Recombinant Human Granulocyte Colony-Stimulating Factor ...

Recombinant Human Granulocyte Colony-Stimulating Factor Significantly Decreases the Expression of CXCR3 and CCR6 on T Cells and Preferentially Induces T helper Cells to a T helper 17 Phenotype in Peripheral Blood Harvests Li-Xia Sun, Han-Yun Ren, Yong-Jin Shi, Li-Hong Wang, Zhi-Xiang Qiu The aim of this study was to investigate the expression of chemokine receptors on T cells and functional changes of T helper (Th) cells in peripheral blood stem cell (PBSC) harvests after treating healthy donors with recombinant human granulocyte colony-stimulating factor (rhG-CSF). Using multiparameter flow cytometry, we analyzed the expression of CXCR3 and CCR6 on T cells and the production of interferon-g (IFN-g), interleukin-4 (IL-4), and IL-17 by CD41 Th cells in PBSC grafts of healthy donors after in vivo rhG-CSF application. Alterations in the relative expression levels of T cell receptor beta variable (TCRBV) family members were determined using real-time polymerase chain reaction (PCR). rhG-CSF mobilization significantly decreased the expression of CXCR3 and CCR6 on T cells. Treating donors with rhG-CSF resulted in decreased IFN-g production and dramatically increased IL-4 and IL-17 secretion by CD41 Th cells, leading to T cell polarization from the Th1 to the Th2 phenotype and a preferential increase in IL-17–producing CD41 Th cells. We did not observe any differences in the relative expression levels of TCRBV family members before and after in vivo rhG-CSF application. Our results suggest that the expression of CXCR3 and CCR6 on donor T cells was dramatically downregulated and an IL-17 phenotype of CD41 Th cells was preferentially induced in PBSC grafts after treating healthy donors with rhG-CSF. The observed effects of rhG-CSF on T cells may be independent of the relative expression levels of TCRBV family members. Biol Blood Marrow Transplant 15: 835-843 (2009) Ó 2009 American Society for Blood and Marrow Transplantation

KEY WORDS: Recombinant human granulocyte colony-stimulating factor, Chemokine receptors, IL-17, TCRBV

INTRODUCTION Currently, recombinant human granulocyte colony-stimulating factor (rhG-CSF) mobilized peripheral blood stem cell (PBSC) grafts are a convenient source of hematopoietic stem cells (HSCs) and have replaced marrow for autologous and most allogeneic transplantations [1,2]. Neither the incidence nor the severity of acute graft-versus-host disease (aGVHD) is higher than with marrow transplantations, although

From the Department of Hematology, Peking University First Hospital, Beijing, People’s Republic of China. Financial disclosure: See Acknowledgments on page 842. Correspondence and reprint requests: Han-Yun Ren, PhD, Department of Hematology, Peking University First Hospital, Steet Xishiku, Beijing, China 100034 (email: [email protected]). Received February 18, 2009; accepted March 18, 2009 Ó 2009 American Society for Blood and Marrow Transplantation 1083-8791/09/157-0001$36.00/0 doi:10.1016/j.bbmt.2009.03.016

rhG-CSF-mobilized PBSC harvests contain many more mature T cells [3,4]. This might be because of the immunoregulatory effects of rhG-CSF on adaptive immunity, such as the ability to switch the T cell cytokine secretion profile to T helper (Th)2 responses and the promotion of regulatory T cell and tolerogenic dendritic cell differentiation [5–9]. However, these mechanisms are not fully understood. Previous studies showed that aGVHD necessitates the migration of donor T cells to secondary lymphoid organs independently of allogeneic disparity, whereupon allospecific T cells can respond to host alloantigens and then traffic to the target organs [10–14]. Chemokines are a family of structurally related chemotactic proteins. Their interactions with chemokine receptors are involved in many fundamental aspects of immunology, including immune system development, homeostasis, host inflammatory response, and GVHD [15–17]. Chemokine receptors have been instrumental in the characterization of subsets of human T cells; CXCR3, CXCR6, and CCR5 are 835

836

L.-X. Sun et al.

expressed in Th1 cells, whereas CCR3, CCR4, and CCR8 are expressed in Th2 cells. Data reported by Anderson et al. and other researchers [18–22] suggest that chemokine receptors, including CXCR3, CCR6, CCR10, CCR2, and CCR5, expressed on donor T cells may be crucial for lymphocyte migration and homing to secondary lymphocyte tissues and target organs during the onset of aGVHD. The important role that CXCR3 and CCR6 play in the onset of aGVHD was confirmed by the finding that blockade of CXCR3 and deletion of the CCR6 gene could alleviate the incidence and severity of aGVHD in human and animal experiments [17,23,24]. Recent studies have investigated the effects of Th17 cells on the development of GVHD [25]. Th17 cells, a newly identified Th cell subset, produce high levels of interleukin (IL)-17A (IL-17), IL-17F, IL-21, and IL-22, and have been shown to contribute to mucosal immunity [26]. Moreover, Th17 cells have been implicated in allograft rejections of solid organs and several autoimmune diseases [27–30]. Data from Yi et al. [31] demonstrate that donor Th17 cells can downregulate Th1 differentiation and ameliorate aGVHD after allogeneic hematopoietic stem cell transplantation (HSCT). Acosta-Rodriguez et al. [32] provided a functional link between CCR6 and IL-17, which have been independently associated with tissue pathology. CCR6 has been shown to be involved in the recruitment of pathogenic T cells in rheumatoid arthritis [33], experimental autoimmune encephalitis [34], allergic pulmonary inflammation, and psoriasis [15], and Th17 cells are increasingly recognized as essential mediators of those diseases [15,35]. Furthermore, an association between T cell receptors (TCR) and T cell anergy has been identified [36]. At present, the effects of rhGCSF on the expression of chemokine receptors, secretion of IL-17 cytokine by CD41 Th cells, and relative expression levels of TCRBV family have not yet been analyzed in detail. Therefore, in this study, we investigated the expression profiles of CXCR3 and CCR6 on T cells, the alteration of IL-17 production by Th cells, and the relative expression levels of TCRBV family members in rhG-CSF-mobilized PBSC harvests. MATERIALS AND METHODS

Biol Blood Marrow Transplant 15:835-843, 2009

samples were washed twice with Iscove’s Modified Dulbecco’s Medium (IMDM) and adjusted to a final concentration of 5-10 106/mL for use. All samples were processed within 6 hours of collection. Flow-Cytometric Analysis of Cell Surface Markers Cells were stained without further separation to minimize selective loss. Monoclonal antibodies (mAbs) used for analyzing surface markers including CD3, CD4, CD8, CXCR3 CCR6, IgG1, and IgG2a, were conjugated with the fluorochromes phycoerythrinTexas Red-X (ECD), phycoerythrincyanin 5.1 (PC5), peridin chlorophyll protein (PerCP), fluorescein isothiocyanate (FITC), phycoerythrin (PE), and allophycocyanin (APC). mAbs were purchased from Becton Dickinson (Fullerton, CA) and Beckman Coulter (Fullerton, CA). One hundred microliters of whole peripheral blood and diluted grafts were incubated with directly conjugated antibodies specific to CD3, CD4, CD8, CXCR3, CCR6, and isotype controls for 20 minutes (concentrations according to manufacturers’ instructions). Red blood cells were then lysed in fluoresceinactivated cell sorter (FACS) solution. After washing in phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA) and 0.1% sodium azide (NaN3), cells were resuspended in PBS for analysis. A minimum of 5000 lymphocyte-gated events were acquired and analyzed with multiparameter flow cytometry (Coulter Epics XL). Intracellular Cytokine Staining Analysis Whole PBSC and diluted grafts were stimulated by phorbol-12-myristate-13-acetate (PMA) (50 ng/mL; Sigma, St. Louis, MO) and ionomycin (1 mg/mL; Sigma) in the presence of monensin (Golgistop 3 mM/mL; Sigma) for 4 hours at 37 C in a 5% CO2 incubator. Erythrocytes were then lysed and the cells were labeled with anti-CD3 and anti-CD8 mAbs. Afterward, the cells were fixed and permeabilized with FACS Permeabilizing Solution (Becton-Dickinson) and stained with anti-IFNg-FITC, anti-IL-4-PE, and anti-IL-17-APC mAbs (Becton Dickinson). As a control, cells were stained with isotype mAbs. Cells were then acquired and analyzed with BD FACSDiva software (Becton-Dickinson).

Sample Collection Steady-state peripheral blood (SS-PB) samples were collected from 15 individual healthy donors (8 males and 7 females, mean age: 31.4 6 11.8 years, range: 14-53 years). Donors received rhG-CSF 5 mg/ kg subcutaneously once daily for 5 to 6 consecutive days. rhG-CSF-mobilized PBSC grafts were collected via apheresis for allogeneic PBSC transplantation. All sample collections had the approval of the Peking University First Hospital Ethical Committee. All graft

Quantitative Real-Time PCR Peripheral blood mononuclear cells (PBMCs) were obtained using density gradient centrifugation with Ficoll-Hypaque (density, 1.077 g/mL). Total RNA was isolated from 1.5-5.0 106 PBMCs using TRIzol Reagent according to the manufacturer’s directions (Invitrogen, Carslbad, CA). cDNA was synthesized using M-MLV reverse transcription, random hexamer primers, and 10 mM dNTP


Effects of rhG-CSF on Chemokine Receptor Expression and IL-17 Production

(Promega, Madison, WI). Primer sequences were developed using the T cell receptor beta variable region (TCRBV) classification system described by the International ImMunoGeneTics database (LeFranc, 2008; http://imgt.cines.fr/IMGT_GENE-DB). Primer and probe design was performed using Primer Express software version 3.0. The relative expression of TCRBV family members was determined using real-time PCR as previous described [37], with some modifications. Briefly, all experiments were performed on an ABI 7300 sequence detection system. Final reaction conditions involved the amplification of 1 mL of cDNA in a 25-mL reaction consisting of 10 PCR buffer containing 2.0 mM magnesium chloride, 2.5 mM dNTPs, BC, and BV primers, Taq DNA polymerase, a 5-mM dual-labeled fluorescein probe, and Rox. Reactions were brought up to final volume with sterile water. The relative expression levels of individual TCRBV transcripts were then calculated as previously described [37,38]. Statistical Analysis All of the normal distribution data were summarized as the mean 6 standard deviation (SD). Comparisons were performed using paired-sample t-tests. A value of P \ .05 was considered statistically significant for 2-sided tests. All analyses were performed with SPSS software (version 11.5).

RESULTS Alteration of T Cell Subsets before and after rhG-CSF Mobilization Not only the total nucleated cells (TNC), but also the percentage of lymphocytes, CD41, and CD81 T cells within TNC were dramatically increased in PBSC harvests after rhG-CSF mobilization (data not shown). The percentage of CD31 T cells was higher in PBSC grafts after rhG-CSF mobilization than in SS-PB (42.93% 6 12.08% versus 25.26 6 5.00%; P \ .001). The percentages of CD31CD41 T cells and CD31CD81 T cells did not differ significantly between PBSC grafts and SS-PB (67.48% 6 7.04% versus 70.71% 6 5.41%, P 5 .087 and 32.52% 6 7.06% versus 29.21% 6 5.45%, P 5 .080; respectively). Furthermore, the ratio of CD41 to CD81 cells was also unchanged (2.22% 6 0.74% versus 2.53% 6 0.61%, P 5 .099). Downregulation of CXCR3 and CCR6 Expression in T Cell Subsets after In Vivo rhGCSF Application To investigate the changes in chemokine receptor phenotypes on T cells after rhG-CSF mobilization, we analyzed the expression of CXCR3 and CCR6 on T cells before and after treating healthy donors with rhG-CSF.

837

Figure 1A shows a representative example of CXCR3 expression on T cells. Compared with SS-PB, the expression of CXCR3 on CD31 T cells was significantly lower in PBSC grafts (12.26% 6 6.58% versus 38.27% 6 4.63%, P \ .001). The expression of CXCR3 on CD41 (6.04% 6 4.80% versus 29.34% 6 4.86%, P \ .001) and CD81 T cells (24.13% 6 10.88% versus 61.76% 6 9.62%, P \ .001) was significantly decreased after rhG-CSF mobilization (Figure 1B). Figure 2A shows a representative example of CCR6 expression on T cells. Compared with SS-PB, expression of CCR6 on CD31 T cells was significantly decreased after in vivo rhG-CSF application (4.07% 6 1.67% versus 14.46% 6 3.50%, P \ .001). There were significant differences between PBSC grafts and SSPB in the percentage of CCR6 expression on CD41 (3.16% 6 2.22% versus 15.12% 6 5.31%, P \ .001) and CD81 T cells (6.53% 6 3.87% versus 12.57% 6 7.99%, P 5 .007) (Figure 2B). Collectively, these experiments show that T cells in PBSC grafts expressed decreased amounts of CXCR3 and CCR6 markers. Furthermore, these data indicate that CXCR3 and CCR6 expression on T cell subsets is downregulated after in vivo rhG-CSF application. rhG-CSF Mobilization Polarizes T Cells from the Th1 to the Th2 Phenotype and Preferentially Increases IL-17-Producing CD41 Th Cells The proportion of IFN-g-, IL-4-, and IL-17-producing CD41 T cells, both in SS-PB (n 5 12) and PBSCs (n 5 12), was determined using intracellular cytokine staining. The results showed that 2.71% and 4.98% of CD41 T cells from PBSC grafts produced IL-4 and IL-17, respectively, upon activation, whereas only 0.83% and 0.85% of Th cells from PB produced these 2 cytokines, respectively (P \ .01). In contrast, the proportion of IFN-g-producing CD41 T cells in PBSC grafts was lower than in SS-PB samples (14.44% 6 7.36% versus 20.18% 6 5.56%, P 5 .017) (Figure 3A). A representative example of IL-17–producing CD41 T cells in PBSC grafts and SS-PB is shown in Figure 3B. A representative example of IFN-g-producing and IL-4-producing CD41 T cells is not shown. We also calculated the ratios of IFN-g-, IL-4-, and IL-17-producing CD41 T cells in PBSC grafts and SSPB. As illustrated in Figure 4, the ratio of Th2:Th1 in PBSC grafts was significantly higher than in SS-PB (0.22 6 0.09 versus 0.04 6 0.01, P \ .01). Similarly, the ratios of Th17:Th2 and Th17:Th1 in PBSC grafts were, respectively, 2-fold and 8-fold higher than in SSPB (1.94 6 0.10 versus 1.02 6 0.40, P 5 .013 and 0.35 6 0.12 versus 0.04 6 0.02, P \ .001). These data indicate that polarization of T cells from the Th1 to the Th2 phenotype could be induced and that IL-17-producing CD41 Th cells were preferentially increased after treating healthy donors with rhG-CSF.

838

L.-X. Sun et al.


Figure 1. CXCR3 expression on T cells in rhG-CSF-mobilized PBSC grafts (light symbols) and SS-PB (dark symbols). Mean values are represented by horizontal lines in each series. (A) Representative example of CXCR3 expression on CD31, CD41, and CD81 T cells in SS-PB and PBSC grafts. (B) Percentages of CXCR3 expression on CD31, CD41, and CD81 T cells before and after in vivo rhG-CSF application.

No Alteration in Relative Expression of TCRBV Family Members in PBSC Grafts after rhG-CSF Administration To investigate whether administering rhG-CSF to mobilize stem cells causes skewing of the expression

levels of genes in the TCRBV family, we determined the relative expression levels of TCRBV family members before and after rhG-CSF mobilization. Figure 5 shows that there were no significant differences in the relative expression of TCRBV family members between PBSC grafts and SS-PB (P . .05).



839

Figure 2. CCR6 expression on T cells in rhG-CSF-mobilized PBSC grafts (light symbols) and SS-PB (dark symbols). Mean values are represented by horizontal lines in each series. (A) Representative example of CCR6 expression on CD31, CD41, and CD81 T cells in SS-PB and PBSC grafts. (B) Percentages of CCR6 expression on CD31, CD41, and CD81 T cells before and after in vivo rhG-CSF application.

DISCUSSION In agreement with previous studies [7], we found that the percentage of CD31 T cells within nuclear cells

in rhG-CSF-mobilized PBSC grafts was higher than in SS-PB, whereas the percentages of CD41 and CD81 T cell subsets, as well as the ratios of CD4:CD8 cells, did not differ significantly before and after in vivo

840

L.-X. Sun et al.


Percentage of CD4+ T cells (%)

A

25

**

20

15

10

*

5

*

0

IL-4

IFN-γ

B

SS-PB 2.45%

IL-17

PBSC grafts 5.60%

Figure 3. Effect of rhG-CSF on IFN-g, IL-4, and IL-17 production by CD41 Th cells. Bars represent percentages (mean 6 SD) in IFN-g-, IL-4-, and IL17–expressing CD41 Th cells before (light bars) and after (black bars) rhG-CSF mobilization. P values were determined using t-tests for paired samples. *P \.01, **P \.05. (A) Mean percentages of IFN-g-, IL-4-, and IL-17-producing CD41 Th cells. (B) Representative example of IL-17-producing CD41 T cells before and after rhG-CSF application.

rhG-CSF application. Our results and those of Tayebi et al. [7] suggest that it was primarily the quality, not the quantity, of T cells in PBSC harvests that changed after in vivo rhG-CSF mobilization. Rutella et al. [5] reported that they found no differences between freshly isolated pre-G-CSF and postG-CSF CD4 T cells in the expression of cell-surface antigens including CXCR3 and CCR6, but they did 2.5

**

Ratio

2 1.5 1 0.5 0

* * Th2 : Th1

Th17 : Th2

Th17 : Th1

Figure 4. Ratios of IFN-g-, IL-4-, and IL-17-producing CD41 Th cells in SS-PB (light bars) and rhG-CSF-mobilized PBSC grafts (dark bars). *P \.01.

not report in detail any changes in the expression of these markers in nonisolated T cell harvests that were transplanted to recipients. In the present study, we found that treating healthy donors with rhG-CSF significantly decreased the expression of CXCR3 and CCR6 on T cells. Recently, using a well-defined experimental bone marrow transplantation model (where aGVHD is mediated by donor CD81 T cells) to investigate the role of CXCR3 on donor cells in aGVHD, Duffner et al. [39] found that CXCR3 is not required for trafficking from venous circulation into secondary lymphoid organs, but is needed for migration into epithelial target tissues. Moreover, blockading CXCR3 receptor/ligand interactions reduces leukocyte recruitment to the lung and the severity of experimental idiopathic pneumonia syndrome [23]. Varona et al. [17] demonstrated that CCR6 may cause T cells to migrate directly to target tissues rather than through secondary lymph nodes, which suggests that CCR6 plays a major role in aGVHD development. Taken together, it is conceivable that downregulation of CXCR3 and CCR6 expression on donor CD41 and CD81 T cells after in vivo rhG-CSF application may alter the migratory ability of these T cells, as demonstrated by other



841

12

10

8

6

4

BV30

BV29

BV28

BV27

BV25

BV24

BV20

BV19

BV18

BV16

BV15

BV14

BV13

BV12

BV11

BV9

BV10

BV7-3

BV7-2

BV6-9

BV5

BV6-1

BV4

0

BV3

2

BV2

TCRBV family relative expression (%)

14

Figure 5. Relative expression levels of TCRBV family members in SS-PB (light bars) and rhG-CSF-mobilized PBSC graft (dark bars). P values were determined using t-tests for paired samples. All P . .05.

researchers [17,23,39]. Also, chemokine receptor CXCR3 and CCR6 expression may be affected by other cytokines such IFN-g, IFN-a, and tumor growth factor (TGF)-b, present during Th cells polarization after rhG-CSF application [40]. This could explain in part the lack of amplification of aGVHD after transplantation, despite the presence of 10-fold more mature T cells in rhG-CSF-mobilized PBSC harvests compared with steady-state bone marrow grafts. The polarization of T cells from the Th1 to the Th2 phenotype has been observed by other researchers [3,8,41-44]. Our results are in agreement with their observations, and show that rhG-CSF-mobilized PBSC harvests contained significantly more IL-4-producing cells (Th2) and less IFN-g-producing cells (Th1). This suggests Th2 polarization, which might contribute to attenuation of aGVHD after rhG-CSF-mobilized PBSC transplantation. To our minds, the most important and interesting finding of our study was that rhG-CSF mobilization preferentially induced a Th17 phenotype, leading to higher ratios of IL-17producing cells (Th17) to IFN-g-producing cells (Th1). This could contribute to the relative low incidence of aGVHD in allogeneic PBSC transplant settings, as demonstrated by Yi et al. [31] in mice models. However, there have been conflicting studies in which researchers found that IL-17 cannot prevent the development of aGVHD and, indeed, may even exacerbate it [45,46]. Therefore, the role of IL-17 in aGVHD warrants further study, especially in clinical settings. With regard to functional changes of CD41 Th cells [47], several possible explanations may account for the increase in IL-4 and IL-17 production and the decrease in IFN-g secretion that we observed. First, rhG-CSF mobilization increased the secretion of IL-

17, resulting in a decrease in IFN-g production by CD41 Th cells as was previously observed in animal models [31]; this led to the polarization of Th cells from the Th1 to the Th2 phenotype. Second, rhGCSF application in vivo simultaneously increased the production of IL-4 and IL-17, both of which suppress the secretion of IFN-g. Third, rhG-CSF treatment could directly increase IL-4 and IL-17 production and decrease IFN-g secretion. Recently, GATA-3 was identified as a T cell-specific transcription factor selectively expressed in Th2 cells [48]. Franzke et al. [41] found that GATA-3 could be upregulated in CD41 T cells upon rhG-CSF stimulation, although GATA-3 directly represses the opposing Th1 immune response most likely by interfering with the IL-12 signal transduction pathway. Therefore, polarization of T cells from Th1 to Th2 could be found after in vivo rhG-CSF application. Furthermore, Th1 cells could negatively regulate the development of Th2 and Th17 cells, so Th17 skewing was expected as demonstrated in the current study [49]. In consideration of the evidence that CXCR3, CXCR6, and CCR5 are ‘‘preferentially’’ expressed in Th1 cells [32], our findings that rhG-CSF in vivo application significantly decreased the percentage of IFN-g-producing Th cells and that CXCR3 expression was significantly reduced suggest that not only the function, but also the migratory characteristics, of T cells were altered after treating healthy donors with rhG-CSF. Another interesting finding of our study was that there were no significant alterations in the relative expression of TCRBV family members after treating healthy donors with rhG-CSF, suggesting that rhG-CSF administration may not cause skewing in the relative expression of TCRBV family members,

842

L.-X. Sun et al.

although rhG-CSF has a direct and indirect immunoregulatory effect exerted by adaptive immunity. This also suggests that a normal immune response could exist after treating healthy donors with rhG-CSF. In conclusion, our results suggest that rhG-CSF may have multiple effects on T cells, including the downregulation of various chemokine receptors, thus influencing the migratory ability of T cells to target organs during aGVHD, and in the alteration of the Th1/ Th2/Th17 paradigm. The association between these changes and transplant outcomes, especially with regard to aGVHD, are currently being investigated in allogeneic transplant settings. This research may lead to a more in-depth understanding of donor T cell migration and Th1/Th2/Th17 imbalance in rhG-CSF-mobilized PBSC transplantation, and may contribute to an effective strategy for GVHD prophylaxis and treatment.

ACKNOWLEDGMENTS The authors thank the faculty members in the Department of Hematology, Peking University First Hospital, for their assistance in sample collection. They also thank Dr. Ding-Fang Bu and Dr. HongJun Hao for their technical support and San Francisco Edit for editing and proofreading the manuscript. Li-Xia Sun performed research, analysis, and interpretation of data; drafted the article; and provided final approval of the version to be published. HanYun Ren conceived and designed the experiments, critically revised the article, and provided final approval of the version to be published. All other authors treated patients at the Department of Hematology, Peking University First Hospital. Financial disclosure: The authors have nothing to disclose.

REFERENCES 1. Anderlini P, Champlin RE. Biologic and molecular effects of granulocyte colony-stimulating factor in healthy individuals: recent findings and current challenges. Blood. 2008;111:1767-1772. 2. Copelan EA. Hematopoietic stem-cell transplantation. N Engl J Med. 2006;354:1813-1826. 3. Guardiola P, Runde V, Bacigalupo A, et al. Retrospective comparison of bone marrow and granulocyte colony-stimulating factor-mobilized peripheral blood progenitor cells for allogeneic stem cell transplantation using HLA identical sibling donors in myelodysplastic syndromes. Blood. 2002;99:4370-4378. 4. Robinet E, Lapierre V, Tayebi H, Kuentz M, Blaise D, Tiberghien P. Blood versus marrow hematopoietic allogeneic graft. Transfus Apher Sci. 2003;29:53-59. 5. Rutella S, Pierelli L, Bonanno G, et al. Role for granulocyte colony-stimulating factor in the generation of human T regulatory type 1 cells. Blood. 2002;100:2562-2571. 6. Rutella S, Bonanno G, Pierelli L, et al. Granulocyte colonystimulating factor promotes the generation of regulatory DC through induction of IL-10 and IFN-alpha. Eur J Immunol. 2004;34:1291-1302.


7. Tayebi H, Kuttler F, Saas P, et al. Effect of granulocyte colonystimulating factor mobilization on phenotypical and functional properties of immune cells. Exp Hematol. 2001;29:458-470. 8. Nawa Y, Teshima T, Sunami K, et al. G-CSF reduces IFNgamma and IL-4 production by T cells after allogeneic stimulation by indirectly modulating monocyte function. Bone Marrow Transplant. 2000;25:1035-1040. 9. Zeng D, Dejbakhsh-Jones S, Strober S. Granulocyte colonystimulating factor reduces the capacity of blood mononuclear cells to induce graft-versus-host disease: impact on blood progenitor cell transplantation. Blood. 1997;90:453-463. 10. Wysocki CA, Panoskaltsis-Mortari A, Blazar BR, Serody JS. Leukocyte migration and graft-versus-host disease. Blood. 2005;105:4191-4199. 11. Beilhack A, Schulz S, Baker J, et al. In vivo analyses of early events in acute graft-versus-host disease reveal sequential infiltration of T-cell subsets. Blood. 2005;106:1113-1122. 12. Ferrara JL, Reddy P. Pathophysiology of graft-versus-host disease. Semin Hematol. 2006;43:3-10. 13. Murai M, Yoneyama H, Ezaki T, et al. Peyer’s patch is the essential site in initiating murine acute and lethal graft-versus-host reaction. Nat Immunol. 2003;4:154-160. 14. von Andrian UH, Mackay CR. T-cell function and migration. Two sides of the same coin. N Engl J Med. 2000;343:1020-1034. 15. Keller M, Spanou Z, Schaerli P, et al. T cell-regulated neutrophilic inflammation in autoinflammatory diseases. J Immunol. 2005;175:7678-7686. 16. Kikly K, Liu L, Na S, Sedgwick JD. The IL-23/Th(17) axis: therapeutic targets for autoimmune inflammation. Curr Opin Immunol. 2006;18:670-675. 17. Varona R, Cadenas V, Gomez L, Martinez AC, Marquez G. CCR6 regulates CD41 T-cell-mediated acute graft-versushost disease responses. Blood. 2005;106:18-26. 18. Anderson BE, Taylor PA, McNiff JM, et al. Effects of donor T-cell trafficking and priming site on graft-versus-host disease induction by naive and memory phenotype CD4 T cells. Blood. 2008;111:5242-5251. 19. Wysocki CA, Jiang Q, Panoskaltsis-Mortari A, et al. Critical role for CCR5 in the function of donor CD41CD251 regulatory T cells during acute graft-versus-host disease. Blood. 2005;106: 3300-3307. 20. Piper KP, Horlock C, Curnow SJ, et al. CXCL10-CXCR3 interactions play an important role in the pathogenesis of acute graft-versus-host disease in the skin following allogeneic stemcell transplantation. Blood. 2007;110:3827-3832. 21. Faaij CM, Lankester AC, Spierings E, et al. A possible role for CCL27/CTACK-CCR10 interaction in recruiting CD4 T cells to skin in human graft-versus-host disease. Br J Haematol. 2006; 133:538-549. 22. Terwey TH, Kim TD, Kochman AA, et al. CCR2 is required for CD8-induced graft-versus-host disease. Blood. 2005;106: 3322-3330. 23. Hildebrandt GC, Corrion LA, Olkiewicz KM, et al. Blockade of CXCR3 receptor:ligand interactions reduces leukocyte recruitment to the lung and the severity of experimental idiopathic pneumonia syndrome. J Immunol. 2004;173:2050-2059. 24. He S, Cao Q, Qiu Y, et al. A new approach to the blocking of alloreactive T cell-mediated graft-versus-host disease by in vivo administration of anti-CXCR3 neutralizing antibody. J Immunol. 2008;181:7581-7592. 25. Chen X, Vodanovic-Jankovic S, Johnson B, Keller M, Komorowski R, Drobyski WR. Absence of regulatory T-cell control of TH1 and TH17 cells is responsible for the autoimmune-mediated pathology in chronic graft-versus-host disease. Blood. 2007;110:3804-3813. 26. Mills KH. Induction, function and regulation of IL-17producing T cells. Eur J Immunol. 2008;38:2636-2649. 27. Antonysamy MA, Fanslow WC, Fu F, et al. Evidence for a role of IL-17 in organ allograft rejection: IL-17 promotes the functional differentiation of dendritic cell progenitors. J Immunol. 1999;162:577-584.



28. Loong CC, Hsieh HG, Lui WY, Chen A, Lin CY. Evidence for the early involvement of interleukin 17 in human and experimental renal allograft rejection. J Pathol. 2002;197:322-332. 29. Singh R, Aggarwal A, Misra R. Th1/Th17 cytokine profiles in patients with reactive arthritis/undifferentiated spondyloarthropathy. J Rheumatol. 2007;34:2285-2290. 30. Yoshida S, Haque A, Mizobuchi T, et al. Anti-type V collagen lymphocytes that express IL-17 and IL-23 induce rejection pathology in fresh and well-healed lung transplants. Am J Transplant. 2006;6:724-735. 31. Yi T, Zhao D, Lin CL, et al. Absence of donor Th17 leads to augmented Th1 differentiation and exacerbated acute graftversus-host disease. Blood. 2008;112:2101-2110. 32. Acosta-Rodriguez EV, Rivino L, Geginat J, et al. Surface phenotype and antigenic specificity of human interleukin 17-producing T helper memory cells. Nat Immunol. 2007;8:639-646. 33. Ruth JH, Shahrara S, Park CC, et al. Role of macrophage inflammatory protein-3alpha and its ligand CCR6 in rheumatoid arthritis. Lab Invest. 2003;83:579-588. 34. Kohler RE, Caon AC, Willenborg DO, Clark-Lewis I, McColl SR. A role for macrophage inflammatory protein-3 alpha/CC chemokine ligand 20 in immune priming during T cell-mediated inflammation of the central nervous system. J Immunol. 2003;170:6298-6306. 35. Schaerli P, Britschgi M, Keller M, et al. Characterization of human T cells that regulate neutrophilic skin inflammation. J Immunol. 2004;173:2151-2158. 36. Salojin KV, Zhang J, Madrenas J, Delovitch TL. T-cell anergy and altered T-cell receptor signaling: effects on autoimmune disease. Immunol Today. 1998;19:468-473. 37. Walters G, Alexander SI. T cell receptor BV repertoires using real time PCR: a comparison of SYBR green and a dual-labelled HuTrec fluorescent probe. J Immunol Methods. 2004;294:43-52. 38. Du G, Qiu L, Shen L, et al. Combined megaplex TCR isolation and SMART-based real-time quantitation methods for quantitating antigen-specific T cell clones in mycobacterial infection. J Immunol Methods. 2006;308:19-35. 39. Duffner U, Lu B, Hildebrandt GC, et al. Role of CXCR3induced donor T-cell migration in acute GVHD. Exp Hematol. 2003;31:897-902.

843

40. Sallusto F, Lenig D, Mackay CR, Lanzavecchia A. Flexible programs of chemokine receptor expression on human polarized T helper 1 and 2 lymphocytes. J Exp Med. 1998;187:875-883. 41. Franzke A, Piao W, Lauber J, et al. G-CSF as immune regulator in T cells expressing the G-CSF receptor: implications for transplantation and autoimmune diseases. Blood. 2003;102:734-739. 42. Pan L, Delmonte J Jr., Jalonen CK, Ferrara JL. Pretreatment of donor mice with granulocyte colony-stimulating factor polarizes donor T lymphocytes toward type-2 cytokine production and reduces severity of experimental graft-versus-host disease. Blood. 1995;86:4422-4429. 43. Pan L, Teshima T, Hill GR, et al. Granulocyte colony-stimulating factor-mobilized allogeneic stem cell transplantation maintains graft-versus-leukemia effects through a perforindependent pathway while preventing graft-versus-host disease. Blood. 1999;93:4071-4078. 44. Sloand EM, Kim S, Maciejewski JP, et al. Pharmacologic doses of granulocyte colony-stimulating factor affect cytokine production by lymphocytes in vitro and in vivo. Blood. 2000;95: 2269-2274. 45. Carlson MJ, West ML, Coghill JM, Panoskaltsis-Mortari A, Blazar BR, Serody JS. In vitro-differentiated TH17 cells mediate lethal acute graft-versus-host disease with severe cutaneous and pulmonary pathologic manifestations. Blood. 2009;113: 1365-1374. 46. Kappel LW, Goldberg GL, King CG, et al. IL-17 contributes to CD4-mediated graft-versus-host disease. Blood. 2009;113: 945-952. 47. Harrington LE, Hatton RD, Mangan PR, et al. Interleukin 17producing CD41 effector T cells develop via a lineage distinct from the T helper type 1 and 2 lineages. Nat Immunol. 2005;6: 1123-1132. 48. Zheng W, Flavell RA. The transcription factor GATA-3 is necessary and sufficient for Th2 cytokine gene expression in CD4 T cells. Cell. 1997;89:587-596. 49. Harrington LE, Mangan PR, Weaver CT. Expanding the effector CD4 T-cell repertoire: the Th17 lineage. Curr Opin Immunol. 2006;18:349-356.