The good and the bad of T cell cross-reactivity: challenges and opportunities for novel therapeutics in autoimmunity and cancer

T cells are main actors of the immune system with an essential role in protection against pathogens and cancer. The molecular key event involved in this absolutely central task is the interaction of membrane-bound specific T cell receptors with peptide-MHC complexes which initiates T cell priming, activation and recall, and thus controls a range of downstream functions. While textbooks teach us that the repertoire of mature T cells is highly diverse, it is clear that this diversity cannot possibly cover all potential foreign peptides that might be encountered during life. TCR cross-reactivity, i.e. the ability of a single TCR to recognise different peptides, offers the best solution to this biological challenge. Reports have shown that indeed, TCR cross-reactivity is surprisingly high. Hence, the T cell dilemma is the following: be as specific as possible to target foreign danger and spare self, while being able to react to a large spectrum of body-threatening situations. This has major consequences for both autoimmune diseases and cancer, and significant implications for the development of T cell-based therapies. In this review, we will present essential experimental evidence of T cell cross-reactivity, implications for two opposite immune conditions, i.e. autoimmunity vs cancer, and how this can be differently exploited for immunotherapy approaches. Finally, we will discuss the tools available for predicting cross-reactivity and how improvements in this field might boost translational approaches

Targeted therapy in renal cell carcinoma: moving from molecular agents to specific immunotherapy

Non-specific immunotherapy has been for a long time a standard treatment option for patients with metastatic renal cell carcinoma but was redeemed by specific targeted molecular therapies, namely the VEGF and mTOR inhibitors. After moving treatment for mRCC to specific molecular agents with a well-defined mode of action, immunotherapy still needs this further development to increase its accuracy. Nowadays, an evolution from a rather non-specific cytokine treatment to sophisticated targeted approaches in specific immunotherapy led to a re-launch of immunotherapy in clinical studies. Recent steps in the development of immunotherapy strategies are discussed in this review with a special focus on peptide vaccination which aims at a tumor targeting by specific T lymphocytes. In addition, different combinatory strategies with immunomodulating agents like cyclophosphamide or sunitinib are outlined, and the effects of immune checkpoint modulators as anti-CTLA-4 or PD-1 antibodies are discussed

More Than Just Tumor Destruction: Immunomodulation by Thermal Ablation of Cancer

Over the past decades, thermoablative techniques for the therapy of localized tumors have gained importance in the treatment of patients not eligible for surgical resection. Anecdotal reports have described spontaneous distant tumor regression after thermal ablation, indicating a possible involvement of the immune system, hence an induction of antitumor immunity after thermoinduced therapy. In recent years, a growing body of evidence for modulation of both adaptive and innate immunity, as well as for the induction of danger signals through thermoablation, has emerged. Induced immune responses, however, are mostly weak and not sufficient for the complete eradication of established tumors or durable prevention of disease progression, and combination therapies with immunomodulating drugs are being evaluated with promising results. This article aims to summarize published findings on immune modulation through radiofrequency ablation, cryoablation, microwave ablation therapy, high-intensity focused ultrasound, and laser-induced thermotherapy

Activated integrins identify functional antigen-specific CD8 T cells within minutes after antigen stimulation

Skin affections after sulfur mustard (SM) exposure include erythema, blister formation and severe inflammation. An antidote or specific therapy does not exist. Anti-inflammatory compounds as well as substances counteracting SM-induced cell death are under investigation. In this study, we investigated the benzylisoquinoline alkaloide berberine (BER), a metabolite in plants like berberis vulgaris, which is used as herbal pharmaceutical in Asian countries, against SM toxicity using a well-established in vitro approach. Keratinocyte (HaCaT) mono-cultures (MoC) or HaCaT/THP-1 co-cultures (CoC) were challenged with 100, 200 or 300 mM SM for 1 h. Post-exposure, both MoC and CoC were treated with 10, 30 or 50 mu M BER for 24 h. At that time, supernatants were collected and analyzed both for interleukine (IL) 6 and 8 levels and for content of adenylate-kinase (AK) as surrogate marker for cell necrosis. Cells were lysed and nucleosome formation as marker for late apoptosis was assessed. In parallel, AK in cells was determined for normalization purposes. BER treatment did not influence necrosis, but significantly decreased apoptosis. Anti-inflammatory effects were moderate, but also significant, primarily in CoC. Overall, BER has protective effects against SM toxicity in vitro. Whether this holds true should be evaluated in future in vivo studies

Two birds with one stone: human SIRPα nanobodies for functional modulation and in vivo imaging of myeloid cells

Signal-regulatory protein α (SIRPα) expressed by myeloid cells is of particular interest for therapeutic strategies targeting the interaction between SIRPα and the “don’t eat me” ligand CD47 and as a marker to monitor macrophage infiltration into tumor lesions. To address both approaches, we developed a set of novel human SIRPα (hSIRPα)–specific nanobodies (Nbs). We identified high-affinity Nbs targeting the hSIRPα/hCD47 interface, thereby enhancing antibody-dependent cellular phagocytosis. For non-invasive in vivo imaging, we chose S36 Nb as a non-modulating binder. By quantitative positron emission tomography in novel hSIRPα/hCD47 knock-in mice, we demonstrated the applicability of 64Cu-hSIRPα-S36 Nb to visualize tumor infiltration of myeloid cells. We envision that the hSIRPα-Nbs presented in this study have potential as versatile theranostic probes, including novel myeloid-specific checkpoint inhibitors for combinatorial treatment approaches and for in vivo stratification and monitoring of individual responses during cancer immunotherapies

Automated Analysis of Flow Cytometry Data to Reduce Inter-Lab Variation in the Detection of Major Histocompatibility Complex Multimer-Binding T Cells

Manual analysis of flow cytometry data and subjective gate-border decisions taken by individuals continue to be a source of variation in the assessment of antigen-specific T cells when comparing data across laboratories, and also over time in individual labs. Therefore, strategies to provide automated analysis of major histocompatibility complex (MHC) multimer-binding T cells represent an attractive solution to decrease subjectivity and technical variation. The challenge of using an automated analysis approach is that MHC multimer-binding T cell populations are often rare and therefore difficult to detect. We used a highly heterogeneous dataset from a recent MHC multimer proficiency panel to assess if MHC multimer-binding CD8+ T cells could be analyzed with computational solutions currently available, and if such analyses would reduce the technical variation across different laboratories. We used three different methods, FLOw Clustering without K (FLOCK), Scalable Weighted Iterative Flow-clustering Technique (SWIFT), and ReFlow to analyze flow cytometry data files from 28 laboratories. Each laboratory screened for antigen-responsive T cell populations with frequency ranging from 0.01 to 1.5% of lymphocytes within samples from two donors. Experience from this analysis shows that all three programs can be used for the identification of high to intermediate frequency of MHC multimer-binding T cell populations, with results very similar to that of manual gating. For the less frequent populations (<0.1% of live, single lymphocytes), SWIFT outperformed the other tools. As used in this study, none of the algorithms offered a completely automated pipeline for identification of MHC multimer populations, as varying degrees of human interventions were needed to complete the analysis. In this study, we demonstrate the feasibility of using automated analysis pipelines for assessing and identifying even rare populations of antigen-responsive T cells and discuss the main properties, differences, and advantages of the different methods tested

Modeling flow cytometry data for cancer vaccine immune monitoring

Flow cytometry (FCM) is widely used in cancer research for diagnosis, detection of minimal residual disease, as well as immune monitoring and profiling following immunotherapy. In all these applications, the challenge is to detect extremely rare cell subsets while avoiding spurious positive events. To achieve this objective, it helps to be able to analyze FCM data using multiple markers simultaneously, since the additional information provided often helps to minimize the number of false positive and false negative events, hence increasing both sensitivity and specificity. However, with manual gating, at most two markers can be examined in a single dot plot, and a sequential strategy is often used. As the sequential strategy discards events that fall outside preceding gates at each stage, the effectiveness of the strategy is difficult to evaluate without laborious and painstaking back-gating. Model-based analysis is a promising computational technique that works using information from all marker dimensions simultaneously, and offers an alternative approach to flow analysis that can usefully complement manual gating in the design of optimal gating strategies. Results from model-based analysis will be illustrated with examples from FCM assays commonly used in cancer immunotherapy laboratories

Harmonization of the intracellular cytokine staining assay

Active immunotherapy for cancer is an accepted treatment modality aiming to reinforce the T-cell response to cancer. T-cell reactivity is measured by various assays and used to guide the clinical development of immunotherapeutics. However, data obtained across different institutions may vary substantially making comparative conclusions difficult. The Cancer Immunotherapy Immunoguiding Program organizes proficiency panels to identify key parameters influencing the outcome of commonly used T-cell assays followed by harmonization. Our successes with IFNγ-ELISPOT and peptide HLA multimer analysis have led to the current study on intracellular cytokine staining (ICS). We report the results of three successive panels evaluating this assay. At the beginning, 3 out of 9 participants (33 %) were able to detect >6 out of 8 known virus-specific T-cell responses in peripheral blood of healthy individuals. This increased to 50 % of the laboratories in the second phase. The reported percentages of cytokine-producing T cells by the different laboratories were highly variable with coefficients of variation well over 60 %. Variability could partially be explained by protocol-related differences in background cytokine production leading to sub-optimal signal-to-noise ratios. The large number of protocol variables prohibited identification of prime guidelines to harmonize the assays. In addition, the gating strategy used to identify reactive T cells had a major impact on assay outcome. Subsequent harmonization of the gating strategy considerably reduced the variability within the group of participants. In conclusion, we propose that first basic guidelines should be applied for gating in ICS experiments before harmonizing assay protocol variables