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

To examine the impact of tumors on the immune system, we compared global gene expression profiles of peripheral blood T cells from previously untreated patients with B cell chronic lymphocytic leukemia (CLL) with those from age-matched healthy donors. Although the cells analyzed were not part of the malignant clone, analysis revealed differentially expressed genes, mainly involved in cell differentiation in CD4 cells and defects in cytoskeleton formation, vesicle trafficking, and cytotoxicity in CD8 cells of the CLL patients. In coculture experiments using CLL cells and T cells from healthy allogeneic donors, similar defects developed in both CD4 and CD8 cells. These changes were induced only with direct contact and were not cytokine mediated. Identification of the specific pathways perturbed in the T cells of cancer-bearing patients will allow us to assess steps to repair these defects, which will likely be required to enhance antitumor immunity.

Development of cancer is associated with immune suppression in the host, contributing to the failure to mount an effective immune response against the cancer cells (1). The mechanisms whereby specific T cell defects occur are not well understood but include production of immune-suppressive factors by cancer cells, direct tumor cell–T cell interactions, and induction of regulatory T cell subsets. Identification of the specific T cell defects that occur in cancer-bearing patients usually requires isolation of tumor-infiltrating lymphocytes, which limits the number of T cells that can be obtained for study. Tumor cells circulate in leukemia, so there is widespread interaction of cancer cells with T cells that can readily be sampled from peripheral blood. Specifically in B cell chronic lymphocytic leukemia (CLL), a number of well-characterized T cell defects have been described, and it is most likely that immunosuppression induced by the malignant B cells plays an important role in the induction of subsequent immune deficiency in this disease. CLL cells express high levels of immunomodulatory factors including TGF-β and IL-10 that suppress response to antigens, T cell activation, expansion, and effector function (2–5). FasL has been detected on a number of tumors, including CLL, and FasL-positive tumor cells can induce apoptosis in vitro (6, 7). T cells from patients with CLL have low levels of expression of CD80, CD86, and CD154 and are Th2-preponderant (8–11). We have observed functional T cell defects and increased expression of Th2-type chemokine receptors on T cells from patients with CLL compared with T cells of healthy donors (12). To examine the mechanisms of T cell defects in tumor-bearing patients, we analyzed the global gene expression profiles of highly purified CD4 and CD8 cells from peripheral blood from individuals with CLL compared with age-matched healthy donors. Similar defects requiring cell-cell contact were induced by coculture of healthy T cells with CLL cells. Therefore, contact with leukemic cells induces specific changes in both CD4 and CD8 T cells, resulting in functional impairment.

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).

Table 1

Patients’ clinical disease characteristics

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).

Figure 1

Differentially expressed genes in CD4 cells from patients with CLL compared with healthy donors. Dendrogram of differentially expressed genes by supervised analysis (P < 0.05). (A) CD4 cells from patients with CLL compared with healthy donors. Twenty-two genes were significantly increased (red) and 68 genes significantly decreased (blue) in CD4 cells from CLL patients. (B) Genes involved in Ras-dependent JNK and p38 MAPK pathways in CD4 cells. The dendrogram represents selected genes from A.

Figure 2

Validation of gene expression observed by microarray. (A) Concordant with data seen on microarray, by quantitative PCR there was decreased expression of NFRKB in CD4 cells and VAMP2 in CD8 cells from CLL patients compared with healthy donors. The figure represents data from CD4 cells from 6 CLL patients and 5 healthy donors and CD8 cells from 4 CLL patients and 6 healthy donors. Statistical significance was assessed in a 2-tailed Student’s t test. (B) Decreased expression of NF-κBp65 in CD4 cells and Rho-GAP p190 proteins in CD8 cells from CLL patients compared with healthy donors. The left 2 lanes represent protein expression in CD4 or CD8 cells from 2 CLL patients (C1 and C2), and the right 2 lanes represent 2 healthy donors (H1 and H2). The expression of proteins was normalized by GAPDH expression level and is shown as protein bands and densitometric intensity of each band. The figure is representative of 3 additional experiments performed on 6 different donors, all showing a similar pattern (P < 0.05). (C) Intracytoplasmic expression of the GP1 gene product granzyme B, detected in CD8 cells from CLL patients and healthy donors by flow cytometry and fluorescent microscopy. To obtain at least 99% CD8 cell population, cells were purified using magnetically labeled negative cell-depletion antibodies. High expression of granzyme B in CD8 cells from healthy donors (CD8-FITC⁺ granzyme B–PE⁺, orange-brown) was observed compared with that in CD8 cells from CLL patients (CD8-FITC⁺ granzyme B–PE^–, green). The figure is representative of experiments performed with 4 different patients and healthy donors (P < 0.05).

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).

Figure 3

Differentially expressed genes in CD8 cells from patients with CLL. Dendrogram of differentially expressed genes by supervised analysis in CD8 cells from patients with CLL compared with healthy donors (P < 0.05). (A) One hundred sixty-eight genes were significantly increased (red) and 105 genes were significantly decreased (blue) in CD8 cells from CLL patients. (B) Dendrogram of differentially expressed genes involved in cytoskeleton formation, vesicle trafficking, and cytotoxicity pathways in CD8 cells. The dendrogram represents selected genes from A.

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.

Figure 4

Impact of T cell–cancer cell contact on healthy T cells. Highly purified T cells from healthy donors were cocultured with B cells from CLL patients or from their HLA-matched healthy donors at a 2:1 (T/B cell) ratio for 48 hours. (A) Decrease in 65-kDa NF-κB and increase in 41-kDa Arp3 in healthy CD4 cells after contact with allogeneic CLL cells (C1 and C2) and healthy B cells (H). (B) Decrease in 190-kDa Rho-GAP and increase in 41-kDa Arp3 in healthy CD8 cells after contact with CLL cells (C1 and C2) and healthy allogeneic B cells (H). (C) Impact of CLL cell contact on cytoskeletal protein expression in allogeneic healthy T cells, confirmed by ICAM1 or LFA1 blocking. Highly purified CD4 or CD8 cells from healthy donors were cocultured with B cells from CLL patients (C1 and C2) or healthy donors (H) with or without blockade of the LFA1 or ICAM1 interaction. Expression of 190-kDa Rho-GAP in healthy CD8 cells was increased after blockade of the ICAM1 during CLL cell–T cell contact, and expression of 41-kDa Arp3 in CD4 T cells and 25-kDa CDC42 in healthy CD8 cells was decreased after blockade of the LFA1 or ICAM1. –, protein expression in nonblocked cells; +, protein expression in blocked cells. Protein expressions were normalized by GAPDH expression level and are shown as protein bands and densitometric intensity of each band. The figure is representative of 3 different experiments performed with 6 different patients with CLL and 6 different healthy donors showing a similar pattern (P < 0.05).

Microarray-based expression profiling has been used most commonly to compare and contrast heterogeneous groups of human tumors to identify expression patterns associated with prognosis and to examine altered expression in tumor cells compared with their normal cellular counterparts. Here we performed gene expression profiling on nonmalignant components in cancer-bearing patients and demonstrate profound changes in gene expression of T cells in patients with CLL compared with healthy donors. Importantly, we demonstrate that these changes can be induced at the protein level in healthy T cells following short-term culture with direct contact with CLL cells.

Analysis of the differentially expressed genes in the T cells in CLL patients demonstrates a number of abnormalities in specific pathways. In CD4 cells, among the most marked changes observed were in the Ras-dependent JNK and p38 MAPK pathways (Figure 5). JNK and p38 MAPK pathways play a major role in regulating CD4 T cell differentiation into Th1 or Th2. JNK2 and p38 MAPKs mediate IFN-γ production and Th1 cell differentiation, and inhibition of p38 MAPK in dnp38 transgenic mice results in decreased IFN-γ production by Th1 cells (15, 36, 37). ADIR encodes a protein involved in protein processing in the endoplasmic reticulum and contains a putative IFN-responsive ATP-binding site involved in regulating expression of genes critical for antigen presentation and immune surveillance against viruses and tumor cells (38). Our data, demonstrating decreased expression in the p38 MAPK pathway activator genes such as MINK, NFRKB, and PIK3CB, are in keeping with our hypothesis that the defects induced by the leukemic cells impair subsequent CD4 differentiation into Th1 cells.

Figure 5

Differentially expressed genes by their involvement in specific signaling pathways. Representative defects in T cell pathways and functions caused by CLL cell–T cell contact are shown in this diagram. Differentially expressed genes involved in cell differentiation, particularly JNK (pink) and p38 MAPK (yellow) pathways and cytoskeleton formation and vesicle transportation (blue), in CD4 T cells from CLL patients compared with healthy donors are represented by selected genes that were increased (rectangles) or decreased (ovals). Differentially expressed genes involved in cytoskeleton formation, vesicle trafficking (blue), and cytotoxicity (red) in CD8 T cells from CLL patients compared with healthy donors are represented by selected increased genes (rectangles) and decreased genes (ovals).

In CD8 cells, our findings are in keeping with the hypothesis that cell contact with CLL cells induces changes in gene expression in genes regulating cytoskeleton formation and vesicle trafficking (Figure 5), thereby resulting in the decreased cytotoxicity and effector function noted in this disease. The cytoskeleton is a cellular network of structural, adaptor, and signaling molecules that regulates most cellular functions during immune responses, including migration, extravasation, antigen recognition, activation, and phagocytosis. CD8 cytotoxic T lymphocytes mediate killing of cancer cells through polarized delivery of vesicles referred to as lytic lysosomes that contain apoptosis-inducing proteins including perforin and granzymes (39–41). Positioning of the secretory cleft and secretory lysosome polarization targeting cancer cells depend on cytoskeletal connections that regulate granule transport to the plasma membrane (40). The altered expression in regulator genes, including increased RAB11B and RAB22A and decreased RAB35, VAMP2, SLC21A11, and SCAMP1, indicated defects in vesicle formation and intracellular trafficking in CD8 cells in CLL patients. We observed decreased expression of GP2 (41), and TPSB1, a gene encoding a tetrameric serine protease, concentrated and stored selectively in secretory granules (40, 42). In CD8 also we observed defects in the p38 MAPK pathway, which also regulates the production of TNF-α, perforin, and granzyme as well as apoptosis in CD8 cells (43–45). Our data suggest that even though CD8 T cells in CLL appear morphologically intact, the production of cytolytic molecules including granzymes and their storage in lysosomes as well as intracellular secretory vesicle transportation are significantly impaired. The decreased expression in activators and increased expression in repressor genes involved in cytoskeleton formation and intracellular vesicle transportation, more specifically the decreased expression in granzyme granules GP2 and TPSB1, likely contribute to the failure of CD8 T cell responses against tumor cells in CLL.

Several studies have shown that CLL cells secrete IL-10, TNF-α, and TGF-β (2–5, 46). The inhibitory cytokine IL-10 initiates a wide variety of activities on binding to its cellular receptor complex. The mechanism of IL-10 inhibition of cytokine production was initially believed to be inhibition of the antigen-presentation capacity of macrophages and DCs (47), but IL-10 also plays important roles in blocking cytokine production, expression of costimulatory molecules, and chemokine secretion. It also modifies chemokine receptor expression, increases integrin ligand (e.g., ICAM1) expression (48, 49), and induces CCR5 expression on monocytes (50). Therefore IL-10 appeared an attractive candidate to induce specific changes in gene expression in T cells in CLL patients. Our results suggest that such changes are largely limited to changes in chemokine expression, but the additive effect of IL-10 production on the changes that are induced by direct contact and in vivo in a murine model is currently under investigation.

Taken together, the results presented here demonstrate that contact with cancer cells can induce changes in gene expression in healthy cells in the cancer-bearing patient. These changes likely contribute to the decreased immune responses observed in these patients and ostensibly may contribute to the lack of autologous antitumor responses. We are currently studying the impact of tumor development in vivo on T cell function and expression profiles using the Eμ-TCL-1 transgenic mouse model of CLL (51). As these mice develop leukemia, there are changes in expression profiles of their CD4 and CD8 cells similar in nature to those observed in patients with CLL (data not shown). Moreover, the observation that CLL cells are capable of inducing similar changes in allogeneic CD4 and CD8 cells has implications for the field of allogeneic stem cell transplantation. As we have observed in the in vitro assay systems, infusion of donor T cells in patients with high tumor burden could induce similar changes in donor T cells with resulting decrease in antitumor immunity, thereby limiting the graft-versus-leukemia effect. Characterization of these defects will now allow us to examine mechanisms to repair T cell function to increase antitumor immunity in both the allogeneic and the autologous setting.

View Supplemental data

We thank Peter Varney for editorial assistance, and Charles Lee, Edward Alvin Fox, and the staff of the Dana-Farber Cancer Institute Microarray Core Facility. This work was supported by grant PO1 CA81538 from the National Cancer Institute to the CLL Research Consortium (J.G. Gribben and D. Neuberg) and the Eskandarian Family CLL Research Fund.

Nonstandard abbreviations used: CLL, chronic lymphocytic leukemia; cRNA, complementary RNA; siRNA, small interfering RNA.

Conflict of interest: The authors have declared that no conflict of interest exists.

1. Houghton, AN, Guevara-Patino, JA. Immune recognition of self in immunity against cancer. J. Clin. Invest. 2004. 114:468-471. doi:10.1172/JCI200422685.
   View this article via: JCI PubMed Google Scholar
2. Benjamin, D, Knobloch, TJ, Dayton, MA. Human B-cell interleukin-10: B-cell lines derived from patients with acquired immunodeficiency syndrome and Burkitt’s lymphoma constitutively secrete large quantities of interleukin-10. Blood. 1992. 80:1289-1298.
   View this article via: PubMed Google Scholar
3. Benjamin, D, Park, CD, Sharma, V. Human B cell interleukin 10. Leuk. Lymphoma. 1994. 12:205-210.
   View this article via: PubMed CrossRef Google Scholar
4. Lotz, M, Ranheim, E, Kipps, TJ. Transforming growth factor beta as endogenous growth inhibitor of chronic lymphocytic leukemia B cells. J. Exp. Med. 1994. 179:999-1004.
   View this article via: PubMed CrossRef Google Scholar
5. Fayad, L, et al. Interleukin-6 and interleukin-10 levels in chronic lymphocytic leukemia: correlation with phenotypic characteristics and outcome. Blood. 2001. 97:256-263.
   View this article via: PubMed CrossRef Google Scholar
6. Hahne, M, et al. Melanoma cell expression of Fas(Apo-1/CD95) ligand: implications for tumor immune escape. Science. 1996. 274:1363-1366.
   View this article via: PubMed CrossRef Google Scholar
7. Tinhofer, I, et al. Differential sensitivity of CD4+ and CD8+ T lymphocytes to the killing efficacy of Fas (Apo-1/CD95) ligand+ tumor cells in B chronic lymphocytic leukemia. Blood. 1998. 91:4273-4281.
   View this article via: PubMed Google Scholar
8. Cantwell, M, Hua, T, Pappas, J, Kipps, TJ. Acquired CD40-ligand deficiency in chronic lymphocytic leukemia. Nat. Med. 1997. 3:984-989.
   View this article via: PubMed CrossRef Google Scholar
9. de Totero, D, et al. IL4 production and increased CD30 expression by a unique CD8+ T-cell subset in B-cell chronic lymphocytic leukaemia. Br. J. Haematol. 1999. 104:589-599.
   View this article via: PubMed CrossRef Google Scholar
10. Kay, NE, Han, L, Bone, N, Williams, G. Interleukin 4 content in chronic lymphocytic leukaemia (CLL) B cells and blood CD8+ T cells from B-CLL patients: impact on clonal B-cell apoptosis. Br. J. Haematol. 2001. 112:760-767.
    View this article via: PubMed CrossRef Google Scholar
11. Cerutti, A, et al. Dysregulation of CD30+ T cells by leukemia impairs isotype switching in normal B cells. Nat. Immunol. 2001. 2:150-156.
    View this article via: PubMed CrossRef Google Scholar
12. Krackhardt, AM, et al. T-cell responses against chronic lymphocytic leukemia cells: implications for immunotherapy. Blood. 2002. 100:167-173.
    View this article via: PubMed CrossRef Google Scholar
13. Rincon, M, Derijard, B, Chow, CW, Davis, RJ, Flavell, RA. Reprogramming the signalling requirement for AP-1 (activator protein-1) activation during differentiation of precursor CD4+ T-cells into effector Th1 and Th2 cells. Genes Funct. 1997. 1:51-68.
    View this article via: PubMed Google Scholar
14. Dong, C, et al. Defective T cell differentiation in the absence of Jnk1. Science. 1998. 282:2092-2095.
    View this article via: PubMed CrossRef Google Scholar
15. Yang, DD, et al. Differentiation of CD4+ T cells to Th1 cells requires MAP kinase JNK2. Immunity. 1998. 9:575-585.
    View this article via: PubMed CrossRef Google Scholar
16. Dan, I, et al. Molecular cloning of MINK, a novel member of mammalian GCK family kinases, which is up-regulated during postnatal mouse cerebral development. FEBS Lett. 2000. 469:19-23.
    View this article via: PubMed CrossRef Google Scholar
17. Bachner, D, Sedlacek, Z, Korn, B, Hameister, H, Poustka, A. Expression patterns of two human genes coding for different rab GDP-dissociation inhibitors (GDIs), extremely conserved proteins involved in cellular transport. Hum. Mol. Genet. 1995. 4:701-708.
    View this article via: PubMed CrossRef Google Scholar
18. Scheffzek, K, Stephan, I, Jensen, ON, Illenberger, D, Gierschik, P. The Rac-RhoGDI complex and the structural basis for the regulation of Rho proteins by RhoGDI. Nat. Struct. Biol. 2000. 7:122-126.
    View this article via: PubMed CrossRef Google Scholar
19. Hoffman, GR, Nassar, N, Cerione, RA. Structure of the Rho family GTP-binding protein Cdc42 in complex with the multifunctional regulator RhoGDI. Cell. 2000. 100:345-356.
    View this article via: PubMed CrossRef Google Scholar
20. Schmitz, ML, Bacher, S, Dienz, O. NF-kappaB activation pathways induced by T cell costimulation. FASEB J. 2003. 17:2187-2193.
    View this article via: PubMed CrossRef Google Scholar
21. Adams, BS, et al. Localization of the gene encoding R kappa B (NFRKB), a tissue-specific DNA binding protein, to chromosome 11q24-q25. Genomics. 1992. 14:270-274.
    View this article via: PubMed CrossRef Google Scholar
22. Conner, SD, Schmid, SL. Identification of an adaptor-associated kinase, AAK1, as a regulator of clathrin-mediated endocytosis. J. Cell Biol. 2002. 156:921-929.
    View this article via: PubMed CrossRef Google Scholar
23. Faundez, V, Horng, JT, Kelly, RB. A function for the AP3 coat complex in synaptic vesicle formation from endosomes. Cell. 1998. 93:423-432.
    View this article via: PubMed CrossRef Google Scholar
24. Rossmann, ED, Lewin, N, Jeddi-Tehrani, M, Osterborg, A, Mellstedt, H. Intracellular T cell cytokines in patients with B cell chronic lymphocytic leukaemia (B-CLL). Eur. J. Haematol. 2002. 68:299-306.
    View this article via: PubMed CrossRef Google Scholar
25. Krugmann, S, et al. Identification of ARAP3, a novel PI3K effector regulating both Arf and Rho GTPases, by selective capture on phosphoinositide affinity matrices. Mol. Cell. 2002. 9:95-108.
    View this article via: PubMed CrossRef Google Scholar
26. Post, PL, Bokoch, GM, Mooseker, MS. Human myosin-IXb is a mechanochemically active motor and a GAP for rho. J. Cell Sci. 1998. 111:941-950.
    View this article via: PubMed Google Scholar
27. Yayoshi-Yamamoto, S, Taniuchi, I, Watanabe, T. FRL, a novel formin-related protein, binds to Rac and regulates cell motility and survival of macrophages. Mol. Cell. Biol. 2000. 20:6872-6881.
    View this article via: PubMed CrossRef Google Scholar
28. Blanchoin, L, Pollard, TD, Mullins, RD. Interactions of ADF/cofilin, Arp2/3 complex, capping protein and profilin in remodeling of branched actin filament networks. Curr. Biol. 2000. 10:1273-1282.
    View this article via: PubMed CrossRef Google Scholar
29. Stowers, L, Yelon, D, Berg, LJ, Chant, J. Regulation of the polarization of T cells toward antigen-presenting cells by Ras-related GTPase CDC42. Proc. Natl. Acad. Sci. U. S. A. 1995. 92:5027-5031.
    View this article via: PubMed CrossRef Google Scholar
30. Labno, CM, et al. Itk functions to control actin polymerization at the immune synapse through localized activation of Cdc42 and WASP. Curr. Biol. 2003. 13:1619-1624.
    View this article via: PubMed CrossRef Google Scholar
31. Buday, L, Wunderlich, L, Tamas, P. The Nck family of adapter proteins: regulators of actin cytoskeleton [review]. Cell. Signal. 2002. 14:723-731.
    View this article via: PubMed CrossRef Google Scholar
32. Yamauchi, J, Nagao, M, Kaziro, Y, Itoh, H. Activation of p38 mitogen-activated protein kinase by signaling through G protein-coupled receptors. Involvement of Gbetagamma and Galphaq/11 subunits. J. Biol. Chem. 1997. 272:27771-27777.
    View this article via: PubMed CrossRef Google Scholar
33. Frost, JA, Xu, S, Hutchison, MR, Marcus, S, Cobb, MH. Actions of Rho family small G proteins and p21-activated protein kinases on mitogen-activated protein kinase family members. Mol. Cell. Biol. 1996. 16:3707-3713.
    View this article via: PubMed Google Scholar
34. Muller, MR, Tsakou, G, Grunebach, F, Schmidt, SM, Brossart, P. Induction of chronic lymphocytic leukemia (CLL)-specific CD4- and CD8-mediated T-cell responses using RNA-transfected dendritic cells. Blood. 2004. 103:1763-1769.
    View this article via: PubMed CrossRef Google Scholar
35. Gitelson, E, et al. Chronic lymphocytic leukemia-reactive T cells during disease progression and after autologous tumor cell vaccines. Clin. Cancer Res. 2003. 9:1656-1665.
    View this article via: PubMed Google Scholar
36. Rincon, M, et al. Interferon-gamma expression by Th1 effector T cells mediated by the p38 MAP kinase signaling pathway. EMBO J. 1998. 17:2817-2829.
    View this article via: PubMed CrossRef Google Scholar
37. Yamashita, M, et al. T cell antigen receptor-mediated activation of the Ras/mitogen-activated protein kinase pathway controls interleukin 4 receptor function and type-2 helper T cell differentiation. Proc. Natl. Acad. Sci. U. S. A. 1999. 96:1024-1029.
    View this article via: PubMed CrossRef Google Scholar
38. Dron, M, et al. Molecular cloning of ADIR, a novel interferon responsive gene encoding a protein related to the torsins. Genomics. 2002. 79:315-325.
    View this article via: PubMed CrossRef Google Scholar
39. Bromley, SK, et al. The immunological synapse and CD28-CD80 interactions. Nat. Immunol. 2001. 2:1159-1166.
    View this article via: PubMed CrossRef Google Scholar
40. Stinchcombe, JC, Bossi, G, Booth, S, Griffiths, GM. The immunological synapse of CTL contains a secretory domain and membrane bridges. Immunity. 2001. 15:751-761.
    View this article via: PubMed CrossRef Google Scholar
41. Waugh, SM, Harris, JL, Fletterick, R, Craik, CS. The structure of the pro-apoptotic protease granzyme B reveals the molecular determinants of its specificity. Nat. Struct. Biol. 2000. 7:762-765.
    View this article via: PubMed CrossRef Google Scholar
42. Baker, E, Sayers, TJ, Sutherland, GR, Smyth, MJ. The genes encoding NK cell granule serine proteases, human tryptase-2 (TRYP2) and human granzyme A (HFSP), both map to chromosome 5q11-q12 and define a new locus for cytotoxic lymphocyte granule tryptases. Immunogenetics. 1994. 40:235-237.
    View this article via: PubMed CrossRef Google Scholar
43. Radoja, S, et al. CD8(+) tumor-infiltrating T cells are deficient in perforin-mediated cytolytic activity due to defective microtubule-organizing center mobilization and lytic granule exocytosis. J. Immunol. 2001. 167:5042-5051.
    View this article via: PubMed Google Scholar
44. Radoja, S, Saio, M, Frey, AB. CD8+ tumor-infiltrating lymphocytes are primed for Fas-mediated activation-induced cell death but are not apoptotic in situ. J. Immunol. 2001. 166:6074-6083.
    View this article via: PubMed Google Scholar
45. Sabapathy, K, et al. c-Jun NH2-terminal kinase (JNK)1 and JNK2 have similar and stage-dependent roles in regulating T cell apoptosis and proliferation. J. Exp. Med. 2001. 193:317-328.
    View this article via: PubMed CrossRef Google Scholar
46. Bojarska-Junak, A, Rolinski, J, Wasik-Szczepaneko, E, Kaluzny, Z, Dmoszynska, A. Intracellular tumor necrosis factor production by T- and B-cells in B-cell chronic lymphocytic leukemia. Haematologica. 2002. 87:490-499.
    View this article via: PubMed Google Scholar
47. Fiorentino, DF, et al. IL-10 acts on the antigen-presenting cell to inhibit cytokine production by Th1 cells. J. Immunol. 1991. 146:3444-3451.
    View this article via: PubMed Google Scholar
48. Willems, F, et al. Interleukin-10 inhibits B7 and intercellular adhesion molecule-1 expression on human monocytes. Eur. J. Immunol. 1994. 24:1007-1009.
    View this article via: PubMed CrossRef Google Scholar
49. Takayama, T, et al. Mammalian and viral IL-10 enhance C-C chemokine receptor 5 but down-regulate C-C chemokine receptor 7 expression by myeloid dendritic cells: impact on chemotactic responses and in vivo homing ability. J. Immunol. 2001. 166:7136-7143.
    View this article via: PubMed Google Scholar
50. Sozzani, S, et al. Interleukin 10 increases CCR5 expression and HIV infection in human monocytes. J. Exp. Med. 1998. 187:439-444.
    View this article via: PubMed CrossRef Google Scholar
51. Bichi, R, et al. Human chronic lymphocytic leukemia modeled in mouse by targeted TCL1 expression. Proc. Natl. Acad. Sci. U. S. A. 2002. 99:6955-6960.
    View this article via: PubMed CrossRef Google Scholar
52. Mutter, GL, et al. Global expression changes of constitutive and hormonally regulated genes during endometrial neoplastic transformation. Gynecol. Oncol. 2001. 83:177-185.
    View this article via: PubMed CrossRef Google Scholar
53. Li, C, Wong, WH. Model-based analysis of oligonucleotide arrays: expression index computation and outlier detection. Proc. Natl. Acad. Sci. U. S. A. 2001. 98:31-36.
    View this article via: PubMed CrossRef Google Scholar