Research article
`
`Chronic lymphocytic leukemia cells
`induce changes in gene expression
`of CD4 and CD8 T cells
`
`Güllü Görgün,1 Tobias A.W. Holderried,1 David Zahrieh,1 Donna Neuberg,1 and John G. Gribben2
`
`1Department of Medical Oncology and Department of Biostatistics and Computational Biology, Dana-Farber Cancer Institute,
`Harvard Medical School, Boston, Massachusetts, USA. 2Cancer Research United Kingdom, Department of Medical Oncology,
`Barts and The London School of Medicine, Queen Mary University of London, London, United Kingdom.
`
`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. Identifi-
`cation 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.
`
`Introduction
`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-infil-
`trating 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 immunosup-
`pression 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 includ-
`ing 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-posi-
`tive 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 
`
`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.
`Citation for this article: J. Clin. Invest. 115:1797–1805 (2005).  
`doi:10.1172/JCI24176.
`
`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.
`
`Results
`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 hier-
`archical 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% ver-
`sus 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) com-
`pared with healthy donors (n = 12) (Figure 1A). The differentially 
`expressed genes were classified by their involvement in specific cel-
`lular pathways, and the full listing of these differentially expressed 
`
`The Journal of Clinical Investigation      http://www.jci.org      Volume 115      Number 7      July 2005 
`
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`research article
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`Table 1
`Patients’ clinical disease characteristics
`
`
`
`Time from
` Age
` Sex
`diagnosis
`
`
` F M
` 14 15 40–80 yr 1–10 yr
`
`Rai stage of disease
`
`Stage Stage Stage Stage
`0
`I
`II
`III
`8
`14
`5
`2
`
`Ig VH
`mutation status
`UM
`M
`
`
`7
`22
`
`Cytogenetics by FISH
`
`Normal Deletion Deletion Deletion Trisomy T(5;14)
`
`13q
`11q
`17p
`12
`12
`11
`1
`1
`3
`
`1
`
`CD4 and CD8 cells were obtained from peripheral blood of patients with CLL. The patients were untreated and were chosen to represent the heterogeneity
`of this disease. Healthy donors were age matched. UM, unmutated; M, mutated.
`
`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 select-
`ed as representative of the defective pathways, changes in RNA 
`expression were confirmed by real-time PCR and changes in pro-
`tein 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 path-
`way, including the activator MINK (MAP4K6) (16); GDI1 (17, 18),  
`which serves as a negative regulator of small GTP-binding pro-
`teins 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 forma-
`tion and vesicle trafficking in CD4 cells from CLL patients includ-
`ed decreases in AAK1, which plays a regulatory role in cell migra-
`
`tion 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 downreg-
`ulated 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 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.
`
`1798
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`The Journal of Clinical Investigation      http://www.jci.org      Volume 115      Number 7      July 2005
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`research article
`
`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).
`
`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 expres-
`sion of CDC42, PIK4CB, RAB35, FLNA, and FMNL, which asso-
`ciate 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 acti-
`vation 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 trans-
`port; 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 dissocia-
`tion 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 vesi-
`cles (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 expres-
`sion 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.
`
`
`
`The Journal of Clinical Investigation      http://www.jci.org      Volume 115      Number 7      July 2005 
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`research article
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`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.
`
`These  changes  would  be  expected  to  result  in  decreased 
`cytotoxicity and effector function. We and others have previ-
`ously 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 cocul-
`ture 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 che-
`mokine 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 trans-
`portation 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.
`
`Discussion
`Microarray-based expression profiling has been used most com-
`monly 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 expres-
`sion profiling on nonmalignant components in cancer-bearing  
`
`1800
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`The Journal of Clinical Investigation      http://www.jci.org      Volume 115      Number 7      July 2005
`
`UPenn Ex. 2069
`Miltenyi v. UPenn
`IPR2022-00855
`
`

`

`research article
`
`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 allo-
`geneic 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 alloge-
`neic 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 dur-
`ing 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 dif-
`ferent healthy donors showing a similar
`pattern (P < 0.05).
`
`patients and demonstrate profound changes in gene expression of 
`T cells in patients with CLL compared with healthy donors. Impor-
`tantly, 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 spe-
`cific pathways. In CD4 cells, among the most marked changes 
`observed were in the Ras-dependent JNK and p38 MAPK path-
`ways (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 differ-
`entiation, 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 criti-
`cal 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.
`
`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 effec-
`tor function noted in this disease. The cytoskeleton is a cellular net-
`work of structural, adaptor, and signaling molecules that regulates 
`most cellular functions during immune responses, including migra-
`tion, extravasation, antigen recognition, activation, and phagocy-
`tosis. 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 secre-
`tory lysosome polarization targeting cancer cells depend on cyto-
`skeletal connections that regulate granule transport to the plasma 
`membrane (40). The altered expression in regulator genes, includ-
`ing 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). 
`
`
`
`The Journal of Clinical Investigation      http://www.jci.org      Volume 115      Number 7      July 2005 
`
`1801
`
`UPenn Ex. 2069
`Miltenyi v. UPenn
`IPR2022-00855
`
`

`

`research article
`
`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  func-
`tion and expression profiles using the  
`Eµ-TCL-1 transgenic mouse model of 
`CLL (51). As these mice develop leuke-
`mia, there are changes in expression pro-
`files 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 capa-
`ble of inducing similar changes in alloge-
`neic CD4 and CD8 cells has implications 
`for the field of allogeneic stem cell trans-
`plantation. As we have observed in the 
`in vitro assay systems, infusion of donor  
`T cells in patients with high tumor bur-
`den  could  induce  similar  changes  in 
`donor T cells with resulting decrease 
`in antitumor immunity, thereby limit-
`ing  the  graft-versus-leukemia  effect. 
`Characterization of these defects will 
`now allow us to examine mechanisms 
`to repair T cell function to increase anti-
`tumor immunity in both the allogeneic 
`and the autologous setting.
`
`Figure 5
`Differentially expressed genes by their involvement in specific signaling pathways. Representa-
`tive 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).
`
`Our data suggest that even though CD8 T cells in CLL appear mor-
`phologically intact, the production of cytolytic molecules includ-
`ing 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 ini-
`tially believed to be inhibition of the antigen-presentation capac-
`ity 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) expres-
`sion (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 chemo-
`kine 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.
`
`Methods
`Cell isolation and RNA extraction. Heparinized venous blood samples from 29 
`CLL patients with Rai stages varying from 0 to 3 (Table 1) and age- and HLA-
`matched healthy donors were obtained after written informed consent. The 
`studies using peripheral blood sample collection from all individuals were 
`approved by the Institutional Review Board of the Dana-Farber Cancer 
`Institute. None of the CLL patients had received chemotherapy before the 
`blood was drawn for these studies. Mononuclear cells were separated by 
`Ficoll-Hypaque density gradient centrifugation, and CD4 T cells from 22 
`patients with CLL and 12 healthy donors, CD8 T cells from 20 patients with 
`CLL and 12 healthy donors, and normal and malignant B cells were nega-
`tively selected by depletion of the following as appropriate: CD4 or CD8  
`T cells, B cells, monocytes, granulocytes, platelets, early erythroid precursor 
`cells, and NK cells. For negative selection, a magnetically labeled cocktail of 
`hapten-modified anti-CD14, -CD16, -CD36, -CD56, -CD123, -TCRγδ, and  
`–glycophorin A, with or without CD4, CD8, or CD19 mAbs (Miltenyi Bio-
`tec), was used. The purity of the isolated T cells and B cells was detected 
`using anti-CD19, anti-CD4, and anti-CD8 antibodies. Frozen or freshly 
`isolated CD4 or CD8 T cells were lysed in TRIzol for total-RNA isolation 
`(Invitrogen Corp.), and 3–15 µg of total RNA was used for gene chip array.
`Gene chip array. Quality control of the RNA samples was performed by 
`spectrophotometric analysis to confirm the concentration and to detect 
`contaminating proteins and other molecules, and a size fractionation pro-
`cedure using a microfluidics instrument (Agilent Technologies) was used 
`to determine whether the RNA was intact.
`
`1802
`
`The Journal of Clinical Investigation      http://www.jci.org      Volume 115      Number 7      July 2005
`
`UPenn Ex. 2069
`Miltenyi v. UPenn
`IPR2022-00855
`
`

`

`research article
`
`RNA conversion of cDNA and subsequent hybridization to gene arrays 
`were performed in the Core Facility at Dana-Farber Cancer Institute, all 
`steps according to the manufacturer’s protocols (Affymetrix Inc.). Briefly,  
`RNA was converted into cDNA using a T7 promoter–tailed oligo-dT 
`primer in the synthesis of the first cDNA strand, and second-strand cDNA 
`synthesis was then carried out. The double-stranded cDNA was used as 
`the template in an in vitro transcription (IVT) reaction catalyzed by T7 
`polymerase and containing biotinylated CTP and UTP in addition to the 4 
`unmodified ribonucleoside triphosphates. The biotinylated complemen-
`tary RNA (cRNA) was purified from the IVT reaction mixture using the 
`RNeasy system (QIAGEN). Purified cRNA was fragmented in order to facil-
`itate the subsequent hybridization step. The cRNA was purified from the 
`fragmentation reaction using phenol/chloroform extraction and ethanol 
`precipitation. The fragmented cRNA was added to a hybridization solu-
`tion containing several biotinylated control oligonucleotides and hybrid-
`ized to an Affymetrix Inc. U133A microarray chip overnight at 45°C. The 
`chips were then washed to remove cRNA that had not hybridized to its 
`complementary oligonucleotide probe. The bound cRNA was fluorescently 
`labeled using PE-conjugated streptavidin (SAPE); additional fluors were 
`then added using biotinylated anti-streptavidin antibody and additional 
`SAPE. Each cRNA bound at its complementary oligonucleotide was excit-
`ed using a confocal laser scanner, and the positions and intensities of the 
`fluorescent emissions were captured. These measures provided the basis of 
`subsequent biostatistical analysis.
`Biostatistical analysis.  Gene  expression  profiling  was  performed  on 
`peripheral blood CD4 and CD8 T cells from 29 previously untreated 
`CLL patients and 25 healthy donors. To identify the genes whose expres-
`sion patterns best distinguished CLL CD4 and CD8 T cells from healthy 
`CD4 and CD8 T cells, the permutation distribution of the maximum t 
`statistic was analyzed using the permax test (52). The customized pro-
`gram Permax 2.1, written by Robert Gray, calculates Permax values and 
`is available free online (http://biowww.dfci.harvard.edu/~gray/permax.
`html). Within the CLL CD4 and CD8 T cells we compared gene expres-
`sion profiles using the permax test according to 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 mm