Innate-like T cells in liver disease.

Mammalian innate-like T cells (ILTCs), including mucosal-associated invariant T (MAIT), natural killer T (NKT), and γδ T cells, are abundant tissue-resident lymphocytes that have recently emerged as orchestrators of hepatic inflammation, tissue repair, and immune homeostasis. This review explores the involvement of different ILTC subsets in liver diseases. We explore the mechanisms underlying the pro- and anti-inflammatory effector functions of ILTCs in a context-dependent manner. We highlight latest findings regarding the dynamic interplay between ILTC functional subsets and other immune and parenchymal cells which may inform candidate immunomodulatory strategies to achieve improved clinical outcomes in liver diseases. We present new insights into how distinct gene expression programs in hepatic ILTCs are induced, maintained, and reprogrammed in a context- and disease stage-dependent manner.

Integrin activation enables rapid detection of functional Vδ1+ and Vδ2+ γδ T cells.

Conformational change of the ß2 integrin lymphocyte function-associated antigen 1 (LFA-1) is an early marker of T cell activation. A protocol using the mAb clone m24 recognizing the active, extended high-affinity conformation has been previously described for the assessment of functional CD4+ and CD8+ T cells in response to MHC-peptide stimulation. We investigated the applicability of the m24 mAb to detect the activation of γδ T cells in response to different soluble and immobilized stimuli. m24 mAb staining was associated with the expression of cytokines and was detectable as early as 10 min after stimulation, but with different kinetics depending on the nature of the stimulus. Hence, we conclude that this assay is suitable for the detection of functional γδ T cells and allows the assessment of activation more rapidly than alternative methods such as cytokine detection. Intracellular staining, protein trafficking inhibitors, or prior knowledge of the stimulating moiety recognized are no longer required for monitoring γδ T cell activation.

Establishing High Dimensional Immune Signatures from Peripheral Blood via Mass Cytometry in a Discovery Cohort of Stage IV Melanoma Patients.

The identification of blood-borne biomarkers correlating with melanoma patient survival remains elusive. Novel techniques such as mass cytometry could help to identify melanoma biomarkers, allowing simultaneous detection of up to 100 parameters. However, the evaluation of multiparametric data generated via time-of-flight mass cytometry requires novel analytical techniques because the application of conventional gating strategies currently used in polychromatic flow cytometry is not feasible. In this study, we have employed 38-channel time-of-flight mass cytometry analysis to generate comprehensive immune cell signatures using matrix boolean analysis in a cohort of 28 stage IV melanoma patients and 17 controls. Clusters of parameters were constructed from the abundance of cellular phenotypes significantly different between patients and controls. This approach identified patient-specific combinatorial immune signatures consisting of high-resolution subsets of the T cell, NK cell, B cell, and myeloid compartments. An association with superior survival was characterized by a balanced distribution of myeloid-derived suppressor cell-like and APC-like myeloid phenotypes and differentiated NK cells. The results of this study in a discovery cohort of melanoma patients suggest that multifactorial immune signatures have the potential to allow more accurate prediction of individual patient outcome. Further investigation of the identified immune signatures in a validation cohort is now warranted.

Accurate quantification of T-cells expressing PD-1 in patients on anti-PD-1 immunotherapy.

Increasing numbers of trials employing anti-PD-1 immunotherapy emphasize the requirement for predictive biomarkers of clinical response. Many studies examine the cell surface expression of PD-1 and other key regulators of T-cell activation and inhibition. Here, we compared common commercially available anti-PD-1 diagnostic antibodies and tested whether they can bind the PD-1 receptor in the presence of the therapeutic antagonists pembrolizumab and nivolumab. We observed that currently no antibodies are available that can reliably stain all PD-1 receptors on T-cells from patients treated with anti-PD-1 antibodies. Furthermore, none of the diagnostic antibodies detected the entire population of PD-1+ T-cells relative to indirect staining using the therapeutic antibodies themselves. To overcome this problem, here we present a reliable method for quantifying PD-1 expression on immune cells from treated patients which can be included in any conventional flow or mass cytometry antibody panel used for patient monitoring.

The Generation of CAR-Transfected Natural Killer T Cells for the Immunotherapy of Melanoma.

Natural killer T (NKT) cells represent a cell subpopulation that combines characteristics of natural killer (NK) cells and T cells. Through their endogenous T-cell receptors (TCRs), they reveal a pronounced intrinsic anti-tumor activity. Thus, a NKT cell transfected with a chimeric antigen receptor (CAR), which recognizes a tumor-specific surface antigen, could attack tumor cells antigen-specifically via the CAR and additionally through its endogenous TCR. NKT cells were isolated from peripheral blood mononuclear cells (PBMCs), expanded, and electroporated with mRNA encoding a chondroitin sulfate proteoglycan 4 (CSPG4)-specific CAR. The CAR expression on NKT cells and their in vitro functionality were analyzed. A transfection efficiency of more than 80% was achieved. Upon stimulation with melanoma cells, CAR-NKT cells produced cytokines antigen-specifically. Compared with conventional CAR-T cells, cytokine secretion of CAR-NKT cells was generally lower. Specific cytotoxicity, however, was similar with CAR-NKT cells showing a trend towards improved cytotoxicity. Additionally, CAR-NKT cells could kill target cells through their endogenous TCRs. In summary, it is feasible to generate CAR-NKT cells by using mRNA electroporation. Their CAR-mediated cytotoxicity is at least equal to that of conventional CAR-T cells, while their intrinsic cytotoxic activity is maintained. Thus, CAR-NKT cells may represent a valuable alternative to conventional CAR-T cells for cancer immunotherapy.

Combined treatment with ipilimumab and intratumoral interleukin-2 in pretreated patients with stage IV melanoma-safety and efficacy in a phase II study.

Treatment of advanced melanoma patients with ipilimumab results in improved survival. However, only about 20% of treated patients experience long-term benefit. Combining treatment of ipilimumab with other drugs may improve immune activation and potentially enhance clinical efficacy. The aims of the phase II clinical trial reported here were to investigate tolerability and efficacy of a combined immunotherapeutic strategy comprising standard systemic ipilimumab at 3 mg/kg four times at 3-week intervals and intratumorally injected IL-2 at 9 MIU daily twice weekly for four weeks in pretreated melanoma patients with distant metastasis. The primary endpoint was the disease control rate according to immune-related response criteria at week 12; tolerability according to Common Terminology Criteria for Adverse Events criteria was secondary endpoint. No objective responses were observed in the 15 enrolled patients. Three patients had stable disease 12 weeks after starting treatment, yielding a disease control rate of 20%. Tolerability of this combination treatment was acceptable. Observed adverse events were those expected from the respective monotherapies. Autoimmune colitis was observed in two patients. Grade III/IV adverse events were observed in 40% of patients, and no treatment-related deaths occurred. Thus, this combined immunotherapy is associated with adverse events similar to those associated with the respective monotherapies. However, this study does not provide any evidence of improved efficacy of the combination over ipilimumab alone.

Phenotypic characterization and prognostic impact of circulating γδ and αß T-cells in metastatic malignant melanoma.

Human T cells carrying γδ T-cell receptors (TCRs) represent a minor population relative to those with αß TCRs. There has been much interest recently in the possibility of using these γδ T-cells in cancer therapy because they can kill tumor cells in vitro in an MHC-unrestricted manner, and possess potential regulatory capability and antigen-presenting capacity. The presence of γδ T-cells in late-stage melanoma patients and their relationship with survival has not been extensively explored, although relatively lower percentages of total γδ T-cells and Vδ2+ cells have been reported. Here, we present a detailed analysis of associations of γδ T-cell subsets and differentiation stages with survival in Stage IV patients, compared with CD4+ and CD8+ αß T-cells. We found an increased Vδ1:Vδ2-ratio and a decreased CD4:CD8-ratio in patients compared to healthy controls, on the basis both of relative frequencies and absolute cell counts per µL blood. Nonetheless, Kaplan-Meier analyses showed that a higher than median frequency of Vδ1+ cells was negatively associated with survival, whereas there were no positive or negative associations with frequencies of Vδ2+ cells. Correlations of cell differentiation status with survival revealed a negative association of early-differentiated Vδ1+ T cells with survival, both on the basis of relative frequencies and absolute counts. There was also a positive correlation between the frequencies of early-differentiated CD8+ αß T-cells and survival. Our findings suggest peripheral blood frequencies of Vδ1+ T-cells as a potential prognostic marker in melanoma. The mechanisms by which higher abundance of Vδ1+ cells are associated with poorer survival require determination.

Peripheral blood T-cell signatures from high-resolution immune phenotyping of γδ and αß T-cells in younger and older subjects in the Berlin Aging Study II.

BACKGROUND: Aging and latent infection with Cytomegalovirus (CMV) are thought to be major factors driving the immune system towards immunosenescence, primarily characterized by reduced amounts of naïve T-cells and increased memory T-cells, potentially associated with higher morbidity and mortality. The composition of both major compartments, γδ as well as αß T-cells, is altered by age and CMV, but detailed knowledge of changes to the γδ subset is currently limited. RESULTS: Here, we have surveyed a population of 73 younger (23-35 years) and 144 older (62-85 years) individuals drawn from the Berlin Aging Study II, investigating the distribution of detailed differentiation phenotypes of both γδ and αß T-cells. Correlation of frequencies and absolute counts of the identified phenotypes with age and the presence of CMV revealed a lower abundance of Vδ2-positive and a higher amount of Vδ1-positive cells. We found higher frequencies of late-differentiated and lower frequencies of early-differentiated cells in the Vδ1+ and Vδ1-Vδ2-, but not in the Vδ2+ populations in elderly CMV-seropositive individuals confirming the association of these Vδ2-negative cells with CMV-immunosurveillance. We identified the highest Vδ1:Vδ2 ratios in the CMV-seropositive elderly. The observed increased CD4:CD8 ratios in the elderly were significantly lower in CMV-seropositive individuals, who also possessed a lower naïve and a larger late-differentiated compartment of CD8+ αß T-cells, reflecting the consensus in the literature. CONCLUSIONS: Our findings illustrate in detail the strong influence of CMV on the abundance and differentiation pattern of γδ T-cells as well as αß T-cells in older and younger people. Mechanisms responsible for the phenotypic alterations in the γδ T-cell compartment, associated both with the presence of CMV and with age require further clarification.

High-dimensional in situ proteomics imaging to assess γδ T cells in spatial biology.

This study presents a high-dimensional immunohistochemistry approach to assess human γδ T cell subsets in their native tissue microenvironments at spatial resolution, a hitherto unmet scientific goal due to the lack of established antibodies and required technology. We report an integrated approach based on multiplexed imaging and bioinformatic analysis to identify γδ T cells, characterize their phenotypes, and analyze the composition of their microenvironment. Twenty-eight γδ T cell microenvironments were identified in tissue samples from fresh frozen human colon and colorectal cancer where interaction partners of the immune system, but also cancer cells were discovered in close proximity to γδ T cells, visualizing their potential contributions to cancer immunosurveillance. While this proof-of-principle study demonstrates the potential of this cutting-edge technology to assess γδ T cell heterogeneity and to investigate their microenvironment, future comprehensive studies are warranted to associate phenotypes and microenvironment profiles with features such as relevant clinical characteristics.

Susceptibility of Melanoma Cells to Targeted Therapy Correlates with Protection by Blood Neutrophils.

Elevated levels of peripheral blood and tumor tissue neutrophils are associated with poorer clinical response and therapy resistance in melanoma. The underlying mechanism and the role of neutrophils in targeted therapy is still not fully understood. Serum samples of patients with advanced melanoma were collected and neutrophil-associated serum markers were measured and correlated with response to targeted therapy. Blood neutrophils from healthy donors and patients with advanced melanoma were isolated, and their phenotypes, as well as their in vitro functions, were compared. In vitro functional tests were conducted through nonadherent cocultures with melanoma cells. Protection of melanoma cell lines by neutrophils was assessed under MAPK inhibition. Blood neutrophils from advanced melanoma patients exhibited lower CD16 expression compared to healthy donors. In vitro, both healthy-donor- and patient-derived neutrophils prevented melanoma cell apoptosis upon dual MAPK inhibition. The effect depended on cell-cell contact and melanoma cell susceptibility to treatment. Interference with protease activity of neutrophils prevented melanoma cell protection during treatment in cocultures. The negative correlation between neutrophils and melanoma outcomes seems to be linked to a protumoral function of neutrophils. In vitro, neutrophils exert a direct protective effect on melanoma cells during dual MAPK inhibition. This study further hints at a crucial role of neutrophil-related protease activity in protection.

Age-associated alterations in γδ T-cells are present predominantly in individuals infected with Cytomegalovirus.

BACKGROUND: Despite the common perception that latent Cytomegalovirus (CMV) infection is usually symptom-free, emerging epidemiological evidence suggests that it may in fact be associated with higher mortality over extended follow-up. Mechanisms responsible for this potentially important effect are unclear. CMV infection is known to have a large impact on the distribution of T cell phenotypes, especially the accumulation of late-stage differentiated CD8+, as well as Vδ2- γδ T-cells, which are the main subset of γδ T-cells involved in anti-CMV immunity. Its impact on γδ T-cells in the aging context is less well-defined. RESULTS: Here, we investigated a group of healthy individuals aged between 21 and 89 years, in order to correlate the frequency and differentiation status of γδ T-cells with age. We found that these parameters were only marginally influenced by age, but were marked in people with a latent CMV infection. Thus, we observed a significant age-associated accumulation of late-differentiated T-cells within the Vδ2- population, but only in CMV-seropositive donors. There was also a strong trend towards reduced frequency of early-differentiated cells within the Vδ2- phenotype. Older people had significantly higher anti-CMV IgG titers, which in turn correlated significantly with a lower Vδ2+/Vδ2- ratio and a shift from early- to a late-differentiated Vδ2- T-cell phenotype. CONCLUSIONS: Our findings demonstrate a strong influence of CMV on γδ T-cells during human ageing, similar to that observed for αß T-cells. Differences between donors of different ages are more marked in CMV-infected individuals. The biological implications of this potent age-associated CMV-mediated immune-modulation require clarification.

Frequencies of activated T cell populations increase in breast milk of HCMV-seropositive mothers during local HCMV reactivation.

Background: Human cytomegalovirus (HCMV) can reactivate in the mammary gland during lactation and is shed into breast milk of nearly every HCMV-IgG-seropositive mother of a preterm infant. Dynamics of breast milk leukocytes during lactation, as well as blood leukocytes and the comparison between both in the context of HCMV reactivation is not well understood. Methods: Here, we present the BlooMil study that aimed at comparing changes of immune cells in blood and breast milk from HCMV-seropositive- vs -seronegative mothers, collected at four time ranges up to two months post-partum. Viral load was monitored by qPCR and nested PCR. Multiparameter flow cytometry was used to identify leukocyte subsets. Results: CD3+ T cell frequencies were found to increase rapidly in HCMV-seropositive mothers' milk, while they remained unchanged in matched blood samples, and in both blood and breast milk of HCMV-seronegatives. The activation marker HLA-DR was more strongly expressed on CD4+ and CD8+ T cells in all breast milk samples than matched blood samples, but HCMV-seropositive mothers displayed a significant increase of HLA-DR+ CD4+ and HLA-DR+ CD8+ T cells during lactation. The CD4+/CD8+ T cell ratio was lower in breast milk of HCMV-seropositive mothers than in the blood. HCMV-specific CD8+ T cell frequencies (recognizing pp65 or IE1) were elevated in breast milk relative to blood, which might be due to clonal expansion of these cells during local HCMV reactivation. Breast milk contained very low frequencies of naïve T cells with no significant differences depending on serostatus. Conclusion: Taken together, we conclude that the distribution of breast milk leukocyte populations is different from blood leukocytes and may contribute to the decrease of breast milk viral load in the late phase of HCMV reactivation in the mammary gland.

S3-CIMA: Supervised spatial single-cell image analysis for identifying disease-associated cell-type compositions in tissue.

The spatial organization of various cell types within the tissue microenvironment is a key element for the formation of physiological and pathological processes, including cancer and autoimmune diseases. Here, we present S3-CIMA, a weakly supervised convolutional neural network model that enables the detection of disease-specific microenvironment compositions from high-dimensional proteomic imaging data. We demonstrate the utility of this approach by determining cancer outcome- and cellular-signaling-specific spatial cell-state compositions in highly multiplexed fluorescence microscopy data of the tumor microenvironment in colorectal cancer. Moreover, we use S3-CIMA to identify disease-onset-specific changes of the pancreatic tissue microenvironment in type 1 diabetes using imaging mass-cytometry data. We evaluated S3-CIMA as a powerful tool to discover novel disease-associated spatial cellular interactions from currently available and future spatial biology datasets.

Early decrease of blood myeloid-derived suppressor cells during checkpoint inhibition is a favorable biomarker in metastatic melanoma.

BACKGROUND: The need for reliable clinical biomarkers to predict which patients with melanoma will benefit from immune checkpoint blockade (ICB) remains unmet. Several different parameters have been considered in the past, including routine differential blood counts, T cell subset distribution patterns and quantification of peripheral myeloid-derived suppressor cells (MDSC), but none has yet achieved sufficient accuracy for clinical utility. METHODS: Here, we investigated potential cellular biomarkers from clinical routine blood counts as well as several myeloid and T cell subsets, using flow cytometry, in two independent cohorts of a total of 141 patients with stage IV M1c melanoma before and during ICB. RESULTS: Elevated baseline frequencies of monocytic MDSCs (M-MDSC) in the blood were confirmed to predict shorter overall survival (OS) (HR 2.086, p=0.030) and progression-free survival (HR 2.425, p=0.001) in the whole patient cohort. However, we identified a subgroup of patients with highly elevated baseline M-MDSC frequencies that fell below a defined cut-off during therapy and found that these patients had a longer OS that was similar to that of patients with low baseline M-MDSC frequencies. Importantly, patients with high M-MDSC frequencies exhibited a skewed baseline distribution of certain other immune cells but these did not influence patient survival, illustrating the paramount utility of MDSC assessment. CONCLUSION: We confirmed that in general, highly elevated frequencies of peripheral M-MDSC are associated with poorer outcomes of ICB in metastatic melanoma. However, one reason for an imperfect correlation between high baseline MDSCs and outcome for individual patients may be the subgroup of patients identified here, with rapidly decreasing M-MDSCs on therapy, in whom the negative effect of high M-MDSC frequencies was lost. These findings might contribute to developing more reliable predictors of late-stage melanoma response to ICB at the individual patient level. A multifactorial model seeking such markers yielded only MDSC behavior and serum lactate dehydrogenase as predictors of treatment outcome.

Tumor-derived GDF-15 blocks LFA-1 dependent T cell recruitment and suppresses responses to anti-PD-1 treatment.

Immune checkpoint blockade therapy is beneficial and even curative for some cancer patients. However, the majority don't respond to immune therapy. Across different tumor types, pre-existing T cell infiltrates predict response to checkpoint-based immunotherapy. Based on in vitro pharmacological studies, mouse models and analyses of human melanoma patients, we show that the cytokine GDF-15 impairs LFA-1/ß2-integrin-mediated adhesion of T cells to activated endothelial cells, which is a pre-requisite of T cell extravasation. In melanoma patients, GDF-15 serum levels strongly correlate with failure of PD-1-based immune checkpoint blockade therapy. Neutralization of GDF-15 improves both T cell trafficking and therapy efficiency in murine tumor models. Thus GDF-15, beside its known role in cancer-related anorexia and cachexia, emerges as a regulator of T cell extravasation into the tumor microenvironment, which provides an even stronger rationale for therapeutic anti-GDF-15 antibody development.

Blood Eosinophils Are Associated with Efficacy of Targeted Therapy in Patients with Advanced Melanoma.

BACKGROUND: Eosinophils appear to contribute to the efficacy of immunotherapy and their frequency was suggested as a predictive biomarker. Whether this observation could be transferred to patients treated with targeted therapy remains unknown. METHODS: Blood and serum samples of healthy controls and 216 patients with advanced melanoma were prospectively and retrospectively collected. Freshly isolated eosinophils were phenotypically characterized by flow cytometry and co-cultured in vitro with melanoma cells to assess cytotoxicity. Soluble serum markers and peripheral blood counts were used for correlative studies. RESULTS: Eosinophil-mediated cytotoxicity towards melanoma cells, as well as phenotypic characteristics, were similar when comparing healthy donors and patients. However, high relative pre-treatment eosinophil counts were significantly associated with response to MAPKi (p = 0.013). Eosinophil-mediated cytotoxicity towards melanoma cells is dose-dependent and requires proximity of eosinophils and their target in vitro. Treatment with targeted therapy in the presence of eosinophils results in an additive tumoricidal effect. Additionally, melanoma cells affected eosinophil phenotype upon co-culture. CONCLUSION: High pre-treatment eosinophil counts in advanced melanoma patients were associated with a significantly improved response to MAPKi. Functionally, eosinophils show potent cytotoxicity towards melanoma cells, which can be reinforced by MAPKi. Further studies are needed to unravel the molecular mechanisms of our observations.

Dynamics of Melanoma-Associated Epitope-Specific CD8+ T Cells in the Blood Correlate With Clinical Outcome Under PD-1 Blockade.

Immune checkpoint blockade (ICB) is standard-of-care for patients with metastatic melanoma. It may re-invigorate T cells recognizing tumors, and several tumor antigens have been identified as potential targets. However, little is known about the dynamics of tumor antigen-specific T cells in the circulation, which might provide valuable information on ICB responses in a minimally invasive manner. Here, we investigated individual signatures composed of up to 167 different melanoma-associated epitope (MAE)-specific CD8+ T cells in the blood of stage IV melanoma patients before and during anti-PD-1 treatment, using a peptide-loaded multimer-based high-throughput approach. Additionally, checkpoint receptor expression patterns on T cell subsets and frequencies of myeloid-derived suppressor cells and regulatory T cells were quantified by flow cytometry. Regression analysis using the MAE-specific CD8+ T cell populations was applied to identify those that correlated with overall survival (OS). The abundance of MAE-specific CD8+ T cell populations, as well as their dynamics under therapy, varied between patients. Those with a dominant increase of these T cell populations during PD-1 ICB had a longer OS and progression-free survival than those with decreasing or balanced signatures. Patients with a dominantly increased MAE-specific CD8+ T cell signature also exhibited an increase in TIM-3+ and LAG-3+ T cells. From these results, we created a model predicting improved/reduced OS by combining data on dynamics of the three most informative MAE-specific CD8+ T cell populations. Our results provide insights into the dynamics of circulating MAE-specific CD8+ T cell populations during ICB, and should contribute to a better understanding of biomarkers of response and anti-cancer mechanisms.

Immunomonitoring of Human Breast Milk Cells During HCMV-Reactivation.

Background: Breast milk leukocytes may play a role in protecting the infant from pathogens. The dynamics and the role of lymphocytes in human cytomegalovirus (HCMV)-seropositive mothers shedding HCMV into breast milk during the first months postpartum (p.p.) are mostly unclear. Methods: Breast milk cells were analyzed by Pappenheim panoptic and alpha-naphthyl acetate esterase staining as well as by imaging and polychromatic flow cytometry to simultaneously establish their morphological and phenotypic properties. The latter were characterized in HCMV-seropositive and seronegative mothers´ breast milk cells at different time points p.p. Results: Panoptic staining of breast milk cells revealed the presence of monocytes/macrophages, granulocytes and lymphocytes. Imaging flow cytometry data combining phenotypic and morphological analysis identified NKT-like cells, NK cells, epithelial cells, T cells and monocytes/macrophages. HCMV-seropositive but not -seronegative mothers had significantly higher T cell frequencies in mature milk. Conclusions: The presence of lymphocyte subsets in breast milk may be more influenced by the HCMV-seropositivity of the mother than previously recognized.

Genetic Influence on the Peripheral Differentiation Signature of Vδ2+ γδ and CD4+ αß T Cells in Adults.

Adaptive as well as innate immune traits are variously affected by environmental and genetic influences, but little is known about the impact of genetics on the diversity, differentiation and functionality of γδ T cells in humans. Here, we analyzed a cohort of 95 middle-aged twins from the Danish Twin Registry. The differentiation status of peripheral αß and γδ T cells was assessed by flow cytometry based on the surface expression of CD27, CD28 and CD45RA. Our data confirm the established associations of latent cytomegalovirus (CMV) infection with an accumulation of late differentiated memory T cells in the αß compartment as well as in the Vδ1+ γδ T cell subset. A comparison of differentiation phenotypes of γδ and αß T cells that were not affected by CMV seropositivity identified a significant correlation of early differentiated (ED) Vδ2+ and intermediate differentiated (ID) CD4+ T cells in monozygotic (MZ), but not in dizygotic (DZ) co-twins. Thus, our data suggest a genetic influence on the differentiation of γδ and αß T cell subsets.