NK) cells are integral to the innate immune system, playing a crucial role in immune surveillance and the rapid response to virally infected and tumor cells. Epigenetic gene expression regulation significantly influences NK cell function and differentiation. Using a high-throughput small-molecule drug screening approach, we identified bromodomain and extra-terminal domain (BET) inhibitors (BETi) as potent modulators of NK cell function, reducing proinflammatory cytokine secretion while increasing markers of NK cell maturation and cytotoxicity. During NK lineage specification from hematopoietic stem cells, we demonstrated that BETi reduced NK cell fate and promoted increased myeloid cell differentiation. Moreover, differentiated NK cell types had more functionally differentiated gene expression programs. Thus, BET proteins are crucial for both mature NK cell functions and controlling NK cell lineage development from progenitors in the bone marrow. These findings suggest that BETi can fine-tune NK cell responses, offering promising therapeutic potential for cancer immunotherapy and the treatment of inflammatory and autoimmune diseases. Our study underscores the critical role of BET inhibitors in regulating NK cell function and opens new avenues for targeted immune modulation.

MHC-E is a highly conserved, non-polymorphic MHC protein that engages inhibitory and activating receptors on natural killer (NK) cells and T cells and can also present antigens to T cell receptors. NK cell responses driven by activating receptor interactions with MHC-E are implicated in controlling chronic viral infections and cancer. Immunotherapeutic targeting of interactions between MHC-E and inhibitory receptors to increase the activation of NK cells and T cells shows promise in improving antitumour immune responses. Furthermore, MHC-E-restricted CD8+ T cells elicited by cytomegalovirus-based vaccines might, for certain infections and cancers, be more effective than CD8+ T cells restricted by classical MHC class I or class II molecules. The ability of MHC-E to regulate or mediate both innate and adaptive immune responses independently of the MHC haplotype of an individual raises the possibility of new, universally effective vaccines and immunotherapies for infectious disease and cancer. Although the therapeutic exploitation of MHC-E is still in its infancy, recent advances in the understanding of MHC-E biology show enormous potential, as described in this Review. The dual nature of non-polymorphic MHC-E as a ligand for innate receptors and as an antigen-presenting protein raises the possibility of new, universally effective vaccines and immunotherapies for infectious disease and cancer that are independent of the MHC haplotype of an individual.

NK cells and CD8+ T cells both contribute to HIV-1 control. These cells not only suppress HIV-1 replication, but also select HIV-1 escape mutant viruses. Most viruses bearing T cell escape mutations are expected to remain susceptible to NK cell suppression, but their inhibition by NK cells is unclear. We investigated the role of HIV-1-specific CD8+ T cells and NK cells recognizing superimposed Pol peptides in selection and control of HIV-1 mutant virus. KIR2DL2+NK cells have an enhanced ability to recognize HIV-1-infected cells after selection of Pol mutant virus by PolIY11-specific HLA-C*12:02-restricted T cells. Mass spectrometry-based immunopeptidome profiling of HIV-1-infected cells and analysis of crystal structures of TCR- and KIR2DL2-HLA-C*12:02-peptide complexes demonstrate the molecular basis for selection and recognition of the escape mutant epitope by TCR and KIR2DL2. The present study elucidates the mechanism for selection and inhibition of an HIV-1 escape virus by T cells and NK cells.

Human Leukocyte Antigen E (HLA-E) is a nonclassical MHC class I molecule that exhibits dual immunological functions in regulating natural killer (NK) cells and T cells through unusual trafficking patterns. We previously reported that HLA-E surface expression is low and transient due to its cytoplasmic tail and dominant VL9 peptide, making it a dynamic indicator of cellular status for NK cell surveillance. Here, we identify a sequence motif in the HLA-E cytoplasmic tail that enables rapid internalization via clathrin-mediated endocytosis (CME) through interaction with the adaptor protein 2 (AP-2) complex. Following internalization, HLA-E is routed to endosomes, where the same cytoplasmic motif and peptide loading together facilitate its reappearance on the cell surface-a process influenced by valosin-containing protein (VCP). Our findings reveal previously unrecognized endosomal trafficking pathways and regulatory mechanisms that distinguish HLA-E from classical HLA class I molecules, with broad implications for understanding the immunoregulatory roles of HLA-E.

Relative to donor CD8+ T cells, the recovery of CD4+ T cell populations is severely delayed following allogeneic hematopoietic stem cell transplantation (allo-HCT). We reasoned CD8+ T cell differentiation would therefore take place under ‘helpless’ conditions and impair anti-tumour immunity. To test whether accelerating CD4+ T cell recovery would improve CD8+ T cell fitness and improve anti-tumour immunity, we conducted a randomized phase II, multicentre trial to evaluate the efficacy of prophylactic transfer of CD4+ T lymphocytes after alemtuzumab-based, reduced intensity HLA-matched related donor transplant for haematological cancers (PROT4; NCT01240525). 106 patients were registered for the trial and tapered cyclosporine from day 40 to day 70. 54 patients without GVHD, disease progression and normal graft function were randomised at a 2:1 ratio to receive 1 x 106 CD4+ T cells/kg between day 100-120. Blood samples were taken at day 90 (pre-infusion) and at 12-week intervals over the next 12 months. Immune population architecture and function were analysed using a combination of high-parameter flow cytometry, effector molecule expression, bulk/ single cell (sc) RNAseq and TCRseq. Although the trial did not meet its primary endpoint of increasing 1-year progression-free survival, CD4+ T cell addback induced greater rates of full donor chimerism without a greater risk of severe graft-versus-host disease. The randomised intervention permitted unbiased measurement of the effects of CD4+ T cell infusion upon T cell population architecture and function. Compared to controls, CD4+ T cell infusion significantly delayed CD8+T cell reconstitution both in terms of frequency and absolute numbers. Although per-cell cytokine generation was similar upon ex vivo stimulation, gene expression profiling showed that CD4+ T cell infusion induced signatures of CD8+ T cellular ‘fitness’ (increased DNA replication, TCR activation and ATP generation). These qualitative changes following CD4+ T cell infusion were matched by a dramatic shift in population architecture towards less differentiated CD8+ T cells with a marked reduction in effector cell accumulation as evaluated by unbiased clustering of flow cytometry and scRNAseq data. By pairing trajectory analysis and TCRseq, we found that CD4+T cell addback restricted CD8+ T cell clonal expansion and redirected differentiation away from terminal effector-like cellular states. In parallel, CD4+ T cell addback remodelled the CD4+ T cell compartment by enriching for circulating PD-1+CXCR5+ T follicular helper (TFH)-like cells with features of central memory cells but antagonized clonal expansion of cytotoxic CD4+ T cells (TCTX). To test whether TFH cells could provide direct help to CD8+ T cells, we cultured human CD8+ T cells in vitro under conditions of repetitive anti-CD3/CD28 stimulation with or without addition of FACS-isolated CD4+ TFH cells or CD4+ TCTXcells. Whereas helpless or CD4+ TCTX-helped CD8+ T cells underwent terminal differentiation, co-incubation with TFH cells promoted early differentiation-like cellular states and proliferative fitness, mirroring the phenotypic effects identified in trial participants receiving CD4+ T cells. To identify molecules which provide helper signals from CD4+ TFHto CD8+ T cells, we performed scRNAseq and Olink proteomics of CD4+ and CD8+ T cell co-cultures to facilitate interactome analyses searching for differential interactions between CD8+ T cells and CD4+ TFH versus CD4+TCTX. Consistent with pre-clinical models of CD4+ TFH help, IL-21-IL21R signalling was found to be the major candidate axis upon which human TFH help to CD8+ T cells was mediated, with IL-21R neutralisation reducing the frequency and absolute number of CD8+ T cells retaining a less differentiated, memory phenotype. Thus, CD4+ T cell infusion after allo-SCT in human patients shifts CD8+T cell clonal expansion and differentiation trajectory to less differentiated cellular states. Our finding that human TFH cells use IL-21 to directly promote fitness in human CD8+ T cells provides a potential mechanism involved and is consistent with recent data from pre-clinical models demonstrating TFH-mediated help for anti-tumour and anti-pathogen CD8+ T cells. These data provide further impetus to explore how the properties of CD4+ T cells can be best exploited in immunotherapeutic targeting of cancer.

A preprint by Xu et al. shows that MHC-pseudotyped retroviruses can reprogramme, activate and expand tumour-specific T cell populations in vivo.

A major natural killer (NK) cell and CD8 + T cell checkpoint is mediated by the inhibitory receptor NKG2A/CD94 and its ligand, HLA-E complexed with 9 amino acid HLA-Ia leader sequence-derived peptides termed VL9 (HLA-E-VL9). Here, we used structure-based design and high throughput library screening to generate antibodies that block NKG2A/CD94 interactions, resulting in direct NK and CD8 + T cell cytotoxicity, and trigger NK cell antibody-dependent cellular cytotoxicity (ADCC). Anti-HLA-E-VL9 antibodies limited HLA-E-VL9+ tumor growth in mice, demonstrating checkpoint inhibition activity in vivo . HLA-E-VL9 was found to be expressed on HIV-infected cells, and its engagement by HLA-E-VL9 antibodies eliminated infected cells by NK-mediated ADCC. HLA-E-VL9 antibodies also enhanced the killing of HIV-infected cells by NKG2A/CD94 + CD8 + T cells targeting a novel HLA-E binding HIV Rev-derived epitope. Therefore, anti-HLA-E-VL9 antibodies represent a novel approach to eliminate pathogenic target cells by enhancing NK and CD8+ T cell function and promoting ADCC.

ABSTRACTThe signal sequences of the human cytomegalovirus (CMV) UL40 protein, and its rhesus CMV counterpart, Rh67, contain a peptide (VMAPRT[L/V][F/I/L/V]L, VL9) that can be presented by Major Histocompatibility Complex (MHC) antigen E. The CMV VL9 peptides replace VL9 peptides derived from classical MHC (Ia) signal sequences, which are lost when CMV disrupts MHC Ia expression, as well as antigen processing and presentation. This allows infected cells to maintain surface expression of MHC-E and escape killing by NK cells expressing the inhibitory NKG2A/CD94 receptor. We demonstrate that processing of the Rh67 VL9 peptide mirrors that of UL40, despite the lack of sequence conservation elsewhere in the two proteins. As previously shown for UL40, up-regulation of MHC-E expression by Rh67 only requires its signal sequence, with sequences upstream of VL9 critical for conferring independence from TAP, the Transporter Associated with Antigen Processing. Additionally, we show that processing of both VL9 peptides depends on cleavage of the signal sequences by the host protease Signal Peptide Peptidase. Notably, our results also reveal that the mature UL40 and Rh67 proteins contribute to CMV immune evasion by down-regulating surface expression of MHC Ia. Unexpectedly, while the Rh67 VL9 peptide is resistant to the effects of the Rh67 protein, the UL40 protein is able to counteract up-regulation of MHC-E expression mediated by its own VL9 peptide. This suggests differences in the mechanisms by which the two VL9 peptides up-regulate MHC-E, which may have implications for translating a RhCMV-vectored SIV vaccine to HIV-1 using HCMV as a vector.IMPORTANCECell surface MHC-E expression, which requires a peptide (VMAPRT[L/V][F/I/L/V]L, “VL9”) from the signal sequences of other MHC class I proteins, prevents cells from being killed by CD94/NKG2A-expressing Natural Killer cells. In cells infected with human CMV, the endogenous VL9 peptide is replaced by one from the signal sequence of the HCMV UL40 protein. We show that processing of the VL9 peptide of Rh67, the rhesus CMV equivalent of UL40, mirrors that of UL40, despite a lack of sequence homology between the two proteins. Of note, we also show that the mature UL40 and Rh67 proteins, which have no previously described function, contribute to CMV immune evasion by reducing classical MHC class I surface expression. Importantly, the mature UL40 protein, but not the mature Rh67 protein, can decrease the up-regulation of MHC-E mediated by its signal sequence VL9 peptide, which may have implications for HCMV as a vaccine vector.

Dynamic regulation of cellular metabolism is important for maintaining homeostasis and can directly influence immune cell function and differentiation, including NK cell responses. Persistent HIV -1 infection leads to a state of chronic immune activation, NK cell subset redistribution, and progressive NK cell dysregulation. In this study, we examined the metabolic processes that characterize NK cell subsets in HIV -1 infection, including adaptive NK cell subpopulations expressing the activating receptor NKG2C, which expand during chronic infection. These adaptive NK cells exhibit an enhanced metabolic profile in HIV -1- individuals infected with human cytomegalovirus (HCMV). However, the bioenergetic advantage of adaptive CD57+NKG2C+ NK cells is diminished during chronic HIV -1 infection, where NK cells uniformly display reduced oxidative phosphorylation (OXPHOS). Defective OXPHOS was accompanied by increased mitochondrial depolarization, structural alterations, and increased DRP-1 levels promoting fission, suggesting that mitochondrial defects are restricting the metabolic plasticity of NK cell subsets in HIV -1 infection. The metabolic requirement for the NK cell response to receptor stimulation was alleviated upon IL15 pretreatment, which enhanced mammalian target of rapamycin complex 1 (mTORC1) activity. IL15 priming enhanced NK cell functionality to anti-CD16 stimulation in HIV -1 infection, representing an effective strategy for pharmacologically boosting NK cell responses.

The commonly used antibodies 3D12 and 4D12 recognise the human leukocyte antigen E (HLA-E) protein. These antibodies bind distinct epitopes on HLA-E and differ in their ability to bind alleles of the major histocompatibility complex E (MHC-E) proteins of rhesus and cynomolgus macaques. We confirmed that neither antibody cross-reacts with classical HLA alleles, and used hybrids of different MHC-E alleles to map the regions that are critical for their binding. 3D12 recognises a region on the alpha 3 domain, with its specificity for HLA-E resulting from the amino acids present at three key positions (219, 223 and 224) that are unique to HLA-E, while 4D12 binds to the start of the alpha 2 domain, adjacent to the C terminus of the presented peptide. 3D12 staining is increased by incubation of cells at 27°C, and by addition of the canonical signal sequence peptide presented by HLA-E peptide (VL9, VMAPRTLVL). This suggests that 3D12 may bind peptide-free forms of HLA-E, which would be expected to accumulate at the cell surface when cells are incubated at lower temperatures, as well as HLA-E with peptide. Therefore, additional studies are required to determine exactly what forms of HLA-E can be recognised by 3D12. In contrast, while staining with 4D12 was also increased when cells were incubated at 27°C, it was decreased when the VL9 peptide was added. We conclude that 4D12 preferentially binds to peptide-free HLA-E, and, although not suitable for measuring the total cell surface levels of MHC-E, may putatively identify peptide-receptive forms.

Understanding the nature and extent of non-canonical human leukocyte antigen (HLA) presentation in tumour cells is a priority for target antigen discovery for the development of next generation immunotherapies in cancer. We here employ a de novo mass spectrometric sequencing approach with a refined, MHC-centric analysis strategy to detect non-canonical MHC-associated peptides specific to cancer without any prior knowledge of the target sequence from genomic or RNA sequencing data. Our strategy integrates M HC binding rank, A verage local confidence scores, and peptide R etention time prediction for improved de novo candidate S election; culminating in the machine learning model MARS. We benchmark our model on a large synthetic peptide library dataset and reanalysis of a published dataset of high-quality non-canonical MHC-associated peptide identifications in human cancer. We achieve almost 2-fold improvement for high quality spectral assignments in comparison to de novo sequencing alone with an estimated accuracy of above 85.7% when integrated with a stepwise peptide sequence mapping strategy. Finally, we utilize MARS to detect and validate lncRNA-derived peptides in human cervical tumour resections, demonstrating its suitability to discover novel, immunogenic, non-canonical peptide sequences in primary tumour tissue.

ABSTRACT The commonly used commercially available antibodies 3D12 and 4D12 recognise the human leukocyte antigen E (HLA-E) protein. 3D12 is known to exhibit minimal cross-reactivity with classical HLA-Ia allotypes and we confirm that this is also the case for 4D12. These antibodies bind different epitopes on HLA-E, and differ in their ability to recognise alleles of the major histocompatibility complex E (MHC-E) proteins of rhesus and cynomolgus macaques. Using hybrids of different MHC-E alleles, we have mapped the regions that are critical for the binding of these two antibodies. 3D12 recognises a region on the alpha 3 domain that is unique to HLA-E, on the opposite side to that bound by β2-microglobulin, while 4D12 recognises the start of the alpha 2 domain, adjacent to the C terminus of the presented peptide. Knowledge of the binding sites of these two antibodies will facilitate selection of the best antibody for a given application, and inform interpretation of the resulting data.

Interest in MHC-E–restricted CD8+ T cell responses has been aroused by the discovery of their efficacy in controlling simian immunodeficiency virus (SIV) infection in a vaccine model. The development of vaccines and immunotherapies utilizing human MHC-E (HLA-E)–restricted CD8+ T cell response requires an understanding of the pathway(s) of HLA-E transport and antigen presentation, which have not been clearly defined previously. We show here that, unlike classical HLA class I, which rapidly exits the endoplasmic reticulum (ER) after synthesis, HLA-E is largely retained because of a limited supply of high-affinity peptides, with further fine-tuning by its cytoplasmic tail. Once at the cell surface, HLA-E is unstable and is rapidly internalized. The cytoplasmic tail plays a crucial role in facilitating HLA-E internalization, which results in its enrichment in late and recycling endosomes. Our data reveal distinctive transport patterns and delicate regulatory mechanisms of HLA-E, which help to explain its unusual immunological functions.

Human immunodeficiency virus type 1 (HIV-1) causes a major burden on global health, and eradication of latent virus infection is one of the biggest challenges in the field. The circadian clock is an endogenous timing system that oscillates with a ~24 h period regulating multiple physiological processes and cellular functions, and we recently reported that the cell intrinsic clock regulates rhythmic HIV-1 replication. Salt inducible kinases (SIK) contribute to circadian regulatory networks, however, there is limited evidence for SIKs regulating HIV-1 infection. Here, we show that pharmacological inhibition of SIKs perturbed the cellular clock and reduced rhythmic HIV-1 replication in circadian synchronised cells. Further, SIK inhibitors or genetic silencing of Sik expression inhibited viral replication in primary cells and in a latency model, respectively. Overall, this study demonstrates a role for salt inducible kinases in regulating HIV-1 replication and latency reactivation, which can provide innovative routes to better understand and target latent HIV-1 infection.

A preprint by Le Coz et al. identifies that human T follicular regulatory cells with differences in T cell suppressive and B cell helper capacities can be derived from the regulatory T cell lineage or the T follicular helper cell lineage.