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Research article BioMed Central Open Access Differential gene expression by integrin 7+ and 7- memory T helper cells
Madeleine W Rodriguez1, Agns C Paquet1, Yee Hwa Yang1 and David J Erle*1,2
Address: 1Department of Medicine, University of California, San Francisco, CA 94143-0854 USA and 2Program in Immunology, University of California, San Francisco, CA 94143-0854 USA Email: Madeleine W Rodriguez - email@example.com; Agns C Paquet - firstname.lastname@example.org; Yee Hwa Yang - email@example.com; David J Erle* - firstname.lastname@example.org * Corresponding author Published: 05 July 2004 BMC Immunology 2004, 5:13 doi:10.1186/1471-2172-5-13 Received: 12 February 2004 Accepted: 05 July 2004 This article is available from: http://www.biomedcentral.com/1471-2172/5/13 2004 Rodriguez et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL. Abstract
Background: The cell adhesion molecule integrin 47 helps direct the migration of blood lymphocytes to the intestine and associated lymphoid tissues. We hypothesized that 7+ and 7blood memory T helper cells differ in their expression of genes that play a role in the adhesion or migration of T cells. Results: RNA was prepared from 7+ and 7- CD4+ CD45RA- blood T cells from nine normal human subjects and analyzed using oligonucleotide microarrays. Of 21357 genes represented on the arrays, 16 were more highly expressed in 7+ cells and 18 were more highly expressed in 7cells (1.5 fold difference and adjusted P < 0.05). Several of the differentially expressed transcripts encode proteins with established or putative roles in lymphocyte adhesion and chemotaxis, including the chemokine receptors CCR9 and CCR10, the integrin 4 subunit, L-selectin, KLRB1 (CD161), NT5E (CD73), LGALS1 and LGALS2 (galectin-1 and -2), and RGS1. Flow cytometry was used to determine whether differences in levels of transcripts encoding cell surface proteins were associated with differential expression of those proteins. Using this approach, we found that surface expression of KLRB1, LAIR1, and NT5E proteins was higher on 7+ memory/effector T cells than on 7- cells. Conclusions: Memory/effector T cells that express integrin 7 have a distinct pattern of expression of a set of gene transcripts. Several of these molecules can affect cell adhesion or chemotaxis and are therefore likely to modulate the complex multistep process that regulates trafficking of CD4+ memory T cell subsets with different homing behaviors. Background
Lymphocyte migration is a multistep process that involves a complex interplay between adhesion molecules and chemokines and their G protein-coupled receptors [1,2]. Nave T cells express the adhesion molecule L-selectin, the chemokine receptor CCR7 and other molecules that allow these cells to migrate preferentially to secondary lymphoid organs where they can encounter antigen-presenting cells. When presented with appropriate antigens, these T cells can differentiate into memory T cells. Some memory cells continue to express L-selectin and/or CCR7 and to migrate efficiently to secondary lymphoid organs, Page 1 of 11
(page number not for citation purposes) BMC Immunology 2004, 5:13 http://www.biomedcentral.com/1471-2172/5/13 whereas others lose expression of these molecules and instead express other molecules that direct migration (or "homing") to other organs [1,2]. Extensive investigation has helped to define the role of some adhesion molecules and chemokine receptors in CD4+ memory T cell homing to the skin and the gut. The adhesion molecule cutaneous lymphocyte antigen (CLA) and the cutaneous T cell-attracting chemokine receptor CCR10 help control T cell homing to the skin [3-8]. The adhesion molecule integrin 47 and the chemokine receptor CCR9 play key roles in homing of lymphocytes to the intestine and Peyer's patches [9-13]. Integrin 47 is a receptor for mucosal addressin cell adhesion molecule-1 (MAdCAM-1), a glycoprotein that is expressed by gut endothelium. CCR9 is a receptor for the chemokine TECK (CCL25), which is expressed by endothelial cells and other cells in the small intestine [14,15]. The adhesion molecule and chemokine receptor expression pattern of memory/effector CD4+ T cells is strongly influenced by whether initial T cell activation takes place in cutaneous or intestinal lymph nodes [16-18]. Recent evidence suggests that T cell homing receptor expression patterns are "imprinted" by dendritic cells during antigen presentation [19-21]. Although many other molecules have been shown to help control lymphocyte adhesion or migration, it is not clear which if any of these are selective expressed in gut homing memory CD4+ T cells. We used DNA microarrays to systematically compare RNA transcript expression in human blood 7+ and 7- CD4+ memory T cells. We identified a substantial number of differentially expressed genes, many of which have been previously shown to have effects on cell adhesion and migration. In addition, we showed that some of these transcript expression differences were reflected in differences in surface expression of the encoded proteins.
Microarray analysis of differential gene expression by 7+ and 7- CD4+ memory T cells As previously reported , human blood CD4+ T cells could be divided into three distinct subsets based upon their surface expression of integrin 7 and the nave cell marker CD45RA (Fig. 1A). CD45RA+ nave cells expressed low levels of 7. CD45RA- memory T cells were divided into a larger population of 7- cells and a smaller population of 7+ cells. The integrin 7 subunit can combine with either integrin 4 (to form the gut homing receptor 47) or E. Integrin E7 is highly expressed on gut intraepithelial lymphocytes but is rarely expressed by blood T cells and its possible role in homing is less clear [23-25]. Consistent with previous reports [22,23,26], staining with antibodies specific for the 47 and E7 complexes (Act-1 and HML-1 respectively) showed that the virtually all 7-expressing CD4+ T cells in the blood were 47+ , and that only 0.5 0.3% (mean SD) of blood CD4+ T cells expressed E7. We were able to obtain highly purified populations of 7+ and 7- CD4+ CD45RA- cells by flow cytometric sorting (Fig. 1B and 1C). We used DNA microarrays to compare RNA transcript expression by 7+ and 7- cells from 9 healthy adult human subjects. Using this approach, we identified 16 genes that were expressed at higher levels in 7+ cells (at least 1.5-fold higher expression and P < 0.05 when adjusted for multiple comparisons). These are listed in Table 1. Our estimates of fold difference are likely to be conservative since the use of amplified RNA (as opposed to unamplified cDNA) tends to underestimate fold differences . The most highly differentially expressed transcript encodes the chemokine receptor CCR9. CCR9 protein has previously been shown to be preferentially expressed on 47+ blood CD4+ T cells . The ITGA4 transcript encodes the subunit of integrin 47, and was expressed at 1.7-fold higher levels on 7+ cells. Other genes listed in Table 1 include genes encoding cell surface receptors (LRRN3, CD1C, KLRB1, LAIR1, IL18RAP, KLRG1), transcription factors (RAM2, SREBF1) and a transcriptional repressor known to play a role in T cell differentiation (GFI1). A complete listing of all gene expression results is provided in Appendix 2 [see additional file 2]. We also identified 18 gene transcripts that were expressed at significantly lower levels in 7+ cells compared with 7cells (at least 1.5 times lower and adjusted P < 0.05). These are listed in Table 2. GPR2, the most highly differentially expressed transcript, encodes CCR10, the receptor for the cutaneous T cell attracting chemokine (CTACK/ CCL27). Hudak et al.  found that CCR10 protein was present on a subset of CD4+ CD45RA- T cells that express the skin homing receptor CLA but do not express integrin 47. We also found that 7- cells expressed more transcript for SELL, which encodes L-selectin (CD62L), a nonintegrin cell adhesion molecule that mediates lymphocyte adhesion to peripheral lymph node high endothelial venules. None of the other genes listed in Table 2 have established roles in organ-specific homing, although both RGS1 and LGALS1 have been reported to modulate lymphocyte adhesion (see Discussion). Given the importance of G protein-coupled receptors in regulating leukocyte migration, we note that in addition to GPR2/CCR10, one other G protein-coupled receptor, P2RY5, was expressed at higher levels on 7- cells. Although classified as a purinergic receptor, expression of P2RY5 did not result in nucleotide-evoked signaling responses, and the function of this receptor remains unknown . Results Page 2 of 11
(page number not for citation purposes) BMC Immunology 2004, 5:13 http://www.biomedcentral.com/1471-2172/5/13 A 10 10 3 B 10 10 3 C 10 10 3 2 2 2 7 10
1 7 10
1 7 10
1 10 0 10 0 10 10 CD45RA 1 2 10 3 10 0 10 0 10 10 CD45RA 1 2 10 3 10 0 10 0 10 10 CD45RA 1 2 10 3 Figure 1 expression on blood T helper cells and on purified 7- and 7+ memory T helper cell subsets Integrin 7 Integrin 7 expression on blood T helper cells and on purified 7- and 7+ memory T helper cell subsets. Dot density plots depicting expression of the nave cell marker CD45RA and integrin 7 on CD4+ blood T cells (A) and on purified 7+ (B) and 7- (C) memory cells in arbitrary fluorescence units. Sorted populations were ~99% pure. Quadrant markers indicate settings used to sort populations based on CD45RA and 7 expression. Colors indicate the relative frequency of cells (from lowest to highest density: green, purple, light blue, orange, dark blue, and yellow). Table 1: Gene transcripts with higher expression in 7+ versus 7- CD4+ CD45RA- T helper cells* Symbol CCR9 CCL5 RAM2 LRRN3 GFI1 ITGA4 CD1C KLRB1 LAIR1 RRM2 -HHL IL18RAP SREBF1 KLRG1 LGALS2 Name chemokine (C-C motif) receptor 9 chemokine (C-C motif) ligand 5 transcription factor RAM2 leucine rich repeat neuronal 3 growth factor independent 1 integrin, alpha 4 (CD49D) CD1C antigen, c polypeptide killer cell lectin-like receptor subfamily B, member 1 leukocyte-associated Ig-like receptor 1 ribonucleotide reductase M2 polypeptide Homo sapiens cDNA FLJ32290 fis, clone PROST2000463 expressed in hematopoietic cells, heart, liver interleukin 18 receptor accessory protein sterol regulatory element binding transcription factor 1 killer cell lectin-like receptor subfamily G, member 1 lectin, galactoside-binding, soluble, 2 (galectin 2) Accession NM_031200 NM_002985 NM_018719 AL442092 NM_005263 NM_000885 NM_001765 NM_002258 NM_002287 NM_001034 AK056852 NM_014857 NM_003853 NM_004176 NM_005810 NM_006498 Fold Difference +3.0 +2.4 +2.2 +2.1 +1.8 +1.7 +1.7 +1.7 +1.7 +1.6 +1.6 +1.6 +1.6 +1.6 +1.5 +1.5 P value < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 0.02 < 0.01 < 0.01 < 0.01 0.01 * Includes all transcripts with fold difference +1.5 and adjusted P < 0.05. Positive fold difference values indicate higher expression on 7+ cells. Comparison of surface protein expression on 7+ and 7CD4+ memory T cells We wished to determine whether differences in expression of selected transcripts were reflected in differences in expression of the cell surface proteins encoded by those transcripts. As previously discussed, the products of the two most highly differentially expressed genes (CCR9 and GPR2/CCR10) have already been shown to be highly selectively expressed on 47+ and 47- CD4+ T cells respectively [8,13]. We selected four other cell surface proteins encoded by transcripts that were expressed more highly on 7+ cells for further analysis based upon the availability of suitable antibodies. Three of these (LAIR1, KLRB1, and KLRG1) are listed in Table 1. A fourth, NT5E (ecto-5'-nucleotidase, also known as CD73), was also significantly higher in 7+ cells (adjusted P < 0.01), Page 3 of 11
(page number not for citation purposes) BMC Immunology 2004, 5:13 http://www.biomedcentral.com/1471-2172/5/13 Table 2: Gene transcripts with higher expression in 7- versus 7+ CD4+ CD45RA- T helper cells* Symbol GPR2 RGS1 TRIM2 C1orf24 CAMTA1 LMNA LGALS1 P2RY5 SELL -ChGn -PTPLA MRC2 PRNP PHLDA1 KRT1 SEMA5A Name G protein-coupled receptor 2 (CCR10) regulator of G-protein signalling 1 tripartite motif-containing 2 chromosome 1 open reading frame 24 calmodulin binding transcription activator 1 lamin A/C lectin, galactoside-binding, soluble, 1 (galectin 1) purinergic receptor P2Y, G-protein coupled, 5 selectin L (lymphocyte adhesion molecule 1) similar to RIKEN cDNA 1700007B22 chondroitin beta1,4 N-acetylgalactosaminyltransferase similar to BcDNA:GH11415 gene product protein tyrosine phosphatase-like, member a mannose receptor, C type 2 prion protein (p27-30) pleckstrin homology-like domain, family A, member 1 keratin 1 semaphorin 5A Accession NM_016602 NM_002922 NM_015271 NM_052966 AB020640 NM_005572 NM_002305 NM_005767 NM_000655 AK021437 NM_018371 AK056276 NM_014241 NM_006039 NM_000311 AK026181 NM_006121 NM_003966 Fold Difference -3.2 -2.0 -1.9 -1.9 -1.9 -1.9 -1.9 -1.8 -1.8 -1.8 -1.8 -1.7 -1.7 -1.6 -1.6 -1.6 -1.5 -1.5 P value < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 0.02 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 * Includes all transcripts with fold difference -1.5 and adjusted P < 0.05. Negative fold difference values indicate higher expression on 7- cells. Table 3: Surface expression of five gene products on 7+ versus 7- CD4+ T cells Mean surface protein expression* Gene LAIR1 KLRB1 KLRG1 NT5E SELL Fold difference: Transcript** +1.7 +1.7 +1.5 +1.3 -1.8 Fold difference: Surface protein*** +2.5 (+1.6 to +3.9) +2.5 (+2.2 to +3.6) 1.0 (-3.2 to +2.1) +1.7 (+1.2 to +3.2) -1.8 (-2.1 to -1.5) CD45RA- 7+ cells 34.6 26.1 17.7 20.3 22.8 CD45RA- 7- cells 15.3 10.7 15.6 11.7 40.2 CD45RA+ cells 37.3 1.1 9.1 6.8 50.7 * Mean fluorescence intensity (MFI, in arbitrary units) was measured for 58 subjects.** As determined using DNA microarrays. *** Values represent mean (range) of the fold difference in MFI between CD45RA- 7+ cells and CD45RA- 7- cells for all subjects. Positive fold difference values indicate higher expression on 7+ cells and negative fold difference values indicate higher expression on 7- cells. although the estimated magnitude of the difference was smaller (1.3-fold). We used flow cytometry to measure expression of each of these four molecules on CD4+ T cell subsets from human blood. For comparison, we also analyzed surface expression of one protein (SELL/CD62L) encoded by a transcript that was expressed at higher levels on 7- cells. We used a different population of study subjects from that used in the microarray studies to ensure that our findings could be validated in an independent sample. For four of the five proteins examined, the extent of differential surface expression was at least as great as predicted from the microarray data (Table 3). The other protein, KLRG1, was not consistently differentially expressed. We used flow cytometry to conduct a more detailed analysis of the expression of KLRB1, NT5E and LAIR1 on T helper cell subsets with distinct homing properties. A small subset of memory cells expressed the skin homing receptor CLA (~4% of CD4+ CD45RA- T cells). These CLA+ cells had low expression of KLRB1, NT5E and LAIR1 (similar to levels found in other 7- CD45RA- cells, data not shown). The adhesion molecule L-selectin (SELL, CD62L) and the chemokine receptor CCR7 both play roles in homing to secondary lymphoid organs and each is Page 4 of 11
(page number not for citation purposes) BMC Immunology 2004, 5:13 http://www.biomedcentral.com/1471-2172/5/13 A 10 10 3 B 10 10 3 2 2 7 10
1 7 10
1 10 0 10 0 10 10 SELL/CD62L 1 2 10 3 10 0 10 0 10 10 CCR7 1 2 10 3 Figure subset of memory/effector T helper cells lacks CD62L and CCR7 A 7high 2 A 7high subset of memory/effector T helper cells lacks CD62L and CCR7. Dot density plots depicting expression of integrin 7 and (A) SELL (CD62L) or (B) CCR7 on CD4+ CD45RA- T cells. Quadrant lines were drawn based on staining obtained with control antibodies. expressed on subsets of memory/effector T cells. Memory/ effector cells that lacked L-selectin and CCR7 included a subset with particularly high 7 expression and a larger subset with no 7 expression (Fig. 2). CD45RA+ cells that expressed L-selectin and CCR7 (central memory cells) also included 7+ and 7- subsets. We found that KLRB1, a member of the killer cell lectin-like receptor superfamily also known as CD161 or NKRP1A , was highly expressed on 7+ CCR7- memory effector T helper cells (Fig. 3). There was less expression on 7- CCR7- memory effector cells and on CCR7+ central memory cells. KLRB1 was essentially undetectable on CD45RA+ nave cells. The expression pattern of NT5E/CD73 was similar, although NT5E expression was limited to a smaller fraction of cells than KLRB1 expression (Fig. 4). In contrast, LAIR1 had a different pattern of expression (Fig. 5). LAIR1 was most highly expressed on nave cells, and was also highly expressed on 7+ CD45RA- cells (both CCR7- and CCR7+ subsets). LAIR1 was less highly expressed on 7- CD45RAcells (especially the CCR7- subset). LAIR1 contains two ITIM domains and can transmit signals following ligation by antibody, however the nature of the LAIR1 ligand(s) and the biological function of LAIR1 on CD4+ T cells remains unknown . Discussion
We used DNA microarrays to identify gene transcripts that were differentially expressed on the subset of CD4+ memory/effector T cells that express integrin 7. Since the overwhelming majority of 7+ cells in blood express the gut homing receptor integrin 47, this approach was designed to identify genes that might play a role in organspecific homing. We identified 16 transcripts that were more highly expressed in 7+ cells and 18 that were more highly expressed on 7- cells. We can relate transcript expression to expression of protein in seven cases (Table 3 and refs. [8,13]). In six of these cases (CCR9, GPR2/ CCR10, KLRB1, LAIR1, NT5E, and SELL), differential transcript expression was accompanied by differential expression of the corresponding protein on the surface of 7+ versus 7- cells. In one case (KLRG1), we did not find evidence for a consistent difference in surface protein expression (Table 3). These data suggest that most of the differentially expressed transcripts correspond to differentially expressed proteins, although it will be important to confirm this directly when suitable reagents become available. Some of the differentially expressed transcripts encode proteins with known roles in organ-specific homing. The Page 5 of 11
(page number not for citation purposes) BMC Immunology 2004, 5:13 http://www.biomedcentral.com/1471-2172/5/13 B 10 MFI 19, 54% positive 7+ CCR7 15 MFI 8, 24% positive 7+ CCR7+ Number of cells A 125 MFI 0, 1% positive CD45RA+ 0 0 10 10 10 10 KLRB1 (CD161)
1 2 3 Number of cells 0 0 10 10 10 10 KLRB1 (CD161) 1 2 3 0 0 10 30 10 10 10 KLRB1 (CD161)
1 2 3 MFI 5, 16% positive 7 CCR7 30 MFI 4, 14% positive 7 CCR7+ Number of cells 0 0 10 10 10 10 KLRB1 (CD161) 1 2 3 0 0 10 10 10 10 KLRB1 (CD161) 1 2 3 Figure 3 Expression of KLRB1 (CD161) on T helper cell subsets Expression of KLRB1 (CD161) on T helper cell subsets. Histograms depict expression of KLRB1 (CD161) on (A) CD45RA+ nave cells and (B) four subsets of CD45RA- memory/effector cells. These subsets were distinguished by their expression of 7 and CCR7, a marker for central memory cells, as shown in Fig. 2B. Black lines indicate KLRB1 staining and filled red lines indicate staining obtained with an isotype control antibody. two most highly differentially expressed genes we detected in our analysis encode chemokine receptors already shown to have important roles in gut and non-gut (skin) homing. These are CCR9, the TECK/CCL25 receptor which is expressed preferentially on 47+ gut homing cells , and GPR2/CCR10, the CTACK/CCL27 receptor which is expressed preferentially on CLA+ skin homing cells . We found that transcripts encoding the subunit (ITGA4) of integrin 47, was more highly expressed in 7+ cells. The microarray experiments indicated that the transcript encoding the 7 subunit itself (ITGB7) was not differentially expressed, consistent with previous results obtained using quantitative RT-PCR (M.W.R and D.J.E, unpublished results). This suggests that surface expression of the 47 heterodimer on CD4+ memory T cells may be determined primarily by the amount of 4 subunit available and not by the amount of 7 produced. The other differentially expressed transcript with a known role in homing is SELL, which encodes the peripheral lymph node homing receptor L-selectin (CD62L). We found that expression of both L-selectin transcript and surface pro- tein was higher in 7- than 7+ memory/effector T cells. Although many 7-expressing memory cells also expressed L-selectin, cells expressing the highest levels of 7 did not express L-selectin (Fig. 2). This presumably contributes to the development of CD4+ T cell subsets with distinct homing properties. We also found evidence that 7+ and 7- cells differ in their expression of other molecules that have been reported to modulate cell adhesion and migration. KLRB1 transcript and surface protein expression was higher on 7+ memory effector T cells. Overexpression of KLRB1 (also known as CD161 or NKRP1A) was previously found to increase transendothelial migration of CD4+ T cells, perhaps by modulating integrin activity . The finding that KLRB1 was preferentially expressed on T cells from human intestine led to a recent suggestion that KLRB1 may help direct intestinal homing . This hypothesis remains to be tested, but would be consistent with our finding of preferential expression of KLRB1 on 7+ blood memory CD4+ T cells. We also found higher expression of Page 6 of 11
(page number not for citation purposes) BMC Immunology 2004, 5:13 http://www.biomedcentral.com/1471-2172/5/13 B MFI 16, 24% positive 10 7+ CCR7 15 MFI 4, 10% positive 7+ CCR7+ Number of cells A MFI 1, 5% positive 125 CD45RA+ 0 0 10 10 10 NT5E (CD73)
1 2 Number of cells 10 3 0 0 10 10 10 NT5E (CD73) 1 2 10 3 MFI 7, 13% positive 30 0 0 10 10 10 NT5E (CD73)
1 2 MFI 4, 10% positive 30 7 CCR7+ 10 3 Number of cells 7 CCR7 0 0 10 10 10 NT5E (CD73) 1 2 10 3 0 0 10 10 10 NT5E (CD73) 1 2 10 3 Figure 4 Expression of NT5E (CD73) on T helper cell subsets Expression of NT5E (CD73) on T helper cell subsets. Histograms depict expression of NT5E (CD73) on (A) CD45RA+ nave cells and (B) four subsets of CD45RA- memory/effector cells. These subsets were distinguished by their expression of 7 and CCR7, a marker for central memory cells, as shown in Fig. 2B. Black lines indicate NT5E staining and filled red lines indicate staining obtained with an isotype control antibody. NT5E (CD73) transcripts and surface protein on 7+ memory effector T cells. NT5E is an ecto 5'-nucleotidase expressed on lymphocytes and on endothelial cells and has been reported to mediate lymphocyte-endothelial adhesion and transmigration by various mechanisms, including promotion of integrin-mediated binding via effects on integrin clustering [33-36]. Transcripts encoding two members of the galectin family were differentially expressed on 7+ versus 7- cells. Both LGALS1/galectin-1 (expressed at higher levels in 7- cells) and LGALS2/galectin-2 (expressed at higher levels in 7+ cells) have been shown to have several effects on cell adhesion in lymphocytes and other cells . Transcripts for RGS1 were expressed at higher levels in 7- cells. RGS1 is a GTPase activating protein that regulates Gi-stimulated pathways. Expression of RGS1 inhibits chemokine-induced cell migration and integrin-mediated adhesion in lymphocytes . Our data suggest that there may be less RGS1-mediated inhibition of chemokine signaling in 7+ cells. We speculate that the differential expression of KLRB1, NT5E, LGALS1, LGALS2, and RGS1 on 7+ and 7- T helper cell subsets may contribute to homing specificity by modulating the complex multistep process of cell adhesion and migration. The primary activation of T cells leads to changes in the expression of integrin 47 and other molecules that control homing. It has recently been shown that upregulation of integrin 47 expression and increased responsiveness to the CCR9 ligand TECK occur within two days after activation of murine nave CD4+ T cells in intestinal, but not cutaneous, lymph nodes  and that activation by dendritic cells from Peyer's patches but not peripheral lymph node or spleen promotes murine CD8+ T cell 47 expression and TECK responsiveness . We found that both KLRB1 and NT5E were virtually absent on CD45RA+ nave CD4+ T cells and were expressed at low levels on CD45RACCR7+ central memory cells and on 7- CD45RA- non-gut homing memory effector cells but were expressed at higher levels in the 7high CCR7- CD45RA- memory effector subset. In contrast, LAIR1 was highly expressed on nave T cells and on both CCR7- and CCR7+ 7+ CD45RA- Page 7 of 11
(page number not for citation purposes) BMC Immunology 2004, 5:13 http://www.biomedcentral.com/1471-2172/5/13 B 10 MFI 20, 66% positive 7+ CCR7 15 MFI 18, 64% positive 7+ CCR7+ Number of cells A 125 MFI 24, 83% positive CD45RA+ 0 0 10 10 10 LAIR1
1 2 Number of cells 10 3 0 0 10 10 10 LAIR1 1 2 10 3 0 0 10 30 10 10 LAIR1
1 2 MFI 6, 26% positive 7 CCR7 30 MFI 9, 40% positive 7 CCR7+ 10 3 Number of cells 0 0 10 10 10 LAIR1 1 2 10 3 0 0 10 10 10 LAIR1 1 2 10 3 Figure 5 Expression of LAIR1 on T helper cell subsets Expression of LAIR1 on T helper cell subsets. Histograms depict expression of LAIR1 on (A) CD45RA+ nave cells and (B) four subsets of CD45RA- memory/effector cells. These subsets were distinguished by their expression of 7 and CCR7, a marker for central memory cells, as shown in Fig. 2B. Black lines indicate LAIR1 staining and filled red lines indicate staining obtained with an isotype control antibody. cells but expression was lower on 7- CD45RA- cells. These results suggest that expression of 47 and expression of KLRB1, NT5E and LAIR1 may be coordinately regulated at the time of human T cell activation, as is apparently the case for 47 and CCR9 in mouse systems. Alternatively, it is possible that differences in expression arise from changes in gene expression that occur after the transition from nave to memory effector cells or from the preferential retention of survival of particular memory effector cell subsets. Although we showed that expression of KLRB1, NT5E and LAIR1 was higher for the 7+ subset as a whole, many 7+ cells did not express these proteins and some 7- cells did. This suggests that the homing properties of individual T cells might be "fine tuned" by complex combinatorial regulation of molecules known to have major roles in determining organ-specific recruitment (e.g., integrin 47 and CCR9) and other molecules that can modulate adhesion and migration (e.g., KLRB1 and NT5E). Conclusions
We used DNA microarrays to identify a set of gene transcripts that were differentially expressed on 7+ versus 7blood memory CD4+ T cells. Some of these encode adhesion receptors (integrin 4 and L-selectin) and chemokine receptors (CCR9 and GPR2/CCR10) already known to be involved in organ-specific lymphocyte migration. Other differentially expressed transcripts encode additional molecules previously reported to affect cell adhesion and migration, including KLRB1, NT5E/CD73, LGALS1 and LGALS2 (galectin-1 and -2) and RGS1. As predicted from the transcript expression data, we found that KLRB1, NT5E and LAIR1 proteins were all preferentially expressed on the surface of 7+ versus 7- memory CD4+ T cells. Our results suggest that expression of several molecules that can modify T cell adhesion, endothelial transmigration and chemotaxis are also selectively regulated on CD4+ T cell subsets, perhaps during the "imprinting" process that takes place during initial T cell activation. These molecules are likely to contribute to the complex multistep process that regulates trafficking of Page 8 of 11
(page number not for citation purposes) BMC Immunology 2004, 5:13 http://www.biomedcentral.com/1471-2172/5/13 CD4+ memory T cell subsets with different homing behaviors. Methods Isolation of 7+ and 7- lymphocytes from human blood This protocol was approved by the Committee on Human Research at the University of California, San Francisco and informed consent was obtained from all subjects. Peripheral blood mononuclear cells (PBMC) were isolated from nine normal adult donors (six females and three males, ages 2442 years) using sodium heparin coated Vacutainer Cell Preparation Tubes (Becton Dickinson) according to the manufacturer's instructions. PBMC were stained with CD4-FITC, CD45RA-Cy5 and anti-integrin 7-PE (all from BD Pharmingen). Anti-FITC microbeads and a MACS bead sorter (Miltenyi Biotec) were used to enrich for the CD4+ cells prior to flow cytometry. CD4+ memory (CD45RA- ) 7+ and 7- T cells were then purified using a FACS Vantage (Becton Dickinson). Microarray analysis of transcript expression Total RNA from 7+ and 7- CD4+ memory T cells was purified using the Mini RNA Isolation Kit (Zymo Research) and DNase treated using the DNA-free RNA Kit (Zymo Research) according to the manufacturer's instructions. RNA quality was evaluated using an Agilent Bioanalyzer. RNA amplification, labeling, and hybridization were performed as previously described with slight modifications . In brief, two rounds of amplification were performed using T7 RNA polymerase (Ambion MessageAmp aRNA Kit). Cy3 or Cy5 was incorporated into the amplified RNA products using amino allyl-modified nucleotides. Fluorescently-labeled amplified RNAs were fragmented using Ambion RNA Fragmentation Reagents and hybridized to DNA microarrays using Ambion SlideHyb Glass Array Hybridization Buffer #1. Each hybridization involved 7+ cell RNA from a single subject (labeled with one dye) and 7- cell RNA from the same subject (labeled with the other dye). A total of 27 arrays were used to analyze samples from the 9 subjects (at least 2 arrays per subject, including a dye swap). determined a log ratio, log2 (7+ sample intensity/7- sample intensity), for each probe on each array. No background subtraction was performed. Complete information about the array platform and data from each of the individual arrays is available from GEO  (GEO accession GSE1039). Appendix 1 [see additional file 1] is a MIAME-compliant description of the array experiments.
Identification of differentially expressed gene transcripts A fixed effects linear model was used to estimate differences in transcript expression between 7+ and 7- CD4+ T cells. For each individual gene (probe) on the array, the model can be described as Y ij = + A i + ij , where Y ij represents the normalized log ratio determined on a single array, i represents the subject number and j represents the array number. The parameter represents the actual log ratio of gene expression between 7+ and 7cells, which we wished to estimate. A i represents the difference between the log ratio for subject i and the average log ratio for all subjects and denotes the experimental error. We fit the model using a zero sum constraint (A i = 0). Fold difference was calculated from the estimated log ratio. We computed moderated t-statistics , log-odds ratios of differential expression (based on empirical Bayes shrinkage of the standard errors towards a common value ), and adjusted p-values (obtained using the Bonferroni correction) using functions in the limma library of the Bioconductor software package [41,42]. Similar results were obtained under a random effects model (not shown).
Analysis of surface protein expression on blood CD4+ T cell subsets Whole blood was collected from nine normal adult subjects (seven females and two males, ages 2338 years, none of whom were included in the microarray experiments described above). Blood was stained with various antibody combinations and analyzed on a FACScalibur equipped for six color analysis. Antibodies recognizing CD4 (SK3), integrin 7 (FIB504), CD62L (DREG-56), CD73 (AD2), and CD161 (DX12) and isotype control antibodies were all from BD Biosciences. Other antibodies used were LAIR1 (DX26, generous gift of L. Lanier), KLRG1 (13A2, generous gift of D. Voehringer and H. Pircher), CD45RA (Immunotech), the anti-47 mAb Act-1 , the anti-aE7 antibody HML-1 (Beckman Coulter), CCR7 (R&D Systems). Mean fluorescence intensity (MFI) was corrected by subtracting the MFI obtained with isotype control antibodies. The percentage of positive cells was determined by setting the threshold so that 1% of cells were considered positive after staining with control antibody. Arrays were produced in our microarray facility using the Operon version 2 set of 70-mer oligonucleotide probes supplemented with some custom-designed 70-mers as described . Arrays included probes for 21357 genes plus control probes. After hybridization, arrays were scanned using an Axon GenePix 4000B scanner and images were processed using GenePix 5.0 software. The "print-tip loess" normalization was used to correct for within-array dye and spatial effects  and single channel quantile normalization was used to facilitate comparison between arrays . We used functions in the library marrayNorm  of the R / Bioconductor package  to perform these normalizations. After normalization we Page 9 of 11
(page number not for citation purposes) BMC Immunology 2004, 5:13 http://www.biomedcentral.com/1471-2172/5/13 Authors' contributions
MWR recruited the subjects, performed the experiments and participated in data analysis and drafting of the manuscript. ACP performed the statistical analysis of the microarray data. YHY supervised the statistical analysis of the microarray data. DJE conceived of the study and participated in design, analysis, and preparation of the manuscript. All authors read and approved the final manuscript. 7. 8. 9. 10. 11. Additional material Additional File 2
Tab-delimited text file with calculated gene expression ratios for each probe on each array. Values represent normalized log2 (Cy5 intensity/Cy3 intensity). Click here for file [http://www.biomedcentral.com/content/supplementary/14712172-5-13-S2.txt] 12. 13. Additional File 1
MIAME-compliant description of the array experiments in rich text format. Click here for file [http://www.biomedcentral.com/content/supplementary/14712172-5-13-S1.rtf] 14. Acknowledgements
Microarray studies were supported by Andrea Barczak, Michael Salazar, and the UCSF/San Francisco General Hospital General Clinical Research Center Core Genomics Laboratory (supported by grant M01-RR00083 from the National Center for Research Resources of the NIH). Flow cytometry was performed in the Flow Cytometry Core Laboratory of the J. David Gladstone Institutes. We thank Gordon Smyth and Mark Segal for helpful discussions regarding the microarray data analysis. The work was supported by NIH grant DK/AI54212. 15. 16. 17. References
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