Lineage-Plasticity and Inflammatory Change of Human FoxP3+ Regulatory T Cells in Acute Viral Infection: Implication in Immune-Mediated Tissue Injury

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4132-4132
Author(s):  
Yoon Seok Choi ◽  
Jeewon Lee ◽  
Ik-Chan Song ◽  
Deog-Yeon Jo ◽  
Eui-Cheol Shin
Keyword(s):  
T Cells ◽  
Viral Infection ◽  
Treg Cell ◽  
Liver Damage ◽  
Cd4 T Cells ◽  
Treg Cells ◽  
Suppressive Activity ◽  
Suppressive Function ◽  
Acute Viral Infection ◽  
Tnf Α

Abstract FoxP3+ CD4+CD25hi regulatory T (Treg) cells play a major role in maintaining the immune homeostasis by preventing the activation of self-reactive T cells as well as in controlling a series of immune responses in viral infections. Recent studies suggest that lineage-commitment of CD4+T cells, including Treg cells, is not a fixed fate, rather a status with a wide range of plasticity. Functional changes and lineage-plasticity of Treg cells during acute viral infection, especially of human, have not been reported so far. Herein, we investigated whether Treg cells show the functional plasticity and whether such changes can affect the regulation of immunopathology in a human acute viral infection. As a model of human acute viral infection, we used a cohort of patients with acute hepatitis A (AHA), since tissue (liver) injury in AHA is mediated exclusively by activated T cells, not by direct cytopathic effect of virus. To assess the plasticity of Treg cell lineage, first, we examined the production of a variety of inflammatory cytokines from Treg cells following T cell receptor (TCR) stimulation of peripheral blood lymphocytes with anti-CD3/CD28 antibody, using intracellular cytokine staining and multicolor flow cytometry. We found that a significant proportion of FoxP3+ CD4+CD25hi Treg cells produced TNF-α following TCR stimulation in patients with AHA, but not in heathy subjects. Analyses at multiple time points during the course of infection showed that TNF-α production from Treg cells decreased in convalescent phase. Likewise, we observed that liver-infiltrating Treg cells also produced TNF-α after TCR stimulation. Moreover, highly-purified CD4+CD25hiCD127lo/-Treg cells could also produce TNF-α following TCR stimulation, indicating that Treg cells of AHA patients can produce TNF-α in direct response to TCR stimulation. Next, to exclude the possibility that TNF-α might be secreted from transiently FoxP3-expressing activated non-Treg CD4+ T cells, we examined the expression level of CD127 on TNF-α-secreting FoxP3+ CD4+ T cells. TNF-α+ Treg cells expressed CD127 in the level similar to conventional TNF-α- counterpart, and CD127 expression levels of both Treg populations were much lower than FoxP3- CD4+ T cells. Furthermore, DNA methylation analysis of Treg cell-specific demethylated region (TSDR) after sorting TNF-α+ Treg cells revealed completely demethylated pattern in highly conserved CpG island of FOXP3 gene. These findings support that TNF-α is produced from bona fide Treg cells, not from FoxP3-expressing activated non-Treg CD4+T cells. In analysis of immunophenotypes, TNF-α+ Treg cells were enriched in CD45RA-FoxP3lo population, implying their reduced in vivo suppressive activity. Along with the lower level of FoxP3, TNF-α+ Treg cells showed lower level of CD39 expression, a surrogate marker of Treg cell suppressive activity, compared to TNF-α- Treg cells. Furthermore, TNF-α+Treg cells showed a robust evidence of lineage-plasticity toward Th17 lineage, expressing a key transcription factor RORγt. Consistently, they expressed CCR6 and co-produced IL-17A following TCR stimulation, which are the hallmark of Th17 effector function. To analyze the clinical implication of attenuate suppressive function and plasticity shown by TNF-α+ Treg cells, we examined correlation between production of proinflammatory cytokines from Treg cells and severity of liver damage in AHA. As a result, proportion of TNF-α-producing Treg cells closely and linearly correlated with severity of liver damage, suggesting the critical role of TNF-α+ Treg cells in the immunopathogenesis of AHA. However, Treg cell suppression assay in the absence or presence of anti-TNF-α antibody showed that Treg cell suppressive function was not affected by TNF-α blockade. This indicates that attenuated function of TNF-α+Treg cells is not attributed simply to production of a kind of inflammatory cytokine, rather to more complicated reprogramming mechanism. Taken together, these data provide a clear evidence of attenuated suppressive activity and Th17-toward lineage plasticity of FoxP3+ Treg cells, represented by TNF-α production, in a human acute viral infection. Also, we suggest one possible mechanism that lineage plasticity and inflammatory changes of Treg cells could be implicated in the immunopathogenesis of human diseases. Disclosures No relevant conflicts of interest to declare.

scholarly journals RORγt-Mediated TNF-α Production from Human FoxP3+ Regulatory T Cells Contributes to Tissue Inflammation in Acute Viral Infection

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1337-1337
Author(s):  
Yoon Seok Choi ◽  
Ji Young Moon ◽  
Seung Woo Baek ◽  
Hyewon Ryu ◽  
Ik Chan Song ◽  
...  
Keyword(s):  
T Cells ◽  
Liver Injury ◽  
Viral Infection ◽  
Inflammatory Cytokines ◽  
Treg Cells ◽  
Suppressive Activity ◽  
Immune Mediated ◽  
Acute Viral Infection ◽  
Tnf Α ◽  
Effector Cytokines

Abstract CD4+CD25+Foxp3+regulatory T (Treg) cells control immune responses, thereby preventing excessive inflammation and homeostasis of the immune system. Paradoxically, in several autoimmune disorders, it has been suggested that Treg cells undergo inflammatory conversion to produce effector cytokines promoting tissue inflammation. However, such inflammatory changes have not been reported so far in human acute viral infection. Herein, we investigated the production of inflammatory cytokines from Treg cells of acute hepatitis A (AHA) patients. We also studied a cellular mechanism for production of effector cytokines by Treg cells and clinical significance of inflammatory conversion of Treg cells. First, we examined the production of a variety of inflammatory cytokines from Treg cells following T cell receptor (TCR) stimulation of peripheral blood lymphocytes with anti-CD3/CD28 antibody, using intracellular cytokine staining. As a result, we found that a remarkable proportion of CD4+CD25+Foxp3+Treg cells of AHA patients produced TNF-α upon TCR stimulation, particularly at the acute phase. TNF-α production by Treg cells was dramatically diminished at convalescent phase of AHA. Next, we investigated the expression of Th cell type-specific transcription factors and chemokine receptors in TNF-α-producing Treg cells of AHA patients, using multicolor flow cytometry. TNF-α-producing Treg cells expressed substantially higher level of RORγt and CCR6 than the TNF-α-counterpart, suggesting that they share various immunophenotypes of helper 17 T (Th17) cells. More strikingly, the TNF-α production was significantly reduced by RORγt inhibition, indicating that TNF-α production from Treg cells of AHA patients is controlled by Th17-specific transcription factor RORγt. We further examined the suppressive activity of TNF-α-producing CD4+CD25+Foxp3+ Treg cells of AHA patients. TNF-α+CD4+CD25+Foxp3+ Treg cells of AHA patients expressed lower level of Foxp3 than the TNF-α- counterpart. Similarly, the percentage of CD39+ cells was lower in TNF-α+CD4+CD25+Foxp3+ Treg cells than in the TNF-α- counterpart. In fact, Treg suppressive activity of AHA patients was significantly reduced compared with that of healthy controls when non-Treg CD4+ T cells or CD8+T cells were used as responder cells. Finally, we examined the clinical significance of TNF-α-producing CD4+CD25+Foxp3+ Treg cells in AHA patients. In particular, we focused on the liver injury, which is mediated by effector T cells during AHA. Intriguingly, the frequency of TNF-α+ cells among circulating Treg cells significantly correlated with the serum ALT level whereas the frequency of IFN-γ+ or IL-17A+cells did not. This result indicates that the pathologic conversion of Treg cells to produce TNF-α contributes to severe immune-mediated liver injury in AHA patients. Taken together, Treg cells undergo functional alteration to produce TNF-α during AHA. TNF-α-producing CD4+CD25+Foxp3+ Treg cells of AHA patients exhibit a Th17-like feature and produce TNF-α in a RORγt-dependent manner. TNF-α-producing Treg cells show reduced suppressive activity and are associated with severe liver injury in AHA. Importantly, we provide new insight into immunopathologic mechanisms in human acute viral infection, by demonstrating inflammatory conversion of Treg cells and its association with immune-mediated liver injury in AHA. Disclosures No relevant conflicts of interest to declare.

517 Regulatory T cell functional identity is sustained by a glucose:lactate axis that is exploited in the tumor microenvironment

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A553-A553
Author(s):  
McLane Watson ◽  
Paolo Vignali ◽  
Steven Mullet ◽  
Abigail Overacre-Delgoffe ◽  
Ronal Peralta ◽  
...  
Keyword(s):  
T Cells ◽  
Tumor Microenvironment ◽  
Treg Cell ◽  
High Glucose ◽  
Cell Function ◽  
Treg Cells ◽  
Effector T Cells ◽  
Flux Analysis ◽  
Major Barrier ◽  
Suppressive Function

BackgroundRegulatory T (Treg) cells are vital for preventing autoimmunity but are a major barrier to robust cancer immunity as the tumor microenvironment (TME) recruits and promotes their function. The deregulated cellular metabolism of tumor cells leads to a metabolite-depleted, hypoxic, and acidic TME. While the TME impairs the effector function of highly glycolytic tumor infiltrating CD8 T cells, Treg cell suppressive function is maintained. Further, studies of in vitro induced and ex vivo Treg cells reveal a distinct metabolic profile compared to effector T cells. Thus, it may be that the altered metabolic landscape of the TME and the increased activity of intratumoral Treg cells are linked.MethodsFlow cytometry, isotopic flux analysis, Foxp3 driven Cre-lox, glucose tracers, Seahorse extracellular flux analysis, RNA sequencing.ResultsHere we show Treg cells display heterogeneity in terms of their glucose metabolism and can engage an alternative metabolic pathway to maintain their high suppressive function and proliferation within the TME and other tissues. Tissue derived Treg cells (both at the steady state and under inflammatory conditions) show broad heterogeneity in their ability to take up glucose. However, glucose uptake correlates with poorer suppressive function and long-term functional stability, and culture of Treg cells in high glucose conditions decreased suppressive function. Treg cells under low glucose conditions upregulate genes associated with the uptake and metabolism of the glycolytic end-product lactic acid. Treg cells withstand high lactate conditions, and lactate treatment prevents the destabilizing effects of high glucose culture. Treg cells utilize lactate within the TCA cycle and generate phosphoenolpyruvate (PEP), a critical intermediate that can fuel intratumoral Treg cell proliferation in vivo. Using mice with a Treg cell-restricted deletion of lactate transporter Slc16a1 (MCT1) we show MCT1 is dispensable for peripheral Treg cell function but required intratumorally, resulting in slowed tumor growth and prolonged survival.ConclusionsThese data support a model in which Treg cells are metabolically flexible such that they can utilize ‘alternative’ metabolites present in the TME to maintain their suppressive identity. Further, our studies support the notion that tumors avoid immune destruction not only by depriving effector T cells of essential nutrients, but also by metabolically supporting regulatory T cells.

scholarly journals Infectious Tolerance

2002 ◽  
Vol 196 (2) ◽  
pp. 255-260 ◽  
Author(s):  
Helmut Jonuleit ◽  
Edgar Schmitt ◽  
Hacer Kakirman ◽  
Michael Stassen ◽  
Jürgen Knop ◽  
...  
Keyword(s):  
T Cells ◽  
Treg Cell ◽  
Cd4 T Cells ◽  
Transforming Growth Factor ◽  
Treg Cells ◽  
Cell Contact ◽  
Th Cells ◽  
Self Tolerance ◽  
Infectious Tolerance ◽  
Membrane Bound

Regulatory CD4+CD25+ T cells (Treg) are mandatory for maintaining immunologic self-tolerance. We demonstrate that the cell-cell contact–mediated suppression of conventional CD4+ T cells by human CD25+ Treg cells is fixation resistant, independent from membrane-bound TGF-β but requires activation and protein synthesis of CD25+ Treg cells. Coactivation of CD25+ Treg cells with Treg cell–depleted CD4+ T cells results in anergized CD4+ T cells that in turn inhibit the activation of conventional, freshly isolated CD4+ T helper (Th) cells. This infectious suppressive activity, transferred from CD25+ Treg cells via cell contact, is cell contact–independent and partially mediated by soluble transforming growth factor (TGF)-β. The induction of suppressive properties in conventional CD4+ Th cells represents a mechanism underlying the phenomenon of infectious tolerance. This explains previously published conflicting data on the role of TGF-β in CD25+ Treg cell–induced immunosuppression.

TNF-α/IFN-γ profile of HBV-specific CD4 T cells is associated with liver damage and viral clearance in chronic HBV infection

2020 ◽  
Vol 72 (1) ◽  
pp. 45-56 ◽  
Author(s):  
Haoliang Wang ◽  
Heng Luo ◽  
Xing Wan ◽  
Xiaolan Fu ◽  
Qing Mao ◽  
...  
Keyword(s):  
T Cells ◽  
Liver Damage ◽  
Cd4 T Cells ◽  
Viral Clearance ◽  
Hbv Infection ◽  
Chronic Hbv Infection ◽  
Tnf Α ◽  
Ifn Γ ◽  
Chronic Hbv

scholarly journals Study on Quantity and Function of Regulatory T Cell Subsets in Multiple Myeloma and MGUS

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3154-3154
Author(s):  
Jinuo Wang ◽  
Jian Li ◽  
Xinxin Cao ◽  
Hao Cai ◽  
Ai-lin Zhao ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Treg Cell ◽  
Cd4 T Cells ◽  
Treg Cells ◽  
Healthy Controls ◽  
Newly Diagnosed ◽  
Inhibition Rate ◽  
Significant Difference ◽  
Normal Controls

Abstract Introduction Almost all multiple myeloma (MM) cases were progressed from a premalignant condition called monoclonal gammopathy of undetermined significance (MGUS). So far, the pathogenesis of myeloma is not yet clear. The immune cells in the tumor microenvironment, such as regulatory T (Treg) cells with a unique immunosuppressive function, play an important role in myelomagenesis. Although there have been reports on Treg cells in MM patients, the results were still in debate. In this study, we performed a comprehensive analysis of peripheral blood (PB) and bone marrow (BM) Treg subsets and aging Treg-like cells in untreated MM patients and individuals with MGUS, which might help further elucidate mechanisms of immune dysfunction during myelomagenesis. Methods Our study included 20 MGUS patients and 26 newly diagnosed MM patients. Flow cytometry was applied to determine the proportion of Treg cell subsets and aging Treg-like cells in PB and BM. Flow sorting technology was used to separate Treg cell subsets and effector T cells in the bone marrow of newly diagnosed MM patients. The inhibitory function was indirectly calculated by detecting proliferation rate of CFSE-labelled effective T cells which were cocultured with different Treg cell subsets. Concentration of IL-10 from the culture supernatants of proliferation assay was measured using ELISA. Results In PB, the proportion of activated Tregs (aTregs, CD4+CD45RA-FoxP3hi) in CD4+ T cells was significantly higher in MGUS and untreated MM patients than healthy controls (P=0.01, P<0.001); there was no difference in the proportion of resting Tregs (rTregs, CD4+CD45RA+FoxP3lo) between MGUS and untreated MM patients compared with healthy adults (P=0.72, P=0.07). There was also no significant difference in the frequencies of non-Tregs (CD4+CD45RA-FoxP3lo) from MGUS and MM patients with normal controls (P=0.22, P=0.67). The proportion of CD4+CD28-FoxP3+ Treg-like cells in CD4+ T cells was gradually increased in MGUS, untreated MM patients than healthy controls (P<0.01, P<0.01); Treg-like cells in newly diagnosed MM patients were significantly higher than those in MGUS patients (P=0.01). In BM, the proportion of aTregs was significantly higher in MGUS, untreated MM patients compared with healthy controls (P<0.01); the proportion of rTregs in MGUS, untreated MM patients was significantly lower than that of controls (P=0.02, P<0.01). However, there was no significant difference in the frequencies of non-Tregs in BM from MGUS and MM patients with normal controls (P=0.14, P=0.88). The proportion of Treg-like cells in CD4+ T cells was significantly higher in MGUS, untreated MM patients compared with healthy controls (P<0.01, P<0.01). Treg-like cells in untreated MM patients were significantly higher than those in MGUS patients (P<0.01). The inhibition rate of aTreg in bone marrow of newly diagnosed MM patients was significantly higher than that of rTreg (P<0.01), while the inhibition rate of non-Treg was significantly lower than that of rTreg cells (P<0.01). The inhibition rates of aTreg (P=0.21), rTreg (P=0.08) and non-Treg (P=0.09) in healthy controls were no difference from those in MM patients. The level of IL-10 secreted by non-Treg in untreated MM patients was notably higher than that of aTreg and rTreg; the ability of cytokine secretion of Treg subsets in MM patients was similar with that of healthy controls. Conclusions There were significant changes in the frequencies of Treg cell subsets and Treg-like cells in peripheral blood and bone marrow of MGUS and MM patients, suggesting that immunomodulatory abnormality has existed in patients at premalignant stage. The immunosuppressive and cytokine secretory functions of Treg subsets in bone marrow of untreated MM patients were intact compared with that in healthy adults. Disclosures No relevant conflicts of interest to declare.

Development of a Modified Skin Explant Assay to Study Treg Suppression of Th17 Cell Mediated GvHD in the Skin

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5434-5434
Author(s):  
Raewyn Broady ◽  
Sarah Q. Crome ◽  
Jessie Yu ◽  
Jan P Dutz ◽  
Megan K Levings
Keyword(s):  
T Cells ◽  
Treg Cell ◽  
Tissue Damage ◽  
Cd4 T Cells ◽  
Th17 Cell ◽  
Th17 Cells ◽  
Acute Gvhd ◽  
Treg Cells ◽  
Haematopoietic Stem Cell ◽  
Graft Versus Host

Abstract Acute graft versus host disease (aGVHD) following haematopoietic stem cell transplantation (HCT) occurs when donor T cells infused with the graft recognise and react to histo-incompatible recipient antigens causing tissue damage. Historically, the inflammatory response in aGVHD was attributed to alloreactive CD4+ T helper and CD8+ cytotoxic T cells and alterations in cytokine production. Recently, a new CD4+ T cell subset, characterised by IL-17 production has been identified. TH17 cells produce high levels of proinflammatory cytokines, including IL-17A, IL-17F, and IL-22, and have been implicated in solid organ rejection and more recently a number of murine studies suggest that Th17 cells play a role in the development of aGVHD. It is well known that FOXP3+ regulatory T cells (Tregs) are critical for the maintenance of self-tolerance, and control the immune response to alloantigens. Murine studies have shown that adoptive transfer of these cells can prevent acute GVHD whereas selective depletion leads to an increased severity. In humans, Tregs also appear to control acute GVHD as they occur at a lower frequency in the peripheral blood patients with aGVHD compared to patients without GVHD. These findings have led to active interest into the use of these cells to prevent or decrease GVHD following allogeneic HCT. It has been reported that in vitro, Th17 cells are resistant to Treg cell mediated suppression of proliferation and IL-17 production, suggesting that the effector functions of Th17 cells might not be susceptible to Treg-cell-mediated inhibition. If true, this would suggest that Treg-based therapies might not be effective at limiting Th17-cell-mediated tissue damage. However, there is currently no evidence regarding whether Treg cells affect the phenotype or function of Th17 cells in tissues. Understanding the interactions between suppressive Tregs and pro-inflammatory T effectors in tissues that are targets of aGVHD, such as the skin, is critical to better define the potential of Tregs as adoptive therapy for the prevention or treatment of aGVHD. In order to address this question, we developed two methods to generate human Th17 cells, one based on over-expression of RORC2 and the other on sorting CCR4+CCR6+CD4+ T cells. We found that ectopic expression of RORC induces a cytokine and chemokine receptor profile analogous to in vivo differentiated Th17 cells. Although expression of RORC2 made CD4+ T cells resistant to Treg-cell mediated suppression of proliferation and IL-17 production, production of IFN-g, TNF-a and IL-6 could be suppressed in these Th17-like cells. In order to further delineate the functional consequence of the interaction between Treg and Th17 cells in tissues we developed a modified the human skin explant model that involves culture of 4 mm punch biopsies of skin with ex vivo Th17 cells (CCR4+CCR6+CD4+ T cells), RORC2 transduced CD4+ T cells, or controls, in the presence or absence of Treg and grading the graft-versus-host reactivity (grades I–IV) histopathologically. Preliminary data suggest that Th17 cells cause significant tissue destruction in this skin explant model, and experiments are ongoing to determine whether Treg cells can counteract these effects.

scholarly journals Bcl6 Preserves the Suppressive Function of Regulatory T Cells during Tumorigenesis

2020 ◽  
Author(s):  
Yiding Li ◽  
Zhiming Wang ◽  
Huayu Lin ◽  
Lisha Wang ◽  
Xiangyu Chen ◽  
...  
Keyword(s):  
T Cells ◽  
Tumor Microenvironment ◽  
Tumor Growth ◽  
Treg Cell ◽  
Synergistic Effects ◽  
Treg Cells ◽  
Immune Checkpoint Blockade ◽  
Effector T Cells ◽  
Tumor Control ◽  
Suppressive Function

AbstractDuring tumorigenesis, tumor infiltrating regulatory T (Treg) cells restrict the function of effector T cells in tumor microenvironment and thereby promoting tumor growth. The anti-tumor activity of effector T cells can be therapeutically unleashed, and is now being exploited for the treatment of various types of human cancers. However, the immune suppressive function of Treg cells remains a major hurdle to broader effectiveness of tumor immunotherapy. In this article, we reported that the deletion of Bcl6 specifically in Treg cells led to stunted tumor growth, which was caused by impaired Treg cell responses. Notably, Bcl6 is essential in maintaining the lineage stability of Treg cells in tumor microenvironment. Meanwhile, we found that the absence of follicular regulatory T (Tfr) cells, which is a result of Bcl6 deletion in Foxp3+ cells, was dispensable for tumor control. Importantly, the increased Bcl6 expression in Treg cells is associated with poor prognosis of human colorectal cancer and lymph node metastasis of skin melanoma. Furthermore, Bcl6 deletion in Treg cells exhibits synergistic effects with immune checkpoint blockade therapy. Collectively, these results indicate that Bcl6 actively participates in regulating Treg cell immune responses during tumorigenesis and can be exploited as a therapeutic target of anti-tumor immunity.

Maintenance of CD8+T Cells during Acute Viral Infection of the Central Nervous System Requires CD4+T Cells But Not Interleukin-2

2005 ◽  
Vol 18 (1) ◽  
pp. 162-169 ◽  
Author(s):  
Jiehao Zhou ◽  
David R. Hinton ◽  
Stephen A. Stohlman ◽  
Chih-Pin Liu ◽  
Lingwen Zhong ◽  
...  
Keyword(s):  
Central Nervous System ◽  
T Cells ◽  
Nervous System ◽  
Viral Infection ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Interleukin 2 ◽  
Acute Viral Infection ◽  
The Central Nervous System

Only Naive CD45RA+CD4+CD25high T Cells from Human Peripheral Blood Give Rise to Homogeneous Regulatory T Cell Lines.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3163-3163
Author(s):  
Petra Hoffmann ◽  
Ruediger Eder ◽  
Tina J. Boeld ◽  
Kristina Doser ◽  
Biserka Piseshka ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
Treg Cell ◽  
Cell Lines ◽  
Treg Cells ◽  
Effector Memory ◽  
Suppressive Activity ◽  
Intracellular Staining ◽  
Memory Type

Abstract The adoptive transfer of donor CD4+CD25high regulatory T (Treg) cells has been suggested for the prevention of graft-versus-host disease (GVHD) after allogeneic stem cell transplantation (SCT). In preparation of such trials we previously described protocols for the efficient in vitro expansion of human Treg cells (Hoffmann et al. 2004, Blood104:895). Strong costimulation provided by immobilized anti-CD3 and anti-CD28 antibodies together with high-dose IL-2 resulted in a more than 3-log polyclonal expansion of Treg cells with strong suppressive activity. In contrast to CD4+CD25− T cells, the majority of the CD4+CD25high Treg cells maintained expression of the lymph node homing receptors CD62L and CCR7 during in vitro culture. Detailed examination of sorted subpopulations from Treg cell lines now revealed that only CD62L+CCR7+ cells combined all phenotypic and functional characteristics of natural Treg cells, such as FOXP3 expression, lack of cytokine secretion and potent suppression of responder T (Tresp) cell proliferation. To elucidate the origin of this cell population, we initiated cultures from CD45RA+ naive as well as CD45RA− effector/memory-type CD4+CD25high Treg cells. The CD45RA+ population initially contained 95 ± 2.5% CD62L+CCR7+ cells and maintained this phenotype with still over 90% CD62L+CCR7+ cells after 2 weeks and appox. 70% after 3 weeks in culture (n=10). In contrast, CD62L and CCR7 expression in CD45RA− Treg cells was less stable and decreased from 58 ± 8% CD62L+CCR7+ cells after isolation to 32 ± 20% after culture for 3 weeks. Similar differences were observed with respect to cytokine production, as determined by intracellular staining: Whereas less than 5% (n=8) of expanded CD45RA+ Treg cells expressed IL-2 and/or IFN-γ, almost 40% of expanded CD45RA− Treg cells produced one or both of these pro-inflammatory cytokines upon stimulation with PMA/ionomycin. Interestingly, expanded CD45RA− Treg cells also contained a defined subpopulation of IL-10-producing cells (6.9 ± 5.6%; n=8), which was absent in expanded CD45RA+ Treg cells. Both subpopulations showed suppressive activity after polyclonal restimulation, however, suppression of Tresp cell proliferation by expanded CD45RA+ Treg cells was much more profound with only 13.6 ± 4% proliferating cells as compared to 35.4 ± 7.7% in the presence of expanded CD45RA− Treg cells and 79.6 ± 18.7% in the absence of Treg cells, as measured in a CFSE-dilution assay at a 1:4 ratio of Treg and Tresp cells (n=7). Most importantly, when determined on a single cell level by intracellular staining, 93.6 ± 1 % (n=3) of expanded CD45RA+ Treg cells still expressed FOXP3 after 3 weeks in culture, whereas only 10.8 ± 6.8% of expanded CD45RA− Treg cells remained FOXP3+. No re-expression could be induced in FOXP3− expanded CD45RA− Treg cells by short-term restimulation via CD3/CD28, whereas expanded FOXP3+ CD45RA+ Treg cells transiently upregulated FOXP3, with peak expression levels as early as 24h after stimulation and return to baseline levels by 48 to 72h. Based on these unexpected findings that only naive CD45RA+, but not memory-type CD45RA− CD4+CD25high T cells give rise to homogeneous Treg cell lines, we suggest that isolation and expansion of CD45RA+ CD4+CD25high T cells is the best strategy for adoptive Treg cell therapies.

Regulatory T Cells Predicts Progression in Previously Untreated Myeloma Patients and Treatment by Cyclophosphamide, Thalidomide Plus Dexamethasone Reduces Regulatory T Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2870-2870 ◽  
Author(s):  
Karthick Raja Muthu Raja ◽  
Lucie Kovarova ◽  
Petra Kaisarova ◽  
Jana Bartonova ◽  
Roman Hajek
Keyword(s):  
T Cells ◽  
Healthy Volunteers ◽  
Cell Count ◽  
Treg Cell ◽  
Cd4 T Cells ◽  
Hematological Malignancies ◽  
Treg Cells ◽  
Cd4 T Cell ◽  
Treatment Responses ◽  
Pre Treatment

Abstract Abstract 2870 Background: Regulatory T (Treg) cells are one of the master regulators in maintaining tolerance and immune homeostasis. Several studies in hematological and non-hematological malignancies revealed that increased level of Treg cells significantly impede the anti-tumor responses. Moreover, in non-hematological malignancies Treg cells correlate inversely with patient's survival (Beyer et al, 2006, Blood). Here in this study we evaluated the prognostic significance of Treg cells in multiple myeloma (MM) patients and influence of chemotherapies on Treg cells. Patients and Method: In this study a cohort of 74 newly diagnosed MM patients were recruited and all patients gave informed consent. Median age of the patient cohort was 67 years (range: 38–88). According to international staging system (ISS) patients were characterized as ISS1=20(27%), ISS2=27(36%), ISS3=25(34%) and undetermined=2(3%). Patients were treated with following treatment regimens: low or high dose combination of cyclophosphamide, thalidomide plus dexamethasone (CTD- 54/74), bortezomib, doxorubicin plus dexamethasone (5/74), and cyclophosphamide, doxorubicin plus dexamethasone (4/74). Eleven patients did not receive any treatment. Using multiparameter flowcytometry all the MM subjects peripheral blood (PB) samples were analyzed for total lymphocytes, CD4 T cells and Treg cells. For comparison, 10 healthy volunteers PB samples were also assessed. Results: Phenotypically, CD4 T cells were assessed based on the expression of CD4 marker and Treg cells were characterized as CD4+CD25hi+FoxP3+. The frequencies of total lymphocytes were significantly reduced in MM than healthy volunteers [median% (range %)= 16.88(2.77–45.93) vs. 36.96(29.42–53.69); P<0.0001]. CD4 T cell numbers were similar between MM patients and healthy volunteers [median% (range %)= 38.17(15.02–81.33) vs. 40.03(30.58–52.16); P=0.34]. Contrasting to lymphocyte and CD4 T cell counts, Treg cells were significantly increased in MM patients compared to healthy volunteers [median% (range %)= 5.22 (2.57–11.73) vs. 3.00 (1.96–3.59); P<0.0001]. Twenty five patients were followed and their PB samples were assessed after CTD treatment (15/25) or transplantation (10/25). Post-treatment or transplantation assessment showed significant reduction in the frequencies of CD4 T cells and Treg cells compared to pre-treatment, whereas lymphocyte numbers were similar (Table: 1). With regard to treatment responses, ≥ very good partial response (VGPR) was achieved in 12 patients (complete response-3/12 plus VGPR-9/12) and 13 patients achieved < VGPR (partial response-7/13, minimal response-1/13 and progressive disease-5/13). Patients with ≥ VGPR showed significant reduction in the frequencies of CD4 T cells and Treg cells after treatment compared to pre-treatment. However, patients with < VGPR showed only significant reduction for CD4 T cells (Table: 2). No significant association was observed between pre- and post-treatment frequencies of Treg cells with treatment responses. Time to progression (TTP) data was available for 44 patients. The median follow-up period of this patient cohort was 11 months (range: 4–18). We separated the patients into two cohorts, such as patients (23/44) with higher Treg cell count (≥ 5%) and patients (21/44) with lower Treg cell count (< 5%). Univariate analysis showed inferior TTP for patients with higher Treg cell count compared to patients with lower Treg cell count (13 months vs. median not reached; P=0.013). Other clinical variables did not hold significance for TTP in this cohort. As well, Cox regression multivariate model ensured inferior TTP for cohort with higher Treg cell count compared to cohort with lower Treg cell count (P=0.045). Conclusions: It is well known that myeloma patients suffer from various immune abnormalities and Treg cells also mediate immune dysfunctions. Unprecedentedly, our data suggest that progression in subgroup of myeloma patients was due to higher infiltration of Treg cells in the peripheral blood. This data also encourages that Treg cells could be a potential target in myeloma patients along with tumor cells. As well our finding showed that CTD efficiently depleted the Treg cells in patients who achieved ≥ VGPR. This study was supported by respective grants MSM0021622434, LC06027, IGA NS10406, IGA NS10408, and GACR P304/10/1395. Disclosures: Off Label Use: cyclophosphamide, thalidomide and dexamethasone.

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