scholarly journals Potential Impact of B Cells on T Cell Function in Multiple Sclerosis

2011 ◽  
Vol 2011 ◽  
pp. 1-9 ◽  
Author(s):  
Sara Ireland ◽  
Nancy Monson
Keyword(s):  
Multiple Sclerosis ◽  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Cell Function ◽  
The Central Nervous System ◽  
Potential Impact ◽  
Anti Cd20 ◽  
The Impact ◽  
Ms Pathogenesis

Multiple sclerosis is a chronic debilitating autoimmune disease of the central nervous system. The contribution of B cells in the pathoetiology of MS has recently been highlighted by the emergence of rituximab, an anti-CD20 monoclonal antibody that specifically depletes B cells, as a potent immunomodulatory therapy for the treatment of MS. However, a clearer understanding of the impact B cells have on the neuro-inflammatory component of MS pathogenesis is needed in order to develop novel therapeutics whose affects on B cells would be beneficial and not harmful. Since T cells are known mediators of the pathology of MS, the goal of this review is to summarize what is known about the interactions between B cells and T cells, and how current and emerging immunotherapies may impact B-T cell interactions in MS.

scholarly journals A New Kid on the Block: IL-10+ Regulatory B Cells and a Possible Role In Psoriasis

2009 ◽  
Vol 14 (3) ◽  
pp. 148-153
Author(s):  
Kamruz Darabi ◽  
Rohit Jaiswal ◽  
Sarah G. Hostetler ◽  
Mark A. Bechtel ◽  
Matthew J. Zirwas ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Cell Population ◽  
Cell Function ◽  
Adolescent Male ◽  
Mechanical Trauma ◽  
Concomitant Treatment ◽  
Anti Cd20

The pivotal role of T cells in the etiology of psoriasis has been elucidated; however, the mechanisms that regulate these T cells are unclear. Recently, it has been shown that an IL-10 producing B cell population may downregulate T cell function and it has been hypothesized that depletion of this B cell population may lead to exacerbation of T-cell mediated autoimmune disease. We present the case of an adolescent male with autoimmune lymphoproliferative syndrome (ALPS) being treated with the anti-CD20 chimeric monoclonal antibody rituximab in addition to intravenous immune globulin (IVIG) for immune thrombocytopenia (ITP) who developed a psoriasiform rash on his hands following mechanical trauma with concomitant severely decreased B cell count. We propose that depletion of the patient's B cells due to rituximab treatment may have led to abrogation of IL-10+ B-cell regulation of T cells. The development of a psoriasiform rash in this predisposed individual may have been triggered by mechanical trauma to his hands (koebnerization). In addition, we believe the patient's rash may have been tempered by concomitant treatment with IVIG, which has been used as treatment in cases of psoriasis. We discuss the immunologic mechanism of psoriasis and the role that a recently described IL-10+ B cell may play in preventing the pathologic process. Further studies are needed to more clearly elucidate this process.

Administration Of Anti-CD20 Mab Is Highly Effective In Preventing But Ineffective In Treating Chronic GVHD While Preserving A Strong GVL Effect

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2010-2010
Author(s):  
Heather F Johnston ◽  
Ya-jing Xu ◽  
Xiong Ni ◽  
Tao Wu ◽  
Jeremy Racine ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Hair Loss ◽  
Cd4 T Cells ◽  
Chronic Gvhd ◽  
Cd8 T Cell ◽  
Effective Prevention ◽  
Anti Cd20 ◽  
The Impact

Abstract Donor T cell-mediated graft versus leukemia/lymphoma (GVL) effect plays a critical role in preventing tumor relapse in leukemia/lymphoma patients treated with allogeneic hematopoietic cell transplantation (HCT). However, these same donor T cells also induce acute and chronic graft versus host disease (GVHD), which remains a major obstacle for allogeneic HCT as a curative therapy for hematological malignancies. Chronic GVHD is a systemic lupus- and scleroderma-like autoimmune syndrome. We and others reported that donor B cells play important roles in augmenting pathogenesis of chronic GVHD in mouse models and humans. Rituxan (an anti-CD20 mAb) has been used in the clinic for the treatment of ongoing chronic GVHD, but the effect is variable and minimum in some reports, and the mechanisms for this ineffectiveness remains unclear. In the current studies, we evaluated the effect of in vivo administration of a depleting anti-CD20 mAb in preventing and treating autoimmune-like chronic GVHD as well as the impact on GVL effect. With the mouse chronic GVHD model of DBA/2 donor to MHC-matched BALB/c recipient established in our lab (Blood, 2006, J. Immunol 2012), we found that one intravenous injection of anti-CD20 (50mg/kg) immediately after HCT effectively prevented induction of autoimmune-like chronic GVHD. While all (12/12) recipients treated with control IgG developed chronic GVHD with proteinuria and hair-loss and died by 30 days, none (0/12) of the anti-CD20-treated recipients developed proteinuria or hair-loss and all survived for more than 50 days after HCT without signs of chronic GVHD. In addition, the anti-CD20-treated recipients eliminated BCL1 leukemia/lymphoma cells without signs of chronic GVHD. The preventative anti-CD20 treatment had little impact on donor CD8+ T cell activation and expansion in the periphery and allowed for the strong CD8+ T cell-mediated GVL effect. The effective prevention of chronic GVHD by anti-CD20 was associated with significant changes in donor B and CD4+ T cells as well as associated with protection of host thymus. Preventive anti-CD20 treatment depleted IL-6-producing donor B cells and increased IL-10-producing B cells. In addition, the treatment also significantly reduced donor CD4+ T cell expansion, the percentage of IFN-g-producing CD4+ T as well as CD4+ T cells expressing CD8αα, the latter of which has recently been reported to represent over-activated pathogenic CD4+ T cells, such that the host thymus was protected from donor T-cell-mediated damage. Anti-CD20 or control IgG treatment of recipients with ongoing chronic GVHD 15-20 days after HCT did not show any difference in disease progress, and all (8/8) in each group died within 30 days after HCT. The ineffectiveness of anti-CD20 therapy was associated with little reduction of CD19+ B or CD4+ T cells, as the majority of CD19+ B cells became CD20- in the recipients with ongoing chronic GVHD. These results indication that anti-CD20 can effectively prevent induction of autoimmune-like chronic GVHD while preserving the GVL effect, but anti-CD20 is ineffective in treating ongoing chronic GVHD. (This study is supported by the Nesvig Lymphoma Foundation). Disclosures: Chan: Genentech Inc.: Employment.

scholarly journals The role of H-2-linked genes in helper T cell function. VII. Expression of I region and immune response genes by B cells in bystander help assays.

1980 ◽  
Vol 152 (5) ◽  
pp. 1274-1288 ◽  
Author(s):  
P Marrack ◽  
J W Kappler
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Cell Function ◽  
High Responder ◽  
Helper T Cells ◽  
Spleen Cells ◽  
Region Gene ◽  
Immunoglobulin Receptor

The mode of action by bystander helper T cells was investigated by priming (responder X nonresponder) (B6A)F1 T cells with poly-L-(Tyr, Glu)-poly-D,L-Ala--poly-L-Lys [(TG)-A--L] and titrating the ability of these cells to stimulate an anti-sheep red blood cell (SRBC) response of parental B cells and macrophages in the presence of (TG)-A--L. Under limiting T cell conditions, and in the presence of (TG)-A--L, (TG)-A--L-responsive T cells were able to drive anti-SRBC responses of high-responder C57BL/10.SgSn (B10) B cells and macrophages (M0), but not of low-responder (B10.A) B cells and M0. Surprisingly, the (TG)-A--L-driven anti-SRBC response of B10.A B cells was not restored by addition of high-responder acessory cells, in the form of (B6A)F1 peritoneal or irradiated T cell-depleted spleen cells, or in the form of B10 nonirradiated T cell-depleted spleen cells. These results suggested that (TG)-A--L-specific Ir genes expressed by B cells controlled the ability of these cells to be induced to respond to SRBC by (TG)-A--L-responding T cells, implying that direct contact between the SRBC-binding B cell precursor and the (TG)-A--L-responsive helper T cells was required. Analogous results were obtained for keyhold limpet hemocyanin (KLH)-driven bystander help using KLH-primed F1 T cells restricted to interact with cells on only one of the parental haplotypes by maturing them in parental bone marrow chimeras. It was hypothesized that bystander help was mediated by nonspecific uptake of antigen [(TG)-A--L or KLH] by SRBC-specific b cells and subsequent display of the antigen on the B cell surface in association with Ir of I-region gene products, in a fashion similar to the M0, where it was then recognized by helper T cells. Such an explanation was supported by the observation that high concentrations of antigen were required to elicit bystander help. This hypothesis raises the possibility of B cell processing of antigen bound to its immunoglobulin receptor and subsequent presentation of antigen to helper T cells.

scholarly journals B cell depletion with anti-CD20 mAb exacerbates anti-donor CD4+ T cell responses in highly sensitized transplant recipients

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Asuka Tanaka ◽  
Kentaro Ide ◽  
Yuka Tanaka ◽  
Masahiro Ohira ◽  
Hiroyuki Tahara ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
T Cell Responses ◽  
Cell Depletion ◽  
B Cell Depletion ◽  
Transplant Recipients ◽  
Cell Responses ◽  
Anti Cd20

AbstractPretransplant desensitization with rituximab has been applied to preformed donor-specific anti-human leukocyte antigen antibody (DSA)-positive recipients for elimination of preformed DSA. We investigated the impact of pretransplant desensitization with rituximab on anti-donor T cell responses in DSA-positive transplant recipients. To monitor the patients’ immune status, mixed lymphocyte reaction (MLR) assays were performed before and after desensitization with rituximab. Two weeks after rituximab administration, the stimulation index (SI) of anti-donor CD4+ T cells was significantly higher in the DSA-positive recipients than in the DSA-negative recipients. To investigate the mechanisms of anti-donor hyper responses of CD4+ T cells after B cell depletion, highly sensitized mice models were injected with anti-CD20 mAb to eliminate B cells. Consistent with clinical observations, the SI values of anti-donor CD4+ T cells were significantly increased after anti-CD20 mAb injection in the sensitized mice models. Adding B cells isolated from untreated sensitized mice to MLR significantly inhibited the enhancement of anti-donor CD4+ T cell response. The depletion of the CD5+ B cell subset, which exclusively included IL-10-positive cells, from the additive B cells abrogated such inhibitory effects. These findings demonstrate that IL-10+ CD5+ B cells suppress the excessive response of anti-donor CD4+ T cells responses in sensitized recipients.

scholarly journals Vitamin D/CD46 Crosstalk in Human T Cells in Multiple Sclerosis

2020 ◽  
Vol 11 ◽  
Author(s):  
Justin Killick ◽  
Joanne Hay ◽  
Elena Morandi ◽  
Sonja Vermeren ◽  
Saniya Kari ◽  
...  
Keyword(s):  
Multiple Sclerosis ◽  
Vitamin D ◽  
T Cells ◽  
T Cell ◽  
Chronic Inflammatory Disease ◽  
Vitamin D Supplementation ◽  
Type I ◽  
T Cell Migration ◽  
The Impact

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS), in which T-cell migration into the CNS is key for pathogenesis. Patients with MS exhibit impaired regulatory T cell populations, and both Foxp3+ Tregs and type I regulatory T cells (Tr1) are dysfunctional. MS is a multifactorial disease and vitamin D deficiency is associated with disease. Herein, we examined the impact of 1,25(OH)2D3 on CD4+ T cells coactivated by either CD28 to induce polyclonal activation or by the complement regulator CD46 to promote Tr1 differentiation. Addition of 1,25(OH)2D3 led to a differential expression of adhesion molecules on CD28- and CD46-costimulated T cells isolated from both healthy donors or from patients with MS. 1,25(OH)2D3 favored Tr1 motility though a Vitamin D-CD46 crosstalk highlighted by increased VDR expression as well as increased CYP24A1 and miR-9 in CD46-costimulated T cells. Furthermore, analysis of CD46 expression on T cells from a cohort of patients with MS supplemented by vitamin D showed a negative correlation with the levels of circulating vitamin D. Moreover, t-Distributed Stochastic Neighbor Embedding (t-SNE) analysis allowed the visualization and identification of clusters increased by vitamin D supplementation, but not by placebo, that exhibited similar adhesion phenotype to what was observed in vitro. Overall, our data show a crosstalk between vitamin D and CD46 that allows a preferential effect of Vitamin D on Tr1 cells, providing novel key insights into the role of an important modifiable environmental factor in MS.

scholarly journals Anti-CD20 therapy depletes activated myelin-specific CD8+T cells in multiple sclerosis

2019 ◽  
Vol 116 (51) ◽  
pp. 25800-25807 ◽  
Author(s):  
Joseph J. Sabatino ◽  
Michael R. Wilson ◽  
Peter A. Calabresi ◽  
Stephen L. Hauser ◽  
Jonathan P. Schneck ◽  
...  
Keyword(s):  
Multiple Sclerosis ◽  
T Cells ◽  
T Cell ◽  
Ex Vivo ◽  
Antibody Therapy ◽  
Myelin Proteins ◽  
T Cell Epitopes ◽  
Control Subjects ◽  
Anti Cd20

CD8+T cells are believed to play an important role in multiple sclerosis (MS), yet their role in MS pathogenesis remains poorly defined. Although myelin proteins are considered potential autoantigenic targets, prior studies of myelin-reactive CD8+T cells in MS have relied on in vitro stimulation, thereby limiting accurate measurement of their ex vivo precursor frequencies and phenotypes. Peptide:MHC I tetramers were used to identify and validate 5 myelin CD8+T cell epitopes, including 2 newly described determinants in humans. The validated tetramers were used to measure the ex vivo precursor frequencies and phenotypes of myelin-specific CD8+T cells in the peripheral blood of untreated MS patients and HLA allele-matched healthy controls. In parallel, CD8+T cell responses against immunodominant influenza epitopes were also measured. There were no differences in ex vivo frequencies of tetramer-positive myelin-specific CD8+T cells between MS patients and control subjects. An increased proportion of myelin-specific CD8+T cells in MS patients exhibited a memory phenotype and expressed CD20 compared to control subjects, while there were no phenotypic differences observed among influenza-specific CD8+T cells. Longitudinal assessments were also measured in a subset of MS patients subsequently treated with anti-CD20 monoclonal antibody therapy. The proportion of memory and CD20+CD8+T cells specific for certain myelin but not influenza epitopes was significantly reduced following anti-CD20 treatment. This study, representing a characterization of unmanipulated myelin-reactive CD8+T cells in MS, indicates these cells may be attractive targets in MS therapy.

scholarly journals Inhibition of normal B-cell function by human immunodeficiency virus envelope glycoprotein, gp120

Blood ◽  
1992 ◽  
Vol 79 (5) ◽  
pp. 1245-1254 ◽  
Author(s):  
N Chirmule ◽  
N Oyaizu ◽  
VS Kalyanaraman ◽  
S Pahwa
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Cell Function ◽  
Cell Contact ◽  
Cell Proliferation And Differentiation ◽  
Cell Clones ◽  
Glycoprotein Gp120

Abstract Despite the occurrence of hypergammaglobulinemia in human immunodeficiency virus (HIV) infection, specific antibody production and in vitro B-cell differentiation responses are frequently impaired. In this study, we have examined the effects of HIV envelope glycoprotein gp120 on T-helper cell function for B cells. In the culture system used, B-cell functional responses were dependent on T-B- cell contact, since separation of T and B cells in double chambers by Transwell membranes rendered the B cells unresponsive in assays of antigen-induced B-cell proliferation and differentiation. Cytokines secreted by T cells were also essential, since anti-CD3 monoclonal antibody (mAb)-activated, paraformaldehyde-fixed T-cell clones failed to induce B-cell proliferation and differentiation. Pretreatment of the CD4+ antigen-specific T cells with gp120 was found to impair their ability to help autologous B cells, as determined by B-cell proliferation, polyclonal IgG secretion, and antigen-specific IgG secretion. The gp120-induced inhibition was specific in that it was blocked by soluble CD4. Furthermore, only fractionated small B cells (which are T-cell-dependent in their function) manifested impaired responses when cultured with gp120-treated T cells. Antigen-induced interleukin (IL)-2 and IL-4, but not IL-6, secretion were markedly reduced in gp120-treated T-cell clones. Addition of exogenous cytokines failed to compensate for defective helper function of gp120-treated T cells. The findings in this study indicate that gp120 impairs helper functions of CD4+ T cells by interfering with T-B-cell contact- dependent interaction; the inhibitory effects of soluble envelope proteins of HIV may contribute to the immunopathogenesis of the HIV- associated disease manifestations.

scholarly journals Ectopic Lymphoid Follicles in Multiple Sclerosis: Centers for Disease Control?

2020 ◽  
Vol 11 ◽  
Author(s):  
Austin Negron ◽  
Olaf Stüve ◽  
Thomas G. Forsthuber
Keyword(s):  
Multiple Sclerosis ◽  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
T Cell Responses ◽  
Memory B Cells ◽  
Lymphoid Follicles ◽  
Cell Responses ◽  
B Cell Responses

While the contribution of autoreactive CD4+ T cells to the pathogenesis of Multiple Sclerosis (MS) is widely accepted, the advent of B cell-depleting monoclonal antibody (mAb) therapies has shed new light on the complex cellular mechanisms underlying MS pathogenesis. Evidence supports the involvement of B cells in both antibody-dependent and -independent capacities. T cell-dependent B cell responses originate and take shape in germinal centers (GCs), specialized microenvironments that regulate B cell activation and subsequent differentiation into antibody-secreting cells (ASCs) or memory B cells, a process for which CD4+ T cells, namely follicular T helper (TFH) cells, are indispensable. ASCs carry out their effector function primarily via secreted Ig but also through the secretion of both pro- and anti-inflammatory cytokines. Memory B cells, in addition to being capable of rapidly differentiating into ASCs, can function as potent antigen-presenting cells (APCs) to cognate memory CD4+ T cells. Aberrant B cell responses are prevented, at least in part, by follicular regulatory T (TFR) cells, which are key suppressors of GC-derived autoreactive B cell responses through the expression of inhibitory receptors and cytokines, such as CTLA4 and IL-10, respectively. Therefore, GCs represent a critical site of peripheral B cell tolerance, and their dysregulation has been implicated in the pathogenesis of several autoimmune diseases. In MS patients, the presence of GC-like leptomeningeal ectopic lymphoid follicles (eLFs) has prompted their investigation as potential sources of pathogenic B and T cell responses. This hypothesis is supported by elevated levels of CXCL13 and circulating TFH cells in the cerebrospinal fluid (CSF) of MS patients, both of which are required to initiate and maintain GC reactions. Additionally, eLFs in post-mortem MS patient samples are notably devoid of TFR cells. The ability of GCs to generate and perpetuate, but also regulate autoreactive B and T cell responses driving MS pathology makes them an attractive target for therapeutic intervention. In this review, we will summarize the evidence from both humans and animal models supporting B cells as drivers of MS, the role of GC-like eLFs in the pathogenesis of MS, and mechanisms controlling GC-derived autoreactive B cell responses in MS.

Immune Characteristics Associated with Lymphomatoid Granulomatosis and Outcome Following Treatment with Interferon-Alpha

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 963-963 ◽  
Author(s):  
Kieron Dunleavy ◽  
Pratip Chattopadhyay ◽  
Junichi Kawada ◽  
Sara Calattini ◽  
Emma Gostick ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Complete Remission ◽  
Interferon Alpha ◽  
Cd8 T Cells ◽  
Cell Function ◽  
Conflicts Of Interest ◽  
Lymphomatoid Granulomatosis ◽  
Grade Iii

Abstract Abstract 963 Lymphomatoid granulomatosis (LYG) is a rare angiocentric/angiodestructive EBV+ B-cell lymphoproliferative disorder. LYG has a spectrum of clinical aggressiveness and histological grading. Grading relates to the number of EBV-positive B-cells with grade I /II being usually polyclonal or oligoclonal and grade III monoclonal. Historical outcomes of patients treated with steroids and/or chemotherapy have been poor with median survivals of 14 months. We have shown that LYG is associated with reduced CD8+ and CD4+ T-cells, and hypothesized that patients have defective immune surveillance of EBV+ B-cells. We are investigating the use of interferon-alpha (IFα) for grade I/II disease and have characterized the maturation, exhaustion, and homeostatic potential of bulk and antigen-specific CD8 T-cells. Patients with grade III disease are treated with DA-EPOCH-R. Characteristics of 53 patients on study include male sex 68%; median age (range) 46 (17-67) and median ECOG P.S. 1 (0-3). Disease sites include lung 98%, CNS 38%, kidney 15%, skin 17% and liver 19%. LYG grades are I –30%, II-26% and III-44%. Prior treatment was none –28%, chemotherapy+/− R-34% and steroids alone – 40% of patients. Herein, we report the outcome of patients with grade I/II LYG treated with IFα. IFα was commenced at 7.5 MIU TIW and dose escalated until best response and then continued for 1 year. Of 31 patients with grade I/II LYG treated with IFα, 28 were evaluable for response. Of these, 17 (60%) achieved a complete remission and 6 (21%) patients progressed with grade III disease and received chemotherapy. Of 10 patients with CNS disease, 9 achieved a CR with IFα. At a median follow-up time of 5 years, the progression-free survival of grade I/II LYG was 56%. The median time to remission was 9 months (3-40) and median IFα dose was 20 MIUs (7-40). Median EBV viral loads at study entry were 18 copies/106 genome equivalents (0-22727) (normal<200). We looked at T-cell kinetics in patients who achieved complete remission and observed statistically significant recovery in both CD4 (p=0.034) and CD8 p=0.034) cells after interferonα. We were interested in further elucidating T-cell function and used polychromatic flow cytometry to characterize CD8 T-cells in the peripheral blood of patients before and after IFα. In 17 patient samples, cells were stained with peptide-MHC I (pMHCI) multimers directed against T-cells specific for epitopes from latent and lytic EBV proteins along with antibodies defining CD8 sub-populations. Influenza or cytomegalovirus-specific pMHCI multimers were controls. We observed no difference in the frequency of EBV specific CD8 T-cells in the blood of LYG patients compared to controls. However, CD27 and PD1 expression appeared to be altered in the bulk CD8+ T-cells and in selected EBV-specific populations in LYG patients; these changes were marginally significant. Following completion of IFα, expression of PD-1, CD27 and CD127 were at normal levels. Evidence from some LYG patients suggests that IL2 production by EBV-specific T-cells is lost during LYG, and normalized after therapy. Our results suggest that LYG, an EBV-associated disease, may arise in the setting of a global deficit in CD8 T-cells with selected defects in EBV-specific immunity that resolve with successful therapy. Disclosures: No relevant conflicts of interest to declare.

Impact of Lenalidomide on Gene Expression Profiles of Malignant and Immune Cells in Patients with Chronic Lymphocytic Leukemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 976-976 ◽  
Author(s):  
John C. Riches ◽  
Ajanthah Sangaralingam ◽  
Shahryar Kiaii ◽  
Tracy Chaplin ◽  
Demet Cekdemir ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Family Member ◽  
Cell Function ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Lymphocytic Leukemia ◽  
Healthy Donors

Abstract Abstract 976 Lenalidomide has recently been demonstrated to have significant activity in chronic lymphocytic leukemia (CLL). Its mechanism of action in this disease is not well understood, but it is thought to act primarily by enhancing anti-tumor immunity and reducing production of pro-tumoral factors in the CLL microenvironment. We have previously demonstrated alterations in the expression of cytoskeletal genes in T-cells from patients with CLL and have subsequently shown that these changes translate into a deficit in T-cell function, due to impaired actin polymerization resulting in defective immunological synapse formation. Treatment of both autologous T-cells and CLL cells with lenalidomide was necessary to repair this defect, suggesting that this may be a key component of this agent's activity in CLL. Therefore we examined the effect of lenalidomide on the global gene expression profiles of isolated B-cells and T-cell subsets from CLL patients and healthy donors. Peripheral blood mononuclear cells from patients with untreated CLL or healthy donors were cultured in the presence of 1 μM lenalidomide or vehicle control for 48 hours. The lymphocyte subsets were isolated, followed by RNA extraction and gene expression profiling using the Affymetrix HGU133Plus2.0 platform. Lenalidomide treatment had similar effects on gene expression in T-cells from both patients with CLL and healthy donors. The most prominent changes in expression were of genes involved in cytoskeletal signaling including a 20-fold increase in WASF1 (Wiskott Aldrich Syndrome protein family, member 1), and greater than 2-fold increases in the expression of Rac-family member RHOC, (Ras homolog gene family, member C), actin binding proteins CORO1B (Coronin 1B), PARVA (Parvin alpha), and the Rho guanine nucleotide exchange factors (GEFs), ARHGEF5 and ARHGEF7. We also observed changes in genes regulating integrin signaling including PXN (Paxilin) and FAK (Focal adhesion kinase), and a shift towards Th1 differentiation with upregulation of TNF, IL-12R, and IL-18R. In addition, we noted increased expression of the transcription factors IKZF1, IKZF4 and IRF4, genes involved in the Ikaros pathways that are essential for hematopoiesis and control of lymphoid proliferation. These changes in gene expression provide further evidence that an important mechanism of action of lenalidomide is the upregulation of the actin cytoskeletal network including Rho-GTPases and integrin activation signaling, and are consistent with our previous observations concerning the functional repair of T-cells in CLL. Initial analysis of the effect of lenalidomide on the gene expression profiles of the CLL B-cells showed similar changes to those previously described in vivo from CLL patients receiving single agent lenalidomide in a clinical trial (Chen et al. JCO 2010). In our system, lenalidomide treatment resulted in a greater than 2-fold upregulation of 189 genes, and a greater than 2-fold downregulation of 85 genes in CLL B-cells. We observed increased expression of several genes belonging to the TNF superfamily including TNF-α, OX40L, and APRIL, and the receptors DR5, DCR2, and OX40. Many of these are known to mediate apoptosis signaling, and we also observed increased expression of pro-apoptotic genes such as FAS, BID (BH3 interacting domain death agonist), HRK (Harakiri), and CFLAR (CASP8 and FADD-like apoptosis regulator), and cell cycle regulators CDKN1A and CDKN1C (Cyclin-dependent kinase inhibitors 1A and 1C). Lenalidomide also upregulated expression of several genes of known importance in the CLL microenvironment, including the chemokines CCL3 and CCL4, CD40, CD274 (PD-L1), CD279 (PD-1), and adhesion molecules LFA3 and ICAM1. The effect of lenalidomide on the gene expression profiles of normal B-cells was less marked, with greater than 2-fold upregulation of 51 genes and downregulation of 12 genes. However, we did observe that lenalidomide treatment induced upregulation of genes involved in cytoskeletal pathways such as RND1 (Rho family GTPase 1), RHOQ (Ras homolog gene family, member Q), and MYO1B (myosin 1B). In conclusion, investigation of the effect of lenalidomide on gene expression profiling in CLL suggests that the drug acts both to enhance T-cell function, and to render the CLL cells more susceptible to immune cell mediated killing. Disclosures: Gribben: Roche: Honoraria; Celgene: Honoraria; GSK: Honoraria; Mundipharma: Honoraria; Gilead: Honoraria; Pharmacyclics: Honoraria.

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