Follicular lymphoma B cells induce the conversion of conventional CD4+T cells to T-regulatory cells

Abstract Advanced stage follicular lymphoma remains an incurable disease with a median survival of 8 to 10 years that has not significantly changed over the last four decades. Therefore, novel treatment options are necessary to improve the clinical outcome in these patients. The observation of spontaneous regressions in a small percentage of patients suggested that augmenting the host immune response could potentially control this malignancy. Strategies using active specific immunotherapy with idiotype vaccines led to induction of clinical and molecular responses in a few patients but have met with only limited success possibly due to the low frequency of antigen-specific T cells induced in the patients. In contrast to active immunization, T cells of a given specificity and function may be selected and expanded in vitro to the desired number for adoptive cell transfer. Towards this goal, we stimulated tumor infiltrating lymphocytes (TILs) or peripheral blood mononuclear cells (PBMCs) from five follicular lymphoma patients with CD40 ligand-activated autologous tumor cells at approximately ten-day intervals in the presence of IL-2 and IL-15. After four rounds of stimulations, T cell lines generated from 3/5 patients recognized autologous unmodified tumor cells by producing significant amounts of TNF-α, GM-CSF and/or IFN-γ. By phenotypic analysis, the T cell lines were predominantly CD4+ T cells (> 70%), and intracellular cytokine assay showed that up to 40% of the CD4+ T cells were tumor-reactive. The inhibition of cytokine production by anti-HLA class II but not class I blocking antibodies confirmed that the CD4+ T cells were tumor-reactive. Further characterization revealed that the T cells from one patient recognized autologous tumor but not autologous normal B cells suggesting that they were tumor-specific. While in a second patient CD4+ T cell clones generated from the T cell line by limiting dilution recognized autologous tumor and autologous normal B cells but not autologous monocytes suggesting that they were B cell lineage-specific. We conclude that follicular lymphoma-specific T cells exist and can be efficiently expanded in vitro from both TILs and PBMCs using CD40 ligand-activated autologous tumor cells for adoptive T cell therapy. Additionally, identification of antigens recognized by these T cells could lead to development of novel immunotherapeutic strategies for lymphomas.

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Multiparameter Microscopy Analysis of the Follicular Lymphoma Microenvironment and Normal Germinal Centers: In Vivo evidence That Follicular Helper T Cells Form Synapses with Neoplastic B Cells and Are Associated with Proliferation and Expression of Activation Induced Cytidine Deaminase

Blood ◽

10.1182/blood.v124.21.144.144 ◽

2014 ◽

Vol 124 (21) ◽

pp. 144-144

Author(s):

William M Townsend ◽

Robert Marcus ◽

Jon Salisbury ◽

Deborah Yallop ◽

Piers EM Patten ◽

...

Keyword(s):

T Cells ◽

B Cells ◽

Follicular Lymphoma ◽

Tumor Cells ◽

Cd4 T Cells ◽

Close Contact ◽

Cd4 Cells ◽

Proliferating Cells ◽

Tfh Cells

Abstract The tumor microenvironment plays a central role in the pathogenesis of follicular lymphoma (FL) and has been shown to influence prognosis. The biological basis for this and the contribution of individual cell types however, remain unclear. In this study we compared the cellular content and structure of neoplastic follicles in FL with their normal counterparts in reactive lymph nodes (LNs). We specifically focused on follicular helper T cells (TFH) which, in normal germinal centers (GCs), form immune synapses with antigen responsive B cells triggering B cell proliferation and expression of activation induced cytidine deaminase (AID), the enzyme required for somatic hypermutation and class switch recombination. This is of relevance because off-target AID activity is thought to play a role in generating the mutations that characterize progressive FL. A limitation of previous studies of the FL microenvironment is the use of either single parameter immunohistochemistry which fails to accurately define the complex populations of cells involved, or flow cytometry on disaggregated cells which results in the loss of architectural information. In this study we used multiparameter confocal immunofluorescent (IF) microscopy to investigate in vivo the phenotype, distribution and interaction of CD4+ T cells in FL and to determine to what extent these are similar to normal GCs. Confocal IF microscopy was performed on multiple sections of formalin fixed paraffin embedded LN biopsy specimens from 20 patients with untreated FL, comparison was made with reactive LNs (n=5) and chronic lymphocytic leukemia (CLL) LN biopsies (n=5). Each section was stained with a combination of up to 4 simultaneously applied primary antibodies against CD3, CD4, CD20, PD1, ICOS, BCL6, AID, and Ki67, and fluorescently labelled secondary antibodies. Microscopy was performed using a Nikon TiE fluorescent microscope equipped with A1R Si Confocal imaging system; images were analyzed using NIS software. Results show that CD4+ T cells in FL are mainly located in the inter-follicular regions but they were also identified within the follicles in all cases. Combination staining with anti-CD4, PD1, and ICOS revealed that 23% (95%CI 18-27) of CD4+ T cells within follicles co-express PD1 and ICOS consistent with a TFH phenotype which is significantly higher than in inter-follicular areas where only 5% (95% CI 3-7) of CD4+ cells had this phenotype (p<0.001). PD1+ ICOS+ T cells were positive for the transcription factor BCL6, further confirming the TFH phenotype. There was no significant difference in the proportion of CD4+ cells that were TFH in FL follicles and reactive LN GCs. In CLL cases, 54% of CD4+ cells expressed PD1 but only 9% co-expressed PD1 and ICOS, significantly lower than either FL follicles or GCs (p<0.001). Automated analysis of 3D z-stacks demonstrated a very close spatial relationship between proliferating tumor cells and TFH in FL with a mean of 42% (95%CI 35-48) Ki67+ tumor cells in direct contact with TFH cells. No association was seen between the extent of co-localization and histological grade. A similar pattern of co-localization of TFH cells next to proliferating B cells was also identified in the light zones of reactive GCs. Of note, we also identified features of synapse formation between TFH cells and proliferating tumor cells; TFH cells demonstrated projections that encompass the tumor cell with distortion of the T cell nucleus and increased CD4 and PD1 expression at sites of cell contact (Figure 1). These findings were similarly present in reactive GCs. Finally, AID was expressed in proliferating GC B cells and in proliferating tumor cells in FL. AID expressing cells were found to be in close contact with PD1+ T cells in both GCs and FL. Our findings show many parallels between the follicles of FL and normal GCs. In particular the proportion of CD4+ T cells with a TFH phenotype and their localization in direct contact with proliferating AID+ B cells were very similar. Of note, features of immune synapses were observed in both GCs and FL. Taken together, the data suggest that TFH cells have an important role in the pathogenesis of FL just as they are vital in the normal GC reaction. Interruption of this interaction is a potential therapeutic target. Figure 1 High power view (x60 zoom) of follicular lymphoma showing proliferating cells in close contact with TFH cells. Ki67 (red), PD1 (white), ICOS (green), DAPI (blue) Figure 1. High power view (x60 zoom) of follicular lymphoma showing proliferating cells in close contact with TFH cells. Ki67 (red), PD1 (white), ICOS (green), DAPI (blue) Disclosures No relevant conflicts of interest to declare.

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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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Ibrutinib Treatment Reduces Both T-Regulatory Cells and B-Regulatory Cell Phenotype in Malignant B Cells in Chronic Lymphocytic Leukemia Patients

Blood ◽

10.1182/blood.v126.23.2940.2940 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2940-2940 ◽

Cited By ~ 3

Author(s):

Meixiao Long ◽

Kyle A. Beckwith ◽

Kami J. Maddocks ◽

Carolyn Cheney ◽

Jennifer A. Woyach ◽

...

Keyword(s):

T Cells ◽

B Cells ◽

Research Funding ◽

T Regulatory Cells ◽

Surface Expression ◽

Lymphocytic Leukemia ◽

Regulatory Cells ◽

Reduced Frequency ◽

Fold Reduction

Abstract Introduction: Chronic lymphocytic leukemia (CLL) has multiple mechanisms of active immune suppression including expansion of T-regulatory cells which increases with progression of the disease. In addition, the malignant CLL cells were found to produce IL-10 in vitro and functionally recapitulate the phenotype of regulatory B cells. These regulatory "B10" (capable of producing IL10 after hours in vitro stimulation) or "B10 pro" (capable of producing IL10 after 2 days in vitro conditioning) are generally rare in healthy individuals, and play an important role in regulating inflammatory and autoimmune process. Similarly, CLL cells can exert tumor specific as well as global immune suppressive effect via IL-10 production. Ibrutinib, the first in class irreversible BTK inhibitor has been proved to be a safe and effective therapy for CLL. Recently we and others have demonstrated favorable cellular immune modulatory effects of ibrutinib through inhibition of an alternative target interleukin-2 induced T-cell kinase (ITK) that promotes Th1 CD4 polarization. Herein, we explore influence of ibrutinib on other immune suppressive features including T-regulatory cells and the B-regulatory phenotype associated with CLL cells. Methods: PBMCs were collected from nine previously treated CLL patients treated with 420mg of ibrutinib daily per clinical trial OSU-11133 (NCT01589302) at the time of pretreatment, cycle 3 day 1 and cycle 6 day 1. For Brief stimulation (B10 condition), cryopreserved PBMCs were thawed and stimulated with PMA/Ionomycin/Golgi-stop plus CpG for 5 hours. For prolonged stimulation (B10-Pro condition), PBMCs were stimulated with CpG plus CD40L for 48 hours, PMA / Ionomycin / Golgi-stop were added for final 5 hours. The cells were then fixed / permeabilized and stained for intracellular IL-10. For FOXP3 staining, PBMCs were permeabilized and fixed with Foxp3 Buffer Set from eBioscience, and were stained with stained with PE conjugated anti-human Foxp3 antibody (clone 259D/C7). Results: Significant IL-10 production was detected in 8 out 9 patient's CLL cells after 48 hours in vitro stimulation. Interestingly, CLL cells collected from patients treated with ibrutinib in vivo were significantly impaired in their capacity to make IL-10 in 7 out of the 8 patients whose CLL cells were capable of producing IL-10. On average, there is more than 4 fold reduction( P< 0.01) in the frequency of cells producing IL-10 by cycle 3, more than 5 fold reduction (P< 0.01) by cycle 6. (Figure 1 A, upper panel). IL-10 production after a brief 5 hour in vitro stimulation was observed in 4 out of the 9 patients studied, though the frequencies of IL-10 producing cells were low (Figure 1 A, lower panel). Samples collected post-ibrutinib treatment showed a trend towards reduced frequency of IL-10 producing CLL cells after 5 hour stimulation. We have also shown that during the first two cycles of ibrutinib, patients' plasma levels of IL-10 decreased. Analysis of potential immunosuppressive molecules revealed a dramatic reduction in surface expression of CD200, BTLA and PD-1 in CLL cells collected post ibrutinib treatment compared to pre-treatment samples (Figure 1B). We also found that for all the patients analyzed, the percentage of CD4+/Foxp3+ and CD4+/CD25+/Foxp3+ regulatory T cells were significantly reduced in samples collected after ibrutinib treatment. The difference is more dramatic for CD25+Foxp3+ cells (figure 1C). Conclusion: Here we demonstrate a significant decrease in the frequency of T-regulatory cells and IL-10 competent "B-reg" like leukemia cells in CLL patients after ibrutinib treatment. Ability of CLL cells to produce IL-10 and their regulatory B cell like features are considered to play a major role in mediating both global and tumor specific immunosuppression in CLL patients. Ibrutinib has been reported to enhance the immune response against B cell lymphoma in a mouse model. Our findings provide potential mechanisms by which ibrutinib treatment relieve the immunosuppressive effect of malignant B cells, thus enhancing global as well as tumor specific immunity. The main mechanisms likely include impaired IL-10 production capability and reduced surface expression of immunosuppressive molecules by CLL cells, as well as reduced frequency of regulatory T cells and IL-10 producing T cells. Figure 1. Figure 1. Figure 2. Figure 2. Figure 3. Figure 3. Disclosures Maddocks: Novartis: Research Funding; Janssen: Research Funding; Pharmacyclics: Consultancy, Research Funding. Byrd:Acerta Pharma BV: Research Funding; Acerta Pharma BV: Research Funding.

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