JCI - Chronic lymphocytic leukemia cells induce changes in gene expression of CD4 and CD8 T cells

Gene expression profiling of CD4 and CD8 T cells from CLL patients and healthy donors. CD4 and CD8 cells were isolated from healthy donors and from previously untreated patients with B cell CLL, who were selected to represent the heterogeneity of this disease (Table 1). Global gene expression profiles were obtained and the microarray data analyzed using both unsupervised and supervised learning. Even though the cells being analyzed were not part of the malignant clone, in an unsupervised analysis, delineation of patients from healthy donors was possible in all cases using hierarchical clustering of CD8 T cells, and in the majority of cases using hierarchical clustering of CD4 T cells (see Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI24176DS1).

In supervised analyses, there were no significant differences between gene expression profiles of CD4 or CD8 T cells from patients with CLL and gene expression profiles of CD4 or CD8 T cells from healthy donors, based on cell purity (less than 85% versus 85% or more), time from diagnosis (1–5 years versus 6–10 years), absolute white blood cell count (less than 20 mm³ versus 20 mm³ or more), stage of disease (0–I versus II–III), Ig heavy chain mutational status (mutated versus unmutated), or cytogenetic abnormalities (deletion 13q versus others). The majority of the contaminating cells in the T cell population were CD19 B cells.

Molecular defects in CD4 cells in tumor-bearing patients. By supervised analysis of CD4 cells, we identified 22 genes that had significantly increased expression and 68 genes that had significantly decreased expression (P < 0.05) in CD4 cells of CLL patients (n = 22) compared with healthy donors (n = 12) (Figure 1A). The differentially expressed genes were classified by their involvement in specific cellular pathways, and the full listing of these differentially expressed genes is shown in Supplemental Table 1. The majority of the genes were involved in cell differentiation and proliferation, survival, cytoskeleton formation, and vesicle trafficking. For genes selected as representative of the defective pathways, changes in RNA expression were confirmed by real-time PCR and changes in protein expression by Western blot (Figure 2, A and B).

In the CD4 cells of CLL patients, there was decreased expression in a number of genes in the Ras-dependent JNK and p38 MAPK pathways. The JNK–p38 MAPK pathway plays major roles in CD4 T cell differentiation into Th1 or Th2 subsets (13–15). There was decreased gene expression in a number of components of this pathway, including the activator MINK (MAP4K6) (16); GDI1 (17, 18), which serves as a negative regulator of small GTP-binding proteins in the Ras-dependent MAPK pathway in induction of NF-κB or actin cytoskeleton remodeling via the Arp2/3 complex; and NFRKB, which binds to several of the κB regulatory elements (17, 19, 20) (Figure 1B). There was also decreased expression of PIK3CB, a regulator of cell growth in response to various mitogenic stimuli through TCR/CD28, IL-1 receptor, G-protein coupled receptor, and members of the TNF receptor family (20, 21).

Differential expression of genes involved in cytoskeleton formation and vesicle trafficking in CD4 cells from CLL patients included decreases in AAK1, which plays a regulatory role in cell migration and clathrin-mediated endocytosis (22), and AP3M2, which facilitates budding of vesicles from the Golgi membrane and trafficking to lysosomes (23). There was increased expression, in CD4 cells from CLL patients, of SPTBN1; of ARPC1, which encodes an actin cytoskeleton–associated protein that plays a role in cell migration/motility or cytokine production/secretory functions by controlling actin polymerization; and of ADIR (Figure 1B).

Functionally, these changes would be expected to result in decreased Th1 differentiation, and we and others have previously demonstrated skewing of T cell responses to Th2 rather than Th1 differentiation in patients with CLL (12, 24).

Molecular defects in CD8 T cells in patients with CLL. By supervised analysis, a larger number of genes (n = 273) had deregulated expression in CD8 cells, including 105 genes that were downregulated and 168 genes upregulated in CD8 T cells from patients with CLL (n = 20) compared with healthy donors (n = 12) (P < 0.05) (Figure 3A). The differentially expressed genes were classified by their involvement in specific cellular pathways, and a number of representative genes of those pathways are listed in Supplemental Table 2. On analysis of these genes, the majority were involved in cytoskeleton formation, intracellular transportation, vesicle trafficking, or cellular secretion as well as cytotoxicity pathways in CD8 T cells (Figure 3B).

Impaired cytoskeleton formation, intracellular transportation, and cytotoxicity in CD8 T cells from CLL patients. We observed decreased expression of ARAP3, a Rho repressor gene that induces PI3K-dependent rearrangements in the cell cytoskeleton (25); myosin IXB, a GTPase-activating protein for the G protein Rho (26); AP3M2; VAMP2; GPR57; and AKAP9. There was increased expression of CDC42, PIK4CB, RAB35, FLNA, and FMNL, which associate with both Rac and profilin and regulate reorganization of the actin cytoskeleton in association with Rac (27, 28). Actin polymerization at the immune synapse is required for T cell activation and effector function, and T cell binding to APCs induces localized activation of CDC42 and WASP at the immune synapse (29, 30). There was increased expression of ARPC1B, required for the formation and stabilization of the immunological synapse at the interface between APCs and T lymphocytes (27–29). We also observed increased expression in SPEC1, which encodes a GTPase inhibitor protein that regulates CDC42 function, and NCK2, which encodes an src homology domain–containing (SH2 and SH3 domain–containing) adaptor protein that couples receptor tyrosine phosphorylation to downstream effector molecules in cytoskeleton formation processes (31).

There was also dysregulation of genes involved in secretory vesicle formation and cytotoxic activity. Such decreased genes included VAMP2; SCAMP1, which encodes a carrier to the cell surface in post-Golgi recycling pathways during vesicular transport; XAB2, a Ras superfamily member involved in controlling a diverse set of essential cellular functions; and GPR57, a GTP-binding protein that activates JNK-, MAPK-, and p38-dependent pathways in the cytotoxic immune response (32). We observed increased expression in inhibitor genes including the Rab family members RAB35, RAB22A, the ral guanine nucleotide dissociation stimulator RALGDS that inhibits binding of Raf to Ras, and RASGRP2, an inhibitor of guanine nucleotide exchange factor. Also increased was AP2B1, an adaptin family member essential for the formation of adaptor complexes of clathrin-coded vesicles (31, 33). Adaptins interact with the cytoplasmic domains of membrane-spanning receptors in the course of their endocytic/exocytic transport. Likely as a consequence of these changes in structural proteins, we observed a decrease in cytotoxicity in CD8 cells of CLL patients compared with healthy donors (data not shown) and a decrease in granzyme B protein in CD8 T cells of CLL patients compared with healthy donors (Figure 2C). Of note, there was no decrease in granzyme B mRNA expression in the CD8 T cells in CLL patients, and we conclude that the decreased granzyme B protein expression reflects failure to package the protein in secretory vesicles.

These changes would be expected to result in decreased cytotoxicity and effector function. We and others have previously demonstrated that such defects occur in the CD8 T cells in patients with CLL (12, 34, 35).

We therefore identified specific pathways with altered expression in CD4 and CD8 cells of CLL patients. From this we developed a representative protein expression panel using Western blot analysis and used this proteomic approach to assess whether CLL cells could induce similar changes in healthy allogeneic T cells and to elucidate the mechanism(s) whereby CLL cells could induce changes in these pathways, using cocultures of healthy T cells with CLL cells.

The CLL B cell–derived soluble factors induce alterations in chemokine and chemokine receptor expression but not cytoskeletal proteins in healthy T cells. CLL cells express cytokines known to inhibit T cell responses, including IL-10. We therefore hypothesized that release of these inhibitory cytokines would induce the changes in gene expression observed in healthy CD4 and CD8 cells. However, following culture of healthy CD4 or CD8 cells with sera from CLL patients or coculture of CLL cells or healthy B cells with healthy CD4 or CD8 cells in transwell culture plates, we did not observe changes in expression of cytoskeleton proteins or other genes that we have shown to be decreased in CD4 or CD8 cells in CLL patients (data not shown). The only defects shown to be induced by culture of healthy T cells with these soluble factors were altered expression of chemokines and chemokine receptors, including decreases in CXCR1, CXCR2, and CXCR4 and increases in CXCR3, CCR4, and CCR5 in CD4 T cells from healthy donors (Supplemental Figure 2). When IL-10 mRNA expression was inhibited by transient transfection of small interfering RNA (siRNA) targeting IL-10 (Supplemental Figure 3) in B cells from both CLL patients and healthy donors or by use of neutralizing anti–IL-10 mAbs, there was no change in expression level of cytoskeletal proteins, but this blocked the changes in chemokine and chemokine receptor expression, suggesting that these alterations were indeed induced by IL-10 and not by other soluble factors (Supplemental Figure 2).

CLL B cells induce alteration in cytoskeleton formation and vesicle transportation pathways in T cells by cell-cell contact. Since soluble factors did not induce changes in healthy T cells, we cocultured CLL cells in direct contact with T cells from healthy donors and analyzed expression of proteins representative of the pathways found to be abnormal in the cancer-bearing patients. By 48 hours of culture of healthy donor CD4 T cells with tumor cells, we observed changes in protein expression patterns consistent with that seen in the CD4 cells of the CLL patients. Such changes included increased expression of Arp3 and decreased expression of NF-κBp65 and GDI1 (Figure 4A). Similarly, in CD8 cells, we observed changes in the expression pattern consistent with that observed by gene expression profiling, including decreased Rho-GAP and increased Arp3 and CDC42 protein (Figure 4B). Induction of these changes required cell-cell contact, and these changes were not observed after blockade of adhesion molecules using anti-CD54 and anti-CD11a mAbs (Figure 4C). These changes were not induced by coculture of allogeneic T cells with healthy B cells from the donors who were HLA matched to the CLL patients.