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T lymphocyte subsets defines unique tissue-specific functions
Department of Veterinary Molecular Biology, Montana State University, Bozeman
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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T cells, in the absence of antigen stimulation, the differential gene expression of two circulating T cell subsets was analyzed. The two subsets, with distinct trafficking phenotypes in young calves, were GD3.5+, CD8-, WC1+ or GD3.5-, CD2+, WC1-, and 90–100% CD8+ and were sorted based on GD3.5 and T cell receptor expression. Results from two different human arrays probed with cDNA from these T cell subsets indicated that they have markedly different tissue-specific functions. The genes preferentially expressed by GD3.5+ (CD8-) T cells demonstrated that they were highly activated, proliferative, and inflammatory, whereas those expressed by GD3.5- (primarily CD8+) T cells were involved in promoting quiescence, consistent with a role for T cells as sentinel mucosal cells, and several were interferon-regulated genes. Gene expression and phenotypic assays indicated that CD8+ T cells were apoptotic, whereas CD8- T cells were apoptosis-resistant. Differential expression of multiple genes was confirmed in both arrays: That of 14 genes was confirmed by quantitative reverse transcriptase-polymerase chain reaction and that of seven proteins was confirmed by flow cytometry. This novel, genomic analysis of circulating T cell subsets, without confounding effects of the tissue microenvironment, offers new insight into the biology and development of neonatal T cells.
Key Words: cDNA array • gene expression
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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T cells have diverse functions and are evolutionarily well conserved and among the first immune cells to develop, yet they are not well understood [1
]. T cells enhance B cell function, present antigen to CD4+ 
ß T cells, and are involved in immunity to infections as well as tissue development and repair [2
]. T cells recognize unprocessed antigen and cellular stress proteins and localize to mucosal sites, which implicates a role in innate immunity [3
]. Studies with T cell receptor -deficient mice have shown that in some infections, T cells are immune modulatory, and their function in protection can be compensated by ß T cells [4
], whereas in others, their immune function is absolutely essential [5
6
].
The study of T cells in rodents and humans is difficult because the frequency of these cells in circulation or within secondary lymphoid organs is small. In contrast, the majority of circulating T cells in young calves is T cells [7
], which greatly facilitates their collection and characterization and provides a powerful animal model in which to study the functions of these cells. Two subsets of circulating T cells in neonatal cattle that have functionally different trafficking phenotypes have distinct T cell receptor (TCR) rearrangements and are GD3.5+, CD8-, CD2-, usually WC1+ or GD3.5-, CD2+, primarily (90–100%) CD8+, and usually WC1- (refs. [8
9
10
]; M. A. Jutila, unpublished observations). As CD8 expression separates the predominant populations within these subsets, and as a result of the functional importance of the CD8 antigen, we have focused on the CD8+ and CD8- T cell subsets. CD8+ T cells develop in and/or localize to mucosal tissue and make up the majority of IELs, whereas CD8- T cells are the predominant T cell in peripheral blood and are specifically recruited to sites of inflammation [2
9
]. Although they can be distinguished with respect to antigenic phenotype, distribution, and function, the global differential gene expression of T cell subsets in healthy neonates is uncharacterized. We hypothesize that tissue-specific functions of circulating T cell subsets will be reflected in their differential gene-expression profiles.
Microarray technology generates data on differential expression of thousands of genes between two cDNA samples and was used to profile tissue IELs in a mouse model of Yersinia infection [11
]. Another analysis was done on mouse versus ß IELs using serial analysis of gene expression (SAGE; ref. [12
]). As these studies analyzed cells isolated from the gut mucosa, they reflected gene regulation events controlled by differences in the individual cell types as well as the tissue microenvironment [11
12
]. In this study, we provide a comprehensive, genomic analysis of CD8+ and CD8- T lymphocytes in circulation using two different human cDNA arrays and identify unique gene expression patterns in these cells before the confounded effects once they enter a tissue. Consistency between the two arrays and analysis of differential expression of several genes and proteins by real time reverse transcriptase-polymerase chain reaction (RT-PCR) and flow cytometry demonstrated that the cross-species array approach is highly reliable. The results indicated that although CD8- T cells were activated, proliferative, and proinflammatory, genes preferentially expressed in CD8+ T cells were involved in promoting quiescence and trafficking to the mucosa and were interferon (IFN)-inducible and consistent with an immune-sentinel role. CD8+ T cells preferentially expressed multiple proapoptotic genes, whereas the CD8- T cell profile suggested they were apoptosis-resistant. By focusing on T cell subsets with a global, genomic approach, potential roles of circulating T cell subsets were defined, and several specific genes, likely integral to their development and function in innate immunity, including genes with heretofore-unknown roles in immunity, were identified.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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TCR [13
]; GD3.5, specific for CD2-, CD8- T cells [9
14
]; CC58, which recognizes the ß form of the CD8 antigen, kindly provided by Chris Howard (Institute for Animal Health, Compton, UK); Fib30, rat mAb specific for human ß7 integrin, which cross-reacts with the bovine molecule [15
]; and DREG56, specific for bovine L-selectin [16
17 ]. Antibodies acquired from commercial sources were specific for ADAM10 (Abcam, Cambridge, UK), interleukin-2 receptor [IL-2R; Veterinary Medical Research & Development (VMRD), Pullman, WA], and vimentin (Serotec, Oxford, UK). Antibodies specific for human CD49f (6 integrin, clone GoH3) and biotin-labeled CXC chemokine receptor (CXCR4; 12G5), which cross-react with bovine cells, and the Apoptosis Detection Kit I were obtained from BD PharMingen (San Diego, CA).
High-speed fluorescence-activated cell sorting (FACS) and flow cytometric analysis
Approximately the same number of bovine peripheral blood leukocytes (PBL) from two different animals collected at three different times was collected by histopaque gradient and centrifugation at 1300 g for 45 min. In an earlier study, we found that CD8 expression on T cells is not of sufficient magnitude beyond background staining to allow efficient, high-speed cell sorting (M. A. Jutila, unpublished observations). Thus, for sorting CD8+ and CD8- T cells, cells were double-stained with GD3.5, indirectly stained with phyco-erythrin (PE)-conjugated anti-mouse immunoglobulin G (IgG) and GD3.8, directly conjugated to fluorescein isothiocyanate (FITC) with a 10-min incubation in mouse serum between the indirect and direct labeling steps, and sorted essentially as described previously [18
]. Cross-linking experiments have indicated that the antibodies GD3.8 and GD3.5 do not activate signaling molecules and thus, do not alter gene expression. CD8- (GD3.8+, GD3.5+) and GD3.8+, GD3.5-, a population that is CD2+, and 90–100% CD8+ (ref. [9
]; M. A. Jutila, unpublished observations) bovine T cells were sorted on a VANTAGE SE cell sorter (BD Immunocytometry Systems, San Jose, CA) to >96% purity. Cells were rested overnight in complete RPMI media (10% fetal bovine serum in RPMI supplemented with 1% each essential amino acids, penicillin/streptomycin, L-glutamine, and 10 mM HEPES) and then treated for 4 h with 5 µg/ml concanavalin A (Con A) and 1 ng/ml recombinant human IL-2 to stimulate transcription of the message down-regulated during the 
8 h staining and sorting process.
For FACS confirmation of the proteins predicted to be differentially expressed by the arrays, cells were similarly isolated and stimulated for 12 h to measure protein expression rather than mRNA with the same concentrations of Con A and IL-2 before labeling. Triple-label flow-cytometry staining was performed as described previously to determine differential mean fluorescence values on the two T cell subsets of antibodies specific for multiple proteins, characterized previously (used as controls) and discovered using the arrays [13
14
]. To detect the ADAM10 protein, cells were first fixed in 2% paraformaldehyde. The vimentin-specific antibody was directly FITC-labeled following standard protocols. For vimentin labeling, cells were first stained for CC58 (conjugated to biotin) and GD3.8 (conjugated to allophyocyanin), then reacted with streptavidin-PE, fixed with 4% paraformaldehyde, permeabilized with 0.2% Tween-20 in phosphate-buffered saline, and then stained with the FITC-conjugated vimentin-specific antibody. Isotype-matched and secondary-only stains controlled for background fluorescence. Levels of expression of some proteins can vary greatly among individuals; thus, FACS stains were performed on lymphocytes isolated from at least three different calves, and results from representative assays are shown.
RNA extraction and array analysis
Total RNA was isolated from sorted subsets of T lymphocytes and used to probe the Incyte Human Drug Discovery LifeArray (Incyte Genomics, Palo Alto, CA; Drug: 8001 unique genes) and Human Unigene LifeArray (Unigene: 8466 unique genes). RNA was extracted from 1 x 107 CD8+ and CD8- T cells (pooled from two calves sampled at three different times) with Trizol (Invitrogen Life Technologies, Carlsbad, CA) according to the manufacturer’s instructions. RNA (5 µg) from each subset was submitted to Incyte for T7 amplification and array hybridization. To correct for variation in data, the average signal from all elements in the Cy3 channel was divided by the average signal from all elements in the Cy5 channel, resulting in the balance coefficient. The Cy5 signal for each element was then multiplied by the balance coefficient, before calculating the balanced differential expression ratio. The balanced, differential expression ratio was calculated as Cy3/Cy5 if the Cy3 signal was greater, reported as a positive number, or Cy5/Cy3 if the Cy5 signal was greater, reported as a negative number. According to Incyte, a balanced, differential expression ratio greater than 1.7 (or less than -1.7) can be considered differentially expressed with 99% confidence. cDNA from CD8+ cells was labeled with Cy5 and cDNA from CD8- cells, with Cy3, so that negative values indicate preferential expression in CD8+ T cells and positive values in CD8- T cells. Array controls included sensitivity controls (ranging from 2 to 2000 pg); variable ratios of labeled cDNA to control for preferential labeling with dye, housekeeping genes, for which there were sufficient signal levels and no differential expression for ribosomal S9, tubulin, and 23-kD HBP;, and buffer-only array spots to control for background hybridization, all performed in quadruplicate.
Real time RT-PCR
Real time RT-PCR was used to confirm the differential expression of several genes. Blood was collected from six different calves to decrease individual variation. Cells were isolated, sorted, cultured, and stimulated exactly as was done for the preparation of RNA for the arrays. The pooled RNA, extracted from T cell subsets, was treated with DNase, extracted again with phenol:chloroform, and used in real time RT-PCR analysis. The RT was performed with Superscript RT random primers (Invitrogen Life Technologies) and 300 ng sample (CD8+ or CD8- T cell) RNA, according to the manufacturer’s protocol. Relative, specific mRNA in the CD8+ and CD8- T cells was quantified by measuring SYBR® Green I double-stranded DNA binding dye incorporation during real time, quantitative RT-PCR using the relative standard curve method. Bovine sequences of 14 genes were analyzed using the Primer Express software (Applied Biosystems, Foster City, CA) to design optimal real time RT-PCR primers (data not shown). Primers specific for bovine 18S RNA were used as the endogenous control. Standard curves were constructed using serially diluted, similarly extracted, total bovine PBL RNA. Each RT reaction (1 µl) was used in the 25 µl real time PCR reactions performed in triplicate. The PCR was set up and cycled, data were collected on the Applied Biosystems GeneAmp 5700 Sequence Detection System (Applied Biosystems), and calculations were performed as described in the manufacturer’s protocol and in User Bulletin #2 for the ABI PRISM 7700 Sequence Detection System. Statistical significance of differential expression was determined for each primer set using the Student’s t-test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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T cells from two calves sampled three different times were sorted to >96% purity, RNA was isolated, and gene expression profiles of pooled RNA were compared on two human Incyte LifeArrays. The Incyte system was chosen for its comprehensive controls and genomic coverage not available on early generation bovine arrays and primarily, as cDNA probes on the arrays are 500–5000 bases, averaging 1000 bases, thus providing an enormous advantage over oligonucleotide arrays for cross-species hybridization. Appropriate results were obtained with the multiple array controls, such that the balanced, differential expression values greater than 1.7 or less than -1.7 indicated differentially expressed genes with 99% confidence. Differential expression between the two populations on the array indicates that cross-reactivity for those genes was sufficient, and only the differentially expressed genes are discussed as significant. Results were highly consistent between the two different arrays and were in excellent agreement with an alternative, genomic approach comparing T cell subsets after phorbol ester and ionomycin stimulation using SAGE (N. Meissner et al., unpublished observations). For example, the genes galectin 1, junB, Bcl-2, Bcl-xL, and pim-1 were strongly up-regulated in GD3.5+ (CD8-) T cells according to both arrays and the SAGE analysis (data not shown).
Before detailed analysis of differentially expressed genes in the two arrays, real time RT-PCR was performed to amplify 14 differentially expressed genes, and flow cytometry was used to compare expression levels on bovine T cell subsets of seven proteins to further validate the array results. T cell subsets were sorted and stimulated, and RNA was isolated exactly as for the array, but RNA isolated from sorted cells from six different calves was pooled for use in the real time RT-PCR. Figure 1A
indicates that RT-PCR data were consistent with the array analysis. The expression of jak1 by CD8+ T cells was indicated on one array but not confirmed on the other or by real time RT-PCR. Detection of the IL-18 message required greater volumes of RNA to detect, and the results were not significantly different between the two subsets. Thus, expression of some mRNAs may vary between RNA samples and depend on message stability; however, real time RT-PCR was highly predictive of array results for all other genes assayed (Fig. 1a)
. Protein expression data assayed by three-color FACS were also highly confirmatory of the results of the array analysis (Fig. 1b)
. FACS stains were performed on lymphocytes from at least three calves, and a representative sample is shown. Mean fluorescence frequently varied among calves, but the relative expression of the proteins remained consistent with the arrays. Fewer antibodies were available to confirm proteins up-regulated by CD8- T cells; thus, mAb specific for the IL-2R, GD3.5, and L-selectin, known to be up-regulated in CD8- T cells, were used as controls. These confirmatory results indicate that the array data likely represent general properties of T cells and are not a result of variation among individuals.
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T cell subsets have distinct expression profiles T cell subsets, and specific trends were immediately obvious. The CD8- T cells preferentially expressed genes involved in proliferation/activation, transcription, and translation (Table 1
). In contrast, the CD8+ T cells were particularly quiescent and antiproliferative, despite equivalent treatment of the two subsets. However, the CD8+ subset preferentially expressed ACT-2, an activation marker specific to bovine CD8+ IELs [19
]. The CD8- and CD8+ T cell subsets differentially expressed several genes that had unknown functions or functions unknown in lymphocytes (Table 1)
. Fully annotated array data, including raw and normalized values of differentially regulated genes, are available at <http://vmbmod10.msu.montana.edu/vmb/jutila-laboratory/array.htm>.
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T cell subsets from the Drug Discovery and Unigene arrays, with genes involved in activation, quiescence, transcription, and translation and genes with unknown functions removed (summarized in Table 1
). Most notably, the two T cell subsets clearly have unique features relating to apoptosis. The up-regulation of proapoptotic genes in the CD8+ T cells (Table 2)
indicates that they were more prone to apoptosis. In contrast, CD8- T cells preferentially expressed multiple antiapoptotic genes such as bcl-2-related genes, bcl-xL, B23, and pim-1 (Table 3)
. The live CD8+ T cells stained with Annexin V, whereas CD8- T cells did not, indicating that the array data correctly reflected a differential apoptosis phenotype (Fig. 1B)
.
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T cells preferentially expressed genes inducible by IFN. Differential expression of IRF-1 and -2, which are directly and indirectly induced by IFNs, respectively, and are responsible for downstream effects of IFN, was confirmed on both arrays and by RT-PCR. To attempt to explain the differential response of IFN-regulated genes, the arrays were mined for genes related to IFN and IFN receptors. IFNs and the IFN receptors were assayed in the arrays, but expression levels were not different between the two subsets (data not shown). Thus, as the genes did not properly cross-react, or they were not differentially expressed, the arrays do not suggest a clear reason for the IFN-sensitive tendency of CD8+ T cells. Expansion of cultured antigen-specific CD8+ T cells upon stimulation with IFN in vitro has been described [20
]. These data suggest that circulating CD8+ T cells from healthy neonates are uniquely IFN-sensitive.
Several genes that suggest specific roles for the two subsets in immunity were apparent in the array analysis. CD8+ T cells preferentially expressed multiple cytoskeletal genes, which were involved in antigen presentation (diubiquitin, cathepsin D, and MHC II), chemokines, and their receptors and adhesion molecules (Table 2)
. Conversely, immune-related genes expressed by CD8- T cells were primarily inflammatory, consistent with their activation and tendency to localize to sites of inflammation (Table 3)
. For example, the arrays indicate that CD8- T cells express binding proteins for the anti-inflammatory drugs FK506 and Sanglifehrin A. Genes involved in innate immunity, such as mannose-binding lectin 1, receptor-interacting serine-threonine kinase 2, and MASP-2, were differentially expressed, as were genes primarily expressed by moncytes and B cells, such as MDNCF (IL-8), B cell receptor-associated protein (BAP37), and related genes, RGS-1 and CD79A. Overall, results of the two arrays indicate exceptionally different and specific functions for two major T cell subsets in circulation.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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T cells as assayed on two different arrays revealed remarkable distinctions between CD8+ and CD8- T cells. Differential expression of multiple genes and proteins was confirmed, are in agreement with those of a similar genomic analysis using SAGE, and are largely consistent with results of genomic studies of differential gene expression between the and ß T cells [11
12
]. In these previous studies, however, specific genes integral to the function of T cell subsets were expected to be identified (e.g., CXCR4 and diaglycerol kinase) but were likely masked by focusing on the group as a whole and perhaps by down-regulation of key genes during processing ex vivo. Our analyses of circulating cells indicate that the CD8- T cells are highly activated, proliferative, and inflammatory, whereas the CD8+ T cells are quiescent, IFN-sensitive, and prone to apoptosis. Antiproliferative genes found in CD8+ T cells are likely involved in down-regulation of localized immune responses. For example, very high-level expression of the TGF-ß-binding protein would facilitate rapid release of this negative mediator, precluding its de novo transcription, as previously suggested for IELs [11
]. The data are consistent with roles of the CD8- and CD8+ T cells in inflammation and as sentinel mucosal cells, respectively, but the extremity of the differential activation states and apoptotic profiles was surprising. Array analysis identified a large number of genes to which a potential function in lymphocytes could not be ascribed; however, some may be involved in promoting the quiescence or proliferation/activation states of the T cell subsets. This set warrants continual monitoring as new information about these genes is discovered.
Recent phylogenetic analysis suggests that T cells are likely ancestors of modern B and ß lymphocytes [21
]. Expression of some B cell genes in these T cell subsets is clearly consistent with this suggestion. T cell subsets expressed genes coding for CD79A, an Ig- gene exclusively expressed in B cells and multiple genes related to BAP37, up-regulated by activation. The role of the T cell subsets in innate immunity is perfectly illustrated by expression of mannose-binding lectin (MBL), a liver-derived serum protein that binds bacterial cell surfaces, and MASP-2, a related downstream protease responsible for cleaving complement component C3 [22
]. Although macrophages express a membrane-bound form of MBL, to our knowledge, expression of MBL and MASP-2 has not been described in lymphocytes.
The two T cell subsets differentially express multiple cytoskeletal genes. Vimentin, for example, is up-regulated in CD8- T cells and functions in retaining rigidity of circulating lymphocytes until specific chemokines trigger their collapse during polarization and transmigration [23
]. Thus, vimentin may be responsible in part for the relative resistance of CD8- T cells to leaving circulation in certain tissues (ref. [16
]; B. Walcheck, E. Wilson, and M. A. Jutila, unpublished observations). Other cytoskeletal proteins expressed by CD8- T cells (MARCKS, moesin, vinculin, ROCKII, RGS-1, and FYB-120/130) may also be involved in regulating their retention in circulation until the proper signal is detected. Conversely, cytoskeletal proteins preferentially expressed by CD8+ T cells, such as ankyrin, -actinin, adducin, talin, spectrin, and Rho-GTPase-related proteins, might be related to a role in antigen presentation/recognition and in the formation of the "immunological synapse" that would facilitate this process [24
]. This dramatic difference in the regulation of cytoskeletal genes warrants further investigation.
The two subsets of T cells clearly have different approaches to relenting to and protecting themselves from apoptosis. The array and flow cytometric data indicate that CD8+ T cells are more prone to apoptosis and perhaps to induction of apoptosis in neighboring cells (e.g., by expressing lymphotoxin-ß) than CD8- T cells. Multiple genes preferentially expressed by CD8- T cells strongly block apoptosis and promote proliferation. Prevention of apoptosis in T cells is a proinflammatory property [25
]; thus, this result is consistent with the respective inflammatory and anti-inflammatory profiles of CD8- and CD8+ T cells. The two circulating populations may also have been exposed to different apoptotic or antiapoptotic stimuli, specific to their trafficking. The death receptors Fas and TNF receptor (types 1 and 2), responsible for antigen-induced, active T cell apoptosis, were equivalently expressed on the two subsets (data not shown). Thus, CD8+ T cells may experience a passive form of apoptosis involving mitochondial mechanisms that are strongly inhibited by bcl-2 and bcl-xL in the CD8- T cells. Alternatively, surface metalloproteases (e.g., ADAM10) up-regulated on the CD8+ T cells can release Fas ligand and TNF, thereby enabling an individual T cell to kill itself [26
].
Adhesion molecules responsible for extravasation of cells from circulation (6 and 5 integrins) and homing to mucosal regions (ß7 integrin; refs. [27
, 28
]) were preferentially expressed by CD8+ T cells. Differential expression of ß7 and CXCR4 has been functionally confirmed [18
]. Preliminary studies of antigen/cytokine-expanded, circulating human T cells show a similar pattern of adhesion-molecule expression on CD8+ versus CD8- T cells (J. F. Hedges, unpublished observations). These data suggest that bovine, and perhaps human, peripheral blood CD8+ T cells are in the process of trafficking to the mucosa. Chemokine expression by these cells (e.g., RANTES and IP-10) might then regulate the balance of lymphocytes attracted to mucosal sites. It seems counter-intuitive that a differentiated cell with a seemingly specific trafficking goal would be highly prone to apoptosis. Several questions thus remain to be answered. Are the circulating CD8+ T cells en route to the mucosa [18
], or do they have a specific role in the peripheral blood of very young animals? Is the apoptotic tendency simply an anti-inflammatory mechanism, which is suggested to be a major role for epithelial-associated T cells [29
], or is apoptosis integral to the life cycle of CD8+ T cells in the peripheral blood? Are apoptotic CD8+ T cells autoreactive [25
26
]? These questions are currently under investigation.
We have taken a global, genomic approach to defining the roles of defined subsets of circulating T cells. The array data indicate defined and divergent activities for the two T cell subsets before their response to signals encountered in tissues, suggest several avenues for detailed investigation, and indicate that definition of a general role for T cells, as a whole, may be misleading. These experiments are difficult in the human, because of the low and variable numbers of T cells that can be isolated from the blood and are nearly impossible to do in the mouse, unless antigen/cytokine-driven cultures are used. However, we have shown that array technology developed for human-functional genomic studies is highly reliable when applied to cattle in which T cells predominate and are likely critical to the immunological health of the animal. The evolutionary conservation of this population of T cells suggests that many findings in this system will be applicable to other species. Only when a clear role for T cell subsets in immunity is established can we then appropriately control them to protect the host. This may be especially necessary for specific age groups, immune states, and in domestic species in which they predominate.
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ACKNOWLEDGEMENTS |
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FOOTNOTES |
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Received September 11, 2002; revised October 22, 2002; accepted October 28, 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cells: a right time and a right place for a conserved third way of protection Annu. Rev. Immunol. 18,975-1026[CrossRef][Medline]
T-cell-deficient mice: protective role of gamma interferon and CD8+ T cells Infect. Immun. 70,5208-5215 T lymphocytes J. Immunol. 162,5033-5036This article has been cited by other articles:
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