Total Treg Research Articles

The SRC Family Kinase, Yes, is Present in Pancreatic Acinar Cells and Activated by Numerous Gastrointestinal Hormones and Growth Factors

Objectives: IgG4-related autoimmune pancreatitis (AIP) is a new clinical entity of pancreatic disorder. There are immunologic and histological abnormalities, including increased serum IgG4 levels and the infiltration of lymphocytes and IgG4-positive plasmacytes. However, the role of IgG4 is unclear. Recently, regulatory T cells (Tregs) were reported to contribute to the development of various autoimmune diseases as well as in B cell shifting to IgG4producing plasmacytes. For this report, we studied regulatory T cells in the pancreas by immunohistochemistory and ICOS+ Tregs and IL-10 producing cells in the peripheral blood lymphocytes by flowcytometry. Methods: We recruited 44 patients with IgG4-related AIP. For comparison, we recruited 37 patients with other pancreatic diseases and 27 healthy subjects as controls. We studied infiltrating cells in the pancreas by immunohistochemistry, and analyzed ICOS+ Tregs and IL-10+ Tregs in the peripheral blood by flow cytometry. Results: The ratio of Foxp3-positive cells to infiltrated mononuclear cells (Foxp3/Mono) in AIP patients (0.091 ± 0.023) was significantly higher than in patients with alcoholic chronic pancreatitis (0.012 ± 0.003; p<0.05). In AIP, Foxp3/Mono and IgG4/Mono were positively correlated (p<0.05; R =0.91). ICOS+ Tregs (%ICOS-positive Tregs of total Tregs) were significantly higher in AIP patients (3.45% ± 1.58%) than in the patients with other pancreatic diseases (alcoholic chronic pancreatitis; 1.71% ± 0.98%, idiopathic chronic pancreatitis; 1.80% ± 0.86%) and the healthy control group (1.57% ± 0.69%, p<0.05). IL-10+ Tregs were significantly higher in AIP patients than in the healthy control group. Conclusions: Increased quantities of ICOS+ Tregs may influence IgG4 production in IgG4-related AIP.

STAT1 and IFNAR1 regulate Treg frequencies and IL-10 expression the tumor microenvironment (66.15)

Abstract The efficacy of cancer immunotherapy is limited by immunosuppression in the tumor microenvironment. Regulatory T cells (Treg) are found at high frequency in tumors, and have greater suppressive activity than Treg from spleen or lymph node tissues. In addition, IL-10 limits the activation of antigen presenting cells and production of proinflammatory cytokines at the tumor site. We examined the cellular sources of IL-10 in the tumor. Using IL-10GFP (VERTX) reporter mice and real-time PCR assays, IL-10 expression was detected in Ly6C+ myeloid cells, macrophages and approximately 50% of tumor Tregs. A similar portion of tumor Treg produced IL-10 following stimulation with PMA/ionomycin. Microarray analyses on IL-10 positive and IL-10 negative tumor Treg and total spleen Treg revealed an activation signature associated with IL-10 expression. This signature correlated strongly with expression of the transcription factor Maf and with induction a type I IFN/STAT1-signature. Using knockout mice we observed that both the type I IFN receptor, IFNAR1 and STAT1 were required for normal tumor Treg frequencies and IL-10 production. This indicates that the STAT1/IFNAR1 axis is required to drive immunosuppressive Treg and their production of IL-10 in the tumor microenvironment. Therefore the immunostimulatory effects of type I IFN are paralleled with an increase in Treg function and IFNAR1/STAT1 may constitute an important therapeutic target for tumor Treg.

Immune Dysregulation after Cardiothoracic Surgery and Incidental Thymectomy: Maintenance of Regulatory T Cells despite Impaired Thymopoiesis

Thymectomy is performed in infants during cardiothoracic surgery leaving many patients with reduced thympopoiesis. An association between immune disorders and regulatory T cells (Treg) after incidental thymectomy has not been investigated. Questionnaires soliciting symptoms of atopic or autoimmune disease and biomarkers were measured in children and adults with congenital heart disease and either reduced or preserved thymopoiesis. Tregs were examined. Atopic or autoimmune-like symptoms and elevated anti-dsDNA antibodies were common after surgery in individuals with low thymopoiesis. Total Treg number and function were maintained but with fewer naïve Treg. TCR spectratypes were similar to other memory T cells. These data suggest that thymectomy does not reduce total Treg number but homeostasis is affected with reduced naïve Treg. Prevalence of autoimmune or atopic symptoms after surgery is not associated with total number or proportion of Tregs but appears to be due to otherwise unknown factors that may include altered Treg homeostasis.

Protection against bronchiolitis obliterans syndrome is associated with allograft CCR7+ CD45RA- T regulatory cells.

Bronchiolitis obliterans syndrome (BOS) is the major obstacle to long-term survival after lung transplantation, yet markers for early detection and intervention are currently lacking. Given the role of regulatory T cells (Treg) in modulation of immunity, we hypothesized that frequencies of Treg in bronchoalveolar lavage fluid (BALF) after lung transplantation would predict subsequent development of BOS. Seventy BALF specimens obtained from 47 lung transplant recipients were analyzed for Treg lymphocyte subsets by flow cytometry, in parallel with ELISA measurements of chemokines. Allograft biopsy tissue was stained for chemokines of interest. Treg were essentially all CD45RA−, and total Treg frequency did not correlate to BOS outcome. The majority of Treg were CCR4+ and CD103− and neither of these subsets correlated to risk for BOS. In contrast, higher percentages of CCR7+ Treg correlated to reduced risk of BOS. Additionally, the CCR7 ligand CCL21 correlated with CCR7+ Treg frequency and inversely with BOS. Higher frequencies of CCR7+ CD3+CD4+CD25hiFoxp3+CD45RA− lymphocytes in lung allografts is associated with protection against subsequent development of BOS, suggesting that this subset of putative Treg may down-modulate alloimmunity. CCL21 may be pivotal for the recruitment of this distinct subset to the lung allograft and thereby decrease the risk for chronic rejection.

IFN-γ expression by regulatory T cells in the CNS of mice persistently infected with neurotropic coronavirus (137.27)

Abstract Mice infected with mouse hepatitis virus, strain JHM develop a chronic infection in the CNS characterized by low levels of viral antigen persistence and retention of virus-specific CD4 and CD8 T cells at the site of infection. We have previously demonstrated that T cell maintenance in the infected CNS is a dynamic process, requiring replenishment from peripheral antigen- experienced and naïve T cell pools. Since mice continued to show clinical improvement, these results raised the possibility that Tregs contributed to a quiescent state in the CNS. Here we show a preferential influx of Tregs to the CNS at very early stage of the infection. They are not converted from Foxp3- cells. Intriguingly, not only IL-10 but also IFN-γ producing Tregs are found in the CNS as early as d 5 p.i. While there are much less (&amp;lt;1%) Tregs making IL-10 during chronic infection, a proportion (&amp;gt;15%) of them continue to express IFN-γ. IFN-γ expression is correlated with T-bet upregulation in Tregs. IFN-γ producing Tregs are induced locally in the CNS from both virus-specific and nonspecific Treg pools. While the frequencies of total Tregs decrease dramatically by d 7 p.i. and remain constant thereafter, two thirds of Tregs lose their Foxp3 expression between d 7 and d 28 p.i. in adoptive transfer experiments. We propose that Tregs acquire effector cell phenotype during type 1 inflammation and the maintenance of Tregs in the persistently infected CNS is also dynamic, requiring recruitment of new Tregs.

Tumor-infiltrating lymphocytes, particularly the balance between CD8 + T cells and CCR4 + regulatory T cells, affect the survival of patients with oral squamous cell carcinoma

The objective of this study was to clarify the prognostic significance of tumor-infiltrating lymphocytes (TILs) in oral squamous cell carcinoma (OSCC); the present study analyzed various TIL-related parameters. Immunohistochemistry was performed in 87 patients with OSCC for the following TIL-related parameters: nest-CD8(+) T cells, stromal CD8(+) T cells, CD4(+) T cells, total regulatory T cells (Tregs), CCR4(+) Tregs, ratio of nest CD8(+) T cells/CCR4(+) Tregs, and ratio of stromal CD8(+) T cells/CCR4(+) Tregs. In univariate analyses, the following parameters were associated with decreased survival: few nest- and stromal CD8(+) T cells and more stromal CCR4(+) Tregs, but not total Tregs. Low ratios of nest and stromal CD8(+) T cell/CCR4(+) Treg were associated with worse survival. In multivariate analysis, the stromal CD8(+) T cell/CCR4(+) Treg ratio was an independent prognostic factor. Host immune responses in the stroma of OSCC affect the survival of the patients. The in situ balance between effector T cells and regulatory T cells is the most important factor predicting survival.

α4β7± Regulatory T Cells (Tregs) at Engraftment Predict Long-Term Graft-Versus-Host Disease (GVHD) Outcomes.

Abstract 2237Poster Board II-214 Background:GVHD occurs unpredictably after allogeneic stem cell transplant (ASCT). GVHD is characterized by profound immune dysregulation, which suggests that abnormalities in suppressor lymphocytes known as Tregs may account for the clinical features of this disease. We have shown that the frequency of Tregs or α4β7± Tregs at neutrophil engraftment predict for recurrent grade II-IV GVHD or recurrent gut GVHD during the first 100 days of transplant, respectively. These data indicate that early lymphocyte populations may determine long-term immune-tolerance following ASCT. We hypothesized that Treg subsets present at engraftment would be associated with the occurrence, National Institutes of Health (NIH) phenotype, and severity of GVHD after day±100. Methods:Patients (pts) undergoing T cell replete ASCT were enrolled. GVHD after day±100 was classified prospectively as either: acute GVHD, overlap GVHD, or classic chronic GVHD using consensus criteria derived from the NIH. Clinical severity was determined by the modified Glucksberg criteria for acute GVHD and the NIH global assessment of severity for overlap/classic chronic GVHD. The frequency of CD45RO±CD25±Foxp3±CD127lo Tregs was quantified within the CD4± T-lymphocyte population at the time of neutrophil engraftment using polychromatic flow cytometry. Gut-homing Tregs were identified by the presence of α4β7±, and their frequency was expressed as a percentage of the total Treg population. Results:Treg analysis was performed at a median of 19 days post-transplant (range, 10-31) on 41 pts undergoing ASCT and surviving until day±100. Twenty-four (59%) pts received ablative conditioning and 17 (41%) pts received a reduced intensity regimen followed by matched related [N= 24 (59%)] or unrelated donor [N= 17 (41%)] ASCT. The stem cell source was peripheral blood, bone marrow, or cord blood for 30 (73%), 9 (22%), and 2 (5%) pts, respectively. GVHD prophylaxis consisted of calcineurin inhibitor and methotrexate [N=23 (56%)] or mycophenolate mofetil [N=18 (44%)]. The majority of patients had grade II-IV acute GVHD prior to day±100 [N=34 (83%)]. After a median follow-up of 15 months (range, 5-24) GVHD after day±100 occurred in 32 (78%) pts at a median of 152 days post ASCT (range, 103-533). GVHD phenotype was classified as: acute (N=19), overlap (N=9), or classic chronic (N=4). Grade III-IV acute and moderate to severe overlap/classic chronic GVHD occurred in 3 (7%) and 11 (27%) pts, respectively. Pts with GVHD after day±100 had lower frequencies of Tregs and α4β7± Tregs at engraftment (4.4% vs. 14.3%; P=0.145) and (9.9% vs. 20.7%; P=0.009), respectively. After excluding pts with gut GVHD, the association between α4β7± Tregs and GVHD after day±100 persisted (P=0.034). Median Treg percentages did not differ among GVHD phenotypes [acute (4.4%) vs. overlap (4.2%) vs. classic chronic (5%) vs. none (14.3%); P=0.464]. However, significant differences between α4β7± Treg frequencies and GVHD subtypes were noted [acute (11%) vs. overlap (9.6%) vs. classic chronic (11.6%) vs. none (20.7%); P=0.002]. α4β7± Treg differences were most apparent when pts with any acute GVHD (acute±overlap) were compared to pts without features of acute GVHD (none±classic chronic) (9.9% vs. 19.4%; P=0.009). Increased frequencies of Tregs and α4β7± Tregs at engraftment tended to be associated with lower grades of acute GVHD after day±100 [odds ratio (OR), 0.95; 95% confidence interval (CI), 0.87-1.04; P=0.255 (Tregs) and OR, 0.81; 95% CI, 0.70-0.92; P=0.002 (α4β7± Tregs)] and less severe overlap/classic chronic GVHD [OR, 0.91; 95% CI, 0.79-1.04; P=0.158 (Tregs) and OR, 0.93; 95% CI, 0.86-1.01; P=0.071 (α4β7± Tregs)]. Using Cox Proportional Hazard regression (adjusting for donor type and acute GVHD prior to day ±100), increased frequencies of α4β7± Tregs at engraftment continued to be associated with decreased risk of GVHD after day ±100 (hazard ratio, 0.93; 95%CI, 0.88-0.99; P=0.013). Conclusion:Frequency of α4β7± Tregs at engraftment is associated with the incidence and severity of GVHD after day ±100. These data suggest that early lymphocyte subsets and gut associated mucosal immunity are important in determining long-term graft tolerance. Treg subsets at engraftment could be used to risk stratify pts prior to development of GVHD or as an endpoint in future clinical trials examining interventions aimed at increasing Tregs and preventing GVHD. Disclosures:No relevant conflicts of interest to declare.

Liposomal KRN7000 (RGI-2001) Synergizes with Low-Dose Rapamycin in Preventing GvHD by promoting expansion of naturally-Occurring CD4+FOXP3+ Regulatory T Cells.

Abstract 4491 IntroductionRGI-2001 is a liposomal formulation of KRN7000 (a synthetic derivative of a-galactosylceramide). We previously demonstrated that RGI-2001 potently reduces GvHD lethality in a mouse model by inducing host-specific tolerance through the expansion of donor-derived CD4+Foxp3+ Regulatory T cells (Tregs) (ASH 2008 Poster III-582). In the present study, we examined if the combination with low-dose rapamycin (LD-RAPA) may further improve the efficacy of RGI-2001. In addition, the origin of Tregs induced by RGI-2001 was investigated. MethodsA fully MHC mismatched GvHD mouse model using lethally-irradiated Balb/c (H-2d) recipients transplanted with C57BL/6 (H-2b) T cell depleted bone marrow cells with total spleen cells were used. In some experiments, Foxp3-GFP reporter mice (C57BL/6 background) were used as the donor. RGI-2001 (0.01 ug/kg to 1 ug/kg) was given on day 0 via single intravenous administration. LD-RAPA (0.1 mg/kg) was given daily from day 0 to day 14 intraperitoneally. Treg frequency was determined on day 15 post-BMT by flow cytometry either by using an antibody for Foxp3 or by detecting GFP as a surrogate marker for Foxp3. ResultsFirst, the pharmacological dose induction of Tregs by RGI-2001 was investigated. Day 15 post-BMT analysis of mice treated with RGI-2001 demonstrated a dose-dependent increase in the % CD4+Foxp3+ Treg cells in the spleen, lymph nodes, and bone marrow. As shown in the Table, frequencies of CD4+Foxp3+ Tregs in the spleen increased in a dose dependent manner, from 3.0 ± 1.4 % in untreated mice to 14.9 ± 5.8% in 1 ug/kg RGI-2001 treated mice. The total Treg cell counts in the spleen also increased in a dose dependent manner with 1 ug/kg RGI-2001 consisting of 4.2×10e5 Tregs as compared with 0.3×10e5 Tregs in untreated mice. The frequency of CD8+ T cell population decreased in a dose dependent manner, suggesting an inverse relationship with frequencies of Tregs. When LD-RAPA was combined, a robust synergy between the two compounds was noted. The frequency of Treg in mice that received combination treatment was significantly higher than that in each monotherapy group (p = .0005 vs. RGI-2001 only, p< .0001 vs. LD-RAPA only). Absolute Treg counts in the spleen were markedly increased in mice received the combination treatment. In three independent experiments, Tregs in combination treatment group were consistently ∼3-fold higher than that in RGI-2001 monotherapy group and ∼9-fold higher than that in LD-RAPA monotherapy group. In contrast, CD8+ T cell frequencies decreased in mice treated with LD-RAPA containing regimen. Efficacy in survival was also improved in mice that received the combination treatment with 80-100% survival over 100 days.Table 1:Cellular composition of Day 15 post-BMT spleen in mice treated by RGI-2001 with or without LD-RAPA.RGI-2001-0.01 ug/kg0.1 ug/kg1 ug/kg-0.1 ug/kgRAPA----0.1 mg/kg0.1 mg/kg%Foxp3+/CD4+3.0 ± 1.4 (n=15)6.9 ± 3.0 (n=17)11.9 ± 4.1 (n=9)14.9 ± 5.8 (n=11)6.2 ± 3.1 (n=12)19.8 ± 4.1 (n=10)%CD8+/CD3+12.3 ± 1.4 (n=9)11.2 ± 4.0 (n=17)9.8 ± 4.7 (n=14)8.5 ± 4.2% (n=11)7.6 ± 2.3% (n=8)6.7 ± 2.6% (n=8)Next, the origin of the Tregs induced by RGI-2001 was investigated using the Foxp3-GFP reporter mice as the spleen cell donor. While 2.5×10e6 total spleen cells from Foxp3-GFP mice gave rise to 7.2 ± 2.4 % Tregs (n= 7) when treated with RGI-2001 (1 ug/kg), equivalent number of GFP+ Treg-depleted spleen cells gave rise to 1.4 ± 0.3 % Tregs (n=5, p < .0006). Likewise, when mice were treated with the combination of RGI-2001 (1 ug/kg) and LD-RAPA, Treg frequencies in mice that received total spleen cells and GFP+ Treg -depleted spleen cells were 17.8 ± 3.8 % (n=7) and 1.5 ± 0.4 % (n=7), respectively (p < .0001). These results together suggest that the majority of Tregs induced by RGI-2001 with or without LD-RAPA were derived from the pre-existing naturally-occurring Treg population, and the contribution of de-novo Tregs was minimal in the GvHD setting. ConclusionsRGI-2001 prevented GvHD by promoting the expansion of naturally-occurring Tregs. LD-RAPA synergistically enhanced the efficacy of RGI-2001 by significantly increasing Treg expansion while suppressing effector T cells. Disclosures:REGiMMUNE employees (OD, AL, JL, TT), Founder (YI), and consultant (RN). YI and RN are stockholders. Disclosures:No relevant conflicts of interest to declare.

Glatiramer acetate improves regulatory T-cell function by expansion of naive CD4 +CD25 +FOXP3 +CD31 + T-cells in patients with multiple sclerosis

Naturally occurring regulatory T-cells (Treg) exhibit impaired function in patients with relapsing-remitting multiple sclerosis (RRMS) resulting from an age-inappropriate disproportion between prevalences of newly generated CD31-coexpressing naive Treg and long-lived memory Treg in the periphery. Recent evidence suggests that the immunomodulatory action of glatiramer acetate (GA) includes effects on Treg function and frequencies. We prospectively assessed suppressive activities and frequencies of Treg and Treg subsets in 15 patients with RRMS undergoing long-term therapy with GA. Treatment for up to six months reconstituted naive Treg and increased total Treg numbers with concomitant reversion of the Treg defect.

Circulating Gut- or Skin-Homing Regulatory T Cells (Tregs) Predict Whether Acute Graft-Versus-Host Disease (aGVHD) Occurs in Gut or Skin Following Allogeneic Stem Cell Transplantation (ASCT)

BACKGROUND: aGVHD occurs unpredictably following ASCT and is a significant cause of morbidity and mortality. Biological factors predicting organ involvement and severity remain to be elucidated. Tregs are a suppressive subset of CD4+ T lymphocytes that are implicated in the prevention of aGVHD. Tregs can express the gut-homing integrin α4β7 or the skin-homing marker cutaneous lymphocyte antigen (CLA), thus potentially conferring organ-specific immune regulation. We hypothesized that at engraftment, increased frequencies of Tregs with either a gut- or skin-homing phenotype are associated with a decrease in gut or skin aGVHD, respectively.METHODS: Patients (pts) undergoing a T cell replete ASCT were enrolled. Overall aGVHD grade and organ stage was determined using the modified Glucksberg criteria. Initial, recurrent, and maximal stage/grade of aGVHD was analyzed. The frequency of CD4+ Tregs, defined as CD25+ Foxp3+CD127lo was quantified from recipient CD4+ T-lymphocytes at the time of neutrophil engraftment using polychromatic flow cytometry. Tregs with a gut- or skin-homing phenotype were identified by the presence of α4β7+ or CLA+, respectively. These subsets were expressed as the percentage of the total Treg population.RESULTS: Forty-three pts underwent ASCT with a matched related donor [N= 26 (60%)] or an unrelated donor [N= 17 (40%)]. The median age at ASCT was 45 years (range, 21–70). Ablative conditioning was utilized in 26 (60%) pts and a reduced intensity regimen was performed in 17 (40%) pts. The stem cell source was peripheral blood (PB), bone marrow (BM), or umbilical cord blood (UCB) for 32 (74%), 9 (21%), and 2 (5%) pts, respectively. Pts received aGVHD prophylaxis with a calcineurin inhibitor and either methotrexate [N=25 (58%)] or mycophenolate mofetil [N=18 (42%)]. aGVHD grade II–IV or grade III–IV occurred in 34 (79%) and 9 (21%) pts, respectively at a median of 23 days post ASCT (range, 9–93). The gastrointestinal (GI) tract was involved in 29 (67%) pts followed by skin in 20 (47%) pts. Twenty (47%) pts previously diagnosed with aGVHD had recurrent grade II–IV aGVHD. Treg engraftment samples were obtained at a median of 19 days post ASCT (range, 10–31). The median percentage of Tregs and their CLA+ skin- or α4β7+ gut-homing subsets were: 4.9% (range, 0.80–35.4%), 1.9% (range, 0.12–9.79), and 16.2% (range, 2.7–47.5), respectively. The percentage of total Tregs at engraftment between pts with or without grade II–IV aGVHD was not different (4.4% vs. 5.0%; P=0.40). Pts with any stage skin aGVHD had a significantly lower frequency of CLA+ Tregs at engraftment (1.7% vs. 2.6%; P=0.04). A trend was noted with regards to the association between α4β7+ Tregs and the occurrence of gut aGVHD [15.2% vs. 20.1%; P=0.11 (gut aGVHD vs. no gut aGVHD)]. Increasing frequencies of CLA+ Tregs and α4β7+ Tregs were associated with lower stages of skin [odds ratio (OR), 0.68; 95% confidence interval (CI), 0.46–0.99; P=0.04] and less severe gut aGVHD (OR, 0.94; 95% CI, 0.88–0.99; P=0.04), respectively. Pts with recurrent grade II–IV aGVHD, any recurrent skin aGVHD, and any recurrent gut aGVHD tended to have lower percentages of total Tregs, CLA+ Tregs, and α4β7+ Tregs when compared to pts without recurrent aGVHD, however these values were not statistically significant (4.1% vs. 7.5%; P=0.07), (1.8% vs. 2.2%; P=0.08), and (10.4% vs. 18.7%; P=0.07), respectively. Pts with recurrent gut aGVHD had significantly lower Tregs (3.1% vs. 7.5%; P<0.01). Using multivariable logistic regression (adjusting for donor type) Tregs became a significant predictor for recurrent grade II–IV aGVHD (OR, 0.88; 95% CI, 0.78–0.99; P=0.04] and CLA+ Tregs conferred a protective effect with the prevention of any stage skin aGVHD (OR, 0.67; 95% CI, 0.45–0.98; P=0.04).CONCLUSIONS: CLA+ Tregs at engraftment are associated with the prevention of skin aGVHD, while total Tregs predict for recurrence of both grade II–IV aGVHD and any stage gut aGVHD. Increased frequencies of CLA+ Tregs and α4β7+ Tregs are associated with less severe skin or gut aGVHD, respectively. Tregs and their subsets could help explain the clinical heterogeneity of aGVHD with respect to aGVHD incidence, severity, and patterns of organ involvement. In the future, Tregs at engraftment could be used to risk stratify pts prior to aGVHD onset for clinical trials examining novel immunosuppressive regimens aimed at increasing Tregs and preventing aGVHD.

Regulatory and Naïve T Cells in Unmanipulated Donor Grafts Are Not Associated with Acute Graft Vs Host Disease in Matched Sibling Transplants for AML

Acute graft-versus-host disease (aGVHD) occurs in approximately 20–50% of matched-related stem cell transplants (HCT). The presence of increased frequencies of CD4+CD25+ regulatory T cells (Treg) in murine donor grafts has been shown to ameliorate aGVHD. In similar models, several groups have demonstrated that the transfer of equivalent numbers of naïve T cells (vs. various memory subsets) confers a relatively greater risk for aGVHD. To test the hypothesis that natural variations in donor graft naïve and Treg content would be associated with the incidence of aGVHD in graft recipients, we conducted a detailed analysis of donor graft T cells within unmanipulated grafts of 70 SCT recipients transplanted for AML/MDS following the administration of a uniform fludarabine/busulfan conditioning regimen. Median follow up time was 38 months. 19% (n=13) of recipients experienced grades II–IV aGVHD (n=3 grades III–IV). Cryopreserved donor grafts samples were analyzed using 9-color flow cytometry (with a panel including a viability dye and MAbs recognizing CD4, CD8, CD25, CD127, CD45RA, CD27, Foxp3, and Ki-67). Tregs were defined as being CD4+CD25+CD127loFoxp3+. CD45RA and CD27 were used to measure naïve (CD45RA+CD27+) and memory conventional CD4+ and CD8+ T cells. In addition, the proliferating fraction of all cells was defined by Ki-67 nuclear-antigen expression. We conducted analyses based on donor graft T cells assessed both continuously and by quartiles, with similar results. No significant associations were found between aGVHD incidence and total Tregs (HR 1.01, 95%CI 0.9–1.2, p 0.8), nor with proliferating Tregs (HR 1.2, 95%CI 0.2–5.9, p 0.8). Since murine models typically fix the dose of Tregs vs. conventional T cells (Tcon), we also analyzed GVHD risk by donor graft Treg:Tcon ratio, finding no association (HR 1.03, 95%CI 0.8–2.1, p 0.3). Although patients in the highest Treg:Tcon quartile received Treg doses known in vitro to induce suppression (>1:21.5), there was no reduction in aGVHD (HR 1.5 Quartile 4 vs others, 95%CI 0.5–4.9, p 0.5). Additionally, no significant association with aGVHD was found with total naïve T cells (HR 1, 95%CI 0.9–1.0, p 0.9), naïve CD4+ (HR 1, 95%CI 0.9–1.03, p 0.6), or naïve CD8+ cells (HR 0.98, 95%CI 0.9–1.03, p 0.6). Because naïve:memory cell ratios were also fixed in murine models demonstrating the importance of naïve T cells in GVHD, we also assessed naïve:memory T cell ratios within donor grafts with respect to recipient aGVHD. No reduction in aGVHD was observed considering naïve:memory ratios analyzed continuously (HR 1.1, 95%CI 0.3–4.2, p 0.8) or in the quartile receiving the lowest naïve:memory ratio (HR 0.5 Quartile 1 vs others, 95%CI 0.1–2.2, p 0.3). Our results demonstrate that donor graft Treg and naïve T cell content, assessed in the context of unmanipulated PBSC transplantation in matched siblings, does not predict recipient aGVHD incidence. These data suggest that the therapeutic efficacy of Treg enrichment and/ or naïve T cell depletion may require grafts to be manipulated to supraphysiologic levels for maximal therapeutic benefit.

The effect of aging on T-regulatory cell frequency in HIV infection

T-regulatory cell (T-reg) frequency is increased in HIV infection and with aging. We evaluated the effect of age on total, memory and naïve T-reg percentages in untreated HIV infection. Older HIV + subjects had a total T-reg percent that is 2.8% ( p = 0.02) higher than among younger HIV +, older HIV − and younger HIV − subjects. In HIV + subjects, the total T-reg percentage is inversely correlated with the lymphocyte proliferative responses to tetanus ( r = − 0.45, p = 0.002) and Candida ( r = − 0.43, p = 0.003) antigens. Similar correlations were seen between memory T-reg percentages and the lymphocyte proliferative response to tetanus and Candida in HIV + subjects. T-reg percentages did not correlate consistently with markers of immune activation. T-reg percentages are increased in the older HIV + population and may play a role in the accelerated disease progression seen in older HIV-infected persons.

The Relationship of T-Regulatory Cell Subsets to Disease Stage, Immune Activation, and Pathogen-Specific Immunity in HIV Infection

Human immunodeficiency virus (HIV) infection is associated with abnormalities in T-regulatory (T-reg) cells, but the effect of HIV on the naive (CD45RO-) and memory (CD45RO) CD25+CD127loCD4+ T-reg cell subsets has not been defined. We measured the absolute number and relative percentage of total, naive, and memory T-reg cells in HIV-infected subjects and compared these parameters with their CD4+ T cells, viral load, levels of immune activation, and pathogen-specific immunity. HIV infection was associated with an increased percentage of memory CD25+CD127loCD4+ T-reg cells and a decreased percentage of naive CD25+CD127loCD4+ T-reg cells as CD4+ T cells declined. The level of HIV viremia inversely correlated with total, memory, and naive CD25+CD127loCD4+ T-reg cell numbers and percentage of naive CD25+CD127loCD4+ T-reg cells. Lower total, memory, and naive CD25+CD127loCD4+ T-cell numbers were associated with higher levels of immune activation, whereas a higher percentage of CD25+CD127loCD4+ T-reg cells was associated with lower Candida- and HIV-specific immune responses. These observations suggest that CD25+CD127loCD4+ T-reg cells contribute to the immunodeficiency seen in HIV disease.

Regulatory T cells in HIV infection: pathogenic or protective participants in the immune response?

Regulatory T cells in HIV infection: pathogenic or protective participants in the immune response?

Prevalence of Newly Generated Naive Regulatory T Cells (Treg) Is Critical for Treg Suppressive Function and Determines Treg Dysfunction in Multiple Sclerosis

The suppressive function of regulatory T cells (T(reg)) is impaired in multiple sclerosis (MS) patients. The mechanism underlying the T(reg) functional defect is unknown. T(reg) mature in the thymus and the majority of cells circulating in the periphery rapidly adopt a memory phenotype. Because our own previous findings suggest that the thymic output of T cells is impaired in MS, we hypothesized that an altered T(reg) generation may contribute to the suppressive deficiency. We therefore determined the role of T(reg) that enter the circulation as recent thymic emigrants (RTE) and, unlike their CD45RO(+) memory counterparts, express CD31 as typical surface marker. We show that the numbers of CD31(+)-coexpressing CD4(+)CD25(+)CD45RA(+)CD45RO(-)FOXP3(+) T(reg) (RTE-T(reg)) within peripheral blood decline with age and are significantly reduced in MS patients. The reduced de novo generation of RTE-T(reg) is compensated by higher proportions of memory T(reg), resulting in a stable cell count of the total T(reg) population. Depletion of CD31(+) cells from T(reg) diminishes the suppressive capacity of donor but not patient T(reg) and neutralizes the difference in inhibitory potencies between the two groups. Overall, there was a clear correlation between T(reg)-mediated suppression and the prevalence of RTE-T(reg), indicating that CD31-expressing naive T(reg) contribute to the functional properties of the entire T(reg) population. Furthermore, patient-derived T(reg), but not healthy T(reg), exhibit a contracted TCR Vbeta repertoire. These observations suggest that a shift in the homeostatic composition of T(reg) subsets related to a reduced thymic-dependent de novo generation of RTE-T(reg) with a compensatory expansion of memory T(reg) may contribute to the T(reg) defect associated with MS.

Oral Tolerance Induction with Antigen Conjugated to Cholera Toxin B Subunit Generates Both Foxp3+CD25+ and Foxp3−CD25− CD4+ Regulatory T Cells

Oral administration of Ag coupled to cholera toxin B subunit (CTB) efficiently induces peripheral immunological tolerance. We investigated the extent to which this oral tolerance is mediated by CD25+CD4+ regulatory T cells (T(reg)). We found that total T(reg), KJ1-26+ T(reg) and CTLA-4+ T(reg) were all increased in Peyer's patches, mesenteric lymph nodes, and, to a lesser extent, in spleen of mice after intragastric administration of OVA/CTB conjugate, which also increased TGF-beta in serum. This could be abolished by co-administering cholera toxin or by treatment with anti-TGF-beta mAb. CD25+ T(reg), but also CD25-CD4+ T cells from OVA/CTB-treated BALB/c or DO11.10 mice efficiently suppressed effector T cell proliferation and IL-2 production in vitro. Following adoptive transfer, both T cell populations also suppressed OVA-specific T cell and delayed-type hypersensitivity responses in vivo. Foxp3 was strongly expressed by CD25+ T(reg) from OVA/CTB-treated mice, and treatment also markedly expanded CD25+Foxp3+ T(reg). Furthermore, in Rag1(-/-) mice that had adoptively received highly purified Foxp3-CD25-CD4+ OT-II T cells OVA/CTB feeding efficiently induced CD25+ T(reg) cells, which expressed Foxp3 more strongly than naturally developing T(reg) and also had stronger ability to suppress effector OT-II T cell proliferation. A remaining CD25- T cell population, which also became suppressive in response to OVA/CTB treatment, did not express Foxp3. Our results demonstrate that oral tolerance induced by CTB-conjugated Ag is associated with increase in TGF-beta and in both the frequency and suppressive capacity of Foxp3+ and CTLA-4+ CD25+ T(reg) together with the generation of both Foxp3+ and Foxp3-CD25- CD4+ T(reg).