Role of CTLA4 in the proliferation and survival of chronic lymphocytic leukemia

Abstract

Earlier, we reported that CTLA4 expression is inversely correlated with CD38 expression in chronic lymphocytic leukemia (CLL) cells. However, the specific role of CTLA4 in CLL pathogenesis remains unknown. Therefore, to elucidate the possible role of CTLA4 in CLL pathogenesis, CTLA4 was down-regulated in primary CLL cells. We then evaluated proliferation/survival in these cells using MTT, (3)H-thymidine uptake and Annexin-V apoptosis assays. We also measured expression levels of downstream molecules involved in B-cell proliferation/survival signaling including STAT1, NFATC2, c-Fos, c-Myc, and Bcl-2 using microarray, PCR, western blotting analyses, and a stromal cell culture system. CLL cells with CTLA4 down-regulation demonstrated a significant increase in proliferation and survival along with an increased expression of STAT1, STAT1 phosphorylation, NFATC2, c-Fos phosphorylation, c-Myc, Ki-67 and Bcl-2 molecules. In addition, compared to controls, the CTLA4-downregulated CLL cells showed a decreased frequency of apoptosis, which also correlated with increased expression of Bcl-2. Interestingly, CLL cells from lymph node and CLL cells co-cultured on stroma expressed lower levels of CTLA4 and higher levels of c-Fos, c-Myc, and Bcl-2 compared to CLL control cells. These results indicate that microenvironment-controlled-CTLA4 expression mediates proliferation/survival of CLL cells by regulating the expression/activation of STAT1, NFATC2, c-Fos, c-Myc, and/or Bcl-2.

Figure 1. Effects of CTLA4 downregulation on CLL cells.

Panel A: RT-PCR showing decreased expression of CTLA4 in low CD38/high CTLA4 CLL cells treated with CTLA4 antisense (AS) compared to control CLL cells or CLL cells treated with irrelevant AS. RPL13A was used as a housekeeping gene to normalize the cDNA. Panel B: Western blot showing a decrease in the expression of CTLA4 at the protein level in CLL (low CD38/high CTLA4) cells. β-Actin was used as a control. Panel C: Real-time PCR results in samples from five different CLL patients with low CD38 and from three different CLL patients with high CD38 confirm CTLA4 downregulation in CLL cells treated with CTLA4 AS compared to control CLL cells or CLL cells treated with irrelevant AS. Panels D–E: ³H-thymidine uptake assays (Panel D) and MTT assays (Panel E) demonstrating cell proliferation levels in CTLA4-downregulated low CD38/high CTLA4 CLL cells compared to untreated CLL cells and to CLL cells treated with irrelevant AS. *p illustrates the statistical difference between control CLL cells and CTLA4-downregulated CLL cells, while ∧p illustrates the difference between CTLA4-downregulated CLL cells and CLL cells treated with irrelevant AS. Panel F–G: Representative results (panel F) of flow cytometry analyses showing the expression of CTLA4, CD38 and Ki-67 among CD19+ gated CLL (low CD38/high CTLA4) cells untreated, treated with scrambled- or CTLA4-siRNA. Panel G showing quantification (fold change of percentage) of flow cytometry analyses results for the expression of CTLA4, CD38 and Ki-67 after downregulation of CTLA4 in three different CLL patient samples.

Figure 2. Upregulation of STAT1/phospho-STAT1, NFATC2, c-Fos/phospho-c-Fos, and c-Myc in CTLA4-downregulated CLL (low CD38/high CTLA4) cells as determined by RT-PCR, real-time PCR, and/or Western blotting.

Panels A: Semi-quantitative RT-PCR showing that downregulation of CTLA4 by AS in CLL cells leads to up-regulation of STAT1, NFATC2, c-Fos, and c-Myc in CLL. Panel B: Real-time PCR results from four patient samples showing a significantly higher expression of c-Myc in CTLA4-downregulated CLL cells compared to control CLL cells or CLL cells treated with irrelevant AS. Panel C–D: Western blot results showing up-regulation and quantification of the expression of STAT1 and its phosphorylation, c-Fos and its phosphorylation, NFATC2, and c-Myc at the protein level in CTLA4 down-regulated CLL patient samples (n = 3). *p indicates the statistical difference between control CLL cells and CTLA4-downregulated CLL cells. β-Actin was used as a control.

Figure 3. Differential expression of CTLA4 and associated molecules in primary CLL cells.

Panel I: Differential expression of CTLA4 and associated molecules in high-CD38/low-CTLA4 and low-CD38/high-CTLA4 CLL subgroups as determined by microarray data. Subpanel A: Mean normalized expression levels of CTLA4 in the CD38-low group compared to the CD38-high group. Subpanels B–D: Mean normalized expression levels of STAT1, NFATC2, and c-Fos (respectively) in the CTLA4-high group compared to CTLA4-low group. Panel II: Overexpression of downstream molecules in BCR proliferation signaling in two prognostic CLL subgroups, as determined by real-time PCR. Subpanels A–C: Real-time PCR showing gene expression for STAT1, NFATC2, and c-Fos (fold change) in the high-CTLA4 CLL group compared to the low-CTLA4 CLL group, respectively. Subpanels D–F: Real-time PCR showing gene expression of STAT1, NFATC2, and c-Fos (fold change) in chromosomal abnormality subgroups, respectively (good outcome includes 13q14 deletion and normal karyotype, while poor outcome includes trisomy12, 11q deletion, and 17p deletion chromosomal abnormality).

Figure 4. Measurement of apoptosis in CTLA4-downregulated low CD38/high CTLA4 CLL cells.

Panel A: Representative results showing the percentage of live, pre-apoptotic, and apoptotic cells in CTLA4-downregulated CLL cells as determined by flow cytometry. Panel B: Mean decrease in apoptotic cell frequency in CTLA4-downregulated CLL cells. The values are derived from primary CLL cells from three different patients using Annexin-V flow cytometry method. Panel C: Expression of Bcl-2 in the CTLA4-high and CTLA4-low subgroups as determined by microarray. Panels D: Expression levels of CTLA4 and Bcl-2 in CTLA4-downregulated CLL cells as determined by semiquantitative RT-PCR. RPL13A, a housekeeping gene, was used as a control. Panels E–F: Bcl-2 expression levels were measured using Western blot in CTLA4-downregulated CLL cells. Panel E: A representative figure for the upregulation of Bcl-2 protein. Panel F: Mean expression of Bcl-2 in (low CD38) patient samples (n = 3) treated with and without CTLA4-siRNA. β-Actin was used as a control.

Figure 5. Microenvironmental influence on the expression of CTLA4 and associated molecules.

Panel A: Supervised cluster analyses of expression of CTLA4 and associated molecules among CLL cells from three different in vivo microenvironments such as peripheral blood (PB-CLL, n = 20), bone marrow (BM-CLL, n = 18) and lymph nodes (LN-CLL, n = 15) using microarray analyses. Panel B: Validation of the differentially expressed genes among PB-CLL (n = 12), BM-CLL (n = 12), and LN-CLL (n = 12) cells using real-time PCR analyses. Panel C: Upregulation of CTLA4, c-Myc, and Bcl-2 in CLL cells (n = 6) co-cultured in vitro with stromal cells including OMA-AD and HMEC cells.

Figure 6. Hypothetical model for the role of CTLA4 in CLL cell proliferation/survival.

Panel A: When CLL cells express low CD38, but high CTLA4, CTLA4 inhibits the CD38/BCR signaling pathway at multiple levels. CTLA4 downregulates NFATC2 and proliferation-associated molecules such as c-Fos and c-Myc. Downregulation of NFATC2 may also be associated with an autoregulatory loop for CTLA4, which would downregulate CTLA4 transcription. CTLA4 also downregulates the expression of Bcl-2, thus decreasing the survival of CLL cells. CTLA4 inhibits the expression of STAT1, thus deregulating the JAK/STAT pathway and inhibiting CLL cell growth. Panel B: When CLL cells express high CD38, but low CTLA4, activated CD38/BCR signaling upregulates downstream molecules in the pathway, such as NFATC2, c-Fos, and Bcl-2. These molecules will increase proliferation and survival of CLL cells. Low expression of CTLA4 does not interfere with the expression of STAT1, which favors CLL cell growth.