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Funder: China Scholarship Council, Despite early clinical successes, the mechanisms of action of low-dose interleukin-2 (LD-IL-2) immunotherapy remain only partly understood. Here we examine the effects of interval administration of low-dose recombinant IL-2 (iLD-IL-2) in type 1 diabetes using high-resolution single-cell multiomics and flow cytometry on longitudinally-collected peripheral blood samples. Our results confirm that iLD-IL-2 selectively expands thymic-derived FOXP3+HELIOS+ regulatory T cells and CD56bright NK cells, and show that the treatment reduces the frequency of IL-21-producing CD4+ T cells and of two innate-like mucosal-associated invariant T and Vγ9Vδ2 CD8+ T cell subsets. The cellular changes induced by iLD-IL-2 associate with an anti-inflammatory gene expression signature, which remains detectable in all T and NK cell subsets analysed one month after treatment. These findings warrant investigations into the potential longer-term clinical benefits of iLD-IL-2 in immunotherapy., This work was supported by the Sir Jules Thorn Trust (13/JTA (OCT2013/DR/1044)), the JDRF (1-SRA-2019-657-A-N), and the NIHR Cambridge Biomedical Research Centre. The Diabetes and Inflammation Laboratory was supported by a strategic award from the Wellcome (107212/A/15/Z) and the JDRF (4-SRA-2017-473-A-A). JYZ was supported by the China Scholarship Council-University of Oxford Scholarship. The research was supported by the Wellcome Trust Core Award Grant Number 203141/Z/16/Z with additional support from the NIHR Oxford BRC. The University of Cambridge has received salary support for ME through the National Health Service in the East of England through the Clinical Academic Reserve. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health.

BACKGROUND: The INNODIA consortium has established a pan-European infrastructure using validated centres to prospectively evaluate clinical data from individuals with newly diagnosed type 1 diabetes combined with centralised collection of clinical samples to determine rates of decline in beta-cell function and identify novel biomarkers, which could be used for future stratification of phase 2 clinical trials. METHODS: In this context, we have developed a Master Protocol, based on the "backbone" of the INNODIA natural history study, which we believe could improve the delivery of phase 2 studies exploring the use of single or combinations of Investigational Medicinal Products (IMPs), designed to prevent or reverse declines in beta-cell function in individuals with newly diagnosed type 1 diabetes. Although many IMPs have demonstrated potential efficacy in phase 2 studies, few subsequent phase 3 studies have confirmed these benefits. Currently, phase 2 drug development for this indication is limited by poor evaluation of drug dosage and lack of mechanistic data to understand variable responses to the IMPs. Identification of biomarkers which might permit more robust stratification of participants at baseline has been slow. DISCUSSION: The Master Protocol provides (1) standardised assessment of efficacy and safety, (2) comparable collection of mechanistic data, (3) the opportunity to include adaptive designs and the use of shared control groups in the evaluation of combination therapies, and (4) benefits of greater understanding of endpoint variation to ensure more robust sample size calculations and future baseline stratification using existing and novel biomarkers.

Background: The INNODIA consortium has established a pan-European infrastructure using validated centres to evaluate prospectively clinical data from individuals with newly diagnosed Type 1 diabetes combined with centralized collection of clinical samples to determine rates of decline in beta-cell function and identify novel biomarkers, which could be used for future stratification of Phase 2 clinical trials. Methods: In this context we have developed a Master Protocol, based on the “backbone” of the INNODIA natural history study, which we believe could improve the delivery of Phase 2 studies exploring the use of single or combinations of Investigational Medicinal Products (IMPs), designed to prevent or reverse declines in beta-cell function in individuals with newly diagnosed Type 1 diabetes. Although many IMPs have demonstrated potential efficacy in Phase 2 studies, few subsequent Phase 3 studies have confirmed these benefits. Currently Phase 2 drug development for this indication is limited by poor evaluation of drug dosage and lack of mechanistic data to understand variable responses to the IMPs. Identification of biomarkers which might permit more robust stratification of participants at baseline has been slow. Discussion: The Master Protocol provides: 1) standardized assessment of efficacy and safety; 2) comparable collection of mechanistic data; 3) the opportunity to include adaptive designs and the use of shared control groups in the evaluation of combination therapies; 4) benefits of greater understanding of endpoint variation to ensure more robust sample size calculations and future baseline stratification using existing and novel biomarkers.

Background The INNODIA consortium has established a pan-European infrastructure using validated centres to prospectively evaluate clinical data from individuals with newly diagnosed type 1 diabetes combined with centralised collection of clinical samples to determine rates of decline in beta-cell function and identify novel biomarkers, which could be used for future stratification of phase 2 clinical trials. Methods In this context, we have developed a Master Protocol, based on the “backbone” of the INNODIA natural history study, which we believe could improve the delivery of phase 2 studies exploring the use of single or combinations of Investigational Medicinal Products (IMPs), designed to prevent or reverse declines in beta-cell function in individuals with newly diagnosed type 1 diabetes. Although many IMPs have demonstrated potential efficacy in phase 2 studies, few subsequent phase 3 studies have confirmed these benefits. Currently, phase 2 drug development for this indication is limited by poor evaluation of drug dosage and lack of mechanistic data to understand variable responses to the IMPs. Identification of biomarkers which might permit more robust stratification of participants at baseline has been slow. Discussion The Master Protocol provides (1) standardised assessment of efficacy and safety, (2) comparable collection of mechanistic data, (3) the opportunity to include adaptive designs and the use of shared control groups in the evaluation of combination therapies, and (4) benefits of greater understanding of endpoint variation to ensure more robust sample size calculations and future baseline stratification using existing and novel biomarkers.

Cytometric immunophenotyping is a powerful tool to discover and implement T-cell biomarkers of type 1 diabetes (T1D) progression and response to clinical therapy. Although many discovery-based T-cell biomarkers have been described, to date, no such markers have been widely adopted in standard practice. The heterogeneous nature of T1D and lack of standardized assays and experimental design across studies is a major barrier to the broader adoption of T-cell immunophenotyping assays. There is an unmet need to harmonize the design of immunophenotyping assays, including those that measure antigen-agnostic cell populations, such that data collected from different clinical trial sites and T1D cohorts are comparable, yet account for cohort-specific features and different drug mechanisms of action. In these Guidelines, we aim to provide expert advice on how to unify aspects of study design and practice. We provide recommendations for defining cohorts, method implementation, as well as tools for data analysis and reporting by highlighting and building on selected successes. Harmonization of cytometry-based T-cell assays will allow researchers to better integrate findings across trials, ultimately enabling the identification and validation of biomarkers of disease progression and treatment response in T1D.

The third edition of Flow Cytometry Guidelines provides the key aspects to consider when performing flow cytometry experiments and includes comprehensive sections describing phenotypes and functional assays of all major human and murine immune cell subsets. Notably, the Guidelines contain helpful tables highlighting phenotypes and key differences between human and murine cells. Another useful feature of this edition is the flow cytometry analysis of clinical samples with examples of flow cytometry applications in the context of autoimmune diseases, cancers as well as acute and chronic infectious diseases. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid. All sections are written and peer-reviewed by leading flow cytometry experts and immunologists, making this edition an essential and state-of-the-art handbook for basic and clinical researchers., European Journal of Immunology, 51 (12), ISSN:0014-2980, ISSN:1521-4141

Defective immune homeostasis in the balance between FOXP3+ regulatory T cells (Tregs) and effector T cells is a likely contributing factor in the loss of self-tolerance observed in type 1 diabetes (T1D). Given the importance of interleukin-2 (IL-2) signaling in the generation and function of Tregs, observations that polymorphisms in genes in the IL-2 pathway associate with T1D and that some individuals with T1D exhibit reduced IL-2 signaling indicate that impairment of this pathway may play a role in Treg dysfunction and the pathogenesis of T1D. Here, we have examined IL-2 sensitivity in CD4+ T-cell subsets in 70 individuals with long-standing T1D, allowing us to investigate the effect of low IL-2 sensitivity on Treg frequency and function. IL-2 responsiveness, measured by STAT5a phosphorylation, was a very stable phenotype within individuals but exhibited considerable interindividual variation and was influenced by T1D-associated PTPN2 gene polymorphisms. Tregs from individuals with lower IL-2 signaling were reduced in frequency, were less able to maintain expression of FOXP3 under limiting concentrations of IL-2, and displayed reduced suppressor function. These results suggest that reduced IL-2 signaling may be used to identify patients with the highest Treg dysfunction and who may benefit most from IL-2 immunotherapy.

Treg cells are critical for the prevention of autoimmune diseases and are thus prime candidates for cell-based clinical therapy. However, human Treg cells are "plastic", and are able to produce IL-17 under inflammatory conditions. Here, we identify and characterize the human Treg subpopulation that can be induced to produce IL-17 and identify its mechanisms. We confirm that a subpopulation of human Treg cells produces IL-17 in vitro when activated in the presence of IL-1β, but not IL-6. "IL-17 potential" is restricted to population III (CD4(+) CD25(hi) CD127(lo) CD45RA(-) ) Treg cells expressing the natural killer cell marker CD161. We show that these cells are functionally as suppressive and have similar phenotypic/molecular characteristics to other subpopulations of Treg cells and retain their suppressive function following IL-17 induction. Importantly, we find that IL-17 production is STAT3 dependent, with Treg cells from patients with STAT3 mutations unable to make IL-17. Finally, we show that CD161(+) population III Treg cells accumulate in inflamed joints of patients with inflammatory arthritis and are the predominant IL-17-producing Treg-cell population at these sites. As IL-17 production from this Treg-cell subpopulation is not accompanied by a loss of regulatory function, in the context of cell therapy, exclusion of these cells from the cell product may not be necessary.

Background: CD4+ T cells are central players of the adaptive immune response. They mediate inflammation via clonal expansion, differentiation into specific response subtypes and production of inflammatory signals that promote cytotoxicity and antibody production by other immune cells. CD4+ T cells may also differentiate into regulatory T cells (Treg) that control autoimmune responses and promote tolerance. Loss of CD4+ T cell tolerance to autoantigen leads to unwanted T cell responses and autoimmunity. Immunomodulation therapies that affect CD4+ T cells have become a paradigm to reverse T cell mediated autoimmunity, either by blocking T cell activation signals (Chamian et al., 2007; Keymeulen et al., 2005; Scheinfeld, 2005) or by enhancing tolerogenic function (Hartemann et al., 2013; Marcovecchio et al., 2020). Some of these single drug therapies are now used in trials or in practice to treat patients with autoimmune diseases. However, their true immunomodulating capacity on CD4+ T cell phenotype is unclear. Scientific Question: The aim of my thesis is to identify immunomodulators that modify the transcriptional profile of antigen-responsive human CD4+ T cells toward anergy or tolerance. I selected compounds targeting critical pathways of T cell activation that have relevance to autoimmunity in type 1 diabetes. I examined the in vitro effect of immunomodulation on naïve and memory CD4+ T cell proliferation upon stimulation with model antigens. Responsiveness of immunomodulated cells to subsequent antigen stimulation was also investigated. Single cell transcriptomic analyses were performed on proliferating and non-proliferating CD4+ T cells stimulated with model antigens in the presence of immunomodulation to detect potential alterations in their profile in comparison to untreated cells. Materials and Methods: CD4+ T cell responses to antigen stimulation were assessed using proliferation assays in the presence of absence of immunomodulating drugs. The drugs assessed were the JAK inhibitor Tofacitinib, the natural phenol Resveratrol, an anti-human IFN-αR monoclonal antibody, the anti-IL-6R monoclonal antibody Tocilizumab, the proteasome inhibitor Bortezomib, the IgG1-CTLA-4 fusion protein Abatacept, a DHODH inhibitor (Compound A) and a RORγT inverse agonist (Compound B). The effects on human peripheral blood memory CD4+ T cells were examined using the recall antigens Flu and Tetanus Toxoid and the superantigen SEB at a concentration that stimulated TCRVβ17+ cells. The effects on naïve CD4+ T cells were assessed using SEB. Responses were measured using flow cytometry. Re-stimulation of inhibited cells in the absence of immunomodulator was performed to examine the legacy of immunomodulated CD4+ T cells post-treatment. For the transcriptomic analyses, single cells were sorted with FACS and their transcriptome was sequenced with RNAseq. For memory CD4+ T cells stimulated with recall antigens, the proliferated responsive cells in cultures with and without immunomodulatory drug were examined and compared. For memory and naïve CD4+ T cells stimulated with SEB, the non-proliferated TCRVβ17+ CD4+ T cells in cultures with and without immunomodulatory drug were examined and compared. The differentially expressed genes were examined and further processed using the Ingenuity Pathway Analysis. Results: All the selected compounds consistently reduced the frequencies of proliferating memory CD4+ T cells responding to recall antigens, with the exception of the anti-IFN-αR and Tocilizumab. Re-stimulation in the absence of inhibitor showed a persistent loss of responsive capacity to the recall antigen for cells that had been exposed to Resveratrol and to the RORγT inverse agonist. The transcriptome of isolated single CD4+ CD25+ T cells that proliferated in the presence of these compounds was comparable to that of not treated proliferative cells, showing minor and inconsistent across PBMC samples differences in gene expression. These data suggest that the immunomodulators induced little change in memory CD4+ T cell phenotype. Resveratrol, the DHODH inhibitor and a RORγT inverse agonist, but none of the other compounds successfully inhibited proliferation of responsive to SEB naïve and memory TCRV17+ cells, without affecting their viability. Responsiveness of these inhibited cells to SEB was restored in the absence of immunomodulators. The transcriptome of isolated non-proliferating TCRV17+ cells exposed to SEB in the absence of immunomodulators was characteristic of stimulated T cells with upregulated pathways related to TCR signaling, anabolic biosynthetic processes and chromosomal replication. Non-proliferating cells that were stimulated but inhibited by the three compounds had a transcriptional profile consistent with downregulation of response to SEB. Resveratrol had the strongest downregulating effect in both naïve and memory cells. The inhibited pathways that were commonly affected by all compounds were associated with cell cycle control of chromosomal replication, tRNA charging, spliceosomal cycle and necroptosis, demonstrating the role of these pathways in the CD4+ T cell response to SEB. The transcriptomic data of immunomodulated dividing or not dividing CD4+ T cells did not provide evidence of an induced Treg phenotype. Conclusions: The established in vitro models identified six inhibitors of memory antigen-specific CD4+ T cell response and three that were additionally able to inhibit memory and naïve response to SEB. The transcriptomic data suggest that the primary effect of the tested immunomodulators is on the T cell responsiveness to antigen and not on the T cell phenotype. I conclude that while the majority of immunomodulators can severely inhibit CD4+ T cell response, none of the tested immunomodulators have a combined capacity to inhibit and shift antigen-responsive CD4+ T cells toward tolerogenic phenotypes in vitro. I interpret these data as indicating that successful therapeutic application is likely to require chronic administration. Hintergrund: CD4+ T-Zellen sind zentrale Koordinatoren der adaptiven Immunantwort. Sie vermitteln Entzündung durch klonale Expansion, durch Ausdifferenzierung in Subtypen der spezifischen Antwort und durch Produktion von Entzündungssignalen, die wiederum die Zytotoxizität und Antikörperproduktion durch andere Immunzellen fördern. CD4+ T-Zellen können sich auch in regulatorische T-Zellen (Treg) differenzieren, die Autoimmunantworten kontrollieren und die Toleranz fördern. Der Verlust der Toleranz von CD4+ T-Zellen gegenüber Autoantigenen führt zu unerwünschten T-Zell-Reaktionen und Autoimmunität. Immunmodulationstherapien, die CD4+ T-Zellen beeinflussen, sind zum Musterbeispiel geworden, um T-Zell-vermittelte Autoimmunität, entweder durch Blockierung von T-Zell-Aktivierungssignalen (Chamian et al., 2007; Keymeulen et al., 2005; Scheinfeld, 2005) oder durch Verbesserung der tolerogenen Funktion (Hartemann et al., 2013; Marcovecchio et al., 2020), umzukehren. Einige dieser auf einzelnen Medikamenten basierenden Therapien werden jetzt in Studien oder in der Praxis zur Behandlung von Patienten mit Autoimmunerkrankungen eingesetzt. Ihr tatsächlicher immunmodulierender Einfluss auf den CD4+ T-Zell-Phänotyp ist jedoch unklar. Fragestellung/Hypothese: Das Ziel meiner Doktorarbeit ist es, Immunmodulatoren zu identifizieren, die das Transkriptionsprofil humaner Antigen-responsiver CD4+ T-Zellen in Richtung Anergie oder Toleranz verändern. Hierzu wählte ich Substanzen aus, die auf maßgebliche Signalwege der T-Zell-Aktivierung wirken und für die Autoimmunität bei Typ-1-Diabetes von Bedeutung sind. Ich untersuchte den in vitro Effekt der Immunmodulation auf die Proliferation von naiven und CD4+ Gedächtnis-T-Zellen nach Stimulation mit Modellantigenen. Das Ansprechverhalten immunomodulierter Zellen auf nachfolgende Antigenstimulation wurde ebenfalls untersucht. Einzelzell-Transkriptomanalysen wurden an proliferierenden und nicht-proliferierenden CD4+ T-Zellen durchgeführt, die mit Modellantigenen in Gegenwart der Immunmodulation stimuliert wurden, um mögliche Veränderungen in ihrem Profil im Vergleich zu unbehandelten Zellen aufzuspüren. Material und Methoden: Die CD4+ T-Zell-Antworten auf die Antigenstimulation wurden mit Hilfe von Proliferationsassays in An- und Abwesenheit von immunmodulierenden Medikamenten untersucht. Bei den untersuchten Medikamenten handelte es sich um den JAK-Inhibitor Tofacitinib, das natürliche Phenol Resveratrol, einen monoklonalen Anti-IFN-αR-Antikörper, den monoklonalen Anti-IL-6R-Antikörper Tocilizumab, den Proteasominhibitor Bortezomib, das IgG1-CTLA-4-Fusionsprotein Abatacept, einen DHODH-Inhibitor (Substanz A) und einen inversen RORγT-Agonisten (Substanz B). Die Effekte auf CD4+ Gedächtnis-T-Zellen des humanen peripheren Blutes wurden mit den Recall-Antigenen Grippe und Tetanus-Toxoid, sowie mit dem Superantigen SEB in einer Konzentration, die TCRVβ17+ T-Zellen stimuliert, untersucht. Die Auswirkungen auf naive CD4+ T-Zellen wurden mit SEB untersucht. Die Antworten wurden mittels Durchflusszytometrie (FACS) gemessen. Eine Re-Stimulation der gehemmten Zellen in Abwesenheit des Immunmodulators wurde durchgeführt, um die längerfristige Wirkung der immunmodulierten CD4+ T-Zellen nach der Behandlung zu untersuchen. Für die Transkriptom-Analysen wurden einzelne Zellen via FACS sortiert und ihr Transkriptom mit von RNAseq sequenziert. Für CD4+ Gedächtnis-T-Zellen, die mit Recall-Antigenen stimuliert wurden, konnten die proliferierten, Antigen-responsiven T-Zellen in Kulturen mit und ohne Immunmodulator untersucht und verglichen werden. Für CD4+ Gedächtnis-T-Zellen und naive CD4+ T-Zellen, bei den die Stimulation mit SEB erfolgte, wurden nicht-proliferierte TCRVβ17+ CD4+ T-Zellen in Kulturen mit und ohne Immunmodulator analysiert. Die differentiell exprimierten Gene wurden ermittelt und mit Hilfe der Ingenuity Pathway Analyse untersucht. Ergebnisse: Alle ausgewählten Substanzen, mit Ausnahme von Anti-IFN-αR und Tocilizumab, reduzierten durchweg die Häufigkeit der proliferierenden CD4+ Gedächtnis-T-Zellen, die auf Recall-Antigene reagierten. Eine erneute Stimulation in Abwesenheit des Inhibitors zeigte einen anhaltenden Verlust des Ansprechverhaltens auf das Recall-Antigen für Zellen, die mit Resveratrol und dem inversen RORγT-Agonisten inkubiert worden waren. Das Transkriptom isolierter einzelner CD4+ CD25+ T-Zellen, die in Gegenwart dieser Substanzen proliferierten, war mit dem von nicht behandelten, proliferativen Zellen vergleichbar und zeigte geringfügige, und über PBMC-Proben hinweg inkonsistente, Unterschiede in der Genexpression. Diese Daten deuten darauf hin, dass die Immunmodulatoren nur eine geringe Veränderung des Phänotyps der CD4+ Gedächtnis-T-Zellen induzierten. Resveratrol, der DHODH-Inhibitor und ein inverser RORγT-Agonist, aber keine der anderen Substanzen hemmten erfolgreich die Proliferation von SEB-responsiven naiven und TCRVβ17+CD4+ Gedächtnis- T-Zellen, ohne deren Viabilität zu beeinträchtigen. Das Ansprechverhalten dieser gehemmten Zellen auf SEB wurde in Abwesenheit von Immunmodulatoren wiederhergestellt. Das Transkriptom von isolierten nicht-proliferierenden TCRV17+ T-Zellen, die SEB in Abwesenheit von Immunmodulatoren ausgesetzt wurden, war charakteristisch für stimulierte T-Zellen und zeigte hochregulierte Signalwege, die mit T-Zell-Rezeptor-Signaling, anabolen biosynthetischen Prozessen und chromosomaler Replikation zusammenhängen. Nicht-proliferierende Zellen, die stimuliert, aber durch die drei Substanzen gehemmt wurden, hatten ein Transkriptionsprofil, das mit einem Herunterregulieren der Reaktion auf SEB übereinstimmt. Resveratrol hatte den stärksten herunterregulierenden Effekt sowohl in naiven als auch in Gedächtnis-T-Zellen. Die gehemmten Signalwege, die von allen Substanzen gemeinsam beeinflusst wurden, waren mit der Zellzykluskontrolle der chromosomalen Replikation, der tRNA-Ladung, dem Spliceprozess und der Nekroptose assoziiert, was die Rolle dieser Signalwege bei der Reaktion der CD4+ T-Zellen auf SEB zeigt. Die Transkriptionsdaten von immunomodulierten, proliferierenden oder nicht-proliferierenden CD4+ T-Zellen lieferten keine Hinweise auf einen induzierten regulatorischen T-Zell-Phänotyp. Schlussfolgerung: Die etablierten in vitro-Modelle identifizierten sechs Inhibitoren der Antigen-spezifischen CD4+ Gedächtnis-T-Zell-Antwort und drei, die zusätzlich die Gedächtnis- und naive T-Zell-Antwort auf SEB hemmen konnten. Die Transkriptom-Daten legen nahe, dass der primäre Effekt der getesteten Immunmodulatoren auf dem T-Zell-Ansprechverhalten auf das jeweilige Antigen beruht und nicht auf Änderungen des T-Zell-Phänotyps zurückzuführen ist. Ich folgere daraus, dass die meisten Immunmodulatoren zwar die CD4+ T-Zell-Antwort stark hemmen können, aber keiner der getesteten Immunmodulatoren in vitro eine kombinierte Fähigkeit zur Hemmung der Antigen-responsiven CD4+ T-Zellen bei gleichzeitiger Verschiebung in Richtung tolerogener Phänotypen hat. Ich interpretiere diese Daten als Hinweis darauf, dass eine erfolgreiche therapeutische Anwendung dieser Substanzen voraussichtlich einer chronischen Verabreichung bedarf.