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.

Cell Contact with CLL Cells Induces Defects in T Cell Differentiation and Effector Pathways: Impact of Silencing Specific Cytokine Production.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 955-955
Author(s):  
Tobias A.W. Holderried ◽  
Gullu Gorgun ◽  
John G. Gribben
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Cd8 T Cells ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Cytoskeletal Proteins ◽  
Cell Contact ◽  
Soluble Factors ◽  
Healthy Donors

Abstract Previous studies have suggested that the development and progression of B cell CLL is dependent on interactions between malignant cells and normal components of the immune system. Although the T cell count may be normal or even increased, T cell dysfunction is a feature of CLL, with abnormal CD4/CD8 ratio, impaired mitogen response, and altered expression of surface antigens in response to antigen presentation. We are evaluating the impact of tumor cells on the immune system and have reported differences in gene expression profiles in T cells in previously untreated CLL patients. Specifically, in both CD4 and CD8 T cells we identified defects in genes regulating cytoskeleton formation, intracellular transportation and control of cytokines and chemokines. Analysis of the abnormal gene expression profiles suggested that many such abnormalities are induced by signaling through surface receptors on T cells by interaction with CLL cells or the microenvironment. To detemine the mechanism, we performed ex vivo tumor cell-T cell interaction assays using patient derived serum, transwell membrane and cell contact assays using CLL and CD4 or CD8 T cells from CLL patients and B cells and CD4 or CD8 T cells from healthy donors. Since the majority of the differentially expressed genes were involved in cell cytoskeleton formation and intracellular vesicle transportation pathways, we examined the impact on those specific pathways using proteomics. In keeping with the gene expression profiles in the T cells of CLL patients, we noted significant decreased expression of NFkB p65 and GDI1 and increase in Arp2/3 in healthy CD4 T cells and decreased expression of Rho-GAP and increased Arp2/3 in healthy CD8 T cells following CLL-T cell contact compared to healthy B cell-T cell contact. In contrast there was no change after exposure to patient sera or tumor cell derived soluble factors for 48h. To further analyze whether tumor cell derived cytokines have an impact on T cells in CLL, we inhibited IL-10 and IL-4 production in CLL cells and healthy B cells using siRNA targeting IL-10 and/or IL-4. The transfection efficiency monitored by flow cytometry with fluorescence labeled non-silencing RNA and observed 60–98 % transfection efficiency and at 48 h observed 40–85% inhibition in IL-10 protein expression. After 48 h incubation of autologous and allogeneic CD4 or CD8 T cells from CLL and healthy donors with non-transfected, mock transfected or IL-10 siRNA transfected CLL or healthy B cells for 48h, T cells were isolated and there was no significant difference in expression of cytoskeletal proteins in both CD4 or CD8 T cells. Addition of anti-IL-10 neutralizing monoclonal antibody also had no effect. Although no effect was noted on cytoskeletal proteins, after incubation with CLL but not healthy B cells, silencing or neutralization of IL-10 induced changes in expression of CXCR1, CXCR2, CXCR3, CXCR4 and CCR5 in CD4 and CCR5 and CCR4 in CD8 cells from healthy donors. We conclude that cell contact with CLL cells induces changes in expression of cytoskeletal proteins in healthy T cells similar to those observed in T cells from CLL patients and this is not induced by soluble factors including IL-10. However, IL-10 and potentially other soluble factors induce changes in chemokines and chemokine receptors on T cells, suggesting multiple mechanisms impair T cell function in CLL cells. Ongoing studies are assessing ways to repair the defects identified here to enhance immune responsiveness in this disease.

Nurselike Cells in Chronic Lymphocytic Leukemia Have Gene Expression Profiles and Functional Characteristics That Are Distinct from Those of Monocytes or Dendritic Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1183-1183
Author(s):  
Davorka Messmer ◽  
Tomoyuki Endo ◽  
Bradley T. Messmer ◽  
Danelle James ◽  
Nathan J. Zvaifler ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cells ◽  
Mononuclear Cells ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Cell Types ◽  
Lymphocytic Leukemia ◽  
Mhc Ii ◽  
Blood Mononuclear Cells

Abstract When CD14+ blood mononuclear cells are cultured with chronic lymphocytic leukemia (CLL) B cells they can differentiate into “nurselike” cells (NLCs), which in turn can support the survival of CLL B cells in vitro and possibly in vivo. While factors that contribute to NLC-mediated support of CLL B cell survival have been identified, it is not clear how this cell type relates to other cell types that also can differentiate from CD14+ blood cells, such as monocyte-derived dendritic cells (DCs). Prior studies have identified some phenotypic differences between NLCs, DCs, and other CD14+ blood mononuclear cells. Thus we hypothesized that these cell types may have different gene expression patterns that may relate to distinctive functional properties. To resolve this we examined the genes expressed by monocytes, NLCs, and immature DCs using Affymetrix U133 microarray analyses. Gene expression profiles were generated from the CD14+ monocyte progenitors, NLC, and DC from three different individuals. The expression profiles of DCs and NLCs differed from the CD14+ progenitors by the expression of many thousands of genes and NLC were distinguished from DCs by the expression of several hundred genes. Some of the genes expressed at higher levels in DCs relative to NLCs encode accessory molecules involved in antigen presentation. Consistent with this, we found that immature DCs were 10 times more effective than NLCs in presenting antigen to allogeneic T cells. DCs express toll like receptors (TLR) on their cell surface that recognize pathogen components and upon exposure to TLR ligands DCs undergo a maturation process, whereby they upregulate surface molecules and gain increased T cell stimulatory capacity. The expression of TLR2, 4, and 9 was analyzed in DCs and NLCs by RT PCR. Both DCs and NLCs were found to express mRNA for TLR2 and 4, but only NLCs expressed TLR9. In concordance with this, NLCs but not DCs unregulated MHC-II after exposure to nonmethylated CpG oligodeoxinucleotides (ODN), a TLR9 agonist, whereas both cell types upregulated MHC-II after exposure to lipopolysaccharide. Given the propensity of CD14+ cells to differentiate down a NLC pathway when co-cultured with leukemic B cells in vitro, we speculate that differentiation of CD14+ cells into NLCs may be favored in patients with CLL over differentiation into DCs. Given the relative differences in APC function of these two cell types, this may account in part for the acquired immune deficiency often observed in patients with this disease. On the other hand a stimulus like CpG ODN, might increase the ability of NLCs to activate T cells and decrease their ability to support CLL B cells survival.

Donor T Cell Gene Expression and GVHD.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. sci-53-sci-53
Author(s):  
Claude Perreault
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Expression Profiling ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Qrt Pcr ◽  
One Year ◽  
Donor Gene

GVHD is initiated by donor T cell responses to host alloantigens. However, the occurrence and severity of GVHD are not determined solely by the level of histoincompatibility between donor and recipient. Two MHC-identical subjects will display over 50 minor histocompatibility antigen differences. If histoincompatibility is sufficient for triggering GVHD, the rate of GVHD in MHC-matched recipients of allogeneic hematopoietic cell transplantation (HCT) that receive no immunosuppressive agents should be 100%. Under these conditions, however, GVHD is found in only 50% and 73% of mouse and human recipients, respectively. Histoincompatibility is thus necessary but not sufficient to elicit GVHD. We tested the hypothesis that some donors may be “stronger alloresponders” than others, and consequently more likely to elicit GVHD. To this end, we studied the gene expression profiles of CD4 and CD8 T cells from 50 HCT donors using microarrays and qRT-PCR. We found that gene expression profiling before HCT was able to distinguish those donors whose cells caused GVHD from those whose cells did not. The “dangerous donor” trait (GVHD+ recipient) is under polygenic control and is shaped by the activity of genes that regulate TGF-β signaling and cell proliferation. The donor gene profile defined on day 0 shows strong correlation with that of recipient CD4 and CD8 T cells harvested one year post-AHCT. The latter correlation provides compelling evidence that a significant portion of the differential gene profiles between GVHD+ and GVHD– donors is imprinted at the hematopoietic stem cell level. Moreover, stability of the gene expression profiles over a one-year period suggests that the profiles result from inherited genetic traits as opposed to environmental factors. The gene with the best GVHD-predictive accuracy was SMAD3, a key component of the TGF-β pathway. By testing a cohort of 450 subjects using qRT-PCR, we found that amounts of SMAD3 transcripts varied over a 6-fold range. In mice and humans, SMAD3 is constitutively activated (as evidenced by phosphorylation and accumulation in the nucleus) in many leukocyte subsets. We found in mice that induction of TGF-β signaling in donor T cells is an early event following AHCT and that Smad3-deficient donors trigger more severe GVHD than wild-type littermates. These findings strongly suggest that the donor gene expression profile has a dominant influence on the occurrence of GVHD. In allogeneic HCT, the ability to discriminate strong and weak alloresponders using gene expression profiling could help select low-risk donors and permit tailoring GVHD prophylaxis regimens according to the probability of GVHD occurrence.

Intraclonal Complexity Of CLL Fractions In Cell Proliferation Rates, Gene Expression Signatures, and Responses To Autologous T-Cell Help In Peripheral blood and Secondary Lymphoid Tissues

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 115-115
Author(s):  
Shih-Shih Chen ◽  
Thomas M. Herndon ◽  
Claire Emson ◽  
John C. Riches ◽  
Fabienne McClanahan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Lymphoid Tissues ◽  
T Cell Help

Abstract Chronic lymphocytic leukemia (CLL) is a disease of resting and dividing B cells. In order to understand the trafficking and transition of proliferative and resting fractions between periphery and lymphoid tissues, we have tried to [1] define the relative levels of CLL B-cell proliferation in distinct anatomic sites, [2] identify and characterize CLL intraclonal fractions from these sites that differ in time since last replication, and [3] model the abilities of these subsets to repopulate immune deficient mice. In the blood, resting and recently-divided CLL cells can be distinguished by surface expression of CXCR4 and CD5. CXCR4dimCD5br (DIM) cells are an activated subset, CXCR4intCD5int (INT) cells are the clonal bulk, and CXCR4brCD5dim (BR) cells are older and more quiescent. Here we compared in vivo proliferation rates, gene expression profiles, and differences in xenografting of these 3 fractions. In addition, we studied the same parameters in 3 other fractions, CXCR4dimCD5dim (dDIM), CXCR4intCD5br (INT/BR) and CXCR4brCD5br (dBR). CLL B-cell kinetics was analyzed in 7 treatment-naïve patients who drank deuterated “heavy” water (2H2O). At day 13, peripheral blood (PB), lymph node (LN), and bone marrow (BM) samples were collected from each patient, and cells from these 3 sites were analyzed for 2H-labeled CD5+CD19+ cells after FACS sorting into 6 fractions: DIM, dDIM, INT, INT/BR, BR and dBR. Overall, more CLL B-cell proliferation was found in LN than PB and BM; only small numbers of divided cells were found in BM. Interestingly, the DIM, dDIM, and INT/BR had the highest 2H-incorporation in LN and PB; and DIM cells from LN and PB showed similar levels of 2H-incorporation. Gene expression profiling using Illumina Human HT12 BeadChips was then performed on the same 6 fractions from PB and LN. Expression value ratios for fractions from each patient were analyzed using R, and sets of significant genes (fold change >1.5 and P<0.05) were determined. Unsupervised hierarchical clustering grouped together the DIM and dDIM fractions from all other fractions. Although the INT/BR had high 2H-incorporation levels, it clustered with the BR, INT and dBR fractions. Genes upregulated in DIM and dDIM included genes involved in cell proliferation and survival, such as DUSP1, PRKCD and BMF, and chemokine genes CCL3, CCL3L1, CCL3L3, CCL4L2. Genes changed in BR, dBR, and INT/BR included negative regulators of cell survival and proliferation, e.g. PRICKLE1 and GRAP. Finally, functional differences in the CLL fractions were analyzed in vivo using NOD/SCID/γcnull (NSG) mice. 3-5x106 PB B cells were injected with 1-1.5x105 resting, autologous T cells. Compared to BR, dBR and INT/BR fractions, DIM and dDIM cells led to more extensive T-cell growth and B-cell expansion in the spleen and BM at week 6. In contrast, INT injected mice exhibited only minimal or no CLL B or T cells. Adding INT cells suppressed DIM and dDIM induced T-cell expansion. This function, which was resistant to irradiation, was associated with greater immunological synapse impairment in vitro. Thus, in vivo kinetics analysis using D2O identified levels of divided cells based on the following ranking: DIM ≥ INT/BR> dDIM > INT> BR=dBR. Although the INT/BR fraction contains recently-divided cells based on 2H-labeling, its genetic signature is similar to fractions containing lesser numbers of recently activated cells, suggesting INT/BR cells have divided but are being inactivated in the microenvironment. Therefore, these cells may represent a transition population between proliferating and resting cells. Because PB and LN DIM fractions are very similar in proliferation rates and gene expression profiles and because little CLL B-cell proliferation occurs in the blood, the circulating DIM fraction accurate reflects LN proliferation. When combining kinetics and gene expression profiling, the DIM and dDIM fractions appear as the most activated intraclonal fractions. In xenografted mice, PB DIM and dDIM cells are better at activating T cells and consequently receiving T-cell help for engraftment and growth. The INT fraction, which constitutes the tumor bulk, inhibits T-cell activation. Finally, the BR and dBR fraction are the older, most quiescent cells in CLL clones. Overall, these results inform about trafficking and transition of CLL cells between lymphoid tissues and periphery, and provide a rationale for preferential therapeutic targeting of these fractions. Disclosures: Riches: Celgene: Research Funding.

scholarly journals Chronic Lymphocytic Leukemia B Cells Can Express CD40 Ligand and Demonstrate T-Cell Type Costimulatory Capacity

Blood ◽  
1998 ◽  
Vol 91 (8) ◽  
pp. 2689-2697 ◽  
Author(s):  
Elaine J. Schattner ◽  
John Mascarenhas ◽  
Inna Reyfman ◽  
Mary Koshy ◽  
Caroline Woo ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Cell Function ◽  
Cd40 Ligand ◽  
Lymphocytic Leukemia ◽  
Similar Degree ◽  
Cd40l Expression

Chronic lymphocytic leukemia (CLL) is characterized by a clonal expansion of CD5+ B cells in the peripheral blood. Associated immune aberrations include abnormal Th-cell function and pathogenic autoantibodies. Under most circumstances, CLL B cells do not proliferate in culture and express a limited repertoire of surface antigens, including CD19, CD20, CD23, CD27, CD40, and CD70. In this report, we demonstrate that freshly isolated B cells from a subset of CLL cases constitutively express CD40 ligand (CD40L, CD154), a member of the tumor necrosis factor family which is normally expressed by activated CD4+ T cells and mediates T-cell–dependent B-cell proliferation and antibody production. The degree of CD40L expression varied considerably among the CLL cases examined. CD40L was detected in purified CLL B cells by immunofluorescence flow cytometry, by RT-PCR, and by immunoprecipitation. To demonstrate that CD40L in the CLL B cells is functional, we used irradiated CLL cells to stimulate IgG production by target, nonmalignant B cells in coculture. The CLL B cells induced IgG production by normal B cells to a similar degree as did purified T cells in a process which was partially inhibited by monoclonal antibody to CD40L. This is one of the first reports of CD40L expression in a B-cell tumor. The data suggest that CD40L in the tumor cells may be a factor in the generation of pathologic antibodies by normal B cells in some patients with CLL.

scholarly journals Gene Expression Profiling of B Cell Chronic Lymphocytic Leukemia Reveals a Homogeneous Phenotype Related to Memory B Cells✪

2001 ◽  
Vol 194 (11) ◽  
pp. 1625-1638 ◽  
Author(s):  
Ulf Klein ◽  
Yuhai Tu ◽  
Gustavo A. Stolovitzky ◽  
Michela Mattioli ◽  
Giorgio Cattoretti ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Variable Region ◽  
Memory B Cells ◽  
Lymphocytic Leukemia ◽  
Cell Subpopulations

B cell–derived chronic lymphocytic leukemia (B-CLL) represents a common malignancy whose cell derivation and pathogenesis are unknown. Recent studies have shown that &gt;50% of CLLs display hypermutated immunoglobulin variable region (IgV) sequences and a more favorable prognosis, suggesting that they may represent a distinct subset of CLLs which have transited through germinal centers (GCs), the physiologic site of IgV hypermutation. To further investigate the phenotype of CLLs, their cellular derivation and their relationship to normal B cells, we have analyzed their gene expression profiles using oligonucleotide-based DNA chip microarrays representative of ∼12,000 genes. The results show that CLLs display a common and characteristic gene expression profile that is largely independent of their IgV genotype. Nevertheless, a restricted number of genes (&lt;30) have been identified whose differential expression can distinguish IgV mutated versus unmutated cases and identify them in independent panels of cases. Comparison of CLL profiles with those of purified normal B cell subpopulations indicates that the common CLL profile is more related to memory B cells than to those derived from naive B cells, CD5+ B cells, and GC centroblasts and centrocytes. Finally, this analysis has identified a subset of genes specifically expressed by CLL cells of potential pathogenetic and clinical relevance.

scholarly journals A HaemAtlas: characterizing gene expression in differentiated human blood cells

Blood ◽  
2009 ◽  
Vol 113 (19) ◽  
pp. e1-e9 ◽  
Author(s):  
Nicholas A. Watkins ◽  
Arief Gusnanto ◽  
Bernard de Bono ◽  
Subhajyoti De ◽  
Diego Miranda-Saavedra ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
Transcription Factors ◽  
Cell Function ◽  
Expression Profiles ◽  
Association Studies ◽  
Cytotoxic T Cells ◽  
Gene Expression Profiles ◽  
Immunoglobulin Superfamily ◽  
Genome Wide Association Studies

Abstract Hematopoiesis is a carefully controlled process that is regulated by complex networks of transcription factors that are, in part, controlled by signals resulting from ligand binding to cell-surface receptors. To further understand hematopoiesis, we have compared gene expression profiles of human erythroblasts, megakaryocytes, B cells, cytotoxic and helper T cells, natural killer cells, granulocytes, and monocytes using whole genome microarrays. A bioinformatics analysis of these data was performed focusing on transcription factors, immunoglobulin superfamily members, and lineage-specific transcripts. We observed that the numbers of lineage-specific genes varies by 2 orders of magnitude, ranging from 5 for cytotoxic T cells to 878 for granulocytes. In addition, we have identified novel coexpression patterns for key transcription factors involved in hematopoiesis (eg, GATA3-GFI1 and GATA2-KLF1). This study represents the most comprehensive analysis of gene expression in hematopoietic cells to date and has identified genes that play key roles in lineage commitment and cell function. The data, which are freely accessible, will be invaluable for future studies on hematopoiesis and the role of specific genes and will also aid the understanding of the recent genome-wide association studies.

scholarly journals Lenalidomide and Dexamethasone Combination in Patients with Chronic Lymphocytic Leukemia (CLL) Relapsing or Resistant to Treatment (LENDEX-LLC-09): A Gene Expression Profiling Study

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4675-4675
Author(s):  
Anna Puiggros ◽  
Pau Abrisqueta ◽  
Lara Nonell ◽  
Marta Bodalo ◽  
Eulalia Puigdecanet ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Cd8 T Cells ◽  
Lymphocytic Leukemia ◽  
Down Regulation ◽  
Tnf Α

Abstract Background. Chronic lymphocytic leukemia (CLL) is a highly heterogeneous disease in which immune evasion of tumoral cells, as well as, an impaired CD4 and CD8 T-cell function have been described. Immunomodulatory drugs, such as lenalidomide, alone or in combination with other treatments are promising strategies for those patients with refractory disease. The combination of lenalidomide with dexamethasone has been investigated in multiple myeloma and has revealed as a highly efficient treatment. Nonetheless, the efficacy and mechanisms of action of this combination in CLL have not been elucidated. Aim. To assess the effect of lenalidomide and dexamethasone combination in gene expression of CLL B cells, as well as CD4+ and CD8+ T cells from CLL patients enrolled in LENDEX-LLC-09 trial. Methods. Four patients included in the LENDEX-LLC-09 trial (NCT01246557) were studied (2M/2F, med age 72). All presented with advanced CLL (2 B and 2 C Binet stages), and were previously treated by a minimum of two chemo-immunotherapy regimens. Peripheral blood samples were taken at the recruitment and the 7th day of the first cycle of lenalidomide (2.5mg/day) and dexamethasone (20mg/day, 4 days). Total RNA was extracted from CLL B cells (CD5+ CD19+) and T cells (CD4+ or CD8+) positively selected by immunomagnetic methods (Miltenyi Biotec). Good quality RNA (RIN>7) was hybridized to Human Gene 2.0 ST array (Affymetrix). Differences between gene expression of pretreated and treated samples were assessed for each cell type using linear models for microarrays. Genes with a |logFC|>1 were considered as potentially relevant. Functional analysis was performed using Ingenuity Pathway Analysis (IPA). Results and discussion. The major effect in the gene expression due to treatment was observed in CD4+ T cells, which presented 290 up-regulated genes and 103 down-regulated. CLL cells showed up-regulation of 189 and down-regulation of 53 genes, while increase and decrease in the expression of 112 and 37 genes, respectively, were found in CD8+ T cells. Globally, the most important involved networks were related to cell-to-cell signaling, cellular growth and proliferation, cell death and survival, as well as inflammatory response and immune cell trafficking. Regarding CLL B cells, TNF-α was the most up-regulated gene, as previously described in lenalidomide treated B cells. Contrarily, we did not observe significant differences in genes involved in the immunologic synapse, as CD80, CD86, CD200, PD-L1, CD276 and CD270, which have been reported as key regulators in lenalidomide mechanism of action. Of note, a general increase of genes associated with binding to cells (CD68, CTLA4, ADAM28, ITGAX, LY96) was detected. In contrast to previous studies that demonstrated a growth arrest and induction of apoptosis by lenalidomide or dexamethasone in monotherapy (Baptista et al, 2012; Fecteau et al, 2014), a global inhibition of the apoptosis (up-regulation of BTK and CD79B and inhibition of SMAD7, among others) were observed when both drugs were combined. Considering CD8+ T cells gene expression, an up-regulation of genes involved in leukocyte activation and cell-to-cell binding was detected. The most remarkable changes were found in TNF-α and IFN-γ induction, as well as in ADAM28, LY96 and CD68. In contrast to CD8+ T cells, an inhibition of CD4+ T cell proliferation was observed after the combined treatment (up-regulation of VSIG4, LILRB4 and down-regulation of ICOS). This observation suggests that dexamethasone administration inhibits the CD4+ activation promoted by lenalidomide, as has been described in multiple myeloma (Hsu et al, 2011). Regarding response to treatment, two patients initially presented a complete response with positive minimal residual disease. However, all patients finally progressed after treatment and one died due to disease progression. No significant differences in gene expression patterns were found among patients. Conclusions. Our results suggest that lenalidomide and dexamethasone combination leads to an anti-tumoral activity displayed by an activation of CD8+ T cells against the tumor, rather than an increase of apoptosis in CLL cells. More studies are needed to confirm these preliminary findings of the combined effect of lenalidomide and dexamethasone in refractory CLL patients. Acknowledgments. This work was funded by Celgene, and supported by PI11/1621, 14SGR585 and Fundació LaCaixa. Disclosures Off Label Use: Lenalidomide and dexamethasone combination in CLL.

scholarly journals Investigation of Cell-Contact Dependent Alterations in Immune Synapse Formation between T Cells and BCP-ALL Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2754-2754
Author(s):  
Britta Wenske ◽  
Lothar Marischen ◽  
Julia Welzenbach ◽  
André Heimbach ◽  
Per Hoffmann ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Board Of Directors ◽  
Cell Lines ◽  
Cd4 T Cells ◽  
Synapse Formation ◽  
Cell Function ◽  
Immune Synapse ◽  
Healthy Donors

Cancer-development is often associated with a state of immune attenuation and impaired antigen-recognition, contributing to the failure of immune surveillance. Defective immunological synapse formation between T cells, B cells and leukemic cells has been demonstrated in patients with chronic lymphocytic leukemia (CLL) (Ramsay, A.G., et al., The Journal of Clinical Investigation, 2008. 118(7): p. 2427-37). Furthermore, CLL cells induce T cell dysfunction in a cell-contact dependent manner. Both actin cytoskeleton changes and alterations in RhoGTPase regulation and activation have been reported in T cells of CLL patients (Ramsay, A.G., et al., Blood, 2013. 121(14): p. 2704-14). Precursor B cell acute lymphoblastic leukemia (BCP-ALL) is the most prevalent immunophenotype of childhood leukemia. We therefore hypothesized that gene expression patterns and actin cytoskeleton reorganization, which is involved in immune synapse formation, may also be altered in T cells of children with BCP-ALL and result in modified T cell function. Comparison of global gene expression profiles of purified peripheral blood CD4+ and CD8+ T cells of untreated BCP-ALL patients and healthy donors using 3´-mRNA-seq technique revealed changes in pathways involved in cell communication, migration, adhesion, DNA damage, mitosis, cell cycle, cell death and signaling, suggesting an alteration of T cell function in the presence of leukemic cells. These results were stressed by in vitro experiments assessing the dynamics of immune synapse formation between allogeneic T cells from healthy donors and sAg activated precursor B-ALL cell lines (Nalm6 and REH) or activated, normal B cells using immunofluorescence staining and confocal microscopy. Cell-interactions were selected at random from imaging, counted and scored for accumulation of F-actin at the immune synapse. We found that CD8+ T cells from healthy donors show a significantly impaired ability to form an immunological synapse after direct short term contact with the cell line Nalm6 compared to normal B cells. Of note, this effect was less pronounced in the cell line REH, known to carry a t(12;21) and express the ETV6-RUNX1 fusion transcript. A decreased number of cell interactions was also seen between CD4+ T cells from healthy donors and the mentioned B-ALL cancer cell lines. However, this effect seemed to be time-dependent, as after slightly prolonged co-incubation of CD4+ T cells with the B-ALL cancer cell lines, synapse formation was improved. Interestingly, the F-actin polymerization pattern at the immunological synapse appeared also qualitatively different between allogeneic CD4+ T cells and normal B cells compared to allogeneic CD4+ T cells and B-ALL cancer cell lines. In normal T cell/B cell interactions improved antigen-recognition and T cell activation is mediated by costimulatory molecules. A lack of costimulatory signals on BCP-ALL cells may contribute to impaired immune recognition. Given the quantitative and qualitative differences in immune synapse formation, we analyzed surface expression of the costimulatory molecules CD70, CD80 and CD86 as well as CD40, one of their key regulators, on the BCP-ALL cell lines by flow cytometry and recognized differences similarly to those previously described in primary BCP-ALL cells, with higher TNFR expression in ETV6-RUNX1 positive leukemia cells compared to negative and more immature BCP-ALL cells (Troeger, A et al., Klin Padiatr., 2014 Nov 226(6-7): p. 332-7; Troeger, A et al., Blood 2008 Aug 15;112(4): p. 1028-34); Troeger A et al., Klin Padiatr., 2008 Nov-Dec 220(6): p. 353-7). These preliminary data suggest that immune synapse formation may be quantitatively altered in a time-dependent manner in T cell/BCP-ALL interactions and result in modified actin cytoskeleton rearrangement. More detailed analysis, also using primary BCP-ALL cells, is warranted to further investigate the underlying mechanism in order to identify novel immunomodulatory approaches that can be used to restore impaired T cell function in the presence of BCP-ALL with the aim to overcome immunosuppressive mechanisms and improve the outcome in high risk patients. Disclosures Williams: Novartis: Membership on an entity's Board of Directors or advisory committees; Orchard Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Co-founder, potential for future royalty/milestone income, Research Funding; bluebird bio: Other: License of certain IP relevant to hemoglobinopathies. Potential for future royalty/milestone income. Received payment in past through BCH institutional licensing agreement., Research Funding; Alerion Biosciences: Membership on an entity's Board of Directors or advisory committees, Other: Co-founder.

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