CD25 High Treg Research Articles

SARS-CoV-2 spike-specific regulatory T cells (Treg) expand and develop memory in vaccine recipients suggesting a role for immune regulation in preventing severe symptoms in COVID-19

We enrolled healthy subjects that received 2 to 4 injections of mRNA-based vaccination to prevent COVID-19 months to a year from the last vaccine boost, and we found numerous SARS-CoV-2 spike-specific regulatory T cell (Treg) that developed T cell memory as effector memory T cells (TEM) and central memory T cells (TCM). CD4+ CD25high Treg expressed the chemokine receptor CCR6 in a considerable percentage, suggesting T cell homing to the vascular endothelium, lung and gut epithelial cells and brain. Treg phenotype was different than peripherally-induced Treg (pTreg) that revert from pro-inflammatory T cells under repeated stimulatory conditions, suggesting that SARS-CoV-2 spike-specific Treg differentiated from naïve T cells in tissues where the SARS-CoV-2 spike proteins were synthetized. Twenty two of 22 subjects studied responded to vaccination developing a spike-specific CD4+ T helper (Th)1 response, and 20 of 22 developing a spike-specific CD8+ cytotoxic T cells (CTL) response. However, in vaccine recipients the expansion of spike-specific pro-inflammatory T cells was less significant than the expansion of spike-specific Treg. Effector (TEM) and central memory (TCM) Treg were numerous as early as after two vaccine doses, with no significant differences following additional vaccine boosts. In co-culture experiments under stimulatory conditions, Treg regulated naïve T cell differentiation toward a pro-inflammatory phenotype and suppressed interferon (IFN)γ production by SARS-CoV-2-specific CD4 + Th1 cells.

Abstract 3239: Selective depletion of regulatory T cells by ALD2510, a novel IL-2-sparing anti-CD25 antibody, synergizes with PD-1 blockade in breast and gynecologic cancers

Abstract Background: PD-1 blockade (αPD-1) has shown limited efficacy in breast & gynecologic cancers. In other types of solid tumor, regulatory T cells (Tregs) harbouring markers of highly suppressive effector Tregs (eTregs) correlates with poor responses to PD-1 blockade, prompting combination therapy based on eTregs depletion. Yet, subsets of Tregs remain to be determined in breast & gynecologic cancers, in order to: (i) evaluate the role of eTregs in resistances to PD-1 blockade, (ii) identify the most selective target for eTregs depletion tailored to tumors context and combination strategy. Methods: We collected public single-cell RNA/TCR-sequencing data from primary tumors and metastases of Triple-Negative Breast Cancer patients (TNBC, N = 28) biopsied before and after αPD-1 (pembrolizumab). Microarray data from primary TNBC (N = 124) biopsied before and after αPD-1 were quired for association with clinical responses. Deep phenotyping of Tregs from normal breast tissues (N = 4) and primary breast tumors (N = 8) or gynecologic tumors (N = 17) was performed by mass cytometry. ALD2510 (αCD25NIB, Alderaan Biotechnology) is a novel non-IL-2 blocking Fc-optimized anti-CD25 mAb (1). αPD-1 and αCD25NIB combination was evaluated using: (i) a humanized αCD25NIB in CD34+ humanized NSG mice grafted with human TNBC cell lines, (ii) a murine surrogate of αCD25NIB in a syngeneic TNBC mouse model. Results: CD25high Tregs were accumulating in breast & gynecologic tumor tissues. In TNBC patients treated with αPD-1, fractions of CD25high Tregs were further increased. CD25high Tregs highly expressed eTregs-related molecules in primary tumors, invaded tumor-draining lymph nodes and distant metastases. Amongst the potential therapeutic targets for Treg depletion, only CD25, 4-1BB and CCR8 were largely restricted to intratumoral CD25high eTregs. Yet, CD25 was the best candidate for CD25high eTregs depletion with limited on-target off-Treg effects, as: (i) CCR8 marked a small fractions of CD25high eTregs in primary tumors and was not detected in metastasis, (ii) 4-1BB marked antigen-experienced αβCD8 T cell revigorated by PD-1 blockade, (iii) only CD25high eTregs expressed 4-1BB and CCR8, yet TCR clonotype analysis revealed that CD25high eTregs originate in part from activated CD25+ Tregs, suggesting that anti-CCR8/-4-1BB mAbs may be ineffective due to CD25high eTregs replenishment in tumors. In murine models resistant to αPD-1 alone, αCD25NIB effectively depleted intratumoral CD25high eTregs, with limited effects on peripheral Tregs, and synergized with αPD-1 by restoring a positive effector αβCD8 T cells/Tregs ratio, leading to tumor clearance without adverse effects. Conclusions: This study supports clinical evaluation of CD25high effector Tregs depletion by ALD2510 in patients with breast or gynecologic cancers resistant to PD-1 blockade. Citation Format: Stéphane FATTORI, Aude Le Roy, Jemila Houacine, Lucie Robert, Riad Abes, Laurent Gorvel, Samuel Granjeaud, Marie-Sarah Rouvière, Amira Ben Amara, Nicolas Boucherit, Carole Tarpin, Jihane Pakradouni, Emmanuelle Charafe-Jauffret, Julien Barrou, Gilles Houvenaeghel, Eric Lambaudie, François Bertucci, Philippe Rochigneux, Anthony Gonçalves, Arnaud Foussat, Anne-Sophie Chrétien, Daniel Olive. Selective depletion of regulatory T cells by ALD2510, a novel IL-2-sparing anti-CD25 antibody, synergizes with PD-1 blockade in breast and gynecologic cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3239.

Abstract 710: Preclinical development of ALD2510, a Next-Gen Fc-enhanced, TREG-selective and IL-2-sparing anti-CD25 antibody for the treatment of solid tumors

Abstract Within the tumor microenvironment (TME), TREGS have been associated with worse prognosis and/or resistance to checkpoint inhibitor (CPI) therapy in various cancer diseases, making these cells a promising target for the development of new immunotherapies. Here, we present recent preclinical development data about ALD2510, a next-gen low-fucose IL-2/TCONV-sparing anti-CD25 antibody designed for the selective depletion of CD25high TREGS and the boost of host anti-tumor immunity. Ultra-humanization of parental anti-CD25 antibody was achieved through phage display and, in parallel, all putative liability sequences were replaced for maximizing developability while minimizing off-target and immunogenicity risks. ALD2510 sequences were re-formatted and used for further CHO cell line development, using the GlymaxX™ technology which boosts Fc effector functions. Early tox ALD2510 material was produced following 10L fed-batch USP/DSP and analytical characterization, including a 6-month early stability study. Preliminary safety and pharmacokinetic/pharmacodynamic (PKPD) profile of ALD2510 were then determined through a non-GLP MTD/DRF study in cynomolgus monkey. ALD2510 demonstrated excellent potency (TREG depletion, ADCC, ADCP), manufacturability and stability together with very low immunogenicity potential. CHO productivity reached ~4g/L in 10L fed-batch bioreactor and characterization revealed excellent purity and activity in line with its low fucose content, together with very low levels of process- or cell-related impurities. This material showed a good behavior during the 6-month stability study and was therefore administered in cynomolgus monkeys during a non-GLP exploratory MTD/DRF study. Increasing doses of ALD2510 (from 0.3 to 100mg/kg) were given to four animals during MTD and same animals received three additional doses at 100mg/kg during DRF. Very good tolerability and safety were reported in animals. ALD2510 half-life and exposure were found consistent with that of humanized IgG1 in cynomolgus. Strong TREG depletion was monitored in the blood in all animals with, importantly, no drop was observed in CD4+ or CD8+ T-cells, confirming ALD2510 capacity to selectively target TREGS while sparing TCONV. Overall, ALD2510 demonstrated excellent potency, manufacturability and stability during the 1st phase of preclinical development. Very good tolerability and safety profile together with satisfying PKPD data were reported during exploratory MTD/DRF study in cynomolgus monkeys. Noteworthy, strong TREG depletion was confirmed while CD4+ or CD8+ T-cells were not impacted, confirming ALD2510 selectivity for TREGS while sparing TCONV. This makes ALD2510 a promising candidate for the treatment of solid tumors, in particular those where the presence of TREGS in the TME is associated with worse prognosis and/or resistance to CPI. Citation Format: Jemila Houacine, Riad Abes, Anne Marie-Cardine, Aude Le Roy, Bettina Serbin, Lucie Robert, Jérôme Giustiniani, Anne-Sophie Chrétien, Stéphane Fattori, Laurent Gorvel, Armand Bensussan, Daniel Olive, Arnaud Foussat. Preclinical development of ALD2510, a Next-Gen Fc-enhanced, TREG-selective and IL-2-sparing anti-CD25 antibody for the treatment of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 710.

Rheumatoid arthritis and the risk of preterm birth.

During pregnancy, many diseases are correlated with different adverse outcomes. In turn, pregnancy affects the body, leading to increased disease susceptibility. This interplay between diseased states and pregnancy outcomes is illustrated in the effect of the chronic autoimmune disorder, rheumatoid arthritis (RA), and the adverse outcome, preterm birth (PTB). RA is a systemic disorder characterized by inflammation of the joints and other body organs. Joint pain and swelling are the most prominent manifestations of RA during pregnancy. However, the exact role of RA on PTB among pregnant women has yet to be established. This review highlighted the immunologic mechanisms involved in PTB in pregnant patients with RA. The immune cell population in pregnant women with RA exhibited higher activity of macrophages, dendritic cells, neutrophils, helper T (Th) 1 cells, and Vδ1 cells, but lower activity of CD4+CD25high T regulatory (CD24+CD25high Treg ), Th2, and Vδ2 cells. Increased pro-inflammatory cytokines IL-6, TNF-α, and IFN-γ and decreased anti-inflammatory cytokines IL-12 and IL-10 are also exhibited by pregnant patients with RA. This review also discussed factors that may predict the risk of PTB in RA. These include disease activity and severity of RA, laboratory parameters (cytokines and immune cell population), and sociodemographic factors such as ethnicity, smoking, alcohol intake, and the level of education. Current findings on the underlying immunological mechanisms of RA can help identify possible strategies to prevent PTB.

Immunopathology of SARS-CoV-2 Infection: A Focus on T Regulatory and B Cell Responses in Children Compared with Adults.

While the clinical impact of COVID-19 on adults has been massive, the majority of children develop pauci-symptomatic or even asymptomatic infection and only a minority of the latter develop a fatal outcome. The reasons of such differences are not yet established. We examined cytokines in sera and Th and B cell subpopulations in peripheral blood mononuclear cells (PBMC) from 40 children (<18 years old), evaluating the impact of COVID-19 infection during the pandemic’s first waves. We correlated our results with clinical symptoms and compared them to samples obtained from 16 infected adults and 7 healthy controls. While IL6 levels were lower in SARS-CoV-2+ children as compared to adult patients, the expression of other pro-inflammatory cytokines such as IFNγ and TNFα directly correlated with early age infection and symptoms. Th and B cell subsets were modified during pediatric infection differently with respect to adult patients and controls and within the pediatric group based on age. Low levels of IgD− CD27+ memory B cells correlated with absent/mild symptoms. On the contrary, high levels of FoxP3+/CD25high T-Regs associated with a moderate–severe clinical course in the childhood. These T and B cells subsets did not associate with severity in infected adults, with children showing a predominant expansion of immature B lymphocytes and natural regulatory T cells. This study shows differences in immunopathology of SARS-CoV-2 infection in children compared with adults. Moreover, these data could provide information that can drive vaccination endpoints for children.

Regulatory T-lymphocyte subsets in children with chronic immune thrombocytopenia after high-dose of dexamethasone

BackgroundImmune thrombocytopenia (ITP) is an acquired autoimmune disease. This study’s objective was to estimate the variations in the population of CD4+CD25+High FoxP3+ cells (CD4+ regulatory T-lymphocytes; Tregs) in previously untreated children with chronic ITP managed in Assiut University Hospitals, as well as to evaluate the efficacy of high-dose dexamethasone (HD-DXM) in these patients.MethodsIn this study, we investigated the frequencies of T-lymphocyte subsets in 27 untreated children with chronic ITP.ResultsPrior to treatment, the percentages of CD4+CD25High cells and Tregs were significantly lower in the chronic ITP group compared to the control group (p = 0.018 and p < 0.0001, respectively). After treatment with HD-DXM, Tregs and platelets were significantly increased in these patients (p < 0.0001 for both).ConclusionsOur results suggest that Tregs are deficient in children with chronic ITP and that HD-DXM immunosuppressive therapy can restore the levels of these cells.ImpactCD4+CD25High cells and Tregs were significantly lower in children chronic ITP compared to healthy control.HD-DXM treatment led to significantly increased Tregs and platelets in these patients.Our results suggest that Tregs are deficient in children with chronic ITP and that HD-DXM immunosuppressive therapy can restore the levels of these cells.

Ex vivo delivery of regulatory T-cells for control of alloimmune priming in the donor lung.

Survival after lung transplantation (LTx) is hampered by uncontrolled inflammation and alloimmunity. Regulatory T-cells (Tregs) are being studied as a cellular therapy in solid organ transplantation. Whether these systemically administered Tregs can function at the appropriate location and time is an important concern. We hypothesised that in vitro-expanded recipient-derived Tregs can be delivered to donor lungs prior to LTx via ex vivo lung perfusion (EVLP), maintaining their immunomodulatory ability. In a rat model, Wistar Kyoto (WKy) CD4+CD25high Tregs were expanded in vitro prior to EVLP. Expanded Tregs were administered to Fisher 344 (F344) donor lungs during EVLP; left lungs were transplanted into WKy recipients. Treg localisation and function post-transplant were assessed. In a proof-of-concept experiment, cryopreserved expanded human CD4+CD25+CD127low Tregs were thawed and injected into discarded human lungs during EVLP. Rat Tregs entered the lung parenchyma and retained suppressive function. Expanded Tregs had no adverse effect on donor lung physiology during EVLP; lung water as measured by wet-to-dry weight ratio was reduced by Treg therapy. The administered cells remained in the graft at 3 days post-transplant where they reduced activation of intra-graft effector CD4+ T-cells; these effects were diminished by day 7. Human Tregs entered the lung parenchyma during EVLP where they expressed key immunoregulatory molecules (CTLA4+, 4-1BB+, CD39+ and CD15s+). Pre-transplant Treg administration can inhibit alloimmunity within the lung allograft at early time points post-transplant. Our organ-directed approach has potential for clinical translation.

Age-Associated Characteristics of CD4+ T-Cell Composition in Patients with Atherosclerosis

Background. We aimed to analyze the contents of the main CD4+ T-cell subsets in patients with atherosclerosis (AS) depending on age. Methods. Male patients with coronary and/or carotid AS, who are non-smokers, and who are receiving statins were divided into three age groups (I—&lt;55 y.o. (n = 23), II—55–64 y.o. (n = 42), III—≥65 y.o. (n = 46)). Leukocyte phenotyping was performed by direct immunofluorescence and flow cytometry. For intracellular cytokine detection, blood mononuclear cells were pre-activated with phorbol 12-myristate 13-acetate and ionomycin in the presence of an intracellular vesicle transport blocker monensin. Results. The groups did not differ in traditional CVD risk factors and AS severity. The content of CD4+ T-cells was lower in group III and II than in group I. The content of CD4+CD25high Treg was lower in group III than in groups I and II. No differences in the quantities of the primed CD39+CD45RA− and CD278high Treg, CD4+INFγ+ Th1, CD4+IL17+ Th17, and CD4+IL17+INFγ+ Th1/17 were observed. There were negative correlations between the values of CD4+ T-cells, CD4+CD45RA+ T-cells, CD4+CD25high Treg, CD4+CD25highCD45RA+ Treg, and age. Conclusion. In patients with AS, the age-related depletion of naive CD4+ T-cells also extends to the regulatory compartment. This phenomenon should be considered when studying the impact of the immune cells on the progression of AS.

Temporal and spatial characterization of negative regulatory T cells in HIV-infected/AIDS patients raises new diagnostic markers and therapeutic strategies.

BackgroundNegative regulatory T cells (Tregs) not only deplete effector T cells but also inhibit the clearance of HIV during infection, which may allow Tregs to be used as informative diagnostic markers. To facilitate both diagnosis and treatment, a thorough understanding of these regulators by characterizing them on temporal and spatial scales is strongly required.MethodsHundred HIV‐infected/AIDS patients, including 87 males, with an average age of 35.8 years, as well as 20 healthy controls, were enrolled. Flow cytometry was used to analyze CD3+T cells, CD4+T cells, and CD8+T cells to evaluate the immune status of the participants. Then, a group of representative negative regulatory T cells, including CD4+PD‐1+T cells, CD4+PD‐1highT cells, CD8+PD‐1+T cells, and CD4+CD25high Tregs was also analyzed to explore their effects on disease progression and intercorrelation.ResultsThe percentages of CD4+PD‐1+T cells and CD4+CD25highTregs increased in patients with the same ultrahigh significance. Temporally, the patients with both intermediate‐stage and late‐stage disease had higher percentages of CD4+PD‐1+T cells; however, the percentage of CD4+CD25highTregs only increased in the patients with late‐stage disease. In addition, CD4+PD‐1+T cells but not CD4+CD25highTregs were negatively correlated with the absolute CD4+T cell count. Spatially, no correlations between CD4+PD‐1+T cells and CD4+CD25highTregs were observed, which suggests these Tregs function differently during immunosuppression.ConclusionsThis study characterized negative regulatory T cells in HIV‐infected/AIDS patients at both temporal and spatial scales and found that CD4+CD25+Tregs and CD4+PD‐1+T cells could be used as potential diagnostic markers for identifying different disease stages and monitoring disease progression.

Aging is accompanied by a decrease in the number of naive Tregs

Abstract Funding Acknowledgements Type of funding sources: None. Background. Age-related changes in the immune system are an important factor contributing to the maintenance of chronic inflammatory status. There are undoubted data on the decrease of the number of T-lymphocytes with age caused by thymus involution, but there are currently no unambiguous data on changes of minor T-cell subpopulations, in particular, regulatory T-cells (Treg). The aim of this study was to analyze the content of effector and regulatory CD4+ T cell subsets in patients with coronary and/or carotid atherosclerosis depending on age. Methods. 111 patients (men, median age 63 (55;69)) with coronary and/or carotid atherosclerosis, without smoking anamnesis, were enrolled. Mononuclear leuocytes were isolated from blood by gradient centrifugation, and CD4 + CD25high and CD4 + Foxp3+ Treg, CD4 + IL17+ T-helpers (Th) 17 and CD4 + INFγ Th1 were evaluated by direct immunofluorescence and flow cytometry. For intracellular cytokine detection cells were pre-activated in vitro in the presence of PMA/ionomycin/brefeldin A. In 74 patients cells were additionally stained with CD39, CD278, CD45RA Mabs to reveal naïve and primed T-cells. Results. According to age the patients formed three groups: I – &amp;lt;55 y.o. (n = 23), II – 55-64 y.o. (n = 42), III - ≥65 y.o. (n = 46). All patients were taking statins at baseline. The groups were comparable in traditional risk factors of CVD (BMI, arterial hypertension, diabetes mellitus, previous myocardial infarction anamnesis). The absolute content of CD4+ T cells was lower in group III (646.3 (516.0;806.4)) compared to groups I (903.0 (585.6;1113.8), p = 0.03) and II (745.4 (502.2;924.0), p = 0.06). The absolute content of CD4 + CD25high Treg was lower in group III (24.2 (18.4;35.2)) compared to groups I (35.0 (28.7;54.4), p = 0.01) and II (31.0 (21.1;43.6), p = 0.03). There were no differences in Th1, Th17, CD39 + CD45RA- and CD278+ Treg content between groups. A negative correlation was found between age and the content of CD4+ T cells (r= -0.28), CD4 + CD25high Treg (r= -0.27), p &amp;lt; 0.05. A negative correlation was found between age and CD4 + CD25highCD45RA+ Treg (r= -0.24) and CD4 + CD45RA+ T cells (r= -0.36), CD4 + CD45RA+/CD4 + CD45RA- T-cells ratio (r= -0.24), p &amp;lt; 0.05. Conclusion. Here we demonstrate an age-dependent decrease of total CD4+ T cell population and Treg subset in patients with atherosclerosis. The changes observed were primary due to the deficiency of CD45RA+ naïve T cells. The effector cell Th1 and Th17 quantities were at the same levels. Future research will show whether the identified immunological patterns can contribute to the progression of atherosclerosis and other chronic inflammatory diseases.

Low-Zone IL-2 Signaling: Fusion Proteins Containing Linked CD25 and IL-2 Domains Sustain Tolerogenic Vaccination in vivo and Promote Dominance of FOXP3+ Tregs in vitro.

Low-zone IL-2 signaling is key to understanding how CD4+ CD25high FOXP3+ regulatory T cells (Tregs) exhibit dominance and overgrow conventional effector T cells (Tcons) that typically express lower levels of the IL-2 receptor alpha chain (i.e., CD25). Thus, modalities such as low-dose IL-2 or IL-2/anti-IL-2 antibody complexes have been advanced in the clinic to selectively expand Treg populations as a treatment for chronic inflammatory autoimmune diseases. However, more effective reagents that efficiently lock IL-2 signaling into a low signaling mode are needed to validate and exploit the low-zone IL-2 signaling niche of Tregs. This study focuses on CD25-IL2 and IL2-CD25 fusion proteins (FPs) that were approximately 32 and 320-fold less potent than IL-2. These FPs exhibited transient binding to transmembrane CD25 on human embryonic kidney (HEK) cells, had partially occluded IL-2 binding sites, and formed higher order multimeric conformers that limited the availability of bioactive IL-2. These FPs exhibited broad bell-shaped concentration ranges that favored dominant Treg outgrowth during continuous culture and were used to derive essentially pure long-term Treg monocultures (∼98% Treg purity). FP-induced Tregs had canonical Treg suppressive activity in that these Tregs suppressed antigen-specific proliferative responses of naïve CD4+ T cells. The in vivo administration of CD25-IL2/Alum elicited robust increases in circulating Tregs and selectively augmented CD25 expression on Tregs but not on Tcons. A single injection of a Myelin Oligodendrocyte Glycoprotein (MOG35-55)-specific tolerogenic vaccine elicited high levels of circulating MOG-specific Tregs in vivo that waned after 2–3 weeks, whereas boosting with CD25-IL2/Alum maintained MOG-specific CD25high Tregs throughout the 30-day observation period. However, these FPs did not antagonize free monomeric IL-2 and lacked therapeutic efficacy in experimental autoimmune encephalomyelitis (EAE). In conclusion, these data reveal that CD25-IL2 FPs can be used to select essentially pure long-term lines of FOXP3+ CD25high Tregs. This study also shows that CD25-IL2 FPs can be administered in vivo in synergy with tolerogenic vaccination to maintain high circulating levels of antigen-specific Tregs. Because tolerogenic vaccination and Treg-based adoptive immunotherapy are limited by gradual waning of Tregs, these FPs have potential utility in sustaining tolerogenic Treg responses in vivo.

Abstract 4553: The CD25 antibody RG6292 selectively depletes Tregs while preserving IL-2 signaling and CTL activity for tumor control

Abstract Despite the advances in cancer immunotherapy, in particular in the field of checkpoint inhibitors (CPI), many patients fail to respond (primary resistance) or initially benefit but then progress upon treatment (secondary resistance). High regulatory T-cell (Treg) counts correlate with poor prognosis and reduced responsiveness to CPI therapy in humans, underscoring their potential as an immunotherapy target. Clinical attempts aiming to lower Treg counts, however, either failed to deliver convincing Treg reduction or lacked specificity for Treg over tumor antigen specific cytotoxic T cells (CTL). CD25 (the interleukin-2 receptor alpha (IL-2Ra) chain) is a recently revisited target for Treg depletion. For privileged access to IL-2, activated CTL up-regulate CD25 expression only transiently during clonal expansion. Further confirmation of an &amp;gt; 20 fold higher cell surface expression of CD25 on Tregs versus CTLs in human malignancies is provided. The novel compound RG6292 was developed as an ADCC and ADCP competent monoclonal antibody of human IgG1 isotype with afucosylated glycans in the Fc region. RG6292 binds with low monovalent affinity (KD 250 nM) to the extracellular domain of CD25 antigen. A high density of CD25 receptors promotes bivalent avidity of RG6292 increasing its binding strength to CD25 by at least 100 fold (KD 2-3 nM). RG6292 selectively favors the depletion of CD25 high Tregs over CD25 low activated CTLs, here shown in comparison to ipilimumab and mogamulizumab in human αCD3 activated PBMC, human tumor explants and immunopharmacodynamic studies in tumor bearing (BxPC-3), stem cell humanized mice and cynomolgus monkeys. IL-2 is an essential prerequisite for clonal expansion of CTLs, which is necessary to generate effective anti-tumor responses. Earlier immunosuppressant anti-CD25 antibodies (e.g. daclizumab and basiliximab) interfered with the formation of the high affinity IL-2R complex. Their evidenced lack of therapeutic activity in immunoncology tempered enthusiasm and highlights the pivotal role of IL-2. RG6292 is the first anti-human CD25 antibody developed to deplete Tregs selectively while fully preserving IL-2 signaling and CTL activity. Pre-clinically, a single administration of the RG6292 surrogate effectively promoted eradication of established tumors in several tumor mouse models and synergized with CPI in models of CPI resistance. RG6292 is expected to unleash the potential of selective Treg depletion while allowing for unrestricted access of IL 2 to CTLs and could therefore result in clinically superiority compared to other Treg depleting antibodies. RG6292 provides a novel therapeutic approach to alleviate a major mechanism of immune suppression in the tumor microenvironment. Clinical testing is currently ongoing to evaluate the safety and tolerability of RG6292 in patients with advanced solid tumors (NCT04158583). Citation Format: Maria Amann, Gabriel Schnetzler, Kolben Theresa, Isabelle Solomon, Christophe Boetsch, Estelle Marrer-Berger, Reto Flury, Claudio Murgia, Vaios Karanikas, Johannes Sam, Roger Sutmuller, Jan Eckmann, Hans Koll, Sara Belli, Frederic Arce Vargas, Dimitrios Zervas, Chen Qing, Mark A. Brown, Josephine Salimu, Anne Goubier, Sebastian Neumann, Karl S. Peggs, Sergio A. Quezada. The CD25 antibody RG6292 selectively depletes Tregs while preserving IL-2 signaling and CTL activity for tumor control [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4553.

Stability and inhibitory function of Treg cells under inflammatory conditions in vitro.

Immunotherapy with transplanted T-regulatory (Treg) cells is currently in use. However, patients have complex internal environments with confounding factors, including the presence of inflammatory cytokines. The present study aimed to detect Treg cell function under simulated inflammatory conditions to provide a foundation for Treg cell-based immunotherapy. CD4+CD25high Treg cells were sorted from peripheral blood mononuclear cells and cultured for 14 days in the presence of recombinant human interleukin-2 (rhIL-2) and anti-CD3/CD28 beads, with or without 25 ng/ml rhIL-6. Next, the absolute count of Treg cells was determined, the stability and activity were detected by measuring the expression levels of forkhead box (Fox)P3 and CD39, and the suppressive function of Treg cells was investigated by assessing the suppression of T-effector cell proliferation by Treg cells after co-culture for 5 days. The number of Treg cells cultured in the presence of 25 ng/ml rhIL-6 for 14 days was reduced by 49.7% when compared with that of cells cultured without rhIL-6. Of the Treg cells continually cultured for 14 days without or with 25 ng/ml rhIL-6, 56.15 and 24.7% expressed FoxP3, respectively. There was no difference in the activity of the FoxP3+ Treg cells after culture for 14 days without or with 25 ng/ml rhIL-6. The suppressive function of Treg cells tended to deteriorate in the presence of rhIL-6. In conclusion, IL-6 inhibited the proliferation and stability of Treg cells, suggesting that administration of increased numbers of Treg cells may be required during Treg cell-based immunotherapy.

Effects of allergen-specific immunotherapy on peripheral blood regulatory T cells and serum concentrations of cytokines and immunoglobulins in horses with allergic dermatitis

The aim of this study was to assess the effect of allergen-specific immunotherapy (ASIT) on the immunological responses of horses. Blood samples were taken from thirty-two horses with allergic dermatitis treated with ASIT and 10 healthy control horses at 0, 3, 6, 9 and 12 months to investigate the evolution of the percentage of regulatory T cells (Treg) in the peripheral blood and the serum levels of cytokines and immunoglobulins. Clinical improvement was appreciated by the majority of the horses' owners (56.6%). No effect of ASIT on CD4+CD25High Treg cells was found during the one year treatment period. No differences in the percentage of CD4+ T cells were observed between the groups, and no effects of ASIT over time were observed. The percentage of CD25+ T cells was always higher in the ASIT group (17.9 ± 11.3%) than in the control group (7.3 ± 4.4%, p < 0.001). We did not detect any effect of ASIT on the serum levels of TGF-β, IL-10 and IFN-γ or on the serum concentrations of IgA and IgG4. A reduction in the serum levels of total IgE in the horses with allergic dermatitis was observed at the 6th month (p < 0.05), but increased again at the end of the study. The results indicate that immunotherapy was insufficient to induce significant changes that could indicate T cell tolerance, a shift in cytokine production to more protective Th1 cells. More studies are needed with new vaccine compositions and administration protocols to improve the immunological responses of the horses with allergic dermatitis.

Evaluation of Exhausted Regulatory T Cells in Preeclampsia.

The development of a maternal immune response to fetal antigens and deficiency in regulatory T-cells (Tregs) may lead to preeclampsia. A plausible explanation for the reduced Treg cell function in women with preeclampsia is the presence of exhausted Treg cells which express CD279 or programmed cell death receptor-1 (PD-1), a negative regulatory molecule associated with limited proliferative capacity and reduced immune suppression. To assess the number of Treg CD4+ CD25 high and exhausted Treg CD4+ CD25 high CD279+ cells in women with preeclampsia (PE group) and healthy pregnant women (HP group) during the third trimester of pregnancy. Three-color flow cytometry was used to determine the proportion of Treg and exhausted Treg cells in 40 women in the PE group and 37 women in the HP group. Participants' blood samples were placed in EDTA blood collection tubes. Peripheral mononuclear cells were separated from the samples and stained with flurochrome-conjugated antibodies against human CD4, CD25 and CD279 markers, and subsequently analyzed by flow cytometry. The PE group had fewer Tregs compared to the HP group (p=0.011). There was a significant increase in the percentage of exhausted PD-1+(CD279) Tregs (p=0.035) in the PE group comparisons with the HP group. The increased number of PD-1 (CD279) molecules on the Treg cells may play a role in preeclampsia, hence it recommendation as a therapeutic target for the disease.

Cytokine Expression and Alloreactivity in a Humanized Mouse Transplant Arteriosclerosis Model Reflects Primary Graft Dysfunction in Lung Transplant Recipients

Purpose An important cause of early mortality after lung transplantation is primary graft dysfunction (PGD). In a humanized mouse model, we studied the impact of the baseline condition of the endothelium of lung donors in combination with recipient PBMC with or without PGD on the development of luminal occlusion. Methods Segments of arteries were implanted into the aorta of immunodeficient mice. Mice were divided into six treatment groups: group A mice with aortic vessels from PGD grade ≥ 2 (PGD+) patients were reconstituted with the respective allogeneic recipient PBMC, group B mice additionally received recipient CD4+CD25high Treg cells. Group C mice with aortic vessels from PGD grade ≤ 1 (PGD-) patients received recipient PBMC and group D mice additionally CD4+CD25high Treg cells. Control group E mice received arteries from PGD+, group F mice from PGD- patients, both without PBMC reconstitution. Luminal occlusion was quantified histologically and systemic immune responses by quantification of plasma cytokine and chemokines etc. Results Luminal occlusion of aortic vessels as sign of TA was significantly more severe in PGD+ group E compared to those from PGD- group F control mice in the absence of PBMC which was paralleled by higher IFN-γ (p Conclusion We conclude that a pre-existing Th1 response of the donor endothelial system contributes to PGD development, which is further enhanced by alloreactive T cells.

Recruitment of CCR6+ Foxp3+ regulatory gastric infiltrating lymphocytes in Helicobacter pylori gastritis.

Helicobacter pylori (H.pylori) infection is associated with an inflammatory response in the gastric mucosa, leading to chronic gastritis, peptic ulcers, and gastric cancer. Increased T-cell infiltration is found at sites of H.pylori infection. The CCR6+ subset of CD4+ regulatory T cells (Tregs), a newly characterized subset of Tregs, has been reported to contribute to local immune inhibition. However, whether CCR6+ Tregs are present in H.pylori gastritis, and what their relationship is to disease prognosis, remains to be elucidated. In this study, gastric infiltrating lymphocytes were isolated from endoscopic biopsy specimens of H.pylori gastritis patients and analyzed. We found that in gastric infiltrating lymphocytes, CCR6+ CD4+ CD25high Tregs, which express high levels of CD45RO, are positively associated with more severe inflammation in gastric mucosa during H.pylori infection. Furthermore, the frequency of CCR6+ Tregs in gastric infiltrating lymphocytes, but not CCR6- Tregs, is significantly increased in inflamed gastric tissues, which is inversely correlated with significantly lower expression of IFN-γ+ CD8+ T cells. We also found that the frequency of CCR6+ Tregs is positively correlated with the frequency of CD4+ IFN-γ+ T cells. In addition, the frequency of CCR6+ Tregs, but not that of CCR6- Tregs, is significantly correlated with increased inflammation in H.pylori gastritis. This study demonstrates that immunosuppression in H.pylori gastritis might be related to the activity of CCR6+ Tregs, which could influence disease prognosis.

Role of cluster of differentiation 39 (CD39) in the immunosuppressive status associated Acute myeloid leukemia (AML)

Background: CD39 is an integral membrane protein (ectoenzyeme) that phosphohydrolyzes ATP in a Ca2+- and Mg2+-dependent fashion to yield AMP, Can be viewed as immunological switch, and is expressed in spleen, thymus, lung, and placenta, and in these organs it is associated primarily with endothelial cells and immune cell populations, such as B cells, NK cells, dendritic cells, Langerhans cells, monocytes, macrophages, mesangial cells, neutrophils, and regulatory T cells. Aim of the study is to assess the expression of CD39 on CD4+ CD25high T-reg cells in patients with acute myeloid leukemia to identify its role in immunosuppressive status of AML patients. Methods: Flow cytometric analysis of T-reg cells using CD4 CD25 and CD39 in 60 AML patients and 15 apparently healthy controls with matched age and sex was done. Results: There was a significant difference between cases and controls as regards expression of CD39 on T-helper cells. A highly significant elevation of expression of CD39 has been obtained on T-reg cells in AML patients compared with Controls with increased expression of CD39 on T-reg cells than on helper T cells. Conclusion: CD39 plays a significant role in immunosuppressive status of AML patients particularly with expressing the receptor of α chain of IL-2 (CD25)

Astragalus Oral Solution Ameliorates Allergic Asthma in Children by Regulating Relative Contents of CD4+CD25highCD127low Treg Cells.

Objective: To explore the effects of Astragalus oral solution (AOS) on allergic asthma in children by investigating relative contents of CD4+CD25highCD127low Treg cells.Methods: The contents of Astragaloside A in AOS were detected by using HPLC. Eighty children with allergic asthma were recruited from February 2016 to June 2017, and randomly assigned into the control group (received placebo, 0.1% quinine chloride in deionized water, daily) and the AOS group (received 10 mL AOS daily). After 6-month treatment, therapeutic results were compared between the two groups. Serum levels of IL-10 and TGF-beta, Th1 cytokines (IL-2 and IFN-γ), and Th2 cytokines (IL-4 and IL-6) were measured by using ELISA kits. Relative contents of CD4+CD25highCD127low Treg cells were determined by using flow cytometry.Results: Astragaloside A was the main ingredient of AOS with 0.216 ± 0.027 mg/mL from six-batch samples. After 6-month therapy, the AOS group showed improved forced expiratory volume in 1 s (FEV1) and the Pediatric Asthma Quality of Life Questionnaire (PAQLQ) scores compared with the control group (P < 0.05). Serum level of IL-10 was higher and the levels of TGF-beta, Th1 cytokines (IL-2 and IFN-γ), and Th2 cytokines (IL-4 and IL-6) were lower in the AOS group than in the control group (P < 0.05). AOS treatment increased the percentage of gated CD4+ T cells, CD4+CD25+ T cells, CD4+CD25high Treg cells, CD4+CD25+FoxP3+ Treg cells and CD4+CD25highCD127low Treg cells when compared with the control group (P < 0.05).Conclusions: Astragaloside A was the main component of AOS, and AOS ameliorated allergic asthma in children by regulating relative contents of CD4+CD25highCD127low Treg cells.

Novel Effector Phenotype of Tim-3+ Regulatory T Cells Leads to Enhanced Suppressive Function in Head and Neck Cancer Patients.

Purpose: Regulatory T (Treg) cells are important suppressive cells among tumor-infiltrating lymphocytes (TIL). Treg cells express the well-known immune checkpoint receptor PD-1, which is reported to mark "exhausted" Treg with lower suppressive function. T-cell immunoglobulin mucin (Tim)-3, a negative regulator of Th1 immunity, is expressed by a sizeable fraction of TIL Tregs, but the functional status of Tim-3+ Tregs remains unclear.Experimental Design: CD4+CTLA-4+CD25high Treg cells were sorted from freshly excised head and neck squamous cell carcinoma (HNSCC) TIL based on Tim-3 expression. Functional and phenotypic features of these Tim-3+ and Tim-3- TIL Tregs were tested by in vitro suppression assays and multi-color flow cytometry. Gene-expression profiling and NanoString analysis of Tim-3+ TIL Treg were performed. A murine HNSCC tumor model was used to test the effect of anti-PD-1 immunotherapy on Tim-3+ Treg.Results: Despite high PD-1 expression, Tim-3+ TIL Treg displayed a greater capacity to inhibit naïve T-cell proliferation than Tim-3- Treg. Tim-3+ Treg from human HNSCC TIL also displayed an effector-like phenotype, with more robust expression of CTLA-4, PD-1, CD39, and IFN-γ receptor. Exogenous IFN-γ treatment could partially reverse the suppressive function of Tim-3+ TIL Treg. Anti-PD-1 immunotherapy downregulated Tim-3 expression on Tregs isolated from murine HNSCC tumors, and this treatment reversed the suppressive function of HNSCC TIL Tregs.Conclusions: Tim-3+ Treg are functionally and phenotypically distinct in HNSCC TIL, and are highly effective at inhibiting T-cell proliferation despite high PD-1 expression. IFN-γ induced by anti-PD-1 immunotherapy may be beneficial by reversing Tim-3+ Treg suppression. Clin Cancer Res; 24(18); 4529-38. ©2018 AACR.