Αβ T-cell Receptor Research Articles

Human γδ T cells recognize CD1b by two distinct mechanisms

γδ T cells form an abundant part of the human cellular immune system, where they respond to tissue damage, infection, and cancer. The spectrum of known molecular targets recognized by Vδ1-expressing γδ T cells is becoming increasingly diverse. Here we describe human γδ T cells that recognize CD1b, a lipid antigen-presenting molecule, which is inducibly expressed on monocytes and dendritic cells. Using CD1b tetramers to study multiple donors, we found that many CD1b-specific γδ T cells use Vδ1. Despite their common use of Vδ1, three CD1b-specific γδ T cell receptors (TCRs) showed clear differences in the surface of CD1b recognized, the requirement for lipid antigens, and corecognition of butryophilin-like proteins. Several Vγ segments were present among the CD1b-specific TCRs, but chain swap experiments demonstrated that CD1b specificity was mediated by the Vδ1 chain. One of the CD1b-specific Vδ1+ TCRs paired with Vγ4 and shows dual reactivity to CD1b and butyrophilin-like proteins. αβ TCRs typically recognize the peptide display platform of MHC proteins. In contrast, our results demonstrate the use of rearranged receptors to mediate diverse modes of recognition across the surface of CD1b in ways that do and do not require carried lipids.

Crystal structure of the ternary complex of TCR, MHC class I and lipopeptides.

The covalent conjugation of a 14-carbon fatty acid (myristic acid) to the N-terminal Gly residue, termed N-myristoylation, occurs in some viral proteins to dictate their pathological function. This protein lipidation reaction, however, is monitored by host cytotoxic T lymphocytes that are capable of recognizing N-terminal lipopeptide fragments in the context of major histocompatibility complex (MHC) class I molecules. In a rhesus model of human AIDS, for example, the classical MHC class I allomorph, Mamu-B*05104, was shown to bind SIV Nef-derived 4-mer lipopeptides (myristic acid-Gly-Gly-Ala-Ile; C14nef4) and present them to the CD8+ T-cell line, SN45. These lipopeptides accommodated in MHC class I molecules expose much shorter peptide chains than conventional MHC class I-presented 8-10-mer peptides, and the molecular mechanisms by which αβ T-cell receptors (TCRs) recognize lipopeptides currently remain unclear. An X-ray crystallographic analysis of the SN45 TCR α and β heterodimer in a form that was co-crystallized with the C14nef4-bound Mamu-B*05104 complex indicated that the amide group of the N-myristoylated glycine residue offered a primary T-cell epitope by establishing a sole hydrogen bond between its nitrogen atom and the side chain of Glu at position 101 of CDR3β. Accordingly, the Glu to Ala mutation at this position resulted in the loss of lipopeptide recognition. On the other hand, TCRs were positioned remotely from the peptide portion of C14nef4, and strong interactions were not observed. Thus, these observations provide novel structural insights into lipopeptide recognition by TCRs, which contrast sharply with the general molecular principle of peptide recognition.

Peptide cargo tunes a network of correlated motions in human leucocyte antigens.

Most biomolecular interactions are typically thought to increase the (local) rigidity of a complex, for example, in drug‐target binding. However, detailed analysis of specific biomolecular complexes can reveal a more subtle interplay between binding and rigidity. Here, we focussed on the human leucocyte antigen (HLA), which plays a crucial role in the adaptive immune system by presenting peptides for recognition by the αβ T‐cell receptor (TCR). The role that the peptide plays in tuning HLA flexibility during TCR recognition is potentially crucial in determining the functional outcome of an immune response, with obvious relevance to the growing list of immunotherapies that target the T‐cell compartment. We have applied high‐pressure/temperature perturbation experiments, combined with molecular dynamics simulations, to explore the drivers that affect molecular flexibility for a series of different peptide–HLA complexes. We find that different peptide sequences affect peptide–HLA flexibility in different ways, with the peptide cargo tuning a network of correlated motions throughout the pHLA complex, including in areas remote from the peptide‐binding interface, in a manner that could influence T‐cell antigen discrimination.

Very rapid cloning, expression and identifying specificity of T-cell receptors for T-cell engineering.

Neoantigens can be predicted and in some cases identified using the data obtained from the whole exome sequencing and transcriptome sequencing of tumor cells. These sequencing data can be coupled with single-cell RNA sequencing for the direct interrogation of the transcriptome, surfaceome, and pairing of αβ T-cell receptors (TCRαβ) from hundreds of single T cells. Using these 2 large datasets, we established a platform for identifying antigens recognized by TCRαβs obtained from single T cells. Our approach is based on the rapid expression of cloned TCRαβ genes as Sleeping Beauty transposons and the determination of the introduced TCRαβs’ antigen specificity and avidity using a reporter cell line. The platform enables the very rapid identification of tumor-reactive TCRs for the bioengineering of T cells with redirected specificity.

A different way for γδ T cells to bind

Immunology The ligands bound by γδ T cell receptors (TCRs) are less well characterized than those of their αβ TCR cousins, which are antigens presented by major histocompatibility complex (MHC) and related proteins. Le Nours et al. identified a phenotypically diverse γδ T cell subset in human tissues that reacts to MHC-related protein 1 (MR1), which presents vitamin B derivatives. A crystal structure of a γδ TCR–MR1–antigen complex revealed that some of these TCRs can bind underneath the MR1 antigen-binding cleft instead of recognizing the presented antigen. This work thus uncovers an additional ligand for γδ T cells and reconceptualizes the nature of T cell antigen recognition. Science , this issue p. [1522][1] [1]: /lookup/doi/10.1126/science.aav3900

The structural basis of T-cell receptor (TCR) activation: An enduring enigma.

T cells are critical for protective immune responses to pathogens and tumors. The T-cell receptor (TCR)-CD3 complex is composed of a diverse αβ TCR heterodimer noncovalently associated with the invariant CD3 dimers CD3ϵγ, CD3ϵδ, and CD3ζζ. The TCR mediates recognition of antigenic peptides bound to MHC molecules (pMHC), whereas the CD3 molecules transduce activation signals to the T cell. Whereas much is known about downstream T-cell signaling pathways, the mechanism whereby TCR engagement by pMHC is first communicated to the CD3 signaling apparatus, a process termed early T-cell activation, remains largely a mystery. In this review, we examine the molecular basis for TCR activation in light of the recently determined cryoEM structure of a complete TCR-CD3 complex. This structure provides an unprecedented opportunity to assess various signaling models that have been proposed for the TCR. We review evidence from single-molecule and structural studies for force-induced conformational changes in the TCR-CD3 complex, for dynamically-driven TCR allostery, and for pMHC-induced structural changes in the transmembrane and cytoplasmic regions of CD3 subunits. We identify major knowledge gaps that must be filled in order to arrive at a comprehensive model of TCR activation that explains, at the molecular level, how pMHC-specific information is transmitted across the T-cell membrane to initiate intracellular signaling. An in-depth understanding of this process will accelerate the rational design of immunotherapeutic agents targeting the TCR-CD3 complex.

On the immune response to barley in celiac disease: Biased and public T-cell receptor usage to a barley unique and immunodominant gluten epitope.

Celiac disease (CeD) is driven by CD4+ T-cell responses to dietary gluten proteins of wheat, barley, and rye when deamidated gluten epitopes are presented by certain disease-associated HLA-DQ allotypes. About 90% of the CeD patients express HLA-DQ2.5. In such patients, five gluten epitopes dominate the anti-gluten T-cell response; two epitopes unique to wheat, two epitopes present in wheat, barley, and rye and one epitope unique to barley. Despite presence of barley in commonly consumed food and beverages and hence being a prominent source of gluten, knowledge about T-cell responses elicited by barley in CeD is scarce. Therefore, in this study, we explored T-cell response toward the barley unique epitope DQ2.5-hor-3 (PIPEQPQPY) by undertaking HLA-DQ:gluten peptide tetramer staining, single-cell T-cell receptor (TCR) αβ sequencing, T-cell cloning, and T-cell proliferation studies. We demonstrate that majority of the CeD patients generate T-cell response to DQ2.5-hor-3, and this response is characterized by clonal expansion, preferential TCR V-gene usage and public TCR features thus echoing findings previously made for wheat gluten epitopes. The knowledge that biased and public TCRs underpin the T-cell response to all the immunodominant gluten epitopes in CeD suggests that such T cells are promising diagnostic and therapeutic targets.

Efficacy Proof of Concept for Allogeneic CD123 Targeting CAR T-Cells Against Primary Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN): Efficient Control of Tumor Progression in PDX Model and Potential Loss of CD123 Expression in Relapsed Disease

Efficacy Proof of Concept for Allogeneic CD123 Targeting CAR T-Cells Against Primary Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN): Efficient Control of Tumor Progression in PDX Model and Potential Loss of CD123 Expression in Relapsed Disease

CD3+CD4–CD8– Double-negative αβ T cells attenuate lung ischemia-reperfusion injury

ObjectiveLung ischemia-reperfusion injury (IRI) is a common complication after lung transplantation, and immune cells have been implicated in modulating outcomes. We hypothesized that a newly described subset of αβ T-cell receptor positive cells; that is, CD4–CD8– (double negative [DN]) T cells, are found in lungs and can protect against lung IRI. MethodsIschemia was induced in C57BL/6 mice by left pulmonary artery and vein occlusion for 30 minutes followed by 180 minutes of reperfusion. These mice were paired with sham hilar dissected surgical controls. In mice undergoing IRI, adoptive transfer of DN T cells or conventional T cells was performed 12 hours before occlusion. Flow cytometry was used to quantify T cells and inflammatory cytokines, and apoptotic signaling pathways were evaluated with immunoblotting. Lung injury was assessed with Evans blue dye extravasation. ResultsDN T cells were significantly higher (5.29% ± 1% vs 2.21% ± 3%; P < .01) in IRI lungs and secreted higher levels of interleukin-10 (30% ± 5% vs 6% ± 1%; P < .01) compared with surgical sham controls. Immunoblotting, hematoxylin and eosin staining and Evans blue dye demonstrated that adoptive transfer of DN T cells significantly decreased interstitial edema (P < .01) and attenuated apoptosis/cleaved caspase-3 expression in the lungs following lung IRI (P < .01). ConclusionsDN T cells traffic into lungs during IRI, and have tissue protective functions regulating inflammation and apoptosis. We propose a potential novel immunoregulatory function of DN T cells during lung IRI.

Abstract 023: γδ T Cells Mediate αβ T Cell Activation in Angiotensin II-Induced Hypertension

Objectives: Both innate and adaptive immune cells have been shown to play a role in hypertension and vascular injury. Recently, we demonstrated that a small subset of “innate-like” T lymphocytes, expressing the γδ T cell receptor (TCR) rather than the αβ TCR, plays a key role in hypertension and vascular injury. We demonstrated an increased number and activation (CD69 + ) of αβ and γδ T cells during the development of hypertension caused by angiotensin (Ang) II infusion, and that deficiency in γδ T cells prevented Ang II-induced hypertension, resistance artery endothelial dysfunction and spleen T-cell activation in mice. We hypothesized that γδ T cells mediate activation of other T cells in hypertension. Methods: C57BL/6 male mice were infused with Ang II (490 ng/kg/min, SC) for 7 and 14 days (n=5-7). All mice were 14-15 week-old at the end of the study. Spleen T cell profile was determined by flow cytometry. γδ and αβ T cells were isolated using magnetic beads from peripheral lymph nodes and spleen from C57BL/6 male mice treated or not with Ang II for 14 days. αβ T cells were cultured alone or with γδ T cells (5:1) in presence of anti-CD3 antibodies plus or minus Ang II, and αβ T cell phenotype was evaluated by flow cytometry. Results: Close correlations were demonstrated between the number (#) of activated CD69 + γδ T cells and CD4 + CD69 + T cells (r 2 =0.74, P &lt;0.01) and CD8 + CD69 + T cells (r 2 =0.64, P &lt;0.01) after 7-day Ang II infusion. Correlations were also shown between the # of CD27 + CD69 + γδ T cells and CD4 + CD69 + T cells (r 2 =0.76, P &lt;0.001) and CD8 + CD69 + T cells (r 2 =0.65, P &lt;0.01) after 7-day Ang II infusion. In vitro , Ang II increased the fraction of CD69 + αβ T cells when αβ T cells were co-cultured with γδ T cells isolated from control mice (% of αβ T cells: 19.9±2.9 vs. 16.4±2.6, P &lt;0.001) but not when cultured alone. The fractions of CD69 + (% of CD69 + αβ T cells: 35.7±2.7 vs. 18.9±1.5, P &lt;0.001) and CCR6 + αβ T cells (% of CCR6 + αβ T cells: 27.6±2.4 vs. 19.2±3.5, P &lt;0.05) were increased when αβ T cells were co-cultured with γδ T cells isolated from Ang II-infused compared to control mice. Conclusions: These results suggest that γδ T cells mediate activation of αβ T cells in Ang II-induced hypertension. Targeting γδ T cells may contribute to reduce the low-grade inflammation found in hypertension.

Abstract 126: Role of V γ6 + γδ T Cells in Angiotensin Il-Induced Hypertension and Vascular Injury

Introduction: Both innate and adaptive immune cells (such as T lymphocytes) have been shown to play a role in hypertension and vascular injury. Recently, we demonstrated that a small subpopulation of T cells considered "innate-like", expressing the γδ T-cell receptor (TCR) rather than the much more frequent αβ TCR, plays a key role in hypertension and vascular injury. The number and activation of γδ T cells were increased after 7 days of angiotensin (Ang) II infusion, and absence of γδ T cells prevented the development of hypertension, endothelial dysfunction of resistance arteries, and activation of CD4 + and CD8 + T cells in mice infused with Ang II for 14 days. γδ T cells can be subdivided according to the TCR variant (V) subtype that is generally specific for a tissue. A subpopulation of γδ T cells in the lungs and skin that are Vγ6 + and produce interleukin (IL)-17A was shown to respond promptly to pneumococcal infection and skin inflammation. However, γδ T cell Vγ subtypes involved in hypertension are still unknown. We hypothesized that γδ T cells involved in hypertension are Vγ6 + . Methods: Eleven- to 13-week old C57BL/6J male mice were infused or not with Ang II (490 ng /kg /min, sc) for 14 days (n = 5-14), and γδ T cell Vγ subtypes were profiled by flow cytometry in the spleen, mesenteric lymph nodes (MLNs), thoracic aortic (TA), perivascular adipose tissue (PVAT) and mesenteric artery (MA) PVAT. Results: In spleen and MLNs the most abundant γδ T cell Vγ subtypes were Vγ1,2 + and Vγ4 + followed by Vγ6 + , Vγ5 + and Vγ7 + . In TA PVAT, the most abundant γδ T cell Vγ subtype was Vγ6 + followed by Vγ4 + , Vγ1,2 + , Vγ5 + and Vγ7 + . In MA PVAT, the most abundant γδ T cell Vγ subtype was Vγ6 + followed by Vγ4 + , Vγ7 + , Vγ5 + and Vγ1,2 + . Ang II infusion increased the frequency of Vγ6 + γδ T cells in the spleen (% of CD3 + T cells: 0.77±0.09 vs. 0.5±0.02, P &lt;0.01) and TA PVAT 5.9±0.8 vs. 3.6±0.4, P &lt;0.01), whereas it only tended to increase in MA PVAT 14.5±1.7 vs. 10.2± 1.9, P =0.07). The Vγ6 + γδ T cell frequency in MLNs was unaffected by Ang II infusion. Conclusion: A differential distribution of γδ T cell Vγ subtypes was observed in lymphoid organs compared to PVAT. Vγ6 + γδ T cells may play a role in hypertension. Targeting Vγ6 + γδ T cells could be a therapeutic approach to reduce inflammation in hypertension.

Unique T-Cell Populations Define Immune-Inflamed Hepatocellular Carcinoma.

Background & AimsThe characterization of T cells infiltrating hepatocellular carcinoma (HCC) provides information on cancer immunity and also on selection of patients with precise indication of immunotherapy. The aim of the study was to characterize T-cell populations within tumor tissue and compare them with non-neoplastic liver tissue as well as circulating cells of the same patients.MethodsThe presence of unique cell populations was investigated in 36 HCC patients by multidimensional flow cytometry followed by t-distributed stochastic neighbor embedding analysis. Functional activity of tumor-infiltrating T cells was determined after activation by phorbol 12-myristate 13-acetate and ionomycin.ResultsWithin the tumor there were more cells expressing CD137 and ICOS than in non-neoplastic liver tissue, possibly after recent antigenic activation. These cells contained several populations, including the following: (1) functionally impaired, proliferating CD4+ cells co-expressing Inducible T-cell costimulator (ICOS) and T cell immunoreceptor with Ig and ITIM domains (TIGIT); (2) functionally active CD8+ cells co-expressing CD38 and Programmed cell-death protein 1 (PD1); and (3) CD4-CD8 double-negative T-cell receptor αβ and γδ cells (both non–major histocompatibility complex–restricted T cells). When the identified clusters were compared with histologic classification performed on the same samples, an accumulation of activated T cells was observed in immune-inflamed HCC. The same analyses performed in 7 patients receiving nivolumab treatment showed a remarkable reduction in the functionally impaired CD4+ cells, which returned to almost normal activity over time.ConclusionsUnique populations of activated T cells are present in HCC tissue, whose antigen specificity remains to be investigated. Some of these cell populations are functionally impaired and nivolumab treatment restores their responsiveness. The finding of ongoing immune response within the tumor shows which lymphocyte populations are impaired within the HCC and identifies the patients who might take benefit from immunotherapy.

The TCR is an allosterically regulated macromolecular machinery changing its conformation while working.

The αβ T-cell receptor (TCR) is a multiprotein complex controlling the activation of T cells. Although the structure of the complete TCR is not known, cumulative evidence supports that the TCR cycles between different conformational states that are promoted either by thermal motion or by force. These structural transitions determine whether the TCR engages intracellular effectors or not, regulating TCR phosphorylation and signaling. As for other membrane receptors, ligand binding selects and stabilizes the TCR in active conformations, and/or switches the TCR to activating states that were not visited before ligand engagement. Here we review the main models of TCR allostery, that is, ligand binding at TCRαβ changes the structure at CD3 and ζ. (a) The ITAM and proline-rich sequence exposure model, in which the TCR's cytoplasmic tails shield each other and ligand binding exposes them for phosphorylation. (b) The membrane-ITAM model, in which the CD3ε and ζ tails are sequestered inside the membrane and again ligand binding exposes them. (c) The mechanosensor model in which ligand binding exerts force on the TCR, inducing structural changes that allow signaling. Since these models are complementary rather than competing, we propose a unified model that aims to incorporate all existing data.

Single T Cell Sequencing Demonstrates the Functional Role of αβ TCR Pairing in Cell Lineage and Antigen Specificity.

Although structural studies of individual T cell receptors (TCRs) have revealed important roles for both the α and β chain in directing MHC and antigen recognition, repertoire-level immunogenomic analyses have historically examined the β chain alone. To determine the amount of useful information about TCR repertoire function encoded within αβ pairings, we analyzed paired TCR sequences from nearly 100,000 unique CD4+ and CD8+ T cells captured using two different high-throughput, single-cell sequencing approaches. Our results demonstrate little overlap in the healthy CD4+ and CD8+ repertoires, with shared TCR sequences possessing significantly shorter CDR3 sequences corresponding to higher generation probabilities. We further utilized tools from information theory and machine learning to show that while α and β chains are only weakly associated with lineage, αβ pairings appear to synergistically drive TCR-MHC interactions. Vαβ gene pairings were found to be the TCR feature most informative of T cell lineage, supporting the existence of germline-encoded paired αβ TCR-MHC interaction motifs. Finally, annotating our TCR pairs using a database of sequences with known antigen specificities, we demonstrate that approximately a third of the T cells possess α and β chains that each recognize different known antigens, suggesting that αβ pairing is critical for the accurate inference of repertoire functionality. Together, these findings provide biological insight into the functional implications of αβ pairing and highlight the utility of single-cell sequencing in immunogenomics.

Coeliac Disease and Mast Cells.

Over the last decades, there has been an impressive progress in our understanding of coeliac disease pathogenesis and it has become clear that the disorder is the final result of complex interactions of environmental, genetic, and immunological factors. Coeliac disease is now considered a prototype of T-cell-mediated disease characterized by loss of tolerance to dietary gluten and the targeted killing of enterocytes by T-cell receptor αβ intraepithelial lymphocytes. Accumulating evidence, however, indicates that the induction of a gluten-specific T helper-1 response must be preceded by the activation of the innate immune system. Mast cells are key players of the innate immune response and contribute to the pathogenesis of a multitude of diseases. Here, we review the results of studies aimed at investigating the role of mast cells in the pathogenesis of coeliac disease, showing that these cells increase in number during the progression of the disease and contribute to define a pro-inflammatory microenvironment.

Genesis of the αβ T-cell receptor.

The T-cell (TCR) repertoire relies on the diversity of receptors composed of two chains, called α and β, to recognize pathogens. Using results of high throughput sequencing and computational chain-pairing experiments of human TCR repertoires, we quantitively characterize the αβ generation process. We estimate the probabilities of a rescue recombination of the β chain on the second chromosome upon failure or success on the first chromosome. Unlike β chains, α chains recombine simultaneously on both chromosomes, resulting in correlated statistics of the two genes which we predict using a mechanistic model. We find that ∼35% of cells express both α chains. Altogether, our statistical analysis gives a complete quantitative mechanistic picture that results in the observed correlations in the generative process. We learn that the probability to generate any TCRαβ is lower than 10−12 and estimate the generation diversity and sharing properties of the αβ TCR repertoire.

NMR: an essential structural tool for integrative studies of T cell development, pMHC ligand recognition and TCR mechanobiology.

Early studies of T cell structural biology using X-ray crystallography, surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) focused on a picture of the αβT cell receptor (αβTCR) component domains and their cognate ligands (peptides bound to MHC molecules, i.e. pMHCs) as static interaction partners. Moving forward requires integrating this corpus of data with dynamic technologies such as NMR, molecular dynamics (MD) simulations and real-time single molecule (SM) studies exemplified by optical tweezers (OT). NMR bridges relevant timescales and provides the potential for an all-atom dynamic description of αβTCR components prior to and during interactions with binding partners. SM techniques have opened up vistas in understanding the non-equilibrium nature of T cell signaling through the introduction of force-mediated binding measurements into the paradigm for T cell function. In this regard, bioforces consequent to T-lineage cell motility are now perceived as placing piconewton (pN)-level loads on single receptor-pMHC bonds to impact structural change and αβT-lineage biology, including peptide discrimination, cellular activation, and developmental progression. We discuss herein essential NMR technologies in illuminating the role of ligand binding in the preT cell receptor (preTCR), the αβTCR developmental precursor, and convergence of NMR, SM and MD data in advancing our comprehension of T cell development. More broadly we review the central hypothesis that the αβTCR is a mechanosensor, fostered by breakthrough NMR-based structural insights. Collectively, elucidating dynamic aspects through the integrative use of NMR, SM, and MD shall advance fundamental appreciation of the mechanism of T cell signaling as well as inform translational efforts in αβTCR and chimeric T cell (CAR-T) immunotherapies and T cell vaccinology.

Human Vδ1+ T Cells in the Immune Response to Plasmodium falciparum Infection.

Naturally acquired protective immunity to Plasmodium falciparum malaria is mainly antibody-mediated. However, other cells of the innate and adaptive immune system also play important roles. These include so-called unconventional T cells, which express a γδ T-cell receptor (TCR) rather than the αβ TCR expressed by the majority of T cells—the conventional T cells. The γδ T-cell compartment can be divided into distinct subsets. One expresses a TCR involving Vγ9 and Vδ2, while another major subset uses instead a TCR composed of Vδ1 paired with one of several types of γ chains. The former of these subsets uses a largely semi-invariant TCR repertoire and responds in an innate-like fashion to pyrophosphate antigens generated by various stressed host cells and infectious pathogens, including P. falciparum. In this short review, we focus instead on the Vδ1 subset, which appears to have a more adaptive immunobiology, but which has been much less studied in general and in malaria in particular. We discuss the evidence that Vδ1+ cells do indeed play a role in malaria and speculate on the function and specificity of this cell type, which is increasingly attracting the attention of immunologists.

Abstract A134: Single-cell roadmap of the evolution of T-cell response during anti-LAG3 and anti-PD1 combination treatment in metastatic melanoma patients

Abstract Despite the impressive impact of CTLA4 and PD1-targeted immuno-oncologic (IO) therapies, a large proportion of patients fail to respond. The observed variance in treatment efficacy has been linked to heterogeneity in the immune cell distribution of individual patients. Lymphocyte activation gene 3 (LAG3, CD223) is one of the newest IO targets entering the clinic. Triggering of LAG3 on T-cells by HLA-DR -ligands has a well-established role in the negative regulation of T-cell function. Although for example on activated regulatory T-cells (Treg) LAG3 is widely expressed, the detailed effects of LAG3 on various cell types are still unknown. We profiled over 30,000 CD45+ lymphocytes cells using a novel paired single-cell RNA and T-cell receptor (TCR) αβ chain (10x Genomics) sequencing method for peripheral blood samples from two patients receiving anti-LAG3 and anti-PD1 combination treatment in multicentre phase I trial. The sequenced cells were from IO-treatment naïve metastatic melanoma patients from before, after 4 weeks and after 12 weeks of the start of therapy. For validation, we performed TCRβ-sequencing and flow cytometry analysis of a larger cohort of melanoma patients (n = 12) enrolled in the same study. To gain in-depth understanding of the immune cell subsets, we used a recently described method to seek matching mutual neighbors between patients to normalize the interpatient variation to enable a systematic comparison across patients. To identify phenotypic clusters, we used a graph theory-based clustering method SNN-Cliq and built predictive machine learning classifiers to assess the reproducibility of learnt clusters. After optimizing our choice of input genes and parameters, we identified 16 distinct clusters that define the T-cell roadmap of anti-LAG3 and anti-PD1 treatment that includes 6 CD8+, 8 CD4+ (including Treg cluster), and 2 other clusters. We identified 4 CD8+ T-cell clusters of increasing cytotoxicity profile using a cytotoxicity score and the pseudotime algorithm Monocle3. The most highly cytotoxic clusters increased and a cluster of lower cytotoxicity score decreased during the treatment. In addition, we defined 2 CD8+ exhaustion clusters. During treatment, we observed a decrease in the exhausted T-cell cluster defined by LAG3 and PDCD1 expression, but an increase in TIGIT+ exhaustion cluster. Furthermore, ordering the cluster of FOXP3+ Treg cells along pseudotime trajectory revealed two different fates for Treg cells, where the other fate showed significantly decreased expression of LAG3 and PDCD1, adding evidence of the effect of the treatment on Treg cells. TCRαβ analysis revealed 19 individual expanded clonotypes of size of 100 sequenced individual cells. The true transcriptomic heterogeneity of identical clonotypes was revealed as most clonotypes spanned several of the 16 different clusters, challenging our view of clonal expansion. Furthermore, we were able to assess the temporal phenotypic changes in individual clones during the course of immunotherapy. Most clonotypes, including cytotoxic and exhausted clones, had more homogenous transcriptomes before the start of the treatment, but diversified during the therapy, suggesting a release of immunologic break in these clones. In summary, we defined the evolution of immunological response to anti-LAG3 and anti-PD1 therapy cell by cell, and described an increase in the heterogeneity of cytotoxicity in CD8+ cells, distinct fates of Treg cells and the release of transcriptomic profiles of individual T-cell clonotypes during IO treatment. Citation Format: Jani Huuhtanen, Henna H.E. Hakanen, Tapio Lönnberg, Olli Dufva, Katriina Peltola, Siru Mäkelä, Micaela Hernberg, Petri Bono, Kreutzman Anna, Satu Mustjoki. Single-cell roadmap of the evolution of T-cell response during anti-LAG3 and anti-PD1 combination treatment in metastatic melanoma patients [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A134.

Abstract A154: Flow cytometric analysis of immune responses in the melanoma tissue biopsies before or during anti-PD1 immunotherapy

Abstract Antibodies directed against the programmed cell death protein 1 (PD-1) immune checkpoint have significantly improved survival of patients with advanced metastatic melanoma. Innate and acquired resistance to anti-PD-1 treatment represents a significant treatment obstacle, yet the mechanisms of resistance are poorly understood and remain difficult to model at the cellular level. Here we used multiparameter flow cytometry to examine the immune profiles of 39 tumor biopsies obtained from 31 patients with stage III-IV metastatic melanoma prior to (baseline, n = 21) or during (n = 18) single-agent anti-PD1 immunotherapy; twelve patients developed resistance to treatment. Samples were enzymatically dissociated and cryopreserved until thawed and stained with fluorescently labeled antibodies to enable a comprehensive analysis of melanoma cells and tumor infiltrating lymphocytes (TILs). Mean melanoma cell content was 57+4.7% (mean+s.e.m.) while mean immune infiltrate (CD45 positive cells) was 31.5+4.5%. Analysis of melanoma cell expression of antigen presenting molecules and PD-1 ligands revealed frequent downregulation of HLA-ABC expression in both baseline (9/21, 43%) and on-treatment tumors (10/18, 56%), including a complete HLA-ABC loss in 2/39 samples. HLA-ABC up-regulation was observed in 9/39 (23%) tumors. Expression of HLA-DR and PD-L1 strongly correlated with that of HLA-ABC (Spearman r=0.68 and =0.89, respectively), indicative of local interferon exposure. Unlike PD-L1, melanoma cell expression of PD-L2 was low in the biopsied tissue. In contrast, PD-L2 expression was readily induced by gamma interferon and correlated with HLA-ABC expression in matching melanoma cell lines established from the dissociated biopsies (9/39). We found multiple correlations between parameters characterizing the immune profiles of tumor cells and TILs in both baseline and on-treatment samples. Among significant treatment-associated changes, conventional T-cells bearing the αβ T-cell receptor were 1.4-fold higher in the on-treatment group compared to baseline (65+2.7% versus 46.2+4.1%, P&amp;lt;0.001), while B cells were 7.7-fold lower (1.5+0.4%, versus 11.4+3.9%, P&amp;lt;0.05). We have identified two unusual “hot melanoma, low TILs” samples (HLA-ABC high HLA-DR positive/high PD-L1 high melanoma cells, &amp;lt;10% CD45 positive TILs) that warrant further investigation, in patients with acquired anti-PD-1 resistance. Our findings suggest that the dynamic downregulation of HLA-ABC expression on melanoma cells is more common than complete loss of HLA-ABC expression and is likely to be a key driver of both innate and acquired resistance to immunotherapy. Functional characterization of melanoma infiltrating T-cells is currently under investigation. Citation Format: Elena Shklovskaya, Jenny Lee, Su Yin Lim, Sara Alavi, John Thompson, Robyn Saw, Matteo Carlino, Richard Scolyer, Alexander Menzies, Georgina Long, Richard Kefford, Helen Rizos. Flow cytometric analysis of immune responses in the melanoma tissue biopsies before or during anti-PD1 immunotherapy [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A154.