Enrichment of Inflammatory IL-17 and TNF-α Secreting CD4+ T Cells within Colorectal Tumors despite the Presence of Elevated CD39+ T Regulatory Cells and Increased Expression of the Immune Checkpoint Molecule, PD-1

Abstract Abstract 586 A major challenge of the immune system is to fight pathogens and tumor antigens while preserving tolerance to self-antigen. T regulatory cells (Treg) are critical extrinsic regulators of immune tolerance and maintenance of lymphoid homeostasis. Recently it was determined that, when used as cell-based immunosuppressive therapy, Treg have a potent effect in preventing GvHD in patients undergoing allogeneic stem cell transplantation. However, several studies suggest that the Treg phenotype is not at end stage of differentiation. Treg can express and produce effector cytokines including IFN-γ and IL-17 under certain conditions, particularly in the context of inflammatory milieu, suggesting that Treg may convert into inflammatory mediators. IL-1β and TNF-α are critical inflammatory cytokines that have been implicated in GvHD. The precise role and the mechanism(s) via which these cytokines may affect development of GvHD remain unclear. In the presence study, we sought to determine whether IL-1β and TNF-α regulate the properties of Treg and specifically whether these cytokines affect Treg expansion and/or conversion into IL-17 producing cells. CD4+CD25+Treg cells were isolated from B6 mice and were stimulated with anti-CD3-plus-anti-CD28 mAbs in the presence of either media, IL-1β or TNF-α. Addition of either cytokine induced Treg proliferation as determined by CFSE. Assessment of intracellular IL-17 expression by flow cytometry and IL-17 production by ELISA revealed that IL-1β but not TNF-α induced conversion of Treg into IL-17 producing cells, suggesting that conversion was mediated via pathways distinct from those that regulate cell cycle progression. To evaluate conversion of Treg to IL-17 producers during antigen stimulation and to determine the role of IL-1β in this process, we used neutral culture conditions in which no exogenous cytokines were supplied. Treg cells isolated from Foxp3GFP-KI mouse were added to cultures of naive conventional CD4+ T cells (Tc) in the presence of APC and anti-CD3 mAb. We found that these conditions preferentially induced conversion of Treg to IL-17 producing cells. To determine the role of IL-1β in this conversion process, we used IL-1β neutralizing antibody. Addition of anti-IL-1β neutralizing antibody reduced IL-17 production to almost undetectable levels. Because it has exogenous IL-6 can induce IL-17 production by both Treg and Tc, we evaluated whether endogenous IL-6 was involved in the conversion of Treg into IL-17 producing cells in our system. Addition of a combination of IL-6 neutralizing and IL-6 receptor blocking antibodies did not affect IL-17 production, suggesting that the conversion process of Treg into IL-17 producing cells was dependent on endogenous IL-1β rather than IL-6. To determine whether IL-1β was mandatory for this process, we used T cells from IL-1R deficient mice. Individual culture of IL−1R−/− Tc or IL-1R−/− Treg with wild type (wt) APC and co-culture of IL-1R−/− Tc and IL-1R−/− Treg with wt APC did not result in detectable IL-17 production. Similarly, no IL-17 production was observed when wt instead of IL-1R−/− Tc were used. In contrast, substitution of IL-1R−/− Treg with wt Treg resulted in abundant IL-17 production. To investigate the in vivo biological relevance of our findings we adoptively transferred Treg cells from either congenic B6.PL mice or IL-1R1−/− mice into IL-1R1−/− recipients, which were then immunized with KLH in IFA. Three days after immunization both IL-1R−/− Treg and IL-1R−/− Tc cells were incapable of producing detectable levels of IL-17 or expressing RORγt, the key transcriptional factor of IL-17. In contrast, a significant percentage of IL-17 and RORγt positive cells were detected within the adoptively transferred Thy1.1+ Treg population. Mechanistic analysis revealed that IL-1β induced activation of p38 and JNK in Treg and addition of pharmacologic inhibitors specific for these MAPKs abrogated IL-17 production. Our studies reveal that although Treg have primarily immunosuppressive functions they may also facilitate pro-inflammatory responses as they can be converted into IL-17 producing cells by IL-1β. These observations may have significant implications on clinical strategies that employ Treg for control of GvHD and suggest that further intervention might be required to prevent attainment of pro-inflammatory properties by Treg while maintaining their suppressive function. Disclosures: No relevant conflicts of interest to declare.

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Tumor-Evoked Regulatory B Cells Promote Breast Cancer Metastasis by Converting Resting CD4+ T Cells to T-Regulatory Cells

Cancer Research ◽

10.1158/0008-5472.can-10-4316 ◽

2011 ◽

Vol 71 (10) ◽

pp. 3505-3515 ◽

Cited By ~ 291

Author(s):

Purevdorj B. Olkhanud ◽

Bazarragchaa Damdinsuren ◽

Monica Bodogai ◽

Ronald E. Gress ◽

Ranjan Sen ◽

...

Keyword(s):

Breast Cancer ◽

T Cells ◽

B Cells ◽

Cd4 T Cells ◽

Cancer Metastasis ◽

T Regulatory Cells ◽

Breast Cancer Metastasis ◽

Regulatory Cells ◽

Regulatory B Cells ◽

Promote Breast Cancer

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Lentiviral Engineered T Regulatory Cells Effectively Inhibit Lymphocytes Alloreactivity across Major HLA Barriers

Blood ◽

10.1182/blood.v112.11.3515.3515 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3515-3515

Author(s):

Dario Sangiolo ◽

Noela Jordaney ◽

Giulia Mesiano ◽

Paola Circosta ◽

Angela Elia ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd4 T Cells ◽

Culture Medium ◽

T Regulatory Cells ◽

Stable Expression ◽

T Cell Subsets ◽

Cell Transplant ◽

Regulatory Cells ◽

Cd4 Cells

Abstract Adoptive immunotherapy strategies enrolling T regulatory cells (Tregs) might have a great potential in modulating donor T cells alloreactivity after Hematopoetic Cell Transplant (HCT). In murine models of HCT Tregs were shown to promote engraftment and contribute controlling graft versus host disease (GVHD) while still not conclusive data are available on humans. Ex-vivo engineering conventional CD4+ T cells to over-express the transcription factor FOXP3 is an intriguing approach to overcome the main difficulty of obtaining large amount of Tregs for experimental studies. Reports of retrovirus-mediated expression of FOXP3 not consistently resulted in functional Tregs while, recently, a lentivirus-mediated strategy was successfully reported to result in homogeneous and stable expression of FOXP3. Lentiviral transduced Tregs were able to suppress a polyclonal proliferation of CD4 purified lymphocytes stimulated with soluble Ab anti-CD3. In our study we generated lentiviral engineered Tregs (eng-Tregs) and investigated their inhibitory effect on unselected lymphocytes alloreactivity across major HLA barriers. Within the bulk lymphocytes population we separately tracked the suppressive influence of eng-Tregs on both CD4+ and CD8+ T cells. To obtain eng-Tregs, CD4+ T cells were purified from healthy donors and transduced with a bidirectional lentiviral vector encoding for FOXP3 and the truncated Nerve Growth Factor Receptor (ΔNGFR). Prior to transduction CD4+ cells were activated for 72 hours with IL2 (100U/ml), IL7 (20ng/ml) and soluble Ab anti-CD3 (200 ng/ml, only IL2 was added to the culture medium after transduction. The lentiviral transduction efficiency ranged from 8 to 25%, ΔNGFR+ T cells were positively selected and tested for their ability to suppress a mixed lymphocyte reaction across major HLA barriers. Effector peripheral blood mononuclear cells (PBMC) were collected from the same donors from whom eng-Tregs were generated. Effector PBMC were stained with CFSE in oder to separately track the alloreactive proliferation of both CD8+ and CD4+ subsets of T cells. Eng-Tregs were added on day 0 and HLA-mismatched irradiated PBMC were used as stimulators; both eng-Tregs and irradiated stimulators were used in a 1:1 ratio with the effectors. No cytokines or additional soluble stimulators were added to the MLR culture medium. The alloreactive proliferation of T cell subsets was determined by evaluating the logarithmic decrease of CFSE fluorescence intensity. The flow cytometry analysis on day +7 showed that alloreactive proliferation of both CD4+ and CD8+ effector cells was significantly inhibited (>75%) by the addition of eng-Tregs compared to controls. In order to rule out a possible role played by the naturally present Tregs (nat-Tregs), the effectors were depleted of the CD4+CD25high subpopulation before the MLR started. The observed alloreactive proliferation was higher after the depletion of nat-Tregs but still it could be significantly inhibited by the addition of eng-Tregs. Eng-Tregs did not significantly expanded when cultured in vitro (up to 2 weeks) with IL2 (100U/ml) but maintained a stable expression of the transgene and retained their suppressive capacity. Our data show that lentiviral engineered Tregs can efficiently down-modulate both CD4+ and CD8+ T cell alloreactivity across major HLA barriers. The observed independence from the presence of nat-Tregs might be important in future experimental HCT settings where the adoptive infusion of eng-Tregs might encounter a great variability in the number and activity of recipient’s nat-Tregs. The possibility of transducing a potentially unlimited number of CD4+ cells makes this strategy appealing for future pre-clinical studies to control GVHD in HCT settings.

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CD4+CD25+Foxp3+ T Regulatory Cells Protect Against Murine Antibody-Mediated Transfusion-Related Acute Lung Injury (TRALI)

Blood ◽

10.1182/blood.v126.23.2342.2342 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2342-2342

Author(s):

Rick Kapur ◽

Michael Kim ◽

Shanjee Shanmugabhavananthan ◽

Edwin R. Speck ◽

Rukhsana Aslam ◽

...

Keyword(s):

T Cells ◽

Cd4 T Cells ◽

T Regulatory Cells ◽

Blood Product ◽

Lung Damage ◽

Chronic Alcohol ◽

Regulatory Cells ◽

Chronic Alcohol Abuse ◽

Murine Antibody

Abstract Transfusion-related acute lung injury (TRALI) is the leading cause of transfusion-related fatalities and is characterized by acute respiratory distress following transfusion of blood products. Frequently, donor antibodies present in the transfused blood product are involved, such as anti-human leukocyte antigen (HLA) antibodies or anti-human neutrophil antigen (HNA) antibodies. Several animal models of TRALI have contributed to understanding the pathogenesis which, however, is still incompletely understood. Several cell types have also been suggested to be involved in antibody-mediated TRALI, including neutrophils, endothelial cells and monocytes. Most of the animal models are based on a two-hit paradigm, where the first hit is based on "patient predisposition", such as sepsis or chronic alcohol abuse, while the second hit is delivered in the form of "transfusion factors", such as antibodies present in the transfused blood product. Although most studies have focused on factors contributing to the development of antibody-mediated TRALI, the factors and mechanisms in place to protect against antibody-mediated TRALI have been underexplored. Adoptive transfer of lymphocytes into recipient severe combined immunodeficient (SCID) mice, in which the well-established TRALI inducing anti-MHC class I antibody clone 34-1-2s was injected, was previously shown to rescue TRALI induction by 34-1-2s. Here we describe, using a murine BALB/c antibody-mediated TRALI model based on injection of 34-1-2s, that CD4 T cells, and more specifically, CD4+CD25+Foxp3+ T regulatory cells (Tregs), are responsible for protection against murine antibody-mediated TRALI. Specific in vivo depletion of CD4+ T cells, or targeted in vivo depletion of Tregs, resulted in severe lung damage after 34-1-2s infusion, as determined by increased lung wet-to-dry ratios (a measure for pulmonary edema), generally greater than 5, indicative of severe pulmonary edema. This was accompanied by significant hypothermia, increased values of the neutrophil chemoattractant macrophage inflammatory protein 2 (MIP-2: equivalent of human IL-8), and increased pulmonary neutrophil accumulation, all compared to control groups. In contrast, systematic in vivo depletion of CD8+ T cells, B cells or monocytes, did not result in significant lung damage. Co-depletion of CD4+ T cells together with monocytes rescued the TRALI induction by 34-1-2s, validating the pathogenic role of monocytes in murine antibody-mediated TRALI induction. Based on MIP-2 values and in vitro studies, we suggest that Tregs suppress monocytes in order to prevent antibody-mediated TRALI. Overall, a novel first hit in TRALI induction could be identified in conditions that cause a decrease in Treg number or function, which could also explain the increased risk for human TRALI in cases of chronic alcohol abuse. In addition, therapies aimed at restoring Treg numbers or function may prove to be a novel therapeutic approach in antibody-mediated TRALI. Disclosures No relevant conflicts of interest to declare.

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