Treg Cell Population Research Articles

Dysregulation of Allergic Airway Inflammation in the Absence of Microbial Colonization

The incidence of allergic disorders is increasing in developed countries and has been associated with reduced exposure to microbes and alterations in the commensal bacterial flora. To ascertain the relevance of commensal bacteria on the development of an allergic response, we used a model of allergic airway inflammation in germ-free (GF) mice that lack any exposure to pathogenic or nonpathogenic microorganisms. Allergic airway inflammation was induced in GF, specific pathogen-free (SPF), or recolonized mice by sensitization and challenge with ovalbumin. The resulting cellular infiltrate and cytokine production were measured. Our results show that the total number of infiltrating lymphocytes and eosinophils were elevated in the airways of allergic GF mice compared with control SPF mice, and that this increase could be reversed by recolonization of GF mice with the complex commensal flora of SPF mice. Exaggerated airway eosinophilia correlated with increased local production of Th2-associated cytokines, elevated IgE production, and an altered number and phenotype of conventional dendritic cells. Regulatory T-cell populations and regulatory cytokine levels were unaltered, but GF mice exhibited an increased number of basophils and decreased numbers of alveolar macrophages and plasmacytoid dendritic cells. These data demonstrate that the presence of commensal bacteria is critical for ensuring normal cellular maturation, recruitment, and control of allergic airway inflammation.

Development of human CD4+FoxP3+ regulatory T cells in human stem cell factor–, granulocyte-macrophage colony-stimulating factor–, and interleukin-3–expressing NOD-SCID IL2Rγnull humanized mice

AbstractHuman hematolymphoid mice have become valuable tools for the study of human hematopoiesis and uniquely human pathogens in vivo. Recent improvements in xenorecipient strains allow for long-term reconstitution with a human immune system. However, certain hematopoietic lineages, for example, the myeloid lineage, are underrepresented, possibly because of the limited cross-reactivity of murine and human cytokines. Therefore, we created a nonobese diabetic/severe combined immunodeficiency/interleukin-2 receptor-γ–null (NOD-SCID IL2Rγnull) mouse strain that expressed human stem cell factor, granulocyte-macrophage colony-stimulating factor, and interleukin-3, termed NSG-SGM3. Transplantation of CD34+ human hematopoietic stem cells into NSG-SGM3 mice led to robust human hematopoietic reconstitution in blood, spleen, bone marrow, and liver. Human myeloid cell frequencies, specifically, myeloid dendritic cells, were elevated in the bone marrow of humanized NSG-SGM3 mice compared with nontransgenic NSG recipients. Most significant, however, was the increase in the CD4+FoxP3+ regulatory T-cell population in all compartments analyzed. These CD4+FoxP3+ regulatory T cells were functional, as evidenced by their ability to suppress T-cell proliferation. In conclusion, humanized NSG-SGM3 mice might serve as a useful model to study human regulatory T-cell development in vivo, but this unexpected lineage skewing also highlights the importance of adequate spatiotemporal expression of human cytokines for future xenorecipient strain development.

Immunology in the heart of the High Tatra mountains

Immunology in the heart of the High Tatra mountains

Inhibition of differentiation, amplification, and function of human TH17 cells by intravenous immunoglobulin

T(H)17 cells play a critical role in the pathogenesis of several autoimmune and allergic diseases. Intravenous immunoglobulin (IVIg), a therapeutic preparation of polyclonal IgG that is increasingly used in the treatment of diverse autoimmune and allergic diseases, might target T(H)17 cells to exert therapeutic effects. We sought to examine whether IVIg interferes with the development and function of human T(H)17 cells. T(H)17 cells were differentiated from naive human CD4(+) T cells in the presence of TGF-β and IL-21. T(H)17 cells were amplified by stimulating memory CD4(+) T cells in the presence of IL-1β and IL-6. The effect of IVIg was examined on the differentiation and amplification of T(H)17 cells, expression of the T(H)17 lineage-specific transcription factor retinoic acid-related orphan receptor C, secretion of T(H)17 effector cytokines, and phosphorylation of signal transducer and activator of transcription 3, a transcription factor that plays an important role in T(H)17 cell development and function. IVIg inhibits the differentiation and amplification of human T(H)17 cells, as well as the production of their effector cytokines IL-17A, IL-17F, IL-21, and CCL20. The inhibitory effects of IVIg on T(H)17 cells are F(ab')(2) dependent and involve interference with the expression of retinoic acid-related orphan receptor C and activation of signal transducer and activator of transcription 3. Also, IVIg significantly enhanced forkhead box protein 3-positive regulatory T cells among the memory CD4(+) T cells. These results reveal a novel mechanism of action of IVIg in achieving a therapeutic effect in autoimmune and allergic diseases, in which T(H)17 cells play a key modulatory role in sustaining the chronic inflammatory response. Our results also suggest a reciprocal regulation of T(H)17 and regulatory T-cell populations by IVIg.

Prolongation of Composite Tissue Allotransplant Survival by Treatment with Bone Marrow Mesenchymal Stem Cells Is Correlated with T-Cell Regulation in a Swine Hind-Limb Model

Recently published reports indicate that treatment with mesenchymal stem cells combined with bone marrow transplantation could prolong survival after composite tissue allotransplantation. This study investigated whether bone marrow mesenchymal stem cells combined with irradiation and short-term immunosuppressant therapy, but without bone marrow transplantation, could prolong composite tissue allotransplantation survival. Correlation with regulatory T-cell populations was also evaluated in a swine hind-limb model. Heterotopic hind-limb transplantation was performed in outbred miniature swine. Group I (n = 4) was the untreated control. Group II (n = 3) received mesenchymal stem cells alone (on days -1, 3, 7, 14, and 21). Group III (n = 5) received cyclosporine A (on days 0 through 28). Group IV (n = 3) received irradiation (on day -1), mesenchymal stem cells (on days 1, 7, 14, and 21), and cyclosporine A (on days 0 to 28). Swine viability and signs of allograft rejection were monitored postoperatively. Histopathologic changes in allografts were examined. The expression and localization of CD4+/CD25+ and CD4+/FoxP3+ T cells were assessed using flow cytometry and immunohistochemistry. Treatment with mesenchymal stem cells along with irradiation and cyclosporine A resulted in significant increases in allograft survival as compared with other groups (>120 days; p = 0.018). Histologic examination revealed the lowest degree of rejection in grafted skin and interstitial muscle layers in the mesenchymal stem cell/irradiation/cyclosporine A group. Flow cytometric analysis revealed a significant increase in the percentage of CD4+/CD25+ and CD4+/FoxP3+ T cells in both the blood and graft in the mesenchymal stem cell/irradiation/cyclosporine A group. These results suggest that prolonged survival after composite tissue allotransplantation induced by treatment with mesenchymal stem cells combined with irradiation/cyclosporine A is correlated with regulatory T cells.

Early lymphocyte recovery after intensive timed sequential chemotherapy for acute myelogenous leukemia: peripheral oligoclonal expansion of regulatory T cells

AbstractFew published studies characterize early lymphocyte recovery after intensive chemotherapy for acute myelogenous leukemia (AML). To test the hypothesis that lymphocyte recovery mirrors ontogeny, we characterized early lymphocyte recovery in 20 consecutive patients undergoing induction timed sequential chemotherapy for newly diagnosed AML. Recovering T lymphocytes were predominantly CD4+ and included a greatly expanded population of CD3+CD4+CD25+Foxp3+ T cells. Recovering CD3+CD4+CD25+Foxp3+ T cells were phenotypically activated regulatory T cells and showed suppressive activity on cytokine production in a mixed lymphocyte reaction. Despite an initial burst of thymopoiesis, most recovering regulatory T cells were peripherally derived. Furthermore, regulatory T cells showed marked oligoclonal skewing, suggesting that their peripheral expansion was antigen-driven. Overall, lymphocyte recovery after chemotherapy differs from ontogeny, specifically identifying a peripherally expanded oligoclonal population of activated regulatory T lymphocytes. These differences suggest a stereotyped immunologic recovery shared by patients with newly diagnosed AML after induction timed sequential chemotherapy. Further insight into this oligoclonal regulatory T-cell population will be fundamental toward developing effective immunomodulatory techniques to improve survival for patients with AML.

The Starting Lineup: Key Microbial Players in Intestinal Immunity and Homeostasis

The complexity of microbiota inhabiting the intestine is increasingly apparent. Delicate balance of numerous bacterial species can affect development of the immune system, how susceptible a host is to pathogenic organisms, and the auto-inflammatory state of the host. In the last decade, with the increased use of germ-free mice, gnotobiotic mice, and animal models in which a germ-free animal has been colonized with a foreign microbiota such as humanized mice, it has been possible to delineate relationships that specific bacteria have with the host immune system and to show what role they may play in overall host health. These models have not only allowed us to tease out the roles of individual species, but have also allowed the discovery and characterization of functionally unknown organisms. For example, segmented filamentous bacteria (SFB) have been shown to play a vital role in expansion of IL-17 producing cells. Prior to linking their key role in immune system development, little was known about these organisms. Bacteroides fragilis can rescue some of the immune defects of gnotobiotic mice after mono-colonization and have anti-inflammatory properties that can alleviate colitis and experimental allergic encephalitis in murine models. Additionally, Clostridium species have most recently been shown to expand regulatory T-cell populations leading to anti-inflammatory conditions. This review will highlight and summarize some of the major findings within the last decade concerning the role of select groups of bacteria including SFB, Clostridium, Bacteroides, Bifidobacterium, and Lactobacillus, and their impact on host mucosal immune systems.

Influenza infection in suckling mice expands an NKT cell subset that protects against airway hyperreactivity

Infection with influenza A virus represents a major public health threat worldwide, particularly in patients with asthma. However, immunity induced by influenza A virus may have beneficial effects, particularly in young children, that might protect against the later development of asthma, as suggested by the hygiene hypothesis. Herein, we show that infection of suckling mice with influenza A virus protected the mice as adults against allergen-induced airway hyperreactivity (AHR), a cardinal feature of asthma. The protective effect was associated with the preferential expansion of CD4-CD8-, but not CD4+, NKT cells and required T-bet and TLR7. Adoptive transfer of this cell population into allergen-sensitized adult mice suppressed the development of allergen-induced AHR, an effect associated with expansion of the allergen-specific forkhead box p3+ (Foxp3+) Treg cell population. Influenza-induced protection was mimicked by treating suckling mice with a glycolipid derived from Helicobacter pylori (a bacterium associated with protection against asthma) that activated NKT cells in a CD1d-restricted fashion. These findings suggest what we believe to be a novel pathway that can regulate AHR, and a new therapeutic strategy (treatment with glycolipid activators of this NKT cell population) for asthma.

Characterization of Interleukin-17–Producing Regulatory T Cells in Inflamed Intestinal Mucosa From Patients With Inflammatory Bowel Diseases

Interleukin (IL)-17-producing CD4(+) helper T cells (Th17) mediate mucosal immunity and are involved in the pathogenesis of inflammatory bowel disease (IBD). They are believed to arise from the same precursor population as regulatory T (Treg) cells, but little is known about how these T-cell subsets interact under chronic inflammatory conditions. We studied Th17 and Treg cells isolated from intestinal lamina propria of patients with IBD to investigate their role in pathogenesis. FoxP3 expression (a marker of Treg cells) and IL-17 production were assessed in CD4(+) lamina propria lymphocytes isolated from IBD patients and healthy subjects. IL-17(+)FoxP3(+) and IL-17(+) CD4(+) T-cell clones were generated by limiting dilution. An in vitro suppression assay was performed to assess the functional capacity of derived T-cell clones. IL-17(+)FoxP3(+) T cells were identified in inflamed intestinal mucosa of patients with Crohn disease (CD), but not in patients with ulcerative colitis (UC) or healthy controls. These cells shared phenotypic characteristics of Th17 and Treg cells, and showed potent suppressor activity in vitro. Transforming growth factor-β was necessary and sufficient to induce development of an IL-17(+) FoxP3(+) cell population in CD4(+) lamina propria lymphocytes derived from patients with UC. The inflammatory environment in the intestinal mucosa of patients with CD contributes to the generation of a distinct population of Treg cells that are FoxP3(+) and produce IL-17. These cells are likely to arise during differentiation of Th17 and Treg cells. Specific microenvironmental cues from tissues are likely to determine their commitment to either lineage and affect the balance between regulation and inflammation in the intestine.

Immunological Aspects of Allogeneic Mesenchymal Stem Cell Therapies

Allogeneic mesenchymal stem or stromal cells (MSCs) are proposed as cell therapies for degenerative, inflammatory, and autoimmune diseases. The feasibility of allogeneic MSC therapies rests heavily on the concept that these cells avoid or actively suppress the immunological responses that cause rejection of most allogeneic cells and tissues. In this article the validity of the immune privileged status of allogeneic MSCs is explored in the context of recent literature. Current data that provide the mechanistic basis for immune modulation by MSCs are reviewed with particular attention to how MSCs modify the triggering and effector functions of innate and adaptive immunity. The ability of MSCs to induce regulatory dendritic and T-cell populations is discussed with regard to cell therapy for autoimmune disease. Finally, we examine the evidence for and against the immune privileged status of allogeneic MSCs in vivo. Allogeneic MSCs emerge as cells that are responsive to local signals and exert wide-ranging, predominantly suppressive, effects on innate and adaptive immunity. Nonetheless, these cells also retain a degree of immunogenicity in some circumstances that may limit MSC longevity and attenuate their beneficial effects. Ultimately successful allogeneic cell therapies will rely on an improved understanding of the parameters of MSC-immune system interactions in vivo.

Methyl Gallate Exhibits Potent Antitumor Activities by Inhibiting Tumor Infiltration of CD4+CD25+ Regulatory T Cells

CD4(+)CD25(+) regulatory T (Treg) cells play crucial roles in the host response to tumors. Increasing evidence supports the existence of elevated numbers of Treg cells in solid tumors and hematologic malignancies. In this study, the effects of methyl gallate on Treg cells were examined. Methyl gallate inhibited Treg cell-suppressive effects on effector CD4(+) T cells and Treg migration toward tumor environment. The expression of Treg surface markers including CTLA-4, CCR4, CXCR4, and glucocorticoid-induced TNFR was significantly suppressed upon methyl gallate treatment. Furthermore, forkhead box P3 (Foxp3) expression was also significantly decreased by methyl gallate, suggesting that the suppressive effects of methyl gallate on Treg were medicated by decrease of Treg-specific transcription factor Foxp3. In tumor-bearing hosts, methyl gallate treatment substantially reduced tumor growth and prolonged the survival rate. In contrast, nu/nu mice did not show decreased tumor progression in response to methyl gallate. In addition, in tumor-bearing Treg-depleted mice, tumor growth and the survival rates were not changed by methyl gallate treatment, strongly suggesting that the main therapeutic target of methyl gallate in tumor suppression was related to modulation of the CD4(+)CD25(+) Treg cell functions. In the spleen of tumor-bearing mice, methyl gallate treatment induced a significant decrease in the CD4(+)CD25(+)Foxp3(high) Treg cell population. Especially, the number of tumor-infiltrating CD25(+)Foxp3(high) Treg cells was significantly lower in methyl gallate-treated mice. These results suggest that methyl gallate can be used to reverse immune suppression and as a potentially useful adjunct for enhancing the efficacy of immune-based cancer therapy.

Preserving and Restoring Optic Nerve Function

Preserving and Restoring Optic Nerve Function

The Impact of Vitamin D on Regulatory T Cells

Epidemiologic studies highlight the increasing prevalence of vitamin D deficiency and insufficiency and its association with an increased risk of autoimmune diseases and poor respiratory function, including asthma. These and additional studies have raised interest in the immunomodulatory properties of vitamin D beyond its well-established role in calcium homeostasis and bone health. Vitamin D has been shown to influence the function of cells intrinsic to innate and adaptive immunity. This review discusses recent evidence that vitamin D promotes--both directly and indirectly--regulatory or suppressor T-cell populations with the capacity to inhibit inappropriate immune responses that cause disease, suggesting that this property may in part underpin the epidemiologic findings.

Immunomodulation by Lymphodepletion and Donor Lymphocyte Infusion to Treat Post Allogeneic Transplantation Relapse

AbstractAbstract 2084Relapse of hematologic malignancies following allogeneic hematopoietic stem cell transplantation (HSCT) remains a major cause of treatment failure. For diseases other than CML, DLI remains relatively disappointing and retreatment with chemotherapy is required before resorting cell therapy. Lymphodepletion (LD) may enhance anti-tumor effects of transferred T cells. To prevent serious toxicity due to severe GvHD, we sequentially escalated both the LD and T cell doses infused with the DLI. LD consisted of Fludarabine (Flu) 25 mg/m2/d for 3 days followed by infusion of 1×107 CD3+ cells/kg. If no GvHD developed and disease persisted or progressed, the same LD was followed by a second DLI of 5×107 CD3+ cells/kg. In a second cohort, LD combined both cyclophosphamide (Cy) 600 mg/m2 for one day and Flu 25 mg/m2/d for 3 days followed by DLI at 5×107 CD3+ cells/kg. Eighteen pts, median age at transplant: 45 y (18-67), were treated for relapses of various non-CML hematologic malignancies (6 AML/MDS, 4 ALL, 3 HD, 2 T-NHL, 2 MM) following allogeneic transplantation (5 myeloablative conditioning, 12 RIC. Donors: 10 siblings, 7 MUD). DLI was the first relapse treatment or given following chemotherapy. Forty four DLIs were performed including 28 LD-DLIs. Prior to LD-DLI, eight patients received DLI not preceded by LD. Ten, 6 and 1 patient(s) received respectively 1, 2 or 3 LD-DLIs. The first LD-DLI was given at a median of 234 days (range 62 d–14y11m) following allogeneic transplantation. The first cohort (n=5) consisted of patients receiving LD with Flu alone. No severe toxicity was observed. In the second cohort (n=4) following a first LD-DLI with Flu alone, Cy was introduced along with Flu prior to DLI. Two patients developed GvHD: one cutaneous Grade II and another cutaneous grade III, liver Grade III and digestive Grade IV. The third cohort included 5 patients who had received prior DLI without LD and who were then treated with LD combining both Flu and Cy followed by high dose DLI (5×107 CD3+ cells/kg). Only one patient developed cutaneous Grade III GvHD. In the fourth group, 4 patients who had not yet received DLI, received LD with Flu and Cy followed by high dose DLI. In this latter group, one case of acute GvHD Grade IV was observed. The cumulative incidence of Grade II-IV acute GvHD was 29.4% with a median onset of acute grade II-IV GvHD following the last LD-DLI was 31 days (range 15–59). In comparison, the cumulative incidence of Grade II-IV GvHD in a control group of 54 patients (35 RIC, 19 myeloablative; donors: 50 siblings, 3 MUD, 1 mismatched unrelated donor) receiving DLI without lymphodepletion was 28.6% with a median onset of 34 days, range 12–120) not statistically different. Twelve patients received cytoreduction chemotherapy prior to DLI. In 9 patients, this failed to result in a complete remission, leaving 3 CRs after chemotherapy (2 AML, 1 MM). Following LD-DLI, 4 additional patients achieved a CR (1 AML, 2 ALL, and 1 T-NHL). After a median follow-up of 8 months (2 – 28.3) from the first LD-DLI, 8 patients (3 HD, 3 ALL, 1 AML, 1 MM) are alive; only 2 remain in CR (1 AML, 1 ALL). Ten patients died, 9 due to progression and 1 from lung cancer. Overall survival from the first LD-DLI is 40% at 2 years. The absolute number of leucocytes and the absolute number of CD4+ cells was compared prior to LD-DLI and 14 days following DLI; in those receiving Flu/Cy and DLI these were found to be decreased but not significantly (202±37 vs 113±38/mm3). The Treg cell population CD4+CD25high also decreased while the number of circulating CD11c+ (myeloid) dendritic cells and BDCA2+ (plasmacytoid) dendritic cells increased, none significantly. IL-7, IL-15 plasma levels remained undetectable at Day 14 post DLI. We conclude that LD-DLI in an escalated manner is not complicated by excessively severe GvHD while transient remission can be achieved. In our cohorts of patients, this did not translated into significant modification of T cells subsets or expansion of homeostatic cytokines. Based on these findings, continued studies with escalating doses are planned. Disclosures:Mohty:Genzyme: Consultancy, Honoraria, None, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria; Janssen Cilag: Consultancy, Honoraria; Celgene: Honoraria.

Clinical Grade Isolation of Regulatory T Cells From G-CSF Mobilized PBSC Improves with Initial Depletion of Monocytes.

AbstractAbstract 1176We have recently demonstrated that G-CSF mobilized peripheral blood stem cells (PBSC) CD4+CD25+FoxP3+ cells (Tregs) prevent anti-CD34+ hematopoietic stem cells T cell alloreactivity in-vitro and co-transplantation of CD34+ cells and Tregs does not affect human stem cell engraftment in NOD/SCID mice (Mahmud D et al. Biol Blood Marrow Transplant, 2010). Since only a small number of Tregs can be isolated from normal peripheral blood we examined whether PBSC can be a useful source of Tregs for future clinical trials. Five leukapheresis products from healthy donors who received rh G-CSF at 10 ug/kg daily for 5 days were processed using the CliniMACS instrument (Miltenyi Biotec, Auburn, CA). CliniMACS CD34 reagent was initially used to isolate CD34+ cells. To isolate Tregs a 2-step procedure was initially utilized. A cocktail of clinical grade CD14, CD8 and CD19 reagents was mixed with the CD34- cells and depletion of monocytes, cytotoxic T cells and B cells was obtained by using the Depletion 2.1 program. The CD4+ cells were then enriched in Tregs by positive selection of CD25+ cells using a clinical grade CD25 reagent (Miltenyi). Because PBSC contain large amount of myeloid cells, and particularly monocytes, this clinical scale 2-step strategy was compared with a 3-step method that included an initial negative selection of CD14+ monocytes, followed by negative selection of CD8+ and CD19+ cells and a positive selection of CD25+ cells. Prior to isolation, the average proportion of CD4+CD25+ cells in PBSC was 0.77±0.26% in 5 separate PBSC products. After the 2-step process the proportion of CD4+CD25+ cells was 35±33% (n=3) vs 72±1% after the 3-step process. Therefore, utilizing the 3-step approach a better yield of Tregs was observed (10 vs 60%). Intracellular expression of FoxP3 was on average 74% in CD4+CD25+ cells obtained with a 3-step process. Contamination of different cell subsets in the final products enriched in Tregs was largely superior following the 2-step as compared to 3-step isolation method. Contaminating monocytes were, on average, 43 vs 5.7%, and contaminating CD8 and CD19+ cells were 12 vs 1.7% and 0.9 vs 0.3%, respectively. In the two procedures using a 3-step approach the final absolute number of Tregs isolated from products containing on average 30 × 109 mononuclear cells, was 95 and 93 × 106, respectively. These findings obtained using clinically available reagents and device, suggest that depletion of monocytes may improve the purity of Treg cell population isolated from PBSC. PBSC may represent a valuable source of Tregs for future clinical trials. Disclosures:No relevant conflicts of interest to declare.

Dynamics of Chimerism In Regulatory T Lymphocytes (Treg; CD4+/CD25+) After Allogeneic Stem Cell Transplantation

AbstractAbstract 1328 Introduction:CD4+CD25+ regulatory T-cells (Treg) play an important role in inducing and maintaining allogeneic tolerance and can inhibit graft-versus-host disease (GVHD) after allogeneic stem cell transplantation (SCT). However, the dynamics of donor and recipient Treg cell populations after Allo-SCT has not been studied yet. Objective:To analyze the dynamics of chimerism in Tregs and compare it to that of T lymphocytes (TL; CD3+) and whole blood leukocytes (WBL). Material and Methods:The study includes 53 patients subjected to Allo-SCT (myeloablative and non-myeloablative). PB samples were obtained weekly during the first month and every 14 days after day +30 until complete chimerism (CC) was achieved, and at fixed time-points (+30, +60, +90, +180, +365) thereafter. TL and Tregs were purified from PB until CC was achieved using inmunomagnetic technology (Miltenyi Biotec; CD3+ Microbeads and CD4+/CD25+ Treg Isolation Kit, respectively). Chimerism analysis was performed by microsatellite PCR (STR-PCR; AmpFlSTR SGM Plus; Applied Biosystems) on genomic DNA obtained from WBL as well as on cell lysates obtained from purified TL and Tregs. Complete chimerism was considered in samples with percentage of recipient cells <1% (sensitivity of the STR-PCR) for WBL samples and <5% (minimum purity of 95% as estimated by flow cytometry) for purified cell lineages. Results:Median follow up for the whole cohort of patients was 338 days. CC was spontaneously achieved in WBL, TL and Tregs in 45/53 patients (85%) in a median time of 35.41, 38.8 and 42.5 days respectively. 5/3 patients (9%) suffered from graft failure/rejection showing mixed chimerism (MC) with increasing percentages of recipient cells both in WBL and leukocyte lineages. 3/53 patients (6%) maintained mixed chimerism (MC) in WBL and leukocyte lineages at day +180, with no signs of graft rejection or disease relapse. Analysis of the chimerism dynamics of those patients who spontaneously achieve CC revealed two different groups: Group 1 included 25/45 patients who achieved CC at the same time in WBL, TL and Tregs. Group 2 included 20/45 patients who achieved CC in WBL while maintaining MC in TL and Tregs. Interestingly enough, 9/20 patients from Group 2 maintained MC in Tregs 7–75 days after achieving CC in both WBL and TL (Figure 1). In a preliminary analysis, the small sample size precluded from obtaining statistically significant associations between the dynamics of chimerism in Tregs and the development of complications such as relapse or GVHD after SCT. [Display omitted] Conclusions:To our knowledge, this is the first study dealing with Treg chimerism after SCT. We have shown it is feasible and can be performed on a routine basis together with standard lineage specific chimerism follow up. Although there is an association between chimerism dynamics in Tregs and TL, this is not absolute and a percentage of patients maintain residual Tregs of recipient origin after WTL and TL have become of complete donor origin. In this small cohort, Treg chimerism did not influence the development of post-SCT complications. Analysis of a larger and more homogeneous cohort would allow establishing the usefulness of Treg chimerism testing for the management of transplanted patients. Disclosures:No relevant conflicts of interest to declare.

Radiation Enhances Regulatory T Cell Representation

Immunotherapy could be a useful adjunct to standard cytotoxic therapies such as radiation in patients with micrometastatic disease, although successful integration of immunotherapy into treatment protocols will require further understanding of how standard therapies affect the generation of antitumor immune responses. This study was undertaken to evaluate the impact of radiation therapy (RT) on immunosuppressive T regulatory (Treg) cells. Treg cells were identified as a CD4(+)CD25(hi)Foxp3(+) lymphocyte subset, and their fate was followed in a murine TRAMP C1 model of prostate cancer in mice with and without RT. CD4(+)CD25(hi)Foxp3(+) Treg cells increased in immune organs after local leg or whole-body radiation. A large part, but not all, of this increase after leg-only irradiation could be ascribed to radiation scatter and Treg cells being intrinsically more radiation resistant than other lymphocyte subpopulations, resulting in their selection. Their functional activity on a per-cell basis was not affected by radiation exposure. Similar findings were made with mice receiving local RT to murine prostate tumors growing in the leg. The importance of the Treg cell population in the response to RT was shown by systemic elimination of Treg cells, which greatly enhanced radiation-induced tumor regression. We conclude that Treg cells are more resistant to radiation than other lymphocytes, resulting in their preferential increase. Treg cells may form an important homeostatic mechanism for tissues injured by radiation, and in a tumor context, they may assist in immune evasion during therapy. Targeting this population may allow enhancement of radiotherapeutic benefit through immune modulation.

Probiotics, Enteric and Diarrheal Diseases, and Global Health

Enteric and diarrheal diseases are a major worldwide cause of death among children under the age of 5. In this age group, diarrhea occurs 2.5 billion times per year [1] and causes 15% of childhood deaths.[2] Diarrheal diseases claim 59 million disability-adjusted life years (DALYs), nearly all from children in low- and middle-income countries.[3] Despite this enormous burden, these numbers fail to capture the full impact of enteric and diarrheal diseases. Early and frequent exposure to intestinal pathogens begins a cycle (Figure 1A) that affects digestion, nutrient absorption, growth, and immunity.[4] Repeated infections, with either overt diarrhea or subclinical enteropathy, produce acute and chronic undernutrition,[5] which leads to more frequent and severe infections.[6] Undernutrition contributes to 53% of childhood deaths [7] and is the leading risk factor for poor health outcomes in childhood;[8] survivors are at risk for developmental deficits in growth, fitness, and cognition that persist into adulthood with devastating consequences.[4] These consequences have a multiplicative effect on calculations of DALYs from diarrheal disease.[9] Open in a separate window Figure 1 The vicious cycle of diarrhea and undernutrition in susceptible children (A) The devastating synergy between enteric infections and undernutrition is influenced by the environment, the human genome, host nutrition, and the human microbiome. Various interventions (red boxes) may inhibit progression to the next step in the cycle, minimizing both acute and chronic morbidities. (B) Employing a spectrum of disease outcome measures would lend greater insight into the pathology underlying enteric and diarrheal diseases, while providing a more complete understanding of interventions targeting basic steps of enteric and diarrheal disease pathogenesis. Adapted with permission from Wiley: Nutrition Reviews,4 copyright 2008. http://www.http://onlinelibrary.wiley.com/journal/10.1111/(ISSN)1753-4887.

Mesenchymal stem cell-mediated immature dendritic cells induce regulatory T cell-based immunosuppressive effect

Immature dendritic cells (imDCs) are increasingly viewed as mediators of T-cell tolerance. We investigated factors enabling induction of regulatory T (Treg) cells through syngeneic imDC/mesenchymal stem cell (MSC) co-cultures in vitro and immunosuppressive effects of MSC-mediated imDCs (MSCs were excluded after 72 h co-culture) in vivo. In these experiments, we found that Foxp3(+) Treg cell population remarkably increased after the T cell priming phase when splenocytes were co-cultured with both imDCs and MSCs, presumably inducing naive T cells into Treg cells by MSCs and imDCs. In parallel, TGF-β secretion was markedly induced from the imDC+MSC+splenocyte culture supernatant to a significant level at 72-h co-culture, compared to the MSC or imDC+splenocyte co-culture. Based on these results, using a murine melanoma tumor model, we confirmed that the subcutaneous injection of B16 cells induced a perfect tumor incidence in allogeneic recipients when MSC-mediated DCs were coinjected. Consequently, these results suggested that immune tolerance with MSC-mediated DCs leads to immunosuppression induced by at least Foxp3-specific Treg cells. This tool may be useful in clinical trials due to the yet unknown side effects of stem cell therapy.

The frequency of CD25+CD4+ and FOXP3+ regulatory T cells in ectopic endometrium and ectopic decidua

BackgroundThe presence of regulatory T (Treg) cells in human endometrium is crucial for maintaining immunological homeostasis within the uterus. For this study we decided to evaluate the subpopulations of Treg cells in conditions where a disturbance in the immunological equilibrium in ectopic endometrium and decidua has been observed, such as in cases of ovarian endometriosis (involving local immune cell suppression) and ectopic pregnancy (involving an increase in local immune system activity). We then compared these findings to what we observed in the normal eutopic endometrium of women during the secretory phase of the menstrual cycle (with immune cells under individual control).MethodsThe endometrium tissue samples evaluated in our study were obtained from 47 women during one of two kinds of laparoscopic procedures. 16 of the women underwent laparoscopies due to Fallopian tube pregnancies (EP), and 16 due to ovarian endometrioma, while 15 women made up a control group. The presence of regulatory T cells in these tissue samples was evaluated by FACS.ResultsIn our study, the percentages of FOXP3+ cells within the subpopulation of CD4+ T lymphocytes found in the decidua of the patients treated for Fallopian tube pregnancies were statistically significantly lower than both those observed in the ovarian endometriosis tissue samples and those found in the secretory eutopic endometrium samples of the control group.ConclusionThe disturbance in the immunological equilibrium observed in ectopic endometrium and decidua would seem to be related to the alteration in the Treg cell population that occurs in these ectopic tissues.