Central Memory Tregs Research Articles

Exhaustion of CD8pos central memory regulatory T cell differentiation is involved in renal allograft rejection

BackgroundThe role of regulatory CD8pos T cells (CD8pos Tregs) and cytotoxic CD8pos responder T cells (CD8pos Tresps) in maintaining stable graft function in kidney transplant recipients (KTR) remains largely unclear. The pathogenesis of graft deterioration in case of rejection involves the exhaustive differentiation of both CD8pos T cell subsets, but the causal mechanisms have not yet been identified.MethodsIn this study, we separately investigated the differentiation of CD8posTregs/Tresps in 134 stable KTR with no evidence of renal graft rejection, in 41 KTR diagnosed with biopsy-confirmed rejection at enrolment and in 5 patients who were unremarkable at enrolment, but developed rejection within three years of enrolment. We were investigating whether changed differentiation of CCR7posCD45RAposCD31pos recent thymic emigrant (RTE) cells via CD45RAnegCD31pos memory (CD31pos memory) cells (pathway 1), via direct proliferation (pathway 2), or via CCR7posCD45RA+CD31neg resting mature naïve (MN) cells (pathway 3) into CD45RAnegCD31neg memory (CD31neg memory) cells affects the CD8pos Treg/Tresp ratio or identifies a CD8pos Treg/Tresp subset that predicts or confirms renal allograft rejection.ResultsWe found that RTE Treg differentiation via pathway 1 was age-independently increased in KTR, who developed graft rejection during the follow-up period, leading to abundant MN Treg and central memory Treg (CM Treg) production and favoring a strongly increased CD8pos Treg/Tresp ratio. In KTR with biopsy-confirmed rejection at the time of enrolment, an increased differentiation of RTE Tregs into CCR7negCD45RAposCD31neg terminally differentiated effector memory (CD31neg TEMRA Tregs) and CD31pos memory Tregs was observed. CD31neg memory Treg production was maintained by alternative differentiation of resting MN Tregs, resulting in increased effector memory Treg (EM Treg) production, while the CD8pos Treg/Treg ratio was unaffected. An altered differentiation of CD8pos Tresps was not observed, shifting the Treg/Tresp ratio in favor of Tregs.ConclusionsOur results show that exhaustive CD8pos Treg differentiation into CM Tregs may lead to future rejection, with a shift towards EM Treg production and an accumulation of CD31neg TEMRA Tregs in KTR with current rejection.

CD38-Directed, Single-Chain T Cell-Engager Targets Leukemia Stem Cells through IFNγ-Induced CD38 Expression

Leukemia stem cells (LSCs), enriched in the CD34+CD38- AML population, are considered the main drivers of relapse in acute myeloid leukemia (AML). While more differentiated progenitors and bulk AML blasts reside in the CD34+CD38+ fraction, targeting CD38 in AML is less pursued, as LSCs are mainly CD38-. We show IFNγ (10 ng/ml) induced an increase in CD38 mRNA (mean fold change IFNγ over vehicle = 7.7, p=0.03, n=3) and surface CD38 (mean CD38 High% population IFNγ 56%±17 vs vehicle 16%±10, p<0.0001) in both CD34+ and CD34- AML primary blasts, and decreased the clonogenic activities of primary AML cells (colony forming unit IFNγ 50±36 vs vehicle 216±117, p=0.002, n=4) regardless of cytogenetics. scRNA and total RNA sequencing data in CD34+ AML primary blasts showed that upon IFNγ treatment, CD38 and Interferon Regulatory Factor 1 (IRF-1) were consistently the most upregulated genes compared to vehicle (>2.5 Log 2 Fold Change; p=0; FDR=0). Furthermore, gene set enrichment analysis (FDR=0) indicated IFNγ induced an upregulation of genes involved in apoptosis and DNA repair, and a suppression of genes in the pro-oncogenic Wnt beta catenin pathways. CD38 luciferase-based reporter assays, chromatin immunoprecipitation, and IRF-1 knockdown experiments in AML cells showed that, upon IFNγ treatment, IRF1 is pivotal in CD38 transcriptional regulation. Because CD4+T helper type 1 and CD8+ cytotoxic T cells release IFNγ once engaged on their target, we hypothesized a CD38-directed T cell engager will convert CD34+CD38- LSC into CD34+CD38+ blasts via the secreted IFNγ to kill LSCs and blasts. We created a single chain construct (BN-CD38), inserting a CD38 nanobody between the light and heavy chains of an anti-CD3 Fab using two short peptide linkers. Based on modeling, BN-CD38 reproduces the distance between T cell receptor and the major histocompatibility complex, mimicking the natural immunological synapse. Surface plasmon resonance and cytometry studies showed BN-CD38 had strong binding affinity to both CD38 (K D 4.77xE -10 M) and CD3 (K D 4.27xE -8 M). The IC 50 for BN-CD38 at 1:1 E:T at 24hrs in different CD38+ AML cell lines (n=6) was within the 0.01 to 0.1 ng/ml range, whereas the IC 50 of a non-binding CD38 construct (BN-CD38Mut) was 100-fold higher. By 24hrs of treatment, we noted >60% CD4+ and CD8+ T cell activation (CD69+, CD25+, IFNγ+) with BN-CD38 (e.g., CD69: 60%±27) in contrast to controls (e.g., CD69: IgG 7%±0.8 and BN-CD38Mut 11%±10, p<0.0001). In primary AML samples (n=10) BN-CD38 activated >70% T cells against autologous leukemia cells (CD69: BN-CD38 73%±16, BN-CD38Mut 10%±9, IgG 11%±10, p<0.0001). CyTOF analysis showed BN-CD38 but not controls significantly reduced CD34+CD38+ AML blasts (%change to IgG range: BN-CD38 -27 to -82% vs BN-CD38Mut 1% to 7%, p = 0.01) and CD34+CD38- LSCs (%change to IgG range: BN-CD38 -78 to -99% vs BN-CD38Mut -7 to 11%, p= 0.0003) and expanded CD8+ effector memory (p<0.01), CD8+ terminally differentiated effector memory CD45RA+ (p<0.05), NKT cells (p<0.01), and central memory Tregs (p<0.0001). Anti-human-IFNγ neutralizing antibody reverted BN-CD38 induced expression of CD38 and BN-CD38 killing activity on CD34+CD38- AML cells (p<0.05). BN-CD38 (2.5 mg/kg, i.v, n=6/group) co-injected weekly with healthy 3x10 6 T cells induced near-complete disease eradication in mice xenografted with luciferase-CD38+ THP1 (p=0.003) and U937 (p=0.004) cells. Long-term survival analysis showed 38% of BN-CD38-treated THP-1 engrafted mice achieved complete cure compared to IgG (OS median: BN-CD38 61.5d, control IgG 29d, p=0.0005). Consistently, BN-CD38 suppressed AML cell engraftment, splenomegaly, and prolonged survival in three patient-derived xenografts (PDX) with complex karyotypes including TP53 mutation (PDX1: OS median BN-CD38 undefined, BN-CD38Mut 28d (p=0.002), and IgG 27d (p=0.002); PDX2: OS median BN-CD38 39d, BN-CD38Mut and control IgG 32d (p<0.01); PDX3: OS median BN-CD38 undefined, BN-CD38Mut 92d (p=0.001), and IgG 81d (p=0.0007)). BN-CD38 did not affect normal hematopoietic stem cell clonogenicity and the development of multi-lineage human immune cells in CD34+ humanized mice. In sum, BN-CD38 induces an effective T cell synapse and IFNγ release, while concomitantly blocking the clonogenicity and inducing CD38 expression on LSCs. These studies suggest a new mechanism to unmask and target LSCs, providing the rationale for using BN-CD38 to effectively treat AML.

TCF-1 controls Treg cell functions that regulate inflammation, CD8+ T cell cytotoxicity and severity of colon cancer.

The transcription factor TCF-1 is essential for the development and function of T regulatory (Treg) cells, however its function is poorly understood. Here, we show that TCF-1 primarily suppresses transcription of genes that are co-bound by Foxp3. Single-cell RNA-seq analysis identified effector- and central-memory Treg-cells with differential expression of Klf2 and memory and activation markers. TCF-1 deficiency did not change the core Treg transcriptional signature, but promoted alternative signaling pathways whereby Treg-cells became activated and gained gut-homing and TH17 characteristics. TCF-1-deficient Treg-cells strongly suppressed T-cell proliferation and cytotoxicity, but were compromised in controlling CD4+ T-cell polarization and inflammation. In mice with polyposis, Treg cell-specific TCF-1 deficiency promoted tumor growth. Consistently, tumor-infiltrating Treg cells of colorectal cancer patients showed lower TCF-1 expression and increased TH17 expression signatures compared to adjacent normal tissue and circulating T-cells. Thus, Treg cell-specific TCF-1 expression differentially regulates TH17-mediated inflammation and T-cell cytotoxicity, and can determine colorectal cancer outcome.

Early Administration of Low-Dose IL-2 Intensify Graft-Versus-Leukemia Effect without Worsening GVHD through Sequential Enhancement of Effector T Cell and CD62L+ Regulatory T Cell Subset

Allogeneic hematopoietic stem cell transplantation (HSCT) is the curative treatment of many hematological malignancies, but the relapse is one of the major causes of treatment failure and mortality. Previous clinical studies have shown that low-dose IL-2 was well tolerated and could lower the relapse rate in human T cell depleted allogeneic BMT. On the other hand, we also demonstrated that low-dose IL-2 could preferentially enhance regulatory T cell (Treg) and improve the symptoms of chronic GVHD (NEJM 2011,Sci Trans Med 2013). In murine bone marrow transplantation (BMT) model showed that Treg can protect from lethal GVHD while preserving graft-versus-leukemia (GVL) effect. Especially, CD62L+ Tregs has a higher capacity in suppressing GVHD than their CD62L- counterpart. Our recent study reported that exogenous low-dose IL-2 induced the expression of PD-1 on Treg preferentially and it was most evident in CD44+CD62L+central-memory Treg (Blood 2016). However, the impact of CD62L+ Treg enhanced by low-dose IL-2 on GVHD and GVL has not been well investigated. In this study, we studied the effect of exogenous IL-2 on the lymphocyte phenotypic profiles including naïve- versus memory-type balance and also examined donor-derived immunity, especially GVHD and GVL, by using leukemia-bearing BMT model. Lethally irradiated B6D2F1 recipients were transplanted spleen cells and BM cells from B6 donors and 5000 IU Teceleukin (recombinant IL-2) was injected once a day for 14 days. The number of CD4+CD25+Foxp3+ Treg, CD4+CD25-Foxp3- conventional T cell (Tcon) and CD8+ T cell was compared between IL2- and vehicle-treated groups. The expressions of CD44, CD62L, CCR4, CCR7, Ki-67, PD-1, CTLA-4, LAG3, GITR, ICOS and TIM-3 in each subset were also examined. Lymphocytes profiling analysis showed that IL-2 treatment in the first 7 days resulted in the dominant expansion of effector T cells without Treg increase (Treg 0.10 vs 0.16,p=0.35, Tcon 2.83 vs 4.20, p=0.01, CD8 T cell 5.52 vs 10.4, p=0.01, million). On the contrary, the extended IL-2 treatment in the next 7 days increased CD62L+ Treg (21.5 vs 48.2 % of total Treg, p<0.001, 0.02 vs 0.07 million, p=0.006) without increasing effector T cells. PD-1 on Treg was very high in BMT-recipients even without receiving IL-2, so further enhancement of PD-1 expression by IL-2 was not observed. However, the expression of other inhibitory molecules including CTLA-4 (MFI, 6.61 vs 9.92, p=<0.001), LAG-3 (1.73 vs 2.17, p=0.04) and ICOS (2.41 vs 3.84, P<0.001) was enhanced by the IL-2 treatment. To elucidate the mechanisms of the increase in CD62L+ Treg, the in vivo violet dye dilution assay was performed. We first sorted out CD62L+ T cells by micro-beads from spleen cells, and then adaptively transferred into recipients. Five days treatment of low-dose IL-2 resulted in the enhancement of the division of CD62L+ Treg (% divided cells; 19.5 vs 29.8 % of Treg, p=0.005, 62.3 vs 108 in number, p=0.002). Interestingly, when CD62L- T cells were transferred, IL-2 treatment also induced the division of the cells and, of note, induced the phenotypic change of CD62L- into CD62L+ Treg (7.63 vs 19.8 % of Treg, p=0.009, 91.3 vs 361, p=0.01). These results suggested that IL-2 have the possibility to enhance the proliferative activity of Treg with the up-regulation of CD62L expression. Lastly, we transplanted lucipherase+P815 leukemia cells into this BMT model and evaluated the clinical impact of low-dose IL-2 on GVL by using in vivo bioluminescence system. To assesse the GVL and GVHD independently, we used two different tumor burden models. In the high tumor burden model, IL-2 injection decreased bioluminescence intensity (BLI) of P815 (BLI at week3, 8.3x10E7 vs 8.1x10E6 photon/second, p<0.001) and significantly extends overall survival. On the other hand, in the low tumor burden model, both treatment groups did not die from leukemia but vehicle-treated control group showed a significant higher clinical GVHD score and died from GVHD. These data demonstrated the clinical benefits on anti-tumor effect of IL-2, which could enhance the GVL activity without the detrimental effect on GVHD. In conclusion, our data indicate multiple effects of IL-2 on post-transplant immunity can coordinate to intensify GVL separately from GVHD on the mechanism of sequential enhancement of effector T cell and CD62L+ Treg in the different phases. These data provide the important information for developing Treg-targeted therapy. DisclosuresMaeda:Toko Pharmaceutical Industries: Other: Investigational drug is provided free of charge.

P.164 Safety and pharmacokinetic study of inhaled Esketamine after a multiple dose in healthy volunteers

CD4+Foxp3+ regulatory T cells (Tregs) play a central role in the maintenance of immune tolerance after hematopoietic stem cell transplantation. We previously reported that low-dose interleukin-2 (IL-2) therapy increased circulating Tregs and improved clinical symptoms of chronic graft-versus-host-disease (cGVHD); however, the mechanisms that regulate Treg homeostasis during IL-2 therapy have not been well studied. To elucidate these regulatory mechanisms, we examined the role of inhibitory coreceptors on Tregs during IL-2 therapy in a murine model and in patients with cGVHD. Murine studies demonstrated that low-dose IL-2 selectively increased Tregs and simultaneously enhanced the expression of programmed cell death 1 (PD-1), especially on CD44+CD62L+ central-memory Tregs, whereas expression of other inhibitory molecules, including CTLA-4, LAG-3, and TIM-3 remained stable. PD-1–deficient Tregs showed rapid Stat5 phosphorylation and proliferation soon after IL-2 initiation, but thereafter Tregs became proapoptotic with higher Fas and lower Bcl-2 expression. As a result, the positive impact of IL-2 on Tregs was completely abolished, and Treg levels returned to baseline despite continued IL-2 administration. We also examined circulating Tregs from patients with cGVHD who were receiving low-dose IL-2 and found that IL-2–induced Treg proliferation was promptly followed by increased PD-1 expression on central-memory Tregs. Notably, clinical improvement of GVHD was associated with increased levels of PD-1 on Tregs, suggesting that the PD-1 pathway supports Treg-mediated tolerance. These studies indicate that PD-1 is a critical homeostatic regulator for Tregs by modulating proliferation and apoptosis during IL-2 therapy. Our findings will facilitate the development of therapeutic strategies that modulate Treg homeostasis to promote immune tolerance.

Increased Regulatory T-Cell Activity and Enhanced T-Cell Homeostatic Signaling in Slow Progressing HIV-infected Children.

Pediatric slow progressors (PSP) are rare ART-naïve, HIV-infected children who maintain high CD4 T-cell counts and low immune activation despite persistently high viral loads. Using a well-defined cohort of PSP, we investigated the role of regulatory T-cells (TREG) and of IL-7 homeostatic signaling in maintaining normal-for-age CD4 counts in these individuals. Compared to children with progressive disease, PSP had greater absolute numbers of TREG, skewed toward functionally suppressive phenotypes. As with immune activation, overall T-cell proliferation was lower in PSP, but was uniquely higher in central memory TREG (CM TREG), indicating active engagement of this subset. Furthermore, PSP secreted higher levels of the immunosuppressive cytokine IL-10 than children who progressed. The frequency of suppressive TREG, CM TREG proliferation, and IL-10 production were all lower in PSP who go on to progress at a later time-point, supporting the importance of an active TREG response in preventing disease progression. In addition, we find that IL-7 homeostatic signaling is enhanced in PSP, both through preserved surface IL-7receptor (CD127) expression on central memory T-cells and increased plasma levels of soluble IL-7receptor, which enhances the bioactivity of IL-7. Combined analysis, using a LASSO modeling approach, indicates that both TREG activity and homeostatic T-cell signaling make independent contributions to the preservation of CD4 T-cells in HIV-infected children. Together, these data demonstrate that maintenance of normal-for-age CD4 counts in PSP is an active process, which requires both suppression of immune activation through functional TREG, and enhanced T-cell homeostatic signaling.

MTOR Complex 1 Signaling Regulates the Generation and Function of Central and Effector Foxp3+ Regulatory T Cells.

The mechanistic/mammalian target of rapamycin (mTOR) has emerged as a critical integrator of signals from the immune microenvironment capable of regulating T cell activation, differentiation, and function. The precise role of mTOR in the control of regulatory T cell (Treg) differentiation and function is complex. Pharmacologic inhibition and genetic deletion of mTOR promotes the generation of Tregs even under conditions that would normally promote generation of effector T cells. Alternatively, mTOR activity has been observed to be increased in Tregs, and the genetic deletion of the mTOR complex 1 (mTORC1)-scaffold protein Raptor inhibits Treg function. In this study, by employing both pharmacologic inhibitors and genetically altered T cells, we seek to clarify the role of mTOR in Tregs. Our studies demonstrate that inhibition of mTOR during T cell activation promotes the generation of long-lived central Tregs with a memory-like phenotype in mice. Metabolically, these central memory Tregs possess enhanced spare respiratory capacity, similar to CD8+ memory cells. Alternatively, the generation of effector Tregs (eTregs) requires mTOR function. Indeed, genetic deletion of Rptor leads to the decreased expression of ICOS and PD-1 on the eTregs. Overall, our studies define a subset of mTORC1hi eTregs and mTORC1lo central Tregs.

Phenotypic characterization of regulatory T cells from antiretroviral-naive HIV-1-infected people.

Regulatory T (Treg) cells play a key role in dampening excessive immune activation. However, antiretroviral therapy (ART) -naive HIV-1 infection maintains the immune system in a sustained state of activation that could alter both Treg cell surface markers and functions. As Treg cell surface markers are directly linked to their functions the overall objective of this study was to determine how ART-naive HIV infection affects the phenotypic properties of Treg cells. Our data showed that in ART-naive HIV-1 infection, Treg cells are dominated by effector (CD45RA+ CD27- CCR7- CD62L- ) and effector memory (CD45RA- CD27- CCR7- CD62L- ) cells. In contrast Treg cells from HIV-negative individuals were mainly naive (CD45RA+ CD27+ CCR7+ CD62L+ ) and central memory (CD45RA- CD27+ CCR7+ CD62L+ ) cells. Whereas effector and effector memory Treg cells showed enhanced expression of CD39 (P<0·05), CD73 (P<0·001), HLA-DR and CD38 (P<0·001); naive and central memory Treg cells showed a significant reduction in the expression of these markers. Overall Treg cell frequencies within total CD4+ T cells correlated positively with plasmatic HIV-1 viral load. As increased viral load is associated with augmented CD4+ T-cell destruction; this could suggest a resistance of peripheral Treg cells to HIV-1 destruction. Hence the modulation of Treg cell phenotype and frequencies could be considered in designing immunotherapeutic strategies targeting immune system restoration during HIV-1 infection.

PD-1 modulates regulatory T-cell homeostasis during low-dose interleukin-2 therapy

AbstractCD4+Foxp3+ regulatory T cells (Tregs) play a central role in the maintenance of immune tolerance after hematopoietic stem cell transplantation. We previously reported that low-dose interleukin-2 (IL-2) therapy increased circulating Tregs and improved clinical symptoms of chronic graft-versus-host-disease (cGVHD); however, the mechanisms that regulate Treg homeostasis during IL-2 therapy have not been well studied. To elucidate these regulatory mechanisms, we examined the role of inhibitory coreceptors on Tregs during IL-2 therapy in a murine model and in patients with cGVHD. Murine studies demonstrated that low-dose IL-2 selectively increased Tregs and simultaneously enhanced the expression of programmed cell death 1 (PD-1), especially on CD44+CD62L+ central-memory Tregs, whereas expression of other inhibitory molecules, including CTLA-4, LAG-3, and TIM-3 remained stable. PD-1–deficient Tregs showed rapid Stat5 phosphorylation and proliferation soon after IL-2 initiation, but thereafter Tregs became proapoptotic with higher Fas and lower Bcl-2 expression. As a result, the positive impact of IL-2 on Tregs was completely abolished, and Treg levels returned to baseline despite continued IL-2 administration. We also examined circulating Tregs from patients with cGVHD who were receiving low-dose IL-2 and found that IL-2–induced Treg proliferation was promptly followed by increased PD-1 expression on central-memory Tregs. Notably, clinical improvement of GVHD was associated with increased levels of PD-1 on Tregs, suggesting that the PD-1 pathway supports Treg-mediated tolerance. These studies indicate that PD-1 is a critical homeostatic regulator for Tregs by modulating proliferation and apoptosis during IL-2 therapy. Our findings will facilitate the development of therapeutic strategies that modulate Treg homeostasis to promote immune tolerance.

Abstract 5417: Expansion of effector regulatory T-cells represents a novel and independent prognostic factor marking escape from immune surveillance in Myleodysplastic Syndrome

Abstract Myelodysplastic syndromes (MDS) refer to a group of pathophysiologically diverse hematopoietic neoplasms associated with cytopenias, myeloid dysplasia, and variable acute myeloid leukemia risk. Response to immunosuppressive therapies (IST) in a subset of younger patients classified as lower-risk by the International Prognostic Scoring System (IPSS) suggests that inflammation and immune reactivity in the bone marrow likely plays a role in early MDS pathogenesis. Conversely, higher-risk patients with more established disease do not respond to IST. Previous reports of increased regulatory T-cells (Tregs) in these patients suggest that immune escape may be required for progression to higher-risk disease. The distinct changes in the role of the immune system between lower and higher-risk disease suggests that MDS may progress according to established principles of cancer immuno-editing involving elimination, equilibrium and escape phases. Because Treg suppressive activity is dependant upon auto-antigen presentation, we hypothesized that analyzing the activation state of Tregs may better reflect the onset of tumor-induced immune suppression in MDS and have better prognostic utility than measuring total Treg numbers alone. Here, we use a panel of surface markers on Tregs that are normally employed to distinguish effector and memory populations among conventional T-cells and analyzed the resulting subpopulations for associations with clinical features including overall survival (OS) among patients predominantly classified as lower-risk. Abnormal numbers of Treg subtypes were seen in 18 (34.6%) of 52 patients compared to age-matched controls. The most prominent and unique change in MDS patients was an expansion of effector Tregs (TregEff) which was significantly associated with anemia (p=0.046) and reduced hemoglobin (p=0.038). This group of patients had increased blast percentage (p=0.006) and displayed worse OS by Cox-regression (HR 4.3, 95% CI 1.6-11.6, p=0.004) independent of IPSS (p=0.002) and the MD Anderson Scoring System (MDAS) (p=0.047). Increased TregEff numbers were not dependant on total Treg numbers. Rather, they came at the expense of central memory Tregs suggesting that Treg “class switching” may reflect active presentation of auto-antigens. When isolated, TregEff cells displayed increased suppressive capacity compared to other Treg subpopulations and heightened TregEff numbers were limited to patients with elevated blasts (tumor burden), suggesting a mechanistic link between immune-evasion and MDS progression. The MDAS significantly refined OS estimates (p&amp;lt;0.001) compared to IPSS. Using the MDAS, OS in higher-risk patients could be further stratified using TregEff numbers (p=0.018), suggesting that enumeration of TregEff cells may improve prognostication and impact therapeutic selection in MDS. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5417. doi:1538-7445.AM2012-5417

A Switch in Regulatory T-Cell Memory Phenotype Is Associated with Poor Survival in Lower Risk Myleodysplastic Syndrome Patients

Abstract 1703Myleodysplastic syndrome (MDS) is a heterogeneous group of clonal hematopoietic diseases marked by various cytopenias including neutropenia and anemia. Hematologic improvement in response to T-cell depleting agents and immunosuppressants in a subset of MDS patients suggests that hematopoietic impairment may result from autoreactive T-cell activation. Conversely, suppression of the T-cell compartment may be permissible to the development of malignant myeloid clones. The suspicion that T-cell homeostatic balance may be disrupted in MDS has spawned numerous studies investigating mechanisms of immune suppression and autoimmune regulation. T-cell autoimmunity is regulated in part by regulatory T-cells (Tregs), which express T cell receptors (TCRs) that recognize self-antigens and function to maintain immune homeostasis. Indeed, higher numbers of Tregs have been found in higher risk MDS patients, and those MDS subtypes with higher blast percentages. However, the role of Tregs in lower risk patients is less clear, but may still represent an important component of pathogenesis in early disease.Because of the autoimmune nature of low risk MDS, we hypothesized that alterations in the Treg compartment may play a role in lower risk MDS disease progression. To address this, we analyzed Treg phenotype and numbers in MDS patients largely classified as low risk by the International Prognostic Scoring System (IPSS) and 41 age-matched controls using cell surface markers as analyzed by flow cytometry. Tregs were identified as CD3+CD4+CD25+CD127dim. The use of CD127 as a Treg marker was validated by intracellular staining with FoxP3. Naïve and memory phenotypes within the Treg compartment were analyzed using CD45RA and CD27 expression. Naïve Tregs (TregN) were characterized as CD45RA+CD27+, central memory Tregs (TregCM) as CD45RA−CD27+, and effector memory Tregs (TregEM) as CD45RA−CD27−. Two distinct subgroups of MDS patients were identified: one with elevated total numbers of Tregs, and a second with a marked shift in Treg memory phenotype away from the common TregCM phenotype and toward the rare TregEM phenotype. These two subgroups of patients were distinct from each other, and appeared to be independent of MDS WHO subtype, IPSS score, karyotype, neutropenic state, and thrombocytopenic state. MDS patients with a shift toward TregEMwere associated with anemia (p=0.0433). This association was not found in MDS patients with total increases in Treg numbers.Next, we sought to determine if there are differences in overall survival (OS) of MDS patients with either increased total numbers of Tregs or in patients with a shift toward TregEM phenotype. Patients with greater than 50 Tregs/μl did have reduced OS compared to patients with less Tregs, but this difference was not statistically significant (p=0.1387). In contrast, a highly significant difference was observed in MDS patients with a shift toward the TregEM phenotype (p=0.0200), with patients having greater than 2.5TregEM/μl displaying the worst OS (Figure 1). [Display omitted] Lastly, because an increased TregEM compartment is associated with reduced OS in MDS patients, we sought to compare the suppressive capacities of TregN, TregCM, and TregEM cells to better understand the functional consequences of Treg memory shift. CD4+ T-cells were isolated by negative selection, and then TregN, TregCM, and TregEM cells were sorted using CD45RA and CD27 expression. Conventional CD4+ responder cells were then labeled with Carboxyfluorescein succinimidyl ester (CFSE) and co-cultured with increasing ratios of each isolated Treg population. Cellular division in response to CD3/CD28 stimulation was measured 5 days later by analyzing CFSE dilution. TregCM appear to be the least suppressive, while TregN and TregEM cells were more potently suppressive. These results indicate that a shift in Treg phenotype toward TregEM may result in a more suppressive Treg compartment without obvious increases in total Treg numbers. Taken together, this data suggests that in lower risk MDS, the phenotypic makeup of the Treg compartment may be more important than total Treg numbers. In addition, this data suggests that increased TregEM cells may be a novel prognostic indicator in low risk MDS. Disclosures:Komrokji:Celgene: Honoraria, Research Funding, Speakers Bureau. List:Celgene: Consultancy.

Ex Vivo IL-1 Receptor Type I Expression in Human CD4+ T Cells Identifies an Early Intermediate in the Differentiation of Th17 from FOXP3+ Naive Regulatory T Cells

IL-17-producing CD4(+) Th (Th17) cells are a unique subset of proinflammatory cells expressing the retinoic acid-related orphan receptor γt and associated with different forms of inflammatory autoimmune pathologies. The development of Th17 cells, mediated by TGF-β and IL-1, is closely related to that of FOXP3(+) suppressor/regulatory T cells (Treg). In this study, we report that ex vivo expression of IL-1RI in human circulating CD4(+) T cells identifies a subpopulation of FOXP3(+) Treg that coexpress retinoic acid-related orphan receptor γt, secrete IL-17, and are highly enriched among CCR7(+) central memory cells. Consistent with the concept that IL-1RI expression in Treg identifies a subpopulation at an early stage of differentiation, we show that, in Th17 populations differentiated in vitro from natural naive FOXP3(+) Treg, IL-1RI(+) IL-17-secreting cells are central memory cells, whereas IL-1RI(-) cells secreting IL-17 are effector memory cells. Together with the absence of detectable IL-1RI and IL-17 expression in resting naive CD4(+) T cells, these data identify circulating CCR7(+) Treg expressing IL-1RI ex vivo as early intermediates along an IL-1-controlled differentiation pathway leading from naive FOXP3(+) Treg to Th17 effectors. We further show that, whereas IL-1RI(+) central memory Treg respond to stimulation in the presence of IL-1 by generating IL-17-secreting effectors, a significant fraction of them maintain FOXP3 expression, consistent with an important role of this population in maintaining the Treg/Th17 memory pool in vivo.

Characterization of Treg Subpopulations in Chronic Lymphocytic Leukemia Using 15 Color Flow Cytometry.

Abstract 1644Poster Board I-670CD4+ regulatory T cells (Treg) act to suppress activation of the immune system and thereby maintain immune system homeostasis and tolerance to self-antigens. Tregs have been shown to be increased in the peripheral circulation of patients with various types of cancer including CLL and have been postulated to play a role in inhibition of anti-tumor immune responses. 15 color staining was performed on mononuclear cells isolated from peripheral blood from 33 healthy controls and 12 patients with chronic lymphoid leukemia (CLL). Treg cells were defined as viable CD45+ CD3+ CD8- CD4+ CD25+ Forkhead box P3+ (FoxP3)+ mononuclear cells. Among the Treg population we defined 4 sub-populations; Naïve Treg (CD45-RA+ CD27+ CD197+), Effector Treg (CD45-RA- CD27- CD197-), Central memory Treg (CD45-RA- CD27+ CD197+) and Effector memory Treg (CD45-RA- CD27+ CD197-). We report level of expression of activation markers CD39, HLA-DR and CD38 and IL-7 receptor CD127 on Treg subsets as defined above. The percentage of Treg was increased significantly in CLL patients compared with healthy controls 12.8±1.3% vs 5.7±0.3% (p<0.0001). In healthy controls, the majority of Treg are CD45-RA- (80.2±2.9 %) whereas in CLL patients, we observed Treg cells in both CD45-RA- and CD45-RA+ populations (62.6±4.9% and 40.4±5% respectively). Inside the CD45-RA+ populations, we showed that there was a significant higher proportion of naïve Treg among CLL patients compare to healthy controls ((83.8±5% and 41.8±5% respectively (p<0.0001)). Among the CD45-RA- populations, there was no significant difference in the proportion of each memory subtypes. We observed a different pattern of activation among the Treg in CLL patients, naïve Treg expressed more CD39 and HLA-DR compare to healthy controls (2x to 3x fold increase, p<0.0001), but less CD38. In central memory subpopulation, CD39 and CD127 expressions were increased in Treg isolated from CLL patients (p=0.04 and p=0.01 respectively), whereas CD38 was decreased (p=0.01). In effector memory population, HLA-DR expression was lower in CLL patients compared to healthy control (3±0.9% and 9.8±0.8% respectively, p<0.0001). Finally, in the effector subpopulation, HLA-DR expression was also decreased in Treg from CLL patients (p<0.0001). In summary, in these studies, we showed proportions of naïve and memory Treg as well as a different activation pattern of Treg cells present in CLL patients compared to healthy controls. The assessment of complex patterns of sub-phenotype expression in diseases vis a vis healthy individuals should expand our ability to understand the intricacies of both adaptive and innate cellular immune responses and their dysregulation in cancer patients. DisclosuresNo relevant conflicts of interest to declare.

Dysregulated Distribution of Naïve and Memory FoxP3 Positive Regulatory T-Cells Associated with Anemia in Myelodysplastic Syndrome (MDS)

Background: An increase in CD4+FoxP3+ regulatory T cells (Tregs) has been associated with disease progression in myelodysplastic syndrome (MDS). Tregs provide peripheral tolerance through naturally occurring negative regulatory mechanism that control self-reactivity and autoimmunity. MDS is a heterogeneous hematopoietic disease linked to several distinct pathophysiologic mechanisms that occurs primarily in older individuals. Through the work of our laboratory and others, the balance of CD4 and CD8 T cells has been shown to be disturbed in MDS patients that respond to immunosuppressive therapy. IST offers hematologic benefit and improved survival in a subpopulation of MDS patients with evidence of a T-cell autodominant immune process. It is critical to clarify the correlation between dysregulated naïve and memory CD4+ T cells and dysfunction of Tregs since it has been reported that the supply and function of naïve Tregs is important for protection against autoimmune diseases mediated by T-cells.Methods: We analyzed the direct correlation among FoxP3, CD25 and CD127 expression, and that of CD45RA and CD27 to define Tregs with naïve, central memory, and effector memory phenotypes in 45 MDS patients and 17 healthy controls. We developed an 8-color flow cytometry staining approach that included the following phenotypic markers to define the cell populations: antibodies to CD3, CD4, CD25, CD127 (IL-7Rα), CD45RA, CD27, FoxP3, and aqua fluorescent dye for live:dead cell discrimination.Results: A total of 23 men and 22 women were enrolled from the Bone Marrow Failure Rare Disease Research Network with a diagnosis of MDS with a median age of 69 years old. Based on International Prognostic Scoring System classification, 90% were in a lower risk category (low and intermediate −1 risk). The controls were 17 healthy individuals that donated blood to the Southwest Florida Blood Services (SFBS) and consisted of 8 men and 9 women (median age 52 years old). We found that bright CD25 expression and dim expression of CD127 were most closely associated with FoxP3 expression in healthy controls. The transcription factor FoxP3, which is the most specific phenotypic marker of Tregs, displayed a distinct association with CD25 and CD127 in naïve and memory cells. Therefore, naïve and memory Tregs were defined by FoxP3 expression and the following phenotypes: naïve Tregs (CD45RA+/CD27+/FoxP3+), central memory Tregs (CD45RA−/CD27+/FoxP3+), and effector memory Tregs (CD45RA−/CD27−/FoxP3+). The mean percentage of FoxP3+ Tregs in MDS patients did not differ from healthy controls (5.24% and 4.83%, respectively P=0.94). Younger age, < 61 years of age, has been strongly associated with responsiveness to IST and T-cell-mediated autoimmunity. When the Tregs were examined in patients divided into two groups based on an age of 60 years old, a higher percentage of FoxP3 positive cells was observed in the older age group compared to the younger group (mean, 7.46% and 4.98%, respectively, P=0.12) of MDS patients. Interestingly, the percentage of FoxP3+ Tregs tended to be inversely correlated with the lymphocyte count (P=0.08) and the CD4/CD8 ratio (P=0.1) in patients. When naïve and memory Tregs were compared between cases and controls, these Treg populations demonstrated distinct skewing with effector memory Tregs dramatically higher in some patients. MDS patients were then divided into two groups based on the percentage of effector memory Tregs. Using a cutpoint equal to the mean + 2 S.D of controls, the patients were grouped by the percentage of effector memory (EM) Tregs into two groups: higher EM-Tregs (mean 39.3% ± 12.5%) and lower EM-Tregs (mean 6.39% ± 5.16%). The patient group with higher EM-Tregs was significantly associated with the presence of anemia (P=0.01) compared to the group with lower effector memory TregsConclusions: Memory Tregs have been reported to express higher levels of integrin alphaE, display differential tissue distribution, and a higher turnover rate compared to naïve Tregs. Memory Tregs differentiate from naïve CD25−FoxP3− non-Tregs in the peripheral blood after antigenic challenge under tolerogenic conditions. Our findings suggest that the conversion of effector memory Tregs may be important during development of anemia in MDS patients with autoimmune-mediated bone marrow failure.