Originally published online as doi:10.1189/jlb.0407263 on October 10, 2007

Published online before print October 10, 2007

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(Journal of Leukocyte Biology. 2008;83:71-79.)
© 2008 by Society for Leukocyte Biology

Differential expression of β₂-integrins and cytokine production between and β T cells in experimental autoimmune encephalomyelitis

Sherry S. Smith^* and Scott R. Barnum^*,,1

Departments of
^* Microbiology and
Neurology, University of Alabama at Birmingham, Birmingham, Alabama, USA

¹ Correspondence: Department of Microbiology, University of Alabama at Birmingham, 845 19th St. S., BBRB/842, Birmingham, AL 35294, USA. E-mail: sbarnum{at}uab.edu

ABSTRACT

The expression of β₂-integrins on T cells in naïve mice or those with experimental autoimmune encephalomyelitis (EAE) remains poorly characterized. We compared β₂-integrin expression and cytokine production between and β T cells over the acute course of EAE. We observed that unlike in β T cells, β₂-integrin expression on T cells increased significantly from baseline, peaked at Day 10, and remained unchanged in the draining lymph nodes or declined in the spleen and CNS by Day 15. In addition, IFN-- and TNF--producing T cells infiltrated the CNS rapidly and produced significantly more of these cytokines than β T cells throughout the course of EAE. These results suggest unique roles for β₂-integrins in the trafficking of versus β T cells during EAE and that T cells infiltrate the CNS rapidly, producing cytokines, which modulate acute disease.

Key Words: adhesion molecules • demyelinating disease • neuroimmunology

INTRODUCTION

Experimental autoimmune encephalomyelitis (EAE) is a T cell-mediated autoimmune disease of the CNS, which shares many features of the human disease multiple sclerosis (MS) [¹ ]. EAE is characterized by mononuclear cell infiltration of the brain and spinal cord and is induced in susceptible animals by immunization with CNS myelin proteins such as myelin oligodendrocyte glycoprotein (MOG) and myelin basic protein (MBP). In EAE, activated T cells and macrophages migrate through the blood brain barrier (BBB) and initiate an inflammatory process, which leads to demyelination in the brain and spinal cord [¹ ].

T cells constitute a small subpopulation of T cells, which bridge innate and adaptive immunity. T cells do not require MHC molecules for antigen recognition, and they recognize self-antigens, making them unique effector cells in autoimmune responses. In the mouse, T cells are present in skin, mucosal, and genitourinary tissues and play an important role in innate immune responses in these tissues [² , ³ ]. The function of T cells in EAE remains controversial. Although several studies have shown protective or regulatory functions for T cells in models of EAE [^{4 5 6} ], others have reported results ranging from no effect on disease [^{7 8 9} ] to a pathogenic role for these cells in EAE and virus-induced demyelination [^{10 11 12 13 14 15} ]. Although these contradictory findings may be, in part, a result of the use of different rodent strains and technical approaches [⁶ , ¹² , ¹⁴ ], they also likely reflect our limited understanding of T cell activation, trafficking, and effector functions in demyelinating disease.

Expression of ₄- and β₂-integrin family members by T cells is critical for the development and progression of EAE and in the case of ₄-integrins, for MS [^{16 17 18 19 20 21 22 23 24} ]. These proteins are important for migration of T cells into lymphoid tissues and sites of inflammation and for adhesive interactions between lymphocytes and APCs [¹⁶ , ^{25 26 27 28 29 30} ]. The functional importance and expression of β₂-integrins on CD4⁺ and CD8⁺ β T cells during EAE have been studied extensively [²³ , ²⁴ , ^{31 32 33 34} ]. In contrast, integrin expression and the role(s) of integrins during EAE on T cells are poorly characterized. T cell expression of VLA integrins has been documented [³⁵ , ³⁶ ]; however, β₂-integrins on T cells have received little attention. β₂-Integrin family members are heterodimers composed of one of four -chains (CD11a, CD11b, CD11c, and CD11d) and a common β-chain (CD18). These integrins play critical roles in leukocyte adhesion events by binding to members of the ICAM family, the complement opsonin iC3b, and fibrinogen [¹⁶ , ²⁶ , ³⁷ , ³⁸ ].

CD11a (LFA-1) is expressed on all leukocytes and functions in leukocyte trafficking and costimulation. EAE studies have shown that in vivo administration of anti-LFA-1 antibody produces results ranging from disease inhibition to exacerbation [³¹ , ^{39 40 41} ], and mice deficient in LFA-1 have attenuated disease [⁴² ] (Dan Bullard, Kari Dugger, Scott R. Barnum, unpublished observations). CD11b [membrane-activated complex 1 (Mac-1)] is expressed on macrophages, microglia, and activated T cells. The best-described function of CD11b in demyelinating disease is microglial or macrophage-mediated myelin phagocytosis [²⁴ , ^{43 44 45 46} ]; however, its expression on T cells is required for full disease development [²⁴ ]. Administration of anti-Mac-1 antibody has been shown to delay and attenuate adoptively transferred EAE with varied protection [³¹ , ⁴⁴ , ⁴⁷ ]. CD11c (p150,95) is expressed on macrophages, dendritic cells, neutrophils, lymphocytes, and microglia and functions in phagocytic clearance of bacteria and apoptotic cells and conjugate formation with cytotoxic T cells [^{48 49 50 51 52 53 54 55 56 57 58 59 60 61 62} ]. Studies have shown that mice deficient in CD11c have attenuated EAE and that CD11c expression on T cells is critical for disease development [²³ ]. CD11d, also known as _Dβ₂, is expressed on a wide variety of leukocytes and functions in macrophage activation and infiltration and T cell activation [³⁴ , ^{63 64 65 66} ]. Recent studies have shown that CD11d-deficient mice are not protected from EAE [³⁴ ], demonstrating that CD11d is the only β₂-integrin family member whose function is redundant in EAE.

The expression pattern of β₂-integrins on T cells in the normal mouse and during EAE serves as an important first step in understanding the function(s) of these integrins on T cells in demyelinating disease. In this report, we examine and compare β₂-integrin expression and cytokine production by T cells and β T cells in C57BL/6 mice throughout the acute course of EAE. We show that T cells differentially express β₂-integrins relative to β T cells in lymph nodes (LN), spleen, and spinal cord throughout disease and produce more IFN- and TNF- in these tissues. Our data suggest that differential regulation of expression of β₂-integrins on and β T cells may affect the trafficking pattern of T cells during EAE and ultimately, the kinetics of EAE development.

MATERIALS AND METHODS

Mice
Inbred wild-type male and female C57BL/6 mice (The Jackson Laboratory, Bar Harbor, ME, USA) were used in all experiments and were between 2 and 4 months of age. All studies were performed with approval from the University of Alabama at Birmingham Institutional Animal Care and Use Committee (Birmingham, AL, USA).

Induction of active EAE
Mice were immunized on Day 0 via s.c. injection of 150 µg MOG_35–55 emulsified in CFA containing 500 µg heat-killed Mycobacterium tuberculosis (Fisher Scientific, Loughborough, UK). On Days 0 and 2, mice received an i.p. injection of pertussis toxin (500 ng, Fisher Scientific). MOG peptide was synthesized by standard 9-fluorenyl-methoxycarbonyl chemistry and was >95% pure, as determined by reversed-phase HPLC (Biosynthesis, Lewisville, TX, USA). Onset and progression of EAE signs were monitored daily using a standard clinical scale ranging from 0 to 5 as follows: 0, asymptomatic; 1, tail paralysis; 2, hind-limb paraparesis; 3, hind-limb paralysis; 4, complete paralysis (tetraplegy); 5, death.

Isolation of leukocytes from draining LN (DLN), spleens, and spinal cords
Following perfusion with 1x PBS, inguinal DLN, spleens, and spinal cords were harvested from EAE-induced mice and processed on Days 0 (uninduced), 6, 10, 12, 15, and 20. Tissues were ground into single cell suspensions with frosted glass slides, put through a cell strainer, and washed in 1x PBS containing 2% FCS. Spleen cells were treated with 1.44% NH₄Cl to lyse RBC. Spinal cord cells were resuspended in 40% Percoll, layered on 70% Percoll, and centrifuged at 2000 rpm (1145 g, Jouan CR3i centrifuge) for 25 min at room temperature. Cells at the interface were removed, washed in 1x PBS containing 2% FCS, and stained as described below.

Flow cytometry and intracellular cytokine staining
Cells obtained from DLN, spleens, and spinal cords were incubated with anti-CD16/32 (FcR block, eBiosciences, San Diego, CA, USA) to prevent nonspecific staining. Cells were incubated for 30 min in the dark at 4°C with PE-conjugated anti-CD11a (M17/4, eBiosciences), APC-conjugated anti-CD11c (HL2, BD PharMingen, San Diego, CA, USA), anti- TCR FITC (GL3, BD PharMingen), or anti-β TCR FITC (H57-597, eBiosciences) and biotin-conjugated anti-CD11b (M1/70, eBiosciences) or purified anti-CD11d (205A, ICOS), followed with biotin-conjugated anti-armenian hamster IgG (eBiosciences). Biotin-conjugated staining was visualized with Sreptavidin-PerCP (BD PharMingen). All antibodies and secondary reagents were diluted in FACS buffer (1x PBS, 2% FCS, 0.1% NaN₃). Intracellular cytokine staining was performed according to the protocol recommended by BD PharMingen Fix and Perm kit (BD Biosciences, San Jose, CA, USA). Cells were first stained for surface receptor expression with biotin conjugated anti-CD3 (145-2C11, eBiosciences) and anti- TCR FITC (GL3, BD PharMingen) or anti-β TCR FITC (H57-597, eBiosciences), as described above. Cells were incubated with 1x fixation/permeabilization solution for 30 min in the dark at 4°C. Cells were washed and incubated with PE-conjugated anti-TNF- (MP6-XT22, eBiosciences) and APC-conjugated anti-IFN- (XMG1.2, eBiosciences) in 1x permeabilization solution for 45 min in the dark at room temperature. Isotype control staining was performed using IgG2a PE (eBR2a) and rat IgG2b APC (eB149/10H5) from eBiosciences. Four-color immunofluorescence analyses were performed using a FACSCalibur and CellQuest software (BD Biosciences).

Statistical analysis
Statistical significance between groups was calculated using the Student’s t-test. Differences were considered significant if P < 0.05.

RESULTS

β₂-Integrin expression on naïve and β T cells
We induced EAE in C57BL/6 mice to compare β₂-integrin expression on and β T cells throughout the acute course of EAE (Days 0–20). Table 1 shows the average (±SD) clinical scores of the mice at each time-point examined. Clinical signs of disease were apparent 10–12 days post-immunization and peaked 20 days post-immunization. We used multiparameter flow cytometric analysis to compare differences in and β T cell β₂-integrin expression in LN and spleen from uninduced mice (Day 0). As expected, T cells constituted only 1–3% of the population of cells in these tissues, and β T cells were abundant (Table 2 ). CD11a, CD11b, and CD11d were expressed on most T cells, and most β T cells expressed CD11a and CD11d and to a much lesser extent, CD11b. CD11c expression was low or undetectable on and β T cells prior to disease induction. Few or no lymphoid cells could be isolated from the spinal cords of uninduced mice, precluding analysis of β₂-integrin expression.

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Table 1. EAE Clinical Scores

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Table 2. β2-Integrin Expression on and β T Cells Following EAE Induction in DLNs, Spleen, and Spinal Corda

β₂-Integrin expression on and β T cells in the DLN, spleen, and spinal cord during EAE
We next analyzed β₂-integrin expression on and β T cells in inguinal DLN and spleen following EAE induction (Figs. 1 and 2 and Table 2 ). In the DLN, T cells expressing all of the β₂-integrins increased significantly from baseline, peaked at Day 10, and remained essentially constant throughout the remaining time-points examined. In contrast, β T cells expressing CD11b and CD11c increased significantly from baseline, peaked at Day 10, and declined slowly. β T cells expressing CD11a and CD11d fluctuated throughout the course of EAE, and expression did not appear to correlate with disease severity or progression (Fig. 1 and Table 2 ). In the spleen, T cells expressing all of the β₂-integrins increased significantly following immunization, peaked at 10–12 days, and declined by Day 15. On β T cells, CD11a expression remained constant in the spleen throughout disease. However, β T cell CD11b and CD11c expression increased on Days 10–12 and declined thereafter. CD11d expression on splenic β T cells fluctuated in no discernable pattern throughout the course of EAE (Fig. 2 and Table 2 ).

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Figure 1. Multiparameter flow cytometric analysis of β2-integrin expression on T cells (A) and β T cells (B) from DLN of EAE-induced mice. DLN were harvested on 0, 10, 12, and 15 days post-EAE induction. Representative dot-plots show TCR expression on the x-axis and β2-integrin expression on the y-axis. Percentages of positive cells are indicated in quadrants of interest. Quadrant settings were based on single color control staining. Cells were pooled from at least four mice per experiment, and data shown are representative of at least three independent experiments.

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Figure 2. Multiparameter flow cytometric analysis of β2-integrin expression on T cells (A) and β T cells (B) from spleens of EAE-induced mice. Spleens were harvested on 0, 10, 12, and 15 days post-EAE induction. Representative dot-plots show TCR expression on the x-axis and β2-integrin expression on the y-axis. Percentages of positive cells are indicated in quadrants of interest. Quadrant settings were based on single color control staining. Cells were pooled from at least four mice per experiment, and data shown are representative of at least three independent experiments.

We next compared the expression of β₂-integrins on and β T cells in the spinal cord during EAE. As shown in Figure 3 and Table 2 , β T cells infiltrated the CNS prior to T cells, and their cell numbers increased gradually during disease. In contrast, T cells migrated rapidly into the CNS, peaked at 10–12 days postimmunization and then declined rapidly by Day 15. T cell β₂-integrin expression followed a similar pattern, peaking at 10–12 days and declining thereafter. It is interesting that CD11a, CD11b, and CD11d expression on T cells is similar during the acute phase of EAE. β T cells expressing CD11a and CD11d increased slightly and remained relatively unchanged throughout the course of EAE, and β T cells expressing CD11b and CD11c peaked at 10 days and declined thereafter (Fig. 3 and Table 2 ). Overall, the expression profile of β₂-integrins on T cells in the spinal cord during EAE is remarkably different than that observed on β T cells.

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Figure 3. Multiparameter flow cytometric analysis of β2-integrin expression on T cells (A) and β T cells (B) from spinal cords of EAE-induced mice. Spinal cords were harvested on 0, 6, 10, 12, 15, and 20 days post-EAE induction. Representative dot-plots show TCR expression on the x-axis and β2-integrin expression on the y-axis. Day 0 staining showed few cells were present in uninduced spinal cord. (A) T cells infiltrated the CNS and peaked 10–12 days postimmunization and declined rapidly by Day 15. The expression of β2-integrins on T cells followed a similar pattern, peaking at Day 12 and declining thereafter. (B) β T cells infiltrated the spinal cord by Day 6, increased in number, and remained relatively unchanged throughout the course of EAE. There were fewer than β T cells in the spinal cord following EAE induction. Percentages of positive cells are indicated in quadrants of interest. Quadrant settings were based on single color control staining. Cells were pooled from at least four mice per experiment, and data shown are representative of at least three independent experiments. Day 6 (n=5) and Day 20 (n=4) data are from one experiment.

Production of IFN- and TNF- by and β T cells during EAE
The role of cytokines in EAE remains a complex and in many cases, controversial area of study (reviewed in refs. [¹ , ^{67 68 69} ]). Even less is known about the role of cytokines produced by T cells during EAE. As such, we performed intracellular cytokine flow cytometric analysis for a limited number of cytokines including IFN-, TNF-, IL-4, and IL-10 on and β T cells in the DLN, spleen (Fig. 4 and Table 3 ), and spinal cord (Fig. 5 and Table 3 ) throughout the course of EAE. Only events within the TCR⁺/CD3⁺ gates were used for analyses (Fig. 4A) . Background staining was low, except for T cells in the DLN and spleen of uninduced mice (Fig. 4B) . The reasons for this are unclear; however, similar findings have been documented for some T cell lines [⁷⁰ ]. LN and β T cells from uninduced mice had a similar cytokine profile compared with mice 10 days postimmunization for EAE. By Day 12, the number of cells producing only TNF- or TNF- and IFN- increased substantially for and β T cells (Fig. 4C) . The majority of T cells produced TNF-, compared with only about half of the β T cells. Early in disease, neither T cell subset had many IFN--only-producing cells, but by Day 15, both subsets were producing IFN-. Similar findings were obtained for and β cells derived from the spleen (Fig. 4D) . Although there were fewer than β T cells in these tissues, T cells produced more cytokine in the DLN and spleen throughout disease (Fig. 4 and Table 3 ). In sharp contrast, there were substantially more IFN-- and TNF--producing T cells in the CNS during EAE than β T cells, and virtually all T cells expressed one or both cytokines (Fig. 5A) . There were few TNF--only-producing T cells of either subset in the spinal cord at any time-point examined. The number of IFN--producing β T cells peaked at Day 12 and declined thereafter. Conversely, T cells produced IFN- up to Day 20, even when their numbers were declining in the spinal cord. Background staining was low (Fig. 5B) . We observed no detectable IL-4 or IL-10 production from either T cell subset in any tissue examined (data not shown).

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Figure 4. Intracellular cytokine flow cytometric analysis of T cells and β T cells in the DLN and spleen 0, 10, 12, and 15 days post-EAE induction. IFN- (x-axis) and TNF- (y-axis) expression was analyzed from gated TCR+/CD3+ or β TCR+/CD3+ (A) cells in the DLN (C) and spleen (D). Although T cells were fewer in number, they produced more TNF- and IFN- compared with β T cells in DLN and spleen throughout disease. Little or no staining was observed using isotype control antibodies (B). Percentages of positive cells are indicated in quadrants of interest. Quadrant settings were based on unstained controls. Cells were pooled from at least four mice per experiment, and data shown are representative of at least three independent experiments.

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Table 3. Cytokine Expression Profiles of Versus β T Cells in DLNs, Spleen, and Spinal Cord Following EAE Inductiona

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Figure 5. Intracellular cytokine flow cytometric analysis of T cells and β T cells in the spinal cord 0, 10, 12, 15, and 20 days post-EAE induction. IFN- (x-axis) and TNF- (y-axis) expression was analyzed from gated TCR+/CD3+ or β TCR+/CD3+ cells in the spinal cord. T cells were fewer in number but produced more TNF- and IFN- compared with β T cells in the spinal cord throughout disease. Day 0 showed no cytokine production. Little or no staining was observed using isotype control antibodies (B). Percentages of positive cells are indicated in quadrants of interest. Quadrant settings were based on unstained controls. Cells were pooled from at least four mice per experiment, and data shown are representative of at least three independent experiments. Day 20 (n=4) data are from one experiment.

DISCUSSION

T cells have been shown to play an important role in immune responses, although their precise effector functions in many disease settings remain elusive [³ , ⁷¹ ]. In EAE, T cells are critical for full disease development based on antibody-depletion studies [^{12 13 14 15} ] and studies using -chain-deleted mice [¹¹ ]. Caution must be used in interpreting studies in which antibodies have been used to deplete T cells in vivo, as some studies have shown that such treatment may affect β T cell activity or exacerbate disease [⁶ , ⁷² ]. Nevertheless, T cells traffic into the CNS during EAE, although the adhesion molecules they use during migration across the BBB remain unclear. Our data demonstrate that T cells express all of the β₂-integrin family members and their expression changes during the course of EAE. These results suggest that β₂-integrins may contribute to T cell trafficking into the CNS.

The functions of adhesion molecules on T cells are diverse and include cellular trafficking, costimulation, and stabilization of the immunological synapse [³⁰ , ⁷³ , ⁷⁴ ]. Understanding these functions is derived largely from studies using CD4⁺ and CD8⁺ β T cell subsets. The function and expression of β₂-integrins on T cells, however, have received little attention. In studies using endothelial cell monolayers designed to examine T cell migration, the expression of CD11a was relatively unchanged before and after migration [³⁵ ]. Similarly, we observed a limited change in CD11a expression on β T cells before and during the acute phase of EAE (Fig. 3 and Table 2 ). In sharp contrast, the expression of CD11a was elevated markedly on T cells, which had migrated into the spinal cord, compared with expression on naïve T cells (Fig. 3 and Table 2 ). We observed similar results for all of the remaining β₂-integrin family members on T cells (but not β T cells) during EAE, and expression peaked between 10 and 12 days after disease induction. The significance of the change in expression of these proteins during EAE on T cells remains unclear; however, deletion of CD11a, CD11b, or CD11c [²³ , ²⁴ , ⁴² ] (D. Bullard, K. Dugger and S. R. Barnum, unpublished observations) results in significantly attenuated disease and cellular infiltration into the CNS. It is interesting that deletion of CD11d had no effect on the course of EAE compared with control mice [³⁴ ]. Taken together, the data with β₂-integrin gene-deleted mice suggest that this family of adhesion molecules, with the exception of CD11d, may be important for migration of T cells into sites of inflammation during EAE.

Although studies have shown that T cells migrate into the CNS during EAE [⁵ , ¹¹ , ¹⁴ , ⁷⁵ , ⁷⁶ ], none have compared the infiltration of T cells with that of β T cells directly. We observed that β T cells reached the CNS quickly (6 days or less) compared with T cells (Fig. 3) . However, by Day 10, the number of T cells in the CNS rose dramatically (over 30-fold) compared with Day 6 levels but were still substantially lower than β T cells. Both T cell subsets remained elevated at Day 12 postimmunization; however, at all later time-points, T cells represented a much smaller fraction of the T cell population in the CNS (Fig. 3) . The reason for the migration/retention differences between these T cell subpopulations as disease progresses is unclear at present. Our findings differ from those reported by Rajan and colleagues [¹⁴ ], in which they observed a peak in T cell numbers at the height of disease, followed by reduced numbers during disease remission and an increase during disease relapse. We observed peak infiltration prior to peak disease severity. The differences between the two studies could be, in part, a result of the use of different mouse strains (C57BL/6 vs. SJL), immunization protocol (active vs. adoptively transferred disease), or even immunogen (MOG peptide vs. MBP). Our data suggest that T cells play a prominent role in early EAE events, perhaps even in modulating β T cell function, as suggested in previous studies [⁴ , ⁵ , ¹⁵ ].

The most remarkable finding in our study was the difference in cytokine production between and β T cells in all tissues examined but particularly, in the CNS (Fig. 5 and Table 3 ). It is interesting that the number of T cells producing IFN- and TNF- in LN and spleen, prior to disease onset, was elevated compared with cytokine production by β T cells. The reason for this is unclear but may suggest a higher baseline state of activation for T cells. We also found more IFN-- and TNF--producing T cells in LN and spleen throughout the course of EAE compared with β T cells. Furthermore, the number of T cells producing both cytokines was also higher than that of β T cells. In the CNS, we observed more IFN--only-producing T cells and a greater percentage of T cells producing both cytokines compared with β T cells. There was little TNF- produced in the spinal cord by these cells at any time-point examined. This is in contrast to a study, which examined the kinetics of TNF- and IFN- production in the CNS following MOG-induced EAE [⁷⁷ ]. In this study, MOG_35–55-stimulated CD4⁺ T cells from the CNS produced similar amounts of TNF- and IFN- by Day 7, peaked at Day 20, and then waned thereafter. We did not examine time-points past Day 20 as a result of the substantial decline in T cell subsets by Day 15. Although our study examined a limited repertoire of cytokines, it is clear that T cells express cytokines differentially in the CNS compared with β T cells and have the potential to modulate the local inflammatory response during EAE.

Numerous studies have examined the production of cytokines by T cells in EAE; however, these studies are limited for several reasons. First, many of these studies quantitated changes in expression of cytokine and chemokine mRNA levels using whole spinal cord as the mRNA source [¹² , ¹³ ]. Thus, neither the actual protein levels nor cell-specific production were assessed, raising the possibility that other cell types were responsible, at least in part, for the reported changes. Where cytokine levels were determined, whole spinal cords were used, again precluding the identification of the cellular source of cytokine production [¹² ]. In a number of studies, cytokine production was assessed using T cells derived from the spleen or LN rather than the CNS [⁵ , ¹¹ , ¹⁵ ]. Our data are the first to demonstrate that IFN- and TNF- production by T cells is markedly different between peripheral lymphoid tissues and the CNS. This suggests that using T cells from LN and spleen to evaluate effector functions of these cells during EAE may be of limited value. Finally, studies have examined cytokine production by T cells by comparing cytokine mRNA or protein levels using mice depleted of T cells [¹¹ , ¹² , ¹⁵ ]. These studies assume that observed deficits in cytokine production are attributable solely to T cells—an assumption, which remains to be proven. It is clear from these studies that many aspects of T cell biology in demyelinating disease remain to be elucidated. The data we present here set the stage for future studies examining the role of adhesion molecules in the trafficking and effector functions of T cells in EAE.

ACKNOWLEDGEMENTS

This work was supported by grants from the National Multiple Sclerosis Society (RG-3437-A-6) and from National Institutes of Health (NS46032) to S. R. B. and (T32 AI07051-30) to S. S. S. We thank Dr. Kohtaro Fujihashi for the use of the FACSCalibur for flow cytometry and Dr. Dan Bullard for helpful discussions.

Received April 27, 2007; revised August 22, 2007; accepted September 17, 2007.

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