Treg Expansion Research Articles

Dynamic tracking of tumor microenvironment modulation using Kaede photoconvertible transgenic mice unveils new biological properties of viral immunotherapy.

CAN-2409 is a replication-defective adenovirus that delivers the herpes simplex virus (HSV)-thymidine kinase gene to infected cells. Intratumoral administration of CAN-2409 followed by prodrug results in the formation of a toxic metabolite able to induce immunogenic cell death, exposure of tumor-associated antigens, and activation of local and systemic immune responses. We used a dynamic labeling model with MC38 tumor cells implanted in photoconvertible Kaede mice. Violet light was used to label the tumor microenvironment, distinguishing retained versus newly entering cells and allowing real-time monitoring of immune compartment changes within tumors. Administration of CAN-2409 + prodrug led to control of tumor growth and a significantly increased effector CD8+ T cell responses. Photolabeling of the tumor microenvironment (TME) revealed that rather than enhancing recruitment of T cells to the tumor, CAN-2409 altered the TME whereby newly entering and retained CD8+ T cells were significantly more proliferative. CAN-2409 supported reinvigoration of tumor associated antigen-specific CD8+ T cells and expansion of Tregs of an altered phenotype. Moreover, the combination of CAN-2409 + prodrug and anti-CTLA-4 antibody treatment further improved control of tumor growth, in part by the enhanced CD8+ T cell-mediated effector function and diminished Treg-mediated immunosuppression. Collectively, these data defined at least two temporally distinct pathways underpinning the mechanism of action of CAN-2409 action that overcome cell exhaustion and decreases immune suppression. The results also support the rationale for future clinical trials of CAN-2409 treatment combined with anti-CTLA-4 antibody therapy.

Fluoride-Mediated Immune Damage Through Cytokine Network Regulation of Tregs

Long-term fluoride exposure can induce inflammatory responses in various tissues of the body, thereby affecting the inflammatory microenvironment. To explore how fluoride induces changes in immune function within this microenvironment, this study collected baseline information and biological samples from participants in areas with the drinking water type of fluorosis, and simultaneously established Wistar rat models with a 12-week and 24-week fluoride exposure, as well as a 12-week fluoride exposure followed by 12-week pure water feeding regimen. Luminex multiplex assays and enzyme-linked immunosorbent assays (ELISAs) were used to measure cytokine expression levels. Subsequently, correlation analysis, multiple linear regression, and mediation analysis were employed to explore the long-term effects induced by the complex cytokine network during fluoride exposure. The population survey results indicated that fluoride suppressed the expression of pro-inflammatory factors such as Interleukin-2 (IL-2), Interleukin-12 (IL-12), Interferon-γ (IFN-γ), Tumor necrosis factor-α (TNF-α), and anti-inflammatory factors such as Interleukin-4 (IL-4), Interleukin-13 (IL-13), and Interleukin-37 (IL-37), while promoting an increase in the proportion of regulatory T cells (Tregs) in peripheral blood. Among these, IL-2 and IFN-γ mediated the fluoride-induced peripheral Tregs expansion. Animal experiments indicate that the proportion of Tregs in peripheral blood and immune organs increases in a time-dependent manner with fluoride exposure. After reducing the fluoride concentration in the drinking water of rats, the number of Tregs remained significantly elevated. The changes in Treg numbers in the 12-week fluoride feeding group, 24-week fluoride feeding group, and 12-week fluoride feeding followed by 12-week water improvement group were related to the cytokine levels. Therefore, the impact of fluoride on the immune homeostasis has cumulative and long-term effects, and may be related to the accumulation and migration of Tregs induced by fluoride in an inflammatory environment, mediated by cytokines.

P1174 Engineering therapeutic Tregs to overexpress GPR15 improves functional fitness for in vivo gut homing

Abstract Background Inflammatory bowel diseases (IBD) like Crohn’s disease and ulcerative colitis, are marked by an imbalance of pro- and anti-inflammatory T cells in the lamina propria of the intestinal tract. Only subgroups of patients respond to current therapeutic approaches designed to restrain pro-inflammatory cells or signalling. Adoptive transfer of autologous ex vivo expanded regulatory T cells (Tregs) has previously been suggested as a promising future therapeutic approach to resolve chronic intestinal inflammation by promoting anti-inflammatory pathways. Methods Using a previously established protocol for the ex vivo expansion of Tregs (Investigational Medicinal Product-Tregs; IMP-Tregs), we characterized the migration and homing capabilities as well as the immunosuppressive function of non-expanded and IMP-Tregs using 3D-motility, in vivo gut homing and T cell suppression assays. Furthermore, via electroporation with GPR15 mRNA, IMP-Tregs were engineered to express the gut homing marker GPR15 and tested for their functional fitness in dynamic adhesion, T cell suppression assays as well as in vivo gut homing assays. Results Our data show that while the expansion protocol generates highly suppressive Tregs, only a fraction of them is equipped with surface molecules for gut homing. In order to overcome this limitation, we successfully engineered IMP-Tregs to express the gut homing marker GPR15. On a functional level, this led to improved adhesion to the cell adhesion molecules MAdCAM-1 and VCAM-1, which are expressed on the intestinal endothelium, as well as to increased gut homing in a humanized in vivo mouse model without affecting the immunosuppressive character of the engineered IMP-Tregs. Conclusion In conclusion, our data indicate superior functional fitness of GPR15-engineered IMP-Tregs for homing to the inflamed gut nominating them a promising further development of autologous Treg transfer therapy for IBD.

Gut-resident Tregs (GTregs) play a pivotal role in maintaining bone health under post-menopausal osteoporotic conditions.

Osteoporosis is a skeletal condition characterized by the deterioration of bone tissue. The immune system plays a crucial role in maintaining bone homeostasis and combating the development of osteoporosis. Immunoporosis is the term used to describe the recent convergence of research on the immune system's role in osteoporosis. The gut harbors the largest component of the immune system and there is growing evidence that intestinal immunity plays a vital role in regulating bone health. Gut-resident regulatory T cells (GTregs) are essential in inhibiting immune responses and preventing various inflammatory manifestations. Our findings show that GTregs are pivotal in the pathophysiology of post-menopausal osteoporosis (PMO). We investigated the potential of GTregs in regulating the development of bone cells in vitro. We observed that GTregs significantly enhance osteoblastogenesis with concomitant inhibition of osteoclastogenesis in a cell-ratio-dependent manner. We further report that the deficiency of short-chain fatty acids (SCFAs) in osteoporotic conditions substantially disrupts the composition of GTregs, leading to a loss of peripherally derived Tregs (pTregs) and an expansion of thymus-derived Tregs (tTregs). Moreover, the administration of probiotics Lactobacillus rhamnosus (UBLR-58) and Bifidobacterium longum (UBBL-64) modulated the GTregs compartment in an SCFA-dependent manner to mitigate inflammatory bone loss in PMO. Notably, SCFAs-primed GTregs were found to be significantly more effective in inhibiting osteoclastogenesis compared to unprimed GTregs. Altogether our results, for the first time, highlight the crucial role of GTregs in the pathophysiology of PMO, with potential clinical implications.

Generation of Human Chimeric Antigen Receptor Regulatory T Cells.

Chimeric antigen receptor (CAR) T-cell therapy has reshaped the face of cancer treatment, leading to record remission rates in previously incurable hematological cancers. These successes have spurred interest in adapting the CAR platform to a small yet pivotal subset of CD4+ T cells primarily responsible for regulating and inhibiting the immune response, regulatory T cells (Tregs). The ability to redirect Tregs' immunosuppressive activity to any extracellular target has enormous implications for creating cell therapies for autoimmune disease, organ transplant rejection, and graft-versus-host disease. Here, we describe in detail methodologies for bona fide Treg isolation from human peripheral blood, genetic modification of human Tregs utilizing either lentivirus or CRISPR/Cas9-aided knock-in using adeno-associated virus-mediated homologous directed repair (HDR) template delivery, and ex vivo expansion of stable human CAR Tregs. Lastly, we describe the assessment of human CAR Treg phenotypic stability and in vitro suppressive function, which provides insights into how the human CAR Tregs will behave in preclinical and clinical applications.

Protocol for a first-in-human feasibility study of T regulatory cells (TR004) for inflammatory bowel disease using (ex vivo) Treg expansion (TRIBUTE)

IntroductionCrohn’s disease (CD) is a chronic, immune-mediated inflammatory bowel disease (IBD), presenting with symptoms of abdominal pain and bleeding from the gastrointestinal tract. There is no known cure. In vitro-expanded...

Low regulatory T-cells frequency is associated with graft rejection after small bowel transplantation: Clinical and experimental evidence.

Intestinal transplantation (ITx) represents the only curative option for patients with irreversible intestinal failure. Nevertheless, its rejection rate surpasses that of other solid organ transplants due to the heightened immunological load of the gut. Regulatory T-cells (Tregs) are key players in the induction and maintenance of peripheral tolerance, suggesting their potential involvement in modulating host vs. graft responses after ITx. Thus, we investigated the association of Tregs with allograft outcomes in pediatric patients and in an experimental model of small bowel transplantation. Treg frequency in human samples was analyzed by Flow cytometry (CD4+CD25highCD127-, blood samples) and immunohistochemistry (FoxP3, graft samples). Experimental allogenic-heterotopic small bowel transplantation was performed in rats and animals divided into 3 groups: non-immunosuppressant treatment, rapamycin (2 mg/kg), and tacrolimus (0.6 mg/kg) treatment. Acute cellular rejection (ACR) was diagnosed based on clinical and histological findings, graft gene expression of pro- and anti-inflammatory mediators assessed by RT-qPCR, serum IL-6 and IL-10 levels by Luminex, and Treg frequency analyzed by flow cytometry (CD4+CD25highFoxP3+). Blood samples from patients undergoing ACR exhibited a significant reduction in the Treg number compared to those with normo-functional grafts. Similarly, a diminished number of FoxP3+ cells was observed in mucosa samples with ACR. In the experimental model, rapamycin-treated animals displayed clinical and histological findings resembling those not receiving immunosuppression treatment. Notably, ACR correlated with a high CD8/CD4 ratio, loss of T-cell chimerism, mRNA upregulation of pro-inflammatory genes and diminished graft Treg frequency. In contrast, tacrolimus treatment prevented ACR and facilitate blood and graft Treg expansion. Remarkably, recipients who achieved Treg expansion within the graft remained free of ACR even after discontinuation of the immunosuppressant treatment and this phenomenon was associated with increased levels of serum IL-10. Our clinical and experimental findings underscore the association between Treg frequency and graft rejection after ITx, advocating for strategies that promote their expansion within the gut mucosa to enhance long-term outcomes.

The IL-2 SYNTHORIN molecule promotes functionally adapted Tregs in a preclinical model of type 1 diabetes.

Deficits in IL-2 signaling can precipitate autoimmunity by altering the function and survival of FoxP3+ regulatory T cells (Tregs) while high concentrations of IL-2 fuel inflammatory responses. Recently, we showed that the non-beta IL-2 SYNTHORIN molecule SAR444336 (SAR'336) can bypass the induction of autoimmune and inflammatory responses by increasing its reliance on IL-2 receptor α chain subunit (CD25) to provide a bona fide IL-2 signal selectively to Tregs, making it an attractive approach for the control of autoimmunity. In this report, we further demonstrate that SAR'336 can support non-beta IL-2 signaling in murine Tregs and limit NK and CD8+ T cells' proliferation and function. Using a murine model of spontaneous type 1 diabetes, we showed that the administration of SAR'336 slows the development of disease in mice by decreasing the degree of insulitis through the expansion of antigen-specific Tregs over Th1 cells in pancreatic islets. Specifically, SAR'336 promoted the differentiation of IL-33-responsive (ST2+), IL-10-producing GATA3+ Tregs over other Treg subsets in the pancreas, demonstrating the ability of this molecule to further orchestrate Treg adaptation. These results offer insight into the capacity of SAR'336 to generate highly specialized, tissue-localized Tregs that promote restoration of homeostasis during ongoing autoimmune disease.

UVB Irradiation Expands Skin-Resident CD81+Foxp3+ Regulatory T Cells with a Highly Activated Phenotype.

Exposure to UVB induces the expansion of regulatory T cells (Tregs) expressing proenkephalin and amphiregulin with a healing function in the skin. It is unclear how this UVB exposure affects the functionally distinct subsets of skin Tregs. In this study, we have demonstrated that skin-resident CD81+Tregs expressing both proenkephalin gene Penk and amphiregulin gene Areg expanded after UVB irradiation. CD81+Tregs in UVB-irradiated skin as well as in normal skin exhibited a highly activated state. Foxp3, BLIMP-1, and IRF4, which transcriptionally enhance Treg function-related molecules, were also highly expressed in UVB-expanded CD81+Tregs. Notably, UVB-expanded skin CD81+Tregs constitutively expressed on their cell surface CTLA-4, a critical molecule for Treg-mediated immune suppression. CD81+Tregs exhibited suppressive activity against CD4+T-cell proliferation. Stimulation of CD81 enhanced the proliferation of Foxp3+Tregs under CD3 and CD28 stimulation invitro, indicating that CD81 acts as a costimulatory molecule. Blocking CD81 partially resulted in reduced Treg expansion in the skin of UVB-irradiated mice. These results suggest that CD81 is a representative marker of highly activated Tregs in normal and UVB-irradiated skin and may represent a functional molecule that controls Treg expansion in the skin in response to UVB irradiation.

Harnessing Regulatory T Cells for Immune Modulation in Transplantation Tolerance

Regulatory T cells (Tregs) are pivotal in maintaining immune tolerance and preventing allograft rejection in transplantation. This review explores the mechanisms by which Tregs mediate immune modulation and their potential therapeutic applications in promoting transplantation tolerance. Tregs are characterized by the expression of CD4, CD25, and the transcription factor FOXP3, and they play a critical role in suppressing effector T cell responses and promoting an anti-inflammatory environment. Various strategies for harnessing Tregs for transplantation include the expansion of Tregs ex vivo, the induction of Tregs in vivo through various approaches, and the transfer of Tregs into transplant recipients. Furthermore, we discuss the impact of the transplant environment on Treg function, the role of dendritic cells and cytokines in Treg activation, and the significance of gut-derived Tregs in fostering tolerance. Additionally, we address the challenges of using Tregs in clinical settings, including potential risks such as opportunistic infections and malignancies. Ultimately, harnessing Tregs represents a promising avenue for achieving transplantation tolerance and improving long-term graft survival, and ongoing research is crucial for developing effective and safe strategies for clinical application. Keywords: Regulatory T cells, Transplantation tolerance, Immune modulation, Allograft rejection, Treg therapy

Regulatory T cell-derived enkephalin gates nociception.

T cells have emerged as sex-dependent orchestrators of pain chronification but the sexually dimorphic mechanisms by which T cells control pain sensitivity is not resolved. Here, we demonstrate an influence of regulatory T cells (Tregs) on pain processing that is distinct from their canonical functions of immune regulation and tissue repair. Specifically, meningeal Tregs (mTregs) express the endogenous opioid, enkephalin, and mTreg-derived enkephalin exerts an antinociceptive action through a presynaptic opioid receptor signaling mechanism that is dispensable for immunosuppression. We demonstrate that mTregs are both necessary and sufficient to suppress mechanical pain sensitivity in female, but not male, mice, with this modulation reliant on sex hormones. These results uncover a fundamental sex-specific, and immunologically-derived endogenous opioid circuit for nociceptive regulation with critical implications for pain biology.

Regulatory T cells require peripheral CCL2-CCR2 signaling to facilitate the resolution of medication overuse headache-related behavioral sensitization

BackgroundMedication overuse headache (MOH) is the most common secondary headache disorder, resulting from chronic and excessive use of medication to treat headaches, for example, sumatriptan. In a recent study, we have shown that the peripheral C-C motif ligand 2 (CCL2), C-C motif chemokine receptor 2 (CCR2) and calcitonin-gene-related peptide (CGRP) signaling pathways interact with each other and play critical roles in the development of chronic migraine-related behavioral and cellular sensitization. In the present study, we investigated whether CCL2-CCR2 and CGRP signaling pathways play a role in the development of sumatriptan overuse-induced sensitization, and whether they are involved in its resolution by the low-dose interleukin-2 (LD-IL-2) treatment.MethodsMice received daily sumatriptan administration for 12 days. MOH-related behavioral sensitization was assessed by measuring changes of periorbital mechanical thresholds for 3 weeks. CCL2-CCR2 and CGRP signaling pathways were inhibited by targeted gene deletion or with an anti-CCL2 antibody. Ca2+-imaging was used to examine whether repetitive sumatriptan treatment enhances CGRP and pituitary adenylate cyclase–activating polypeptide (PACAP) signaling in trigeminal ganglion (TG) neurons. LD-IL-2 treatment was initiated after the establishment of sumatriptan-induced sensitization. Immunohistochemistry and flow cytometry analyses were used to examine whether CCL2-CCR2 signaling controls regulatory T (Treg) cell proliferation and/or trafficking.ResultsCCL2, CCR2 and CGRPα global KO mice exhibited robust sumatriptan-induced behavioral sensitization comparable to wild-type controls. Antibody neutralization of peripheral CCL2 did not affect sumatriptan-induced behaviors either. Repeated sumatriptan administration did not enhance the strength of CGRP or PACAP signaling in TG neurons. Nevertheless, LD-IL-2 treatment, which facilitated the resolution of sumatriptan-induced sensitization in wild-type and CGRPα KO mice, was completely ineffective in mice with compromised CCL2-CCR2 signaling. In CCL2 KO mice, we observed normal LD-IL-2-induced Treg expansion in peripheral blood, but the increase of Treg cells in dura and TG tissues was significantly reduced in LD-IL-2-treated CCL2 KO mice relative to wild-type controls.ConclusionsThese results indicate that the endogenous CCL2-CCR2 and CGRP signaling pathways are not involved in sumatriptan-induced behavioral sensitization, suggesting that distinct molecular mechanisms underlie chronic migraine and MOH. On the other hand, peripheral CCL2-CCR2 signaling is required for LD-IL-2 to reverse chronic headache-related sensitization.Graphical abstract

Effect of mitochondrial oxidative stress on regulatory T cell manufacturing for clinical application in transplantation: Results from a pilot study

The manufacturing process of regulatory T (Treg) cells for clinical application begins with the positive selection of CD25+ cells using superparamagnetic iron oxide nanoparticle (SPION)-conjugated anti-CD25 antibodies (spCD25) and immunomagnetic cell separation technology. Our findings revealed that the interaction of spCD25 with its cell target induced the internalization of the complex spCD25–interleukin-2 receptor. Accumulation of intracellular spCD25 triggered oxidative stress, causing delayed Treg expansion and temporary reduction in suppressor activity. This activation delay hindered the efficient generation of clinically competent cells. During this early phase, Treg cells exhibited elevated mitochondrial superoxide and lipid peroxidation levels, with a concomitant decrease in mitochondrial respiration rates. The results uncovered the increased mitochondrial unfolded protein response. This protective, redox-sensitive activity is inherent in Tregs when contrasted with homologous, spCD25-treated, conventional T cells. Although the temporary effects of spCD25 on clinically competent cells did not impede their use in a safety/feasibility pilot study with kidney transplant recipients, it is reasonable to anticipate a potential reduction in their therapeutic efficacy. The mechanistic understanding of the adverse effects triggered by spCD25 is crucial for improving the manufacturing process of clinically competent Treg cells, a pivotal step in the successful implementation of immune cell therapy in transplantation.

Ovalbumin-induced food allergy suppression via regulatory T cell expansion mediated by a TNFR2 agonist in mice

Food allergies represent a growing health concern worldwide, characterized by abnormal immune responses to specific dietary antigens. This condition is often associated with a dysregulation of immune tolerance, especially within the intestinal mucosa. Regulatory T cells (Tregs), a crucial subset of lymphocytes, play a central role in maintaining peripheral immune tolerance and are abundant in the intestinal lamina propria. Recent studies have highlighted Treg dysfunction in patients with food allergies, suggesting a potential connection between impaired Treg function and allergy onset. Therefore, strategies to adequately control and activate Tregs could offer new avenues for the prevention and treatment of food allergies. Our research focuses on targeting the regulatory molecule, tumor necrosis factor receptor type 2 (TNFR2), a key modulator of Treg function. We have developed a TNFR2 agonist, scR2agoTNF-Fc, characterized by high TNFR2-stimulating activity and enhanced blood retention in vivo for Treg expansion. In this study, we utilized an ovalbumin (OVA)-induced food allergy mouse model to verify the therapeutic potential of scR2agoTNF-Fc in modulating allergic responses and restoring immune balance. The results showed that scR2agoTNF-Fc promoted the expansion of Treg population in vivo in mice. In addition, scR2agoTNF-Fc reduced diarrhea caused by the food allergy. This was consistent with the molecular mechanisms of suppression of blood immunoglobulins and Th2 cells. Therefore, it was shown that quantitative and functional enhancement of Tregs by the TNFR2 agonist, scR2agoTNF-Fc, may be effective in treating food allergies.

Prostate cancer cell-derived exosomes ZNF667-AS1 reduces TGFBR1 mRNA stability to inhibit Treg expansion and DTX resistance by binding to U2AF1

BackgroundDocetaxel (DTX) resistance attenuates anti-tumor effects of DTX on prostate cancer (mCRPC) and drug resistance was related to Treg expansion in tumors. ZNF667-AS1 played a suppressing role in various tumors and tumor-derived exosomes carry lncRNAs to participate in tumor progression. Here, the effects of ZNF667-AS1 on malignant characteristics and DTX resistance in PC and the effect and its underlying molecular mechanism of tumor-derived exosomes carrying ZNF667-AS1 on Treg expansion were investigated.MethodsThe identification of exosomes were determined using TEM, NTA and western blot. The abundance of genes and proteins were evaluated using IHC, RT-qPCR, western blot and FISH. Malignant phenotypes of PC cells were evaluated by means of Edu, scratch test, transwell, CCK-8 and flow cytometry. The percentage of CD4+CD25+Foxp3+ Tregs was detected using flow cytometry. The location of ZNF667-AS1 was detected using nuclear-cytoplasmic fractionation. The co-location of ZNF667-AS1 and U2AF1 protein was detected using IF-FISH assay. The interactions among ZNF667-AS1, TGFBR1 and U2AF1 were verified using RNA pull-down, RIP and dual luciferase activity.ResultsZNF667-AS1 expression in PC samples was lowered, which was negatively relative to poor prognosis and DTX resistance. ZNF667-AS1 overexpression inhibited malignant phenotypes of PC cells, tumor growth and DTX resistance. Besides, DTX resistant cell-derived exosomes expressed lower ZNF667-AS1 expression. Exosomes carrying exogenously high ZNF667-AS1 expression derived PC cells or serum of mice suppressed Treg expansion. On the mechanism, ZNF667-AS1 interacted with U2AF1 to destabilize TGFBR1 mRNA and reduce TGFBR1 expression in CD4+T cells.ConclusionZNF667-AS1 suppressed cell growth of PC cells, tumor growth of mice and DTX resistance to PC cells and exogenously high ZNF667-AS1 expression in tumor-derived exosomes destabilized TGFBR1 mRNA and reduce TGFBR1 expression through interacting with U2AF1, thus resulting in attenuated Treg expansion, which was related to DTX resistance.

Immunomodulatory Effect of Adipose Stem Cell-Derived Extra-Cellular Vesicles on Cytokine Expression and Regulatory T Cells in Patients with Asthma.

Although mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) are as effective as MSCs in the suppression of allergic airway inflammation, few studies have evaluated the immunomodulatory capacity of MSC-derived EVs in patients with asthma. Thus, we assessed the effects of adipose stem cell (ASC)-derived EVs on cytokine expression and regulatory T cells (Tregs) in peripheral blood mononuclear cells (PBMCs) of asthmatic patients. PBMCs (1 × 106 cells/mL) were isolated from asthmatic patient and healthy controls and co-cultured with 1 μg/mL of ASC-derived EVs. Th (T helper) 1-, Th2-, and Treg-related cytokine expression, fluorescence-activated cell sorting analysis of CD4+CD25+FOXP3+ T cells, and co-stimulatory molecules were analyzed before and after ASC-derived EV treatment. The expression levels of IL-4 and costimulatory molecules such as CD83 and CD86 were significantly higher in PBMCs of asthmatic patients than in control PBMCs. However, ASC-derived EV treatment significantly decreased the levels of interleukin (IL)-4 and co-stimulatory molecules such as CD83 and CD86 in the phytohemagglutinin (PHA)-stimulated PBMC of asthmatic patients. Furthermore, ASC-derived EVs remarkably increased the transforming growth factor-β (TGF-β) levels and expression of Tregs in the PBMC of asthmatic patients. ASC-derived EVs induce Treg expansion and have immunomodulatory effects by downregulating IL-4 and upregulating TGF-β in PBMCs of asthmatic patients.

Distinct characteristics of BTLA/HVEM axis expression on Tregs and its impact on the expansion and attributes of Tregs in patients with active pulmonary tuberculosis.

Pulmonary tuberculosis (PTB) remains one of the deadliest infectious diseases. Understanding PTB immunity is of potential value for exploring immunotherapy for treating chemotherapy-resistant PTB. CD4+CD25+Foxp3+ regulatory T cells (Tregs) are key players that impair immune responses to Mycobacteria tuberculosis (MTB). Currently, the intrinsic factors governing Treg expansion and influencing the immunosuppressive attributes of Tregs in PTB patients are far from clear. Here, we employed flow cytometry to determine the frequency of Tregs and the expression of B and T lymphocyte attenuator (BTLA) and its ligand, herpesvirus entry mediator (HVEM), on Tregs in patients with active PTB. Furthermore, the expression of conventional T cells and of programmed death-ligand 1 (PD-L1) and programmed death-1 (PD-1) on Tregs in patients with active PTB was determined. We then examined the characteristics of BTLA/HVEM expression and its correlation with Treg frequency and PD-L1 and PD-1 expression on Tregs in PTB patients. The frequency of Tregs was increased in PTB patients and it had a relevance to PTB progression. Intriguingly, the axis of cosignal molecules, BTLA and HVEM, were both downregulated on the Tregs of PTB patients compared with healthy controls (HCs), which was the opposite of their upregulation on conventional T cells. Unexpectedly, their expression levels were positively correlated with the frequency of Tregs, respectively. These seemingly contradictory results may be interpreted as follows: the downregulation of BTLA and HVEM may alleviate BTLA/HVEM cis-interaction-mediated coinhibitory signals pressing on naïve Tregs, helping their activation, while the BTLA/HVEM axis on effector Tregs induces a costimulatory signal, promoting their expansion. Certainly, the mechanism underlying such complex effects remains to be explored. Additionally, PD-L1 and PD-1, regarded as two of the markers characterizing the immunosuppressive attributes and differentiation potential of Tregs, were upregulated on the Tregs of PTB patients. Further analysis revealed that the expression levels of BTLA and HVEM were positively correlated with the frequency of PD-1+Tregs and PD-L1+Tregs, respectively. Our study illuminated distinct characteristics of BTLA/HVEM axis expression on Tregs and uncovered its impact on the expansion and attributes of Tregs in patients with active PTB. Therefore, blockade of the BTLA/HVEM axis may be a promising potential pathway to reduce Treg expansion for the improvement of anti-MTB immune responses.

A PD-1-targeted, receptor-masked IL-2 immunocytokine that engages IL-2Rα strengthens T cell-mediated anti-tumor therapies

The clinical use of interleukin-2 (IL-2) for cancer immunotherapy is limited by severe toxicity. Emerging IL-2 therapies with reduced IL-2 receptor alpha (IL-2Rα) binding aim to mitigate toxicity and regulatory Tcell (Treg) expansion but have had limited clinical success. Here, we show that IL-2Rα engagement is critical for the anti-tumor activity of systemic IL-2 therapy. A "non-α" IL-2 mutein induces systemic expansion of CD8+ Tcells and natural killer (NK) cells over Tregs but exhibits limited anti-tumor efficacy. We develop a programmed cell death protein 1 (PD-1)-targeted, receptor-masked IL-2 immunocytokine, PD1-IL2Ra-IL2, which attenuates systemic IL-2 activity while maintaining the capacity to engage IL-2Rα on PD-1+ Tcells. Mice treated with PD1-IL2Ra-IL2 show no systemic toxicities observed with unmasked IL-2 treatment yet achieve robust tumor growth control. Furthermore, PD1-IL2Ra-IL2 can be effectively combined with other Tcell-mediated immunotherapies to enhance anti-tumor responses. These findings highlight the therapeutic potential of PD1-IL2Ra-IL2 as a targeted, receptor-masked, and "α-maintained" IL-2 therapy for cancer.

The Interactions of T Cells with Myeloid-Derived Suppressor Cells in Peripheral Blood Stem Cell Grafts.

The interaction of myeloid-derived suppressor cells (MDSCs) with T cells within G-CSF-mobilized peripheral blood stem cell (PBSC) grafts in patients undergoing autologous or allogeneic hematopoietic stem cell transplantation remains to be elucidated. Through studying allo- and auto-PBSC grafts, we observed grafts containing large numbers of T cells and MDSCs with intergraft variability in their percentage and number. T cells from autologous grafts compared to allografts expressed relative higher percentages of inhibitory receptors PD-1, CTLA-4, TIM-3, LAG-3, TIGIT and BTLA. Autograft T cells had decreased cell proliferation and IFN-γ secretion, which supported the possible presence of T cell exhaustion. On the contrary, graft monocytic MDSCs (M-MDSCs) expressed multiple inhibitory receptor ligands, including PD-L1, CD86, Galectin-9, HVEM and CD155. The expression of inhibitory receptor ligands on M-MDSCs was correlated with their corresponding inhibitory receptors on T cells in the grafts. Isolated M-MDSCs had the ability to suppress T cell proliferation and IFN-γ secretion and/or promote Treg expansion. Blocking the PD-L1-PD-1 signaling pathway partially reversed the functions of M-MDSCs. Taken together, our data indicated that T cells and M-MDSCs in PBSC grafts express complementary inhibitory receptor-ligand pairing, which may impact the quality of immune recovery and clinical outcome post transplantation.

Propionic acid supplementation promotes the expansion of regulatory T cells in patients with end-stage renal disease but not in renal transplant patients.

In a previous study, we showed an anti-inflammatory effect of propionic acid supplementation in dialysis patients. The present study intends to analyze the effect of propionic acid on the chronic inflammatory state and T-cell composition in kidney transplant patients compared to dialysis patients. A total of 10 dialysis patients and 16 kidney transplant patients under immunosuppressive standard triple immunosuppressive therapy received 2 × 500 mg propionic acid per day for 30 days. The cellular immune system was analyzed before and after the propionic acid supplementation and 30-90 days thereafter as a follow-up. We measured the main immune cell types and performed an in-depth characterization of T cells including regulatory T cells (Tregs), B cells, and dendritic cells. In addition, we assessed the functional activity and antigenic responsiveness by analysis of third-party antigen-specific T cells after their stimulation by recall (tetanus diphtheria vaccine) antigen. In dialysis patients, we observed an expansion of CD25highCD127- Tregs after propionic acid intake. In contrast, the same supplementation did not result in any expansion of Tregs in transplant patients under immunosuppressive therapy. We also did not observe any changes in the frequencies of the main immune cell subsets except for CD4+/CD8+ distribution with an increase of CD4+ T cells and decrease of CD8+ T cells in the transplant population. Our data suggest that dietary supplements containing propionate might have a beneficial effect decreasing systemic inflammation in dialysis patients through Treg expansion. However, this effect was not observed in transplant patients, which could be explained by counteracting effect of immunosuppressive drugs preventing Treg expansion.