Author Correction: Tissue patrol by resident memory CD8+ T cells in human skin.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Tissue patrol by resident memory CD8+ T cells in human skin.

Emerging data show that tissue-resident memory T (TRM) cells play an important protective role at murine and human barrier sites. TRM cells in the epidermis of mouse skin patrol their surroundings and rapidly respond when antigens are encountered. However, whether a similar migratory behavior is performed by human TRM cells is unclear, as technology to longitudinally follow them in situ has been lacking. To address this issue, we developed an ex vivo culture system to label and track T cells in fresh skin samples. We validated this system by comparing in vivo and ex vivo properties of murine TRM cells. Using nanobody labeling, we subsequently demonstrated in human ex vivo skin that CD8+ TRM cells migrated through the papillary dermis and the epidermis, below sessile Langerhans cells. Collectively, this work allows the dynamic study of resident immune cells in human skin and provides evidence of tissue patrol by human CD8+ TRM cells.

Single-cell analysis of regions of interest (SCARI) using a photosensitive tag.

The functional activity and differentiation potential of cells are determined by their interactions with surrounding cells. Approaches that allow unbiased characterization of cell states while at the same time providing spatial information are of major value to assess this environmental influence. However, most current techniques are hampered by a tradeoff between spatial resolution and cell profiling depth. Here, we develop a photocage-based technology that allows isolation and in-depth analysis of live cells from regions of interest in complex ex vivo systems, including primary human tissues. The use of a highly sensitive 4-nitrophenyl(benzofuran) cage coupled to a set of nanobodies allows high-resolution photo-uncaging of different cell types in areas of interest. Single-cell RNA-sequencing of spatially defined CD8+ T cells is used to exemplify the feasibility of identifying location-dependent cell states. The technology described here provides a valuable tool for the analysis of spatially defined cells in diverse biological systems, including clinical samples.

Checkpoint blockade cancer immunotherapy targets tumour-specific mutant antigens.

The immune system influences the fate of developing cancers by not only functioning as a tumour promoter that facilitates cellular transformation, promotes tumour growth and sculpts tumour cell immunogenicity, but also as an extrinsic tumour suppressor that either destroys developing tumours or restrains their expansion. Yet, clinically apparent cancers still arise in immunocompetent individuals in part as a consequence of cancer-induced immunosuppression. In many individuals, immunosuppression is mediated by cytotoxic T-lymphocyte associated antigen-4 (CTLA-4) and programmed death-1 (PD-1), two immunomodulatory receptors expressed on T cells. Monoclonal-antibody-based therapies targeting CTLA-4 and/or PD-1 (checkpoint blockade) have yielded significant clinical benefits-including durable responses--to patients with different malignancies. However, little is known about the identity of the tumour antigens that function as the targets of T cells activated by checkpoint blockade immunotherapy and whether these antigens can be used to generate vaccines that are highly tumour-specific. Here we use genomics and bioinformatics approaches to identify tumour-specific mutant proteins as a major class of T-cell rejection antigens following anti-PD-1 and/or anti-CTLA-4 therapy of mice bearing progressively growing sarcomas, and we show that therapeutic synthetic long-peptide vaccines incorporating these mutant epitopes induce tumour rejection comparably to checkpoint blockade immunotherapy. Although mutant tumour-antigen-specific T cells are present in progressively growing tumours, they are reactivated following treatment with anti-PD-1 and/or anti-CTLA-4 and display some overlapping but mostly treatment-specific transcriptional profiles, rendering them capable of mediating tumour rejection. These results reveal that tumour-specific mutant antigens are not only important targets of checkpoint blockade therapy, but they can also be used to develop personalized cancer-specific vaccines and to probe the mechanistic underpinnings of different checkpoint blockade treatments.

Antigen-specific TIL therapy for melanoma: A flexible platform for personalized cancer immunotherapy.

Tumor infiltrating lymphocyte (TIL) therapy has shown objective clinical response rates of 50% in stage IV melanoma patients in a number of clinical trials. Nevertheless, the majority of patients progress either directly upon therapy or after an initial period of tumor control. Recent data have shown that most TIL products that are used for therapy contain only low frequencies of T cells reactive against known melanoma-associated epitopes. Because of this, the development of a technology to create T-cell products that are enriched for reactivity against defined melanoma-associated antigens would seem valuable, both to evaluate the tumoricidal potential of T cells directed against different antigen classes and to potentially increase response rates. Here, we developed and validated a conditional MHC streptamer-based platform for the creation of TIL products with defined antigen reactivities. We have used this platform to successfully enrich both high-frequency (≥1%) and low-frequency (<1%) tumor-specific CD8(+) T-cell populations, and thereby created T-cell products with enhanced tumor recognition potential. Collectively, these data demonstrate that selection of antigen-specific T-cell populations can be used to create defined T-cell products for clinical use. This strategy thus forms a highly flexible platform for the development of antigen-specific cell products for personalized cancer immunotherapy.

Altered peptide ligands revisited: vaccine design through chemically modified HLA-A2-restricted T cell epitopes.

Virus or tumor Ag-derived peptides that are displayed by MHC class I molecules are attractive starting points for vaccine development because they induce strong protective and therapeutic cytotoxic T cell responses. In thus study, we show that the MHC binding and consequent T cell reactivity against several HLA-A*02 restricted epitopes can be further improved through the incorporation of nonproteogenic amino acids at primary and secondary anchor positions. We screened more than 90 nonproteogenic, synthetic amino acids through a range of epitopes and tested more than 3000 chemically enhanced altered peptide ligands (CPLs) for binding affinity to HLA-A*0201. With this approach, we designed CPLs of viral epitopes, of melanoma-associated Ags, and of the minor histocompatibility Ag UTA2-1, which is currently being evaluated for its antileukemic activity in clinical dendritic cell vaccination trials. The crystal structure of one of the CPLs in complex with HLA-A*0201 revealed the molecular interactions likely responsible for improved binding. The best CPLs displayed enhanced affinity for MHC, increasing MHC stability and prolonging recognition by Ag-specific T cells and, most importantly, they induced accelerated expansion of antitumor T cell frequencies in vitro and in vivo as compared with the native epitope. Eventually, we were able to construct a toolbox of preferred nonproteogenic residues with which practically any given HLA-A*02 restricted epitope can be readily optimized. These CPLs could improve the therapeutic outcome of vaccination strategies or can be used for ex vivo enrichment and faster expansion of Ag-specific T cells for transfer into patients.

HLA micropolymorphisms strongly affect peptide-MHC multimer-based monitoring of antigen-specific CD8+ T cell responses.

Peptide-MHC (pMHC) multimers have become one of the most widely used tools to measure Ag-specific T cell responses in humans. With the aim of understanding the requirements for pMHC-based personalized immunomonitoring, in which individuals expressing subtypes of the commonly studied HLA alleles are encountered, we assessed how the ability to detect Ag-specific T cells for a given peptide is affected by micropolymorphic differences between HLA subtypes. First, analysis of a set of 10 HLA-A*02:01-restricted T cell clones demonstrated that staining with pMHC multimers of seven distinct subtypes of the HLA-A*02 allele group was highly variable and not predicted by sequence homology. Second, to analyze the effect of minor sequence variation in a clinical setting, we screened tumor-infiltrating lymphocytes of an HLA-A*02:06 melanoma patient with either subtype-matched or HLA-A*02:01 multimers loaded with 145 different melanoma-associated Ags. This revealed that of the four HLA-A*02:06-restricted melanoma-associated T cell responses observed in this patient, two responses were underestimated and one was overlooked when using subtype-mismatched pMHC multimer collections. To our knowledge, these data provide the first demonstration of the strong effect of minor sequence variation on pMHC-based personalized immunomonitoring, and they provide tools to prevent this issue for common variants within the HLA-A*02 allele group.

Distinct spatiotemporal dynamics of CD8+ T cell-derived cytokines in the tumor microenvironment.

Cells in the tumor microenvironment (TME) influence each other through secretion and sensing of soluble mediators, such as cytokines and chemokines. While signaling of interferon γ (IFNγ) and tumor necrosis factor α (TNFα) is integral to anti-tumor immune responses, our understanding of the spatiotemporal behavior of these cytokines is limited. Here, we describe a single cell transcriptome-based approach to infer which signal(s) an individual cell has received. We demonstrate that, contrary to expectations, CD8+ T cell-derived IFNγ is the dominant modifier of the TME relative to TNFα. Furthermore, we demonstrate that cell pools that show abundant IFNγ sensing are characterized by decreased expression of transforming growth factor ß (TGFß)-induced genes, consistent with IFNγ-mediated TME remodeling. Collectively, these data provide evidence that CD8+ T cell-secreted cytokines should be categorized into local and global tissue modifiers, and describe a broadly applicable approach to dissect cytokine and chemokine modulation of the TME.

Development of a hypersensitive periodate-cleavable amino acid that is methionine- and disulfide-compatible and its application in MHC exchange reagents for T cell characterisation.

Incorporation of cleavable linkers into peptides and proteins is of particular value in the study of biological processes. Here we describe the synthesis of a cleavable linker that is hypersensitive to oxidative cleavage as the result of the periodate reactivity of a vicinal amino alcohol moiety. Two strategies directed towards the synthesis of a building block suitable for solid-phase peptide synthesis were developed: a chemoenzymatic route, involving L-threonine aldolase, and an enantioselective chemical route; these led to α,γ-diamino-ß-hydroxybutanoic acids in diastereoisomerically mixed and enantiopure forms, respectively. Incorporation of the 1,2-amino alcohol linker into the backbone of a peptide generated a conditional peptide that was rapidly cleaved at very low concentrations of sodium periodate. This cleavable peptide ligand was applied in the generation of MHC exchange reagents for the detection of antigen-specific T cells in peripheral blood cells. The extremely low concentration of periodate required to trigger MHC peptide exchange allowed the co-oxidation of methionine and disulfide residues to be avoided. Conditional MHC reagents hypersensitive to periodate can now be applied without limitations when UV irradiation is undesired or less practical.

Design and use of conditional MHC class I ligands.

Major histocompatibility complex (MHC) class I molecules associate with a variety of peptide ligands during biosynthesis and present these ligands on the cell surface for recognition by cytotoxic T cells. We have designed conditional MHC ligands that form stable complexes with MHC molecules but degrade on command, by exposure to a defined photostimulus. 'Empty MHC molecules' generated in this manner can be loaded with arrays of peptide ligands to determine MHC binding properties and to monitor antigen-specific T-cell responses in a high-throughput manner. We document the value of this approach by identifying cytotoxic T-cell epitopes within the H5N1 influenza A/Vietnam/1194/04 genome.

An early HIV mutation within an HLA-B*57-restricted T cell epitope abrogates binding to the killer inhibitory receptor 3DL1.

Mutations within MHC class I-restricted epitopes have been studied in relation to T cell-mediated immune escape, but their impact on NK cells via interaction with killer Ig-like receptors (KIRs) during early HIV infection is poorly understood. In two patients acutely infected with HIV-1, we observed the appearance of a mutation within the B*57-restricted TW10 epitope (G9E) that did not facilitate strong escape from T cell recognition. The NK cell receptor KIR3DL1, carried by these patients, is known to recognize HLA-B*5703 and is associated with good control of HIV-1. Therefore, we tested whether the G9E mutation influenced the binding of HLA-B*5703 to soluble KIR3DL1 protein by surface plasmon resonance, and while the wild-type sequence and a second (T3N) variant were recognized, the G9E variant abrogated KIR3DL1 binding. We extended the study to determine the peptide sensitivity of KIR3DL1 interaction with epitopes carrying mutations near the C termini of TW10 and a second HLA-B*57-restricted epitope, IW9. Several amino acid changes interfered with KIR3DL1 binding, the most extreme of which included the G9E mutation commonly selected by HLA-B*57. Our results imply that during HIV-1 infection, some early-emerging variants could affect KIR-HLA interaction, with possible implications for immune recognition.

QPCTL regulates macrophage and monocyte abundance and inflammatory signatures in the tumor microenvironment.

The enzyme glutaminyl-peptide cyclotransferase-like protein (QPCTL) catalyzes the formation of pyroglutamate residues at the NH2-terminus of proteins, thereby influencing their biological properties. A number of studies have implicated QPCTL in the regulation of chemokine stability. Furthermore, QPCTL activity has recently been shown to be critical for the formation of the high-affinity SIRPα binding site of the CD47 "don't-eat-me" protein. Based on the latter data, interference with QPCTL activity -and hence CD47 maturation-may be proposed as a means to promote anti-tumor immunity. However, the pleiotropic activity of QPCTL makes it difficult to predict the effects of QPCTL inhibition on the tumor microenvironment (TME). Using a syngeneic mouse melanoma model, we demonstrate that QPCTL deficiency alters the intra-tumoral monocyte-to-macrophage ratio, results in a profound increase in the presence of pro-inflammatory cancer-associated fibroblasts (CAFs) relative to immunosuppressive TGF-ß1-driven CAFs, and leads to an increased IFN and decreased TGF-ß transcriptional response signature in tumor cells. Importantly, the functional relevance of the observed TME remodeling is demonstrated by the synergy between QPCTL deletion and anti PD-L1 therapy, sensitizing an otherwise refractory melanoma model to anti-checkpoint therapy. Collectively, these data provide support for the development of strategies to interfere with QPCTL activity as a means to promote tumor-specific immunity.

T cells targeted to TdT kill leukemic lymphoblasts while sparing normal lymphocytes.

Unlike chimeric antigen receptors, T-cell receptors (TCRs) can recognize intracellular targets presented on human leukocyte antigen (HLA) molecules. Here we demonstrate that T cells expressing TCRs specific for peptides from the intracellular lymphoid-specific enzyme terminal deoxynucleotidyl transferase (TdT), presented in the context of HLA-A*02:01, specifically eliminate primary acute lymphoblastic leukemia (ALL) cells of T- and B-cell origin in vitro and in three mouse models of disseminated B-ALL. By contrast, the treatment spares normal peripheral T- and B-cell repertoires and normal myeloid cells in vitro, and in vivo in humanized mice. TdT is an attractive cancer target as it is highly and homogeneously expressed in 80-94% of B- and T-ALLs, but only transiently expressed during normal lymphoid differentiation, limiting on-target toxicity of TdT-specific T cells. TCR-modified T cells targeting TdT may be a promising immunotherapy for B-ALL and T-ALL that preserves normal lymphocytes.

Preclinical development of highly effective and safe DNA vaccines directed against HPV 16 E6 and E7.

To allow vaccination irrespective of HLA type, DNA vaccines encoding full-length antigens are required. However, here, we demonstrate that the immunogenicity of DNA vaccines encoding the full-length human papillomavirus (HPV) type 16 E7 and E6 proteins is highly reduced compared to vaccines encoding only the immunodominant epitope. Furthermore, the low remaining immunogenicity is essentially lost for both E7 and E6 when a nononcogenic "gene-shuffled" variant is utilized. To address these issues, we tested whether alterations in transgene design can restore the immunogenicity of full-length and gene-shuffled DNA vaccines. Remarkably, genetic fusion of E7 with tetanus toxin fragment C (TTFC) resulted in a dramatic increase in immunogenicity both for the full-length and the gene-shuffled version of E7. Moreover, the TTFC fusion vaccines were more immunogenic than a vaccine encoding a fusion of E7 and mycobacterial heat shock protein-70, which has recently been tested in a clinical trial. Interestingly, vaccination with these TTFC fusion vaccines also resulted in extremely persistent T-cell responses. The E7-specific CD8(+) T cells induced by TTFC fusion vaccines were functional in terms of IFN-γ production, formation of immunological memory, in vivo cytolytic activity and tumor eradication. Finally, we show that genetic fusion with TTFC also improves the immunogenicity of a gene-shuffled E6 DNA vaccine. These data demonstrate that genetic fusion with tetanus toxin fragment C can dramatically improve the immunogenicity of full-length and gene-shuffled DNA vaccines. The DNA fusion vaccines developed here will be evaluated for the treatment of HPV-positive carcinomas in future studies.

Conditional MHC class I ligands and peptide exchange technology for the human MHC gene products HLA-A1, -A3, -A11, and -B7.

Major histocompatibility complex (MHC) class I multimer technology has become an indispensable immunological assay system to dissect antigen-specific cytotoxic CD8(+) T cell responses by flow cytometry. However, the development of high-throughput assay systems, in which T cell responses against a multitude of epitopes are analyzed, has been precluded by the fact that for each T cell epitope, a separate in vitro MHC refolding reaction is required. We have recently demonstrated that conditional ligands that disintegrate upon exposure to long-wavelength UV light can be designed for the human MHC molecule HLA-A2. To determine whether this peptide-exchange technology can be developed into a generally applicable approach for high throughput MHC based applications we set out to design conditional ligands for the human MHC gene products HLA-A1, -A3, -A11, and -B7. Here, we describe the development and characterization of conditional ligands for this set of human MHC molecules and apply the peptide-exchange technology to identify melanoma-associated peptides that bind to HLA-A3 with high affinity. The conditional ligand technology developed here will allow high-throughput MHC-based analysis of cytotoxic T cell immunity in the vast majority of Western European individuals.

CRASH-IT Switch Enables Reversible and Dose-Dependent Control of TCR and CAR T-cell Function.

Adoptive transfer of genetically modified or donor-derived T cells can efficiently eradicate human tumors but is also frequently associated with major toxicity. There are several switches that can be used to kill the infused cell pool in the case of major toxicity, but the irreversible nature of these suicide switches means that the therapeutic effect is lost when they are used. To address this issue, we engineered a small-molecule responsive genetic safety switch that in the absence of drug robustly blocked cytotoxicity and cytokine expression of primary human T cells. Upon administration of drug, T-cell functions were restored in a reversible and titratable manner. We showed that this T-cell switch was universal, as it could be combined with endogenous or transduced T-cell receptors (TCR), as well as chimeric antigen receptors. The modular nature of the Chemically Regulated - SH2-delivered Inhibitory Tail (CRASH-IT) switch concept, in which inhibitory domains are brought to activating immune receptors in a controlled manner, makes it a versatile platform to regulate the activity of cell products that signal through immunoreceptor tyrosine-based activation motif (ITAM)-containing receptors.

Labeling and tracking of immune cells in ex vivo human skin.

Human skin harbors various immune cells that are crucial for the control of injury and infection. However, the current understanding of immune cell function within viable human skin tissue is limited. We developed an ex vivo imaging approach in which fresh skin biopsies are mounted and then labeled with nanobodies or antibodies against cell surface markers on tissue-resident memory CD8+ T cells, other immune cells of interest, or extracellular tissue components. Subsequent longitudinal imaging allows one to describe the dynamic behavior of human skin-resident cells in situ. In addition, this strategy can be used to study immune cell function in murine skin. The ability to follow the spatiotemporal behavior of CD8+ T cells and other immune cells in skin, including their response to immune stimuli, provides a platform to investigate physiological immune cell behavior and immune cell behavior in skin diseases. The mounting, staining and imaging of skin samples requires ~1.5 d, and subsequent tracking analysis requires a minimum of 1 d. The optional production of fluorescently labeled nanobodies takes ~5 d.

Identification and characterization of a SARS-CoV-2 specific CD8+ T cell response with immunodominant features.

The COVID-19 pandemic caused by SARS-CoV-2 is a continuous challenge worldwide, and there is an urgent need to map the landscape of immunogenic and immunodominant epitopes recognized by CD8+ T cells. Here, we analyze samples from 31 patients with COVID-19 for CD8+ T cell recognition of 500 peptide-HLA class I complexes, restricted by 10 common HLA alleles. We identify 18 CD8+ T cell recognized SARS-CoV-2 epitopes, including an epitope with immunodominant features derived from ORF1ab and restricted by HLA-A*01:01. In-depth characterization of SARS-CoV-2-specific CD8+ T cell responses of patients with acute critical and severe disease reveals high expression of NKG2A, lack of cytokine production and a gene expression profile inhibiting T cell re-activation and migration while sustaining survival. SARS-CoV-2-specific CD8+ T cell responses are detectable up to 5 months after recovery from critical and severe disease, and these responses convert from dysfunctional effector to functional memory CD8+ T cells during convalescence.

Long-distance modulation of bystander tumor cells by CD8+ T cell-secreted IFNγ.

T cell-secreted IFNγ can exert pleiotropic effects on tumor cells that include induction of immune checkpoints and antigen presentation machinery components, and inhibition of cell growth. Despite its role as key effector molecule, little is known about the spatiotemporal spreading of IFNγ secreted by activated CD8+ T cells within the tumor environment. Using multiday intravital imaging, we demonstrate that T cell recognition of a minor fraction of tumor cells leads to sensing of IFNγ by a large part of the tumor mass. Furthermore, imaging of tumors in which antigen-positive and -negative tumor cells are separated in space reveals spreading of the IFNγ response, reaching distances of >800 µm. Notably, long-range sensing of IFNγ can modify tumor behavior, as both shown by induction of PD-L1 expression and inhibition of tumor growth. Collectively, these data reveal how, through IFNγ, CD8+ T cells modulate the behavior of remote tumor cells, including antigen-loss variants.

UV-induced ligand exchange in MHC class I protein crystals.

High-throughput structure determination of protein-ligand complexes is central in drug development and structural proteomics. To facilitate such high-throughput structure determination we designed an induced replacement strategy. Crystals of a protein complex bound to a photosensitive ligand are exposed to UV light, inducing the departure of the bound ligand, allowing a new ligand to soak in. We exemplify the approach for a class of protein complexes that is especially recalcitrant to high-throughput strategies: the MHC class I proteins. We developed a UV-sensitive, "conditional", peptide ligand whose UV-induced cleavage in the crystals leads to the exchange of the low-affinity lytic fragments for full-length peptides introduced in the crystallant solution. This "in crystallo" exchange is monitored by the loss of seleno-methionine anomalous diffraction signal of the conditional peptide compared to the signal of labeled MHC beta2m subunit. This method has the potential to facilitate high-throughput crystallography in various protein families.