Regulatory Cell Populations Research Articles

Minnelide suppresses GVHD and enhances survival while maintaining GVT responses.

Allogeneic hematopoietic stem cell transplantation (aHSCT) can cure patients with otherwise fatal leukemias and lymphomas. However, the benefits of aHSCT are limited by graft-versus-host disease (GVHD). Minnelide, a water-soluble analog of triptolide, has demonstrated potent antiinflammatory and antitumor activity in several preclinical models and has proven both safe and efficacious in clinical trials for advanced gastrointestinal malignancies. Here, we tested the effectiveness of Minnelide in preventing acute GVHD as compared with posttransplant cyclophosphamide (PTCy). Strikingly, we found Minnelide improved survival, weight loss, and clinical scores in an MHC-mismatched model of aHSCT. These benefits were also apparent in minor MHC-matched aHSCT and xenogeneic HSCT models. Minnelide was comparable to PTCy in terms of survival, GVHD clinical score, and colonic length. Notably, in addition to decreased donor T cell infiltration early after aHSCT, several regulatory cell populations, including Tregs, ILC2s, and myeloid-derived stem cells in the colon were increased, which together may account for Minnelide's GVHD suppression after aHSCT. Importantly, Minnelide's GVHD prevention was accompanied by preservation of graft-versus-tumor activity. As Minnelide possesses anti-acute myeloid leukemia (anti-AML) activity and is being applied in clinical trials, together with the present findings, we conclude that this compound might provide a new approach for patients with AML undergoing aHSCT.

Fecal microbiota transplantation stimulates type 2 and tolerogenic immune responses in a mouse model

ObjectivesClostridioides difficile infection (CDI) is the leading hospital-acquired infection in North America. While previous work on fecal microbiota transplantation (FMT), a highly effective treatment for CDI, has focused on colonization resistance mounted against C. difficile by FMT-delivered commensals, the effects of FMT on host gene expression are relatively unexplored. This study aims to identify transcriptional changes associated with FMT, particularly changes associated with protective immune responses. MethodsGene expression was assessed on day 2 and day 7 after FMT in mice after antibiotic-induced dysbiosis. Flow cytometry was also performed on colon and mesenteric lymph nodes at day 7 to investigate changes in immune cell populations. ResultsFMT administration after antibiotic-induced dysbiosis successfully restored microbial alpha diversity to levels of donor mice by day 7 post-FMT. Bulk RNA sequencing of cecal tissue at day 2 identified immune genes, including both pro-inflammatory and Type 2 immune pathways as upregulated after FMT. RNA sequencing was repeated on day 7 post-FMT, and expression of these immune genes was decreased along with upregulation of genes associated with restoration of intestinal homeostasis. Immunoprofiling on day 7 identified increased colonic CD45+ immune cells that exhibited dampened Type 1 and heightened regulatory and Type 2 responses. These include an increased abundance of eosinophils, alternatively activated macrophages, Th2, and T regulatory cell populations. ConclusionThese results highlight the impact of FMT on host gene expression, providing evidence that FMT restores intestinal homeostasis after antibiotic treatment and facilitates tolerogenic and Type 2 immune responses.

The balance between memory and regulatory cell populations in kidney transplant recipients with operational tolerance.

Donor-reactive memory cells represent a barrier to long-term kidney graft survival. A better understanding of regulatory mechanisms that counterbalance alloreactive memory responses may help to identify patients with operational tolerance. This prospective study investigated the equilibrium between memory T-cell subsets and regulatory T or B cells (Tregs, Bregs) in peripheral blood of kidney transplant recipients with operational tolerance (N = 8), chronic rejection (N = 8), and different immunosuppressive treatment regimens (N = 81). Patients on hemodialysis and healthy individuals served as controls (N = 50). In addition, the expression of Treg- and Breg-associated molecule genes was analyzed. Patients with chronic rejection showed a disrupted memory T-cell composition with a significantly higher frequency of circulating CD8+ terminally differentiated effector memory (TEMRA) T cells than patients with operational tolerance, patients on hemodialysis, or healthy controls (P < 0.001). Low frequency of CD8+ TEMRA and high frequency of Tregs and transitional Bregs were found in operationally tolerant patients. Consequently, operationally tolerant patients showed, as compared to all other transplant recipients with different immunosuppressive regiments, the lowest ratios between CD8+ TEMRA T cells and Tregs or Bregs (for both P < 0.001). Moreover, a specific peripheral blood transcription pattern was found in operationally tolerant patients with an increased expression of Breg- and Treg-associated genes CD22 and FoxP3 and a decreased FcγRIIA/FcγRIIB transcript ratio (for all P < 0.001). In conclusion, monitoring the balance between circulating CD8+ TEMRA T cells and regulatory cell subsets and their transcripts may help to distinguish transplant recipients with operational tolerance from recipients at risk of graft loss.

CD56 brightCD16 -Perforin - regulatory Natural Killer Cells Associated with the Suppression of Chronic Graft-Versus-Host Disease Prevent CD4 + T Cell Proliferation through PD-1 and LAG-3 Dependent Pathways

Background: Chronic graft-versus-host disease (cGvHD) is a major cause of morbidity and mortality after Hematopoietic Stem Cell Transplantation (HSCT). Previously, in large cohorts of HSCT patients we identified increased numbers of CD56 bright, granzyme B -, perforin - NK cells (NK reg) to be associated with a lack of cGvHD development. Transcriptome analysis of HSCT patient samples identified a unique transcriptome among the CD56 bright NK reg cells which associated with patients who failed to develop cGvHD, including overexpression of genes IL7R, GPR183, and Granzyme K, and low expression of genes such as perforin, granzyme B, and CD16. We then utilized the transcriptome information and further cell phenotyping to determine the optimal sorting approach for viable NK reg cells to allow for functional analysis by the sorting of CD56 brightCD3 -CD16 - cells. Functional analysis suggested that the NK reg population suppresses CD4 + T cells through a non-cytolytic, and direct cell-cell contact-dependent mechanism, though the exact receptor and ligand interaction of which CD4 + T cell populations are impacted is unknown. We hypothesized that the NK reg cell suppressive mechanism may be more specific than the more widely studied regulatory cell population, T reg cells, by suppressing CD4 + T cell subpopulations involved in inflammatory responses. Methodology: To investigate the suppressive capacity of NK reg cells, the cells were isolated from healthy donor peripheral blood and co-cultured with allogeneic CD4 + T cells, CD8 + T cells, T reg cells, T reg cells and dendritic cells, B cells, or NK cells for 96hrs. After 96hrs, the proliferation and viability of the responder cells were evaluated via proliferation dye dilution, and 7-AAD dye, respectively. To determine if the CD4 + T cell suppression is specific to a T helper (T h) cell subset we stained the co-cultured cells with a T h1/T h2/T h17 Phenotyping Kit. Further, PD-1 and LAG-3 neutralizing antibodies were added to the NK reg/CD4 + T cell co-culture to determine the receptor dependence. All samples were acquired with the BD FACSymphony Flow Cytometer and the data was analyzed via Kaluza software. Results: CD56 brightCD16 -perforin - NK regcellsstrongly suppress CD4 + T cell proliferation (approximately 96% suppression of CD4 + T cell proliferation at the 1:1 ratio of NK reg cells to CD4 + T cells). The contact-dependent mechanism of NK reg suppression of CD4 + T cell proliferation was significantly decreased when blocking either the PD-1 (15% decrease in suppression, p=0.03), or LAG-3 (29% decrease in suppression, p=0.04) receptors, at the 1:2 ratio of NK reg cells to CD4 + T cells. When both receptors are blocked the inhibition of suppressive effect is comparable to that of the LAG-3 blocking antibody being added individually (30% decrease in suppression, p=0.03). The suppressive mechanism of NK reg cells was observed to be selective in that they strongly suppress CD4 + T cell proliferation, but do not result in statistically significant suppression of CD8 + T cell, T reg cell, B cell, NK cell, or a specific T h cell subset proliferation (p&amp;gt;0.05). Further, though the average proliferation of the T reg cells co-cultured with NK reg cells and dendritic cells compared to just NK reg cells was greater (127% proliferation compared to 100% proliferation, respectively), we did not observe NK reg cells to induce a statistically significant proliferative effect towards the T reg cells in the presence of dendritic cells. Conclusion: As a result of our studies, we have confirmed the NK reg cell immune suppressive function towards CD4 + T cell proliferation, a main contributor to cGvHD development. Further, we demonstrated a PD-1/LAG-3-dependent direct contact mechanism of NK reg cell suppression, which is selective of total CD4 + T cells, with a lack of suppressive effect towards CD8 + T cells, T reg cells, B cells, and NK cells. The results of these studies contribute to our better understanding of how NK reg cells may induce a cell-specific suppressive function to promote immune tolerance, providing potential cell therapeutic applications for enhancing NK reg cell suppressive function through increasing PD-1/LAG-3 ligand or receptor expression on NK reg cells.

The Non-Hematopoietic Content in Paediatric Autologous Hematopoietic Stem Cell Grafts Is Skewed Towards Regulatory Myeloid and Exhausted Phenotypes

Autologous hematopoietic stem cell transplant (aHSCT) is a central component of paediatric brain tumours and high-risk neuroblastoma management. Standardly, chemotherapy and G-CSF are used to mobilize the CD34+ hematopoietic stem/progenitor cells (HSC), which are used to support hematopoietic recovery following dose chemotherapy. Clinically, the graft infused is defined by solely by the CD34+ content. The non-hematopoietic passenger cells makeup most of the graft and are a mixture of myeloid and lymphoid cells that are potentially biologically active in the post-transplant setting. Here we study the content of the graft, focusing on the phenotypes of the cell populations, to begin to look at how the non-hematopoietic cells may facilitate anti-tumor activity, affect the tumour microenvironment, and/or immune recovery post-transplant. High parameter mass cytometry, with a custom 40-marker panel, was used to characterize 43 autologous cryopreserved grafts from children (median age 39 months, range 14-255 months) undergoing apheresis following chemotherapy and G-CSF mobilization. Healthy G-CSF-mobilized allogeneic donors (allograft) were used as a comparator group (n=10). Young children frequently have robust mobilization (peripheral blood CD34 count range from 100-1800 CD34/µL on day of collection) and generally one day collection will support multiple cycles of high dose therapy (target collections 20 x10 6 CD34+ cells/kg and often over collecting in 2-3 blood volume processed). This results in a product that is rich in CD34+ cells, with intergraft heterogeneity (median 7%; range 0.7-31%), compared to the CD34+ content of allografts (median 1.1%; range 0.3-2.4%) (autograft vs allograft median CD34 difference p&amp;lt;0.00005; Wilcoxon unpaired). There was variability in the non-hematopoietic component in the autografts (median T cells 10.8%; range 0.6 - 51.7%, and median monocytes 18.8%; range 4.1 - 51.4%). The allogeneic products were found to have less variability (T cells: 19.9 - 56%; monocytes: 7.3 - 19.6%). Autografts had increased expression of T cell exhaustion markers, with a median 30.8% (range 14.7 - 88.1%) of T cells expressing TIGIT and 35.1% (range 13.1 - 75.9%) expressing PD1 compared to the allografts (median TIGIT: 15.7%; range 9.3 - 28.7% and median PD1: 11%; range 4.2 - 19.7%). When grafts from high mobilizers (N= 21; &amp;gt;7% CD34+ cells) were analyzed by the actual number of cells infused they had had fewer regulatory cells: monocytic-myeloid derived-suppressor cells (M-MDSCs) (3.6x10 7 cells/kg; range 0.5 - 73.8x10 7; p&amp;lt; 0.0003), non-classical monocytes (0.9x10 7 cells/kg; range 0.5 - 21.3x10 7 cells/kg; p&amp;lt; 0.002) compared to the grafts from lower mobilizers (N = 22; &amp;lt;7.1% CD34+ cells) M-MDSC: median 7.7x10 7/kg; range 0.6 - 21.3x10 7 cells/kg, non-classical monocytes: 1.7x10 7 cells/kg; range 0.05 - 3.7x10 7 cells/kg). T cells and NK cells did not differ between high and low mobilizers. We are seeing significant differences in the regulatory cell populations infused in paediatric aHSCT based on the how robustly a patient mobilizes CD34+ cells after chemotherapy and G-CSF. It is not known whether these differences are clinically significant or affect anti-tumour immunity, tumour microenvironment perturbation, or immune recovery. With this high parameter mass cytometry strategy, we will be able to correlate engraftment kinetics and transplant outcomes (survival, relapse), which will allow us to re-envision how grafts should be optimally manufactured. Controllable variables include the timing of collection (earlier or later in the clinical course), alternative growth factor usage, enrichment or depletion of cell populations in the graft, or post-transplant growth factor administration. We suggest that the aHSCT graft can be thought of as an immunotherapy tool and not just for hematopoietic recovery.

CD8+ Treg cells play a role in the obesity-associated insulin resistance

Obesity-related chronic low-grade inflammation may trigger insulin resistance and type 2 diabetes (T2D) development. Cells with regulatory phenotype have been shown to be reduced during obesity, especially CD4+ Treg cells. However, little is known about the CD8+ Treg cells. Therefore, we aim to characterize the CD8+ Treg cells in human peripheral blood and adipose tissue, specifically, to address the effect of obesity and insulin resistance in this regulatory immune cell population. A group of 42 participants with obesity (OB group) were recruited. Fourteen of them were evaluated pre- and post-bariatric surgery. A group of age- and sex-matched healthy volunteers (n = 12) was also recruited (nOB group). CD8+ Treg cell quantification and phenotype were evaluated by flow cytometry, in peripheral blood (PB), subcutaneous (SAT), and visceral adipose tissues (VAT). The OB group displayed a higher percentage of CD8+ Treg cells in PB, compared to the nOB. In addition, they were preferentially polarized into Tc1- and Tc1/17-like CD8+ Treg cells, compared to nOB. Moreover, SAT displayed the highest content of CD8+ Tregs infiltrated, compared to PB or VAT, while CD8+ Tregs infiltrating VAT displayed a higher percentage of cells with Tc1-like phenotype. Participants with pre-diabetes displayed a reduced percentage of TIM-3+CD8+ Tregs in circulation, and PD-1+CD8+ Tregs infiltrated in the VAT. An increase in the percentage of circulating Tc1-like CD8+ Treg cells expressing PD-1 was observed post-surgery.In conclusion, obesity induces significant alterations in CD8+ Treg cells, affecting their percentage and phenotype, as well as the expression of important immune regulatory molecules.

Phenotype Alterations in the Cecal Ecosystem Involved in the Asymptomatic Intestinal Persistence of Paratyphoid Salmonella in Chickens.

The gastrointestinal ecosystem involves interactions between the host, gut microbiota, and external environment. To colonize the gut of poultry, Salmonella must surmount barriers levied by the intestine including mucosal innate immune responses and microbiota-mediated niche restrictions. Accordingly, comprehending Salmonella intestinal colonization in poultry requires an understanding of how the pathogen interacts with the intestinal ecosystem. In chickens, the paratyphoid Salmonella have evolved the capacity to survive the initial immune response and persist in the avian ceca for months without triggering clinical signs. The persistence of a Salmonella infection in the avian host involves both host defenses and tolerogenic defense strategies. The initial phase of the Salmonella-gut ecosystem interaction is characteristically an innate pro-inflammatory response that controls bacterial invasion. The second phase is initiated by an expansion of the T regulatory cell population in the cecum of Salmonella-infected chickens accompanied by well-defined shifts in the enteric neuro-immunometabolic pathways that changes the local phenotype from pro-inflammatory to an anti-inflammatory environment. Thus, paratyphoid Salmonella in chickens have evolved a unique survival strategy that minimizes the inflammatory response (disease resistance) during the initial infection and then induces an immunometabolic reprogramming in the cecum that alters the host defense to disease tolerance that provides an environment conducive to drive asymptomatic carriage of the bacterial pathogen.

239 - CD39 Delineates Chimeric Antigen Receptor T Regulatory Cell Populations with Different Cytotoxic and Immunoregulatory Potential Against Monocytes and Pancreatic Islet Beta Cells

239 - CD39 Delineates Chimeric Antigen Receptor T Regulatory Cell Populations with Different Cytotoxic and Immunoregulatory Potential Against Monocytes and Pancreatic Islet Beta Cells

AGING-DEPENDENT CHANGES IN ADIPOSE PRECURSORS

Abstract Adipose tissue mass and adiposity change throughout the lifespan. During aging, while visceral adipose tissue (VAT) tends to increase, peripheral subcutaneous adipose tissue (SAT) decreases significantly. Unlike VAT, which is linked to metabolic diseases, including type 2 diabetes, SAT has beneficial effects. However, the molecular details behind the aging-associated loss of SAT remain unclear. Here, by comparing scRNA-seq of total stromal vascular cells of SAT from young and aging mice, we identify an aging-dependent regulatory cell (ARC) population that emerges only in SAT of aged mice and humans. ARCs express adipose progenitor markers but lack adipogenic capacity; they secrete high levels of pro-inflammatory chemokines, including Ccl6, to inhibit proliferation and differentiation of neighboring adipose precursors. We also found Pu.1 to be a driving factor for ARC development. We identify an ARC population and its capacity to inhibit differentiation of neighboring adipose precursors, correlating with aging-associated loss of SAT.

Altered meningeal immunity contributing to the autism-like behavior of BTBR T+Itpr3tf/J mice

Autism spectrum disorder (ASD) is a complicated neurodevelopmental disorder, which is categorized by deficiency of social contact and communication, and stereotyped forms of performance. Meningeal immunity conditions the immune reflection and immune defense in the meningeal area involving meningeal lymphatic organization, glymphatic structure, immune cells, and cytokines. The development of meningeal immunity dysfunction might be the leading cause for many neural diseases including ASD. The inbred mouse strain BTBRT + Itpr3tf/J (BTBR) shows multiple ASD-like behavioral phenotypes, thus making this strain a widely used animal model for ASD. In our previous study, we reported an altered peripheral immune profile in BTBR mice. Herein, we are investigating immunological and neural interactions associated with the aberrant behavior of BTBR mice. BTBR mice have an increased level of immune cell deposition in the meninges along with a higher level of CD4+ T cells expressing CD25 and of B and myeloid cells expressing more MHCII than C57BL/6 (B6) mice, which have normal behaviors. BTBR mice also have higher levels of autoantibodies to dsDNA, Aquaporin-4, NMDAR1, Pentraxin/SAP and Caspr2 than B6 mice, which may affect neural functions. Interestingly, the T regulatory (Treg) cell population and their function was significantly reduced in the meninges and brain draining lymph nodes, which may explain the increased level of activated B and T cells in the meninges of BTBR mice. A low level of Treg cells, less IL-10 production by Treg, and activated T and B cells in meninges together with higher autoantibody levels might contribute to the development of autism-like behavior through neuroinflammation, which is known to be increased in BTBR mice.

Depletion of Foxp3+ regulatory T cells but not the absence of CD19+IL-10+ regulatory B cells hinders tumor growth in a para-orthotopic neuroblastoma mouse model.

Adaptive immune cells with regulatory function reportedly mediate immune escape in a variety of tumors. Little is known regarding the relevance of the most prominent regulatory cell populations, namely Foxp3+ T regulatory cells (Tregs) and CD19+IL-10+ B regulatory cells (Bregs), for neuroblastoma (NB) survival. After establishing a novel immunocompetent syngeneic NB mouse model where orthotopic tumors can be generated after intrarenal injection of NB975A cells, we studied the importance of Tregs and Bregs in Foxp3-DTR mice whose Tregs can be depleted by diphtheria toxin (DT) application as well as in CD19-specific IL-10 deficient mice that lack IL-10+ Bregs (CD19cre+/- × IL-10fl/fl mice). We observed Foxp3 Treg cells in tumors from wild type mice. On the contrary, Bregs or B cells were scarce. Specific depletion of Tregs in Foxp3-DTR mice resulted in an 85% reduction of tumor volume and weight compared to DT-treated wild type mice and untreated Foxp3-DTR mice. In contrast, NB tumor growth was not affected in CD19-specific IL-10 deficient mice. Similarly, mice lacking mature B cells (μMT mice) and CD19 deficient mice (CD19cre mice) showed no change in growth pattern of NB tumors. In Treg-depleted mice, reduced tumor growth was associated with an increased concentration of IFN-gamma, TNF-alpha, IL-4, IL-6, and IL-10 in isolated splenocytes. In summary, transient ablation of Tregs but not absence of Bregs hindered the growth of NB, strongly suggesting the therapeutic potential of targeting Tregs for this aggressive childhood tumor.

The Potential Clinical Application of Induced Tolerogenic Macrophages.

The Potential Clinical Application of Induced Tolerogenic Macrophages.

P1270: IMMUNE REGULATORY CELLS IN B-CELL LYMPHOMAS: SIGNIFICANT HETEROGENEITY ACROSS HISTOLOGIC TYPES.

Background: Immune regulatory cell populations play a fundamental role in tumor development and tumor immune evasion. Myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs) considered as dominant suppressor cells which are significantly enriched in blood, bone marrow (BM), or tumor sites of patients with hematologic malignancies and often associate with high tumor burden, adverse clinical features, and outcome. Studies in lymphoma patients however, have generated conflicting results, regarding the function and phenotype of MDSC and Tregs mainly due to large heterogeneity of the disease as well as due to the lack of a unanimous nomenclature of these cell subsets in human samples. Furthermore, the molecular mechanisms via which MDSCs and Tregs contribute to clinical outcomes of disease and how they interfere with response to therapy remain elusive. Aims: To investigate the prevalence of immune suppressive population in B-cell lymphomas according to histologic, subtype providing the basis for further analysis of their biologic properties and association with clinical features and outcome of specific lymphoma subtypes. Methods: Peripheral blood (PB) and BM samples were collected from healthy individuals (n= 22) and lymphoma patients (n=50). Specifically, lymphoma subtypes were as follows:Marginal Zone (MZL, N=19), Diffuse Large B-cell Lymphoma (DLBCL, N=18) and Follicular Lymphoma (FL, N=13). Peripheral blood mononuclear cells (PBMCs) were isolated with ficoll gradient and subjected to flow cytometry analysis. MDSC characterization was based on HLADR-/lowCD11b+CD33- expression with CD14+CD15- for M-MDSCs and CD14-CD15+ for PMN-MDSCs. In addition, Treg cells were denoted as CD4+CD25highCD127-Foxp3+. Expression of PDL1 and PD1 on MDSCs and Tregs respectively, was also determined by flow cytometry. M-MDSCs and PMN-MDSCs were sorted from BM samples, RNA was extracted in order to subjected to transcriptomic analysis (RNAseq). Informed consent was obtained. Results: Frequencies of MDSCs were significantly increased in PB of all lymphoma patients compared to healthy individuals, with MZL subtype exhibiting the most prominent increase of MDSCs (p<.0001). Notably numbers of M-MDSCs were increased in FL and MZL patients and to a lesser extend in DLBCL patients while PMN-MDSCs were elevated in all subtypes with MZL displaying the higher frequencies. Regarding the Treg cells, although all lymphoma patients exhibited increased absolute numbers of CD4+CD25highCD127- Treg cells, Foxp3-expressing Tregs were significantly increased in the PB of DLBCL and MZL patients, but not in FL patients. Of interest, CD4+ and CD8+ T cells in the PB of DLBCL patients exhibited increase expression of PD1 compared to respective cell subsets from healthy individuals. Summary/Conclusion: Our findings comprehensively demonstrate enhanced numbers of MDSC and Treg immune suppressive cells in the PB of B-cell lymphoma patients while an increased heterogeneity was observed among the different histological lymphoma types. Functional characterization of MDSCs and Tregs by transcriptomic (RNAseq) and proteomic analysis are ongoing.

Autoimmunity and Cancer-Two Sides of the Same Coin.

Autoimmune disease results from the immune response against self-antigens, while cancer develops when the immune system does not respond to malignant cells. Thus, for years, autoimmunity and cancer have been considered as two separate fields of research that do not have a lot in common. However, the discovery of immune checkpoints and the development of anti-cancer drugs targeting PD-1 (programmed cell death receptor 1) and CTLA-4 (cytotoxic T lymphocyte antigen 4) pathways proved that studying autoimmune diseases can be extremely helpful in the development of novel anti-cancer drugs. Therefore, autoimmunity and cancer seem to be just two sides of the same coin. In the current review, we broadly discuss how various regulatory cell populations, effector molecules, genetic predisposition, and environmental factors contribute to the loss of self-tolerance in autoimmunity or tolerance induction to cancer. With the current paper, we also aim to convince the readers that the pathways involved in cancer and autoimmune disease development consist of similar molecular players working in opposite directions. Therefore, a deep understanding of the two sides of immune tolerance is crucial for the proper designing of novel and selective immunotherapies.

The role of ionotropic AMPA receptors in T cell tolerance

Abstract The ability of T cells to mount effective immune responses to pathogens and cancer while remaining tolerant to self-tissue is critical for maintaining immune homeostasis. Previously, our group identified neuritin, a neuronal protein, as a highly differentially expressed gene in anergic and regulatory T cells. Furthermore, our results indicate neuritin deficient mice exhibit enhanced autoimmunity due, in part, to dysfunctional regulatory T cells. Neuritin was recently identified as an accessory component of the ionotropic AMPA receptor (AMPAR) complex in neurons. AMPAR complexes are typically found on the post-synaptic neuronal cell and mediate glutamate dependent cation flux. Given our previous findings involving neuritin in autoimmunity and this newly identified interaction between neuritin and AMPAR, we sought to evaluate whether there is a role for the AMPAR in T cell tolerance. Here, we show that the AMPAR is expressed in CD4+ T cells and that the specific deletion of the AMPAR in T cells (AMPAR KO) leads to significantly reduced disease pathogenesis in an experimental autoimmune encephalomyelitis (EAE) model. The spinal cord inflammatory immune infiltrates from AMPAR KO mice are markedly reduced in cell number, proliferative state, and expression of proinflammatory cytokines compared to wild type (WT) mice. In addition, we have observed an increase in the CD4+FoxP3+ regulatory cell population among spinal cord infiltrates from AMPAR KO EAE mice. Consistent with the EAE model, AMPAR KO T cells also exhibit increased induced regulatory T cell development during in vitro differentiation. Taken together our results support a previously unappreciated role for ionotropic AMPA receptors in regulating T cell tolerance.

Mechanistic and Biomarker Studies to Demonstrate Immune Tolerance in Multiple Sclerosis.

The induction of specific immunological tolerance represents an important therapeutic goal for multiple sclerosis and other autoimmune diseases. Sound knowledge of the target antigens, the underlying pathomechanisms of the disease and the presumed mechanisms of action of the respective tolerance-inducing approach are essential for successful translation. Furthermore, suitable tools and assays to evaluate the induction of immune tolerance are key aspects for the development of such treatments. However, investigation of the mechanisms of action underlying tolerance induction poses several challenges. The optimization of sensitive, robust methods which allow the assessment of low frequency autoreactive T cells and the long-term reduction or change of their responses, the detection of regulatory cell populations and their immune mediators, as well as the validation of specific biomarkers indicating reduction of inflammation and damage, are needed to develop tolerance-inducing approaches successfully to patients. This short review focuses on how to demonstrate mechanistic proof-of-concept in antigen-specific tolerance-inducing therapies in MS.

The Balance Between Memory and Regulatory Cell Populations in Kidney Transplant Recipients with Operational Tolerance

The Balance Between Memory and Regulatory Cell Populations in Kidney Transplant Recipients with Operational Tolerance

Reduced infiltration of regulatory T cells in tumours from mice fed daily with gamma-tocotrienol supplementation.

Gamma-tocotrienol (γT3) is an analogue of vitamin E with beneficial effects on the immune system, including immune-modulatory properties. This study reports the immune-modulatory effects of daily supplementation of γT3 on host T helper (Th) and T regulatory cell (Treg ) populations in a syngeneic mouse model of breast cancer. Female BALB/c mice were fed with either γT3 or vehicle (soy oil) for 2weeks via oral gavage before they were inoculated with syngeneic 4T1 mouse mammary cancer cells (4T1 cells). Supplementation continued until the mice were euthanized. Mice (n=6) were euthanized at specified time-points for various analysis (blood leucocyte, cytokine production and immunohistochemistry). Tumour volume was measured once every 7days. Gene expression studies were carried out on tumour-specific T lymphocytes isolated from splenic cultures. Supplementation with γT3 increased CD4+ (p<0.05), CD8+ (p<0.05) T-cells and natural killer cells (p<0.05) but suppressed Treg cells (p<0.05) in peripheral blood when compared to animals fed with the vehicle. Higher interferon (IFN)-γ and lower transforming growth factor (TGF)-ꞵ levels were noted in the γT3 fed mice. Immunohistochemistry findings revealed higher infiltration of CD4+ cells, increased expression of interleukin-12 receptor-beta-2 (IL-12ꞵ2R), interleukin (IL)-24 and reduced expression of cells that express the forkhead box P3 (FoxP3) in tumours from the γT3-fed animals. Gene expression studies showed the down-regulation of seven prominent genes in splenic CD4+ T cells isolated from γT3-fed mice. Supplementation with γT3 from palm oil-induced T cell-dependent cell-mediated immune responses and suppressed T cells in the tumour microenvironment in a syngeneic mouse model of breast cancer.

In vitro Immunomodulatory Effects of Human Umbilical Cord-Derived Mesenchymal Stem Cells on Peripheral Blood Cells from Warm Autoimmune Hemolytic Anemia Patients

Introduction: Autoimmune hemolytic anemia is a potentially lethal disease characterized by autoimmune hemolysis. Although human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) have been reported as a promising therapy, there is limited evidence regarding warm autoimmune hemolytic anemia (wAIHA) patients. This study aimed to investigate the potential therapeutic effects of hUC-MSCs via immune regulation in wAIHA patients. Methods: Peripheral blood mononuclear cells (PBMCs) from 10 wAIHA patients and 8 healthy controls were isolated from peripheral blood and cultured for 3 days with or without the presence of hUC-MSCs; PBMCs were co-cultured with hUC-MSCs using Transwell assays. The supernatant cytokine levels were measured after culture through AimPlex Multiple Immunoassays for Flow, including IL-2, IL-4, IL-10, IFN-γ, TNF-α, and IL-17A. The percentages of regulatory T cells, regulatory B cells, and Th1/Th2 in PBMCs were also assessed before and after culturing. Results: In the wAIHA group, hUC-MSCs could upregulate the Treg and Breg proportions after culturing for 3 days, and the Treg and Breg percentages increased after co-culturing with hUC-MSCs in the wAIHA group compared with PBMC cultured alone for 3 days (8.29 ± 8.59 vs. 6.82 ± 1.32, 3.82 ± 1.87 vs. 1.75 ± 1.20, respectively). Compared with the PBMC wAIHA group, the levels of TNF-α (2.13 ± 2.07 vs. 16.20 ± 21.13 pg/mL, p = 0.019) and IL-10 (10.51 ± 18.42 vs. 37.78 ± 44.20 pg/mL, p = 0.012) were significantly elevated in the PBMC + hUC-MSCs wAIHA group. Conclusion: The hUC-MSCs contributed to the increasing proportion of regulatory cell populations in PBMCs of wAIHA patients, thereby potentially regulating autoimmune response; thus, hUC-MSCs may be a promising approach for wAIHA treatment.

Immune Cells Profiling in ANCA-Associated Vasculitis Patients-Relation to Disease Activity.

Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAV) are a group of necrotizing multiorgan autoimmune vasculitides that predominantly affect small blood vessels and are associated with the presence of ANCAs. The aim was to assess regulatory and effector cell populations accompanied by the suPAR biomarker level and link the so-defined immune state to the AAV disease activity. The research involved a multicomponent description of an immune state encompassing a range of B and T cell subsets such as transitional/regulatory B cells (CD19+CD24++CD38++), naïve B cells (CD19+CD24INTCD38INT), Th17 cells, T regulatory cells (CD4+CD25+FoxP3+) and cytotoxic CD4+CD28− cells by flow cytometry. The suPAR plasma level was measured by ELISA. The results indicate that AAV is associated with an increased suPAR plasma level and immune fingerprint characterized by an expansion of Th17 cells and T cells lacking the costimulatory molecule CD28, accompanied by a decrease of regulatory populations (Tregs and transitional B cells) and NK cells. Decreased numbers of regulatory T cells and transitional B cells were shown to be linked to activation of the AAV disease while the increased suPAR plasma level—to AAV-related deterioration of kidney function. The observed immune fingerprint might be a reflection of peripheral tolerance failure responsible for development and progression of ANCA-associated vasculitides.