Functional maturation of proteolipid protein139–151-specific Th1 cells in the central nervous system in experimental autoimmune encephalomyelitis

Journal of Neuroimmunology 155 (2004) 127 – 135 www.elsevier.com/locate/jneuroim

Functional maturation of proteolipid protein139–151-specific Th1 cells in the central nervous system in experimental autoimmune encephalomyelitis Mani Mohindru, Bongsu Kang, Byung S. Kim* Department of Microbiology-Immunology and Institute for Neuroscience, Northwestern University Feinberg Medical School, 303 East Chicago Ave, Chicago, IL 60611, United States Received 24 March 2004; received in revised form 28 June 2004; accepted 28 June 2004

Abstract Experimental autoimmune encephalomyelitis (EAE) is a widely adopted animal model system for studying human multiple sclerosis that affects the central nervous system (CNS). To understand the underlying pathogenic mechanisms of the autoimmune T cell response, localization, enumeration and characterization of autoreactive T cells are essential. We assessed encephalitogenic proteolipid protein epitope (PLP139–151)-specific T cells in the periphery and CNS of SJL/J mice using MHC class II I-As multimers during both pre-clinical and clinical phases of PLP-induced EAE in conjunction with T cell function. Our results strongly suggest that PLP139–151-specific CD4+ T cells first expand primarily in the CNS-draining cervical lymph nodes and then migrate to the CNS. In the CNS, these PLP-specific CD4+ T cells accumulate, become activated and differentiate into effector cells that produce IFN-g in response to the self-peptide. D 2004 Elsevier B.V. All rights reserved. Keywords: Class II tetramers; EAE/MS; T cell receptor; Autoimmunity; MHC

1. Introduction The experimental autoimmune encephalomyelitis (EAE) has been widely used to investigate immune-mediated inflammatory demyelinating disease in various animals as a model of human multiple sclerosis (Martin et al., 1992). This experimental model provides important information regarding the immune cell types involved in the pathogenesis of the demyelinating disease. Although autoimmune T cell responses of this disease model have been widely studied in the peripheral lymphoid organs, the fate and function of autoreactive T cells in the target CNS are still poorly understood, mainly due to the relatively low frequencies of autoreactive T cells and their low affinities to autoantigens. Therefore, understanding the process of activation, expansion and functional maturation of autoreactive effector T cells is critical for clinical applications. However, the lack of appropriate tools has * Corresponding author. Tel.: +1 312 503 8693; fax: +1 312 503 1339. E-mail address: [email protected] (B.S. Kim). 0165-5728/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2004.06.012

hindered the in vivo detection and characterization of autoreactive T cells during the development of autoimmune diseases such as EAE. Soluble major histocompatibility complex (MHC)–peptide tetramers/multimers are widely used for the enumeration and characterization of antigen-specific T cells (Hackett and Sharma, 2002). These tetramers allow direct enumeration of T cells based on antigen specificity without functional bias. Since the development of first MHC tetramers (Altman et al., 1996), a vast majority of studies have focused on studying CD8+ T cell responses using MHC class I multimers. The generation and use of class II MHC multimers have proven to be difficult mainly because of low T cell receptor (TCR) avidities of CD4+ T cells and low frequencies of antigen specific CD4+ T cells (Cameron et al., 2002; Kwok, 2003). There have been studies using human (Kwok et al., 2000; Novak et al., 1999; Quarsten et al., 2001) and mouse MHC class II multimers (Crawford et al., 1998; Homann et al., 2001; Mallet-Designe et al., 2003; Radu et al., 2000; Reddy et al., 2003). Most of these reports involve ex vivo antigen-driven expansion of T cells before

128

M. Mohindru et al. / Journal of Neuroimmunology 155 (2004) 127–135

class II MHC multimer staining. Despite being useful, this complicates the assessment of frequencies of antigenspecific T cells in vivo. In particular, detection of selfantigen specific CD4+ T cells using MHC multimers has been especially challenging because of the low affinity of TCRs reactive to MHC/autoantigen complexes. In order to analyze antigen-specific CD4+ T cells in myelin proteolipid protein (PLP)-induced EAE, we generated MHC class II I-As/PLP139–151 multimers. Our PLP multimer preparation differs significantly from that reported recently (Reddy et al., 2003) in the generation method as well as detection sensitivity. Unlike the covalent linkage of PLP139–151 peptide by Reddy et al. (2003), our multimers have the same peptide loaded in vitro onto pre-assembled IAs class II molecules. We have been successful in using this reagent to track autoreactive CD4+ T cells in various organs during the course of PLP139–151-induced EAE without ex vivo expansion or manipulation of isolated T cells. Here we report that PLP139–151-specific CD4+ T cells first accumulate and expand in the cervical lymph nodes before migrating to the CNS. In the CNS, these autoreactive T cells differentiate into effector Th1 cells and produce cytokines such as IFN-g. These activated T cells appear to down-regulate TCRs to undetectable levels. It appears that external peptide loading onto class II MHC molecules may be an important consideration for sensitive and accurate enumeration of antigen-specific T cells.

2. Materials and methods 2.1. Animals Female SJL/J mice (6–8 week old) were purchased from Charles River Laboratories (Wilmington, MA.) through the National Cancer Center and were housed in the Animal Care Facility at Northwestern University Medical School. 2.2. Antigen and induction of EAE PLP139–151 (HSLGKWLGHPDKF) peptide was synthesized by Genemed Synthesis (San Francisco, CA). Mice were immunized with PLP139–151 in modified complete Freund’s adjuvant (CFA) as described previously (McRae et al., 1992). Briefly, each animal received 100 Al of CFA emulsion containing 200 Ag of Myobacterium tuberculosis and 50 Ag of PLP139–151 subcutaneously. Mice were scored according to the clinical severity as described (McRae et al., 1992). By day 14 post-immunization, mice showed partial to complete hind limb paralysis. 2.3. Cell preparation CNS infiltrating lymphocytes were isolated as described previously (Kang et al., 2002). Briefly, brains and spinal cords were removed from mice after intracardiac perfusion,

forced through a wire mesh and incubated at 37 8C for 45– 60 min in 250 Ag/ml collagenase type 4 (Worthington Biochemical, Lakewood, NJ). CNS lymphocytes were enriched by continuous Percoll (Pharmacia, Piscataway, NJ) gradient centrifugation for 30 min at 27,000g. Single cell suspensions from spleens and lymph nodes were prepared by gentle teasing through a wire mesh. 2.4. Construction of hybrid I-As a and b chain expression vectors Hybrid cassettes containing the extracellular domain sequences of MHC-I-As a chain and basic leucine zipper (LZ) sequences as well as I-As h chain and acidic LZ were generated by PCR-mediated gene splicing by overlap extension (Horton et al., 1990). For a chain, the first PCR involved amplification from As a plasmid using sense primer, 5V-AGAATTCATGCCGTGCAGCAGAGCTCTG3V and antisense primer, 5V-CTGTAGTGGATCCGCGAGTCTCTGTCAGCTCTGACATGG-3V. For the second PCR, LZ-containing plasmid, pCRII base, was used as a template and primary PCR product was used as a primer along with the sense As a chain primer and the antisense p r i me r : 5V- C T G GTA C C ATCC TAC TG G G C G A GTTTCTTC-3V. Histidine tag was added by a final amplification using the above sense primer and antisense primer, 5 V- C T G G TA C C AT C C TA G T G AT G G T G AT G G TGATGCTGGGCGAGTTTCTTCTTGAGGGC-3V . The sequence-verified product was cloned into the EcoRI and KpnI sites of insect cell expression plasmid, pRmHa-3. To generate recombinant h chain, the primary PCR was performed using As h plasmid and the sense primer, 5VACTCGAGATGGCTCTGCAGATCCCCAGCC-3V and antisense primer, 5V-CTGTAGTGGATCCGCGAGACTGTGCCCTCCACTCCACAG-3V. For the second PCR, another LZ plasmid, pCRII acid, was used as a template and first As h PCR product was used as primer along with the same sense primer and the antisense primer, 5V-ACAAGCTTGCCTGAGCCAGTTCTTTTTCC-3V. The PCR product was subcloned into the XhoI and HindIII sites of pAC-4 plasmid (Avidity, Denver, CO, contains C-terminal bacterial biotinylation sequence). The chimeric As h-acid LZbiotinylation sequence was amplified from the pAC-4 plasmid using the sense, 5V-AGAATTCATGGCTCTGCAG-ATCCCCAGCC-3V and antisense 5V-CTGGTACCTTATTCGTGCCATTCGATTTTCTG-3V primers. After sequence verification, the PCR product was subcloned into the EcoRI and KpnI sites of pRmHa-3. 2.5. Generation of I-As multimers The recombinant I-As a and h chain expression vectors were cotransfected into Drosophila Schneider S2 cells (Invitrogen, Carlsbad, CA) along with hygromycin resistance vector, pCoHYGRO (Invitrogen) using calcium phosphate transfection method. The transfectants were

M. Mohindru et al. / Journal of Neuroimmunology 155 (2004) 127–135

selected in hygromycin containing media, subcloned and induced with 1 mM CuSO4 to express the recombinant proteins. The culture supernatant was concentrated and the recombinant I-As a/h dimers were purified using nickel columns. The purified protein was biotinylated using bacterial BirA enzyme (Avidity) according to the manufacturer’s protocol and excess biotin was removed by dialysis. The purified biotinylated MHC molecules were multimerized overnight with streptavidin–R-phycoerythrin (Biosource, Camarillo, CA) in a 8:1 molar ratio. The mixture was dialyzed in a 100-kDa cutoff membrane to remove the potential monomers and unconjugated streptavidin. The multimerized product was loaded with 50-fold molar excess of peptides in 0.4% n-octylglucopyranoside with gentle shaking. 2.6. Staining with I-As/PLP139–151 multimers and flow cytometry Fc receptors on lymphocytes were blocked by incubating the cells in 50 Al of 2.4G2 hybridoma supernatant (ATCC, Rockville, MD) for 30 min at 4 8C. Multimers were added to the supernatant to a final concentration of 1 AM and the cells were incubated for 2 h at 4 8C. In the last 20 min of incubation, fluorochrome-labeled antibodies were added (i.e., anti-CD4 (clone RM4-5, BD Biosciences, San Diego, CA) and anti-a/h TCR (clone H57-597, Cedarlane Laboratories, Horby, Ontario, Canada). Cells were washed and analyzed on a Becton DickinsonFACSCalibur flow cytometer. 2.7. Intracellular cytokine staining (ICS) ICS was performed using the Cytofix/Cytoperm kit (BD Biosciences) according to manufacturer’s instructions. Briefly, isolated lymphocytes were incubated in the presence of 10 AM PLP139–151 peptide or 10 Ag/ml immobilized anti-CD3 antibody (hybridoma 145-2C11, ATCC) plus 2 Ag/ml anti-CD28 antibody (hybridoma PV-1, ATCC) and Monensin for 6–8 h at 37 8C. Fc receptors were blocked with 2.4G2 hybridoma supernatant and the cells were stained with Allophycocyanin conjugated anti-CD4 (clone RM4-5, BD Biosciences) antibody. The cells were then permeabilized and stained for intracellular IFN-g with phycoerythrin-labeled anti-IFN-g antibody (clone XMG1.2). Stained cells were analyzed on a Becton DickinsonFACSCalibur flow cytometer.

3. Results 3.1. Generation of I-As/PLP139–151 and I-As/VP272–86 multimers Accurate assessments of CD4+ T cell populations involved in a variety of inflammatory diseases following

129

infections and/or autoimmune diseases are very important for understanding the disease as well as for potential intervention. In order to analyze antigen-specific CD4+ T cells in PLP-induced EAE, we generated two different MHC class II (I-As) multimers loaded with encephalitogenic PLP139–151 or Theiler’s murine encephalomyelitis virus (TMEV) VP272–86 peptide using a modified procedure (Fig. 1A). Specifically, we introduced His tag in the expressed MHC protein, which facilitates isolation of the pre-assembled MHC class II molecules using nickel column rather than conventional isolation using antibodies specific for individual haplotypes of class II molecules. We then generated stably assembled class II (I-As) multimer complexes by mixing biotin-conjugated class II molecules with the fluorochrome-labeled streptavidin. The pre-assembled I-As multimers were subsequently loaded with peptides (see Materials and methods). For most previously generated tetramers/multimers, peptide-loaded class II monomers were first prepared and then multimerized by biotin–avidin interaction followed by fluorochrome conjugation. 3.2. Specificity of I-As/PLP139–151 and I-As/VP272–86 multimers We first examined the binding specificities of I-As/ PLP139–151 and I-As/VP272–86 multimers to CD4+ T cell lines or hybridomas with different antigen specificities. The VP272–86 multimer which binds specifically to T cell hybridomas reactive to Theiler’s virus VP272–86 epitope (data not shown) did not react with a PLP139–151-specific hybridoma, 5B6 (Fig. 1B,a). However, the PLP139–151 multimer stained 62% of 5B6 hybridoma cells (Fig. 1B,a) but not 14C7 hybridoma cells specific for VP272–86 (not shown). In addition, an I-As multimer without external peptide load (bemptyQ multimer) did not stain PLP-specific hybridoma similar to the multimer loaded with unrelated VP2 peptide (Fig. 1B,a). PLP139–151 multimers also stained 70% of I-As-restricted PLP-reactive T cells maintained by repeated stimulation with PLP139–151 peptide (six to eight cycles). However, we did not observe PLP multimer binding to ConA-stimulated polyclonal T cells (Fig. 1B,b). These results indicate that the PLP139–151 multimer is indeed specific for their respective TCRs. 3.3. Emergence of I-As/PLP multimer positive, CD4+ T cells in mice with EAE It is important to assess self-reactive T cells in the periphery as well as in the CNS to understand the immunemediated pathogenic mechanisms. Hence, we employed PLP139–151 multimers to enumerate PLP139–151-specific CD4+ T cells in lymph nodes (cervical lymph nodes (Lider et al., 1989), as well as brachial and inguinal lymph nodes, denoted as bother lymph nodesQ), spleen and CNS of female SJL/J mice post-immunization with either

130

M. Mohindru et al. / Journal of Neuroimmunology 155 (2004) 127–135

Fig. 1. (A) Schematic representation of the procedures for PLP multimer generation. The external domains of I-As a chain was linked to a basic-leucine zipper and a His tag. Similarly, the I-As h chain was linked to an acidic leucine-zipper and a biotylation sequence. Monomeric I-As molecules isolated from transfected S2 cultures were then isolated using a nickel column. These molecules were biotinylated and tetramerized with PE-conjugated streptavidin. The tetramers/ multimers were then loaded peptides. (B) Specificity of PLP139–151 multimer positive CD4+ T cells. (a) The PLP139–151, VP272–86 or empty multimers were used for staining PLP139–151 hybridoma, 5B6 as described in Materials and methods. (b) PLP139–151 multimer was used to stain ConA-stimulated T cells from C57BL/6 (H-2b) mice or polyclonal PLP139–151-specific T cell line derived from PLP139–151-immunized SJL/J (H-2s) mice.

PLP139–151 peptide in CFA or CFA alone as control. We analyzed PLP multimer+ CD4+ T cells by flow cytometry during pre-clinical (day 3) and clinical disease (day 14 post-immunization) (Fig. 2). At day 3, no T cells are detected in the CNS but a significant level of PLP-specific CD4+ T cells (N2% of CD4+ T cells) can be seen in the cervical lymph nodes (CLN) of PLP-immunized mice. At the peak of clinical disease (day 14), about 4% of all CD4+ T cells in the CNS of PLP-immunized mice are specific to PLP139–151 (Fig. 2A). It is not yet clear whether nonmultimer+ T cells include anergized/TCR-down-regulated PLP-specific CD4+ T cells. If this is the case, the number and proportion of PLP-specific CD4+ T cells in mice with

EAE could be drastically increased. Interestingly, the proportion of PLP139–151 multimer+ CD4+ T cells in the CNS of control CFA-immunized mice is relatively high (2.9%). This may reflect the presence of relatively high levels of PLP139–151-reactive Th cells (based on T cell proliferation assays) in the periphery of naRve SJL/J mice (Anderson et al., 2000; Klein et al., 2000). However, the overall CNS-infiltrating CD4+ T cell number is far greater in PLP-immunized (5.4F1.7105/CNS) mice than in CFA-control (6.6F1.5103/CNS) mice. Therefore, N80fold higher PLP139–151 multimer+ T cells are present in the CNS of PLP-immunized SJL mice compared to CFAimmunized control mice.

M. Mohindru et al. / Journal of Neuroimmunology 155 (2004) 127–135

131

Fig. 2. Specificity and emergence of PLP139–151 multimer positive CD4+ T cells in various organs during the course of EAE. (A) Percentage of multimerpositive CD4+ T cells in different organs during pre-clinical (day 3) and clinical EAE (day 14) isolated from PLP/CFA or control CFA-immunized mice. The results are shown with the gated population for CD4+ T cells. (B) The PLP139–151 multimer positive cells were followed in immune organs (cervical or other lymph nodes, LNs, and spleen) and the target organ, CNS, up to the induction of clinical EAE. The cells from PLP/CFA- or CFA-immunized mice were isolated and stained with PLP multimers and anti-CD4 antibody, and the results are expressed as total multimer positive cells per 104 cells (for immune organs) or as total multimer positive cells per CNS. A representative or the meanFstandard error of the mean of three separate experiments is shown here. **pb0.01 between CNS CD4+ T cells from PLP/CFA- and CFA-immunized mice based on unpaired (two-tailed) t-test.

To further determine the fate of PLP-specific CD4+ T cells in the periphery and CNS, we compared PLP139–151 multimer+ T cells in the CLN, other lymph nodes, spleen and CNS during the course of EAE. PLP139–151-specific T cells could be detected in the CLN and other lymph nodes as early as 1 day post-immunization (Fig. 2B). Our data suggest that PLP139–151-specific T cells accumulate in the CLN until day 5 and start declining by day 9 post-immunization (Fig. 2B). The numbers of PLP139–151-specific T cells continue to increase day 5 in the CNS with time. By day 14 (peak of clinical EAE), the CNS accumulates a large proportion of T cells specific for PLP139–151. In contrast, there is no significant expansion of PLP multimer+ cells in the lymph nodes of healthy, CFA-immunized control animals (not shown) and very few, if any, overall as well as PLP multimer+ CD4+ T cells in the CNS (Fig. 2B). Although other lymph nodes contain a higher number of PLP multimer+ T cells than the spleen, there is no indication of expansion of these T cells in peptide-immunized mice. These results indicate that PLP-specific Th cells expand/accumu-

late first in the CLN (3–5 days post-immunization) and then migrate to the CNS during 5–14 days post-immunization. 3.4. Higher PLP multimer binding by TCRlow CD4+ T cell population It is known that highly activated T cells down-regulate the expression of TCRs on their surface (Alcover and Alarcon, 2000). To correlate the specificity of T cells with the activation state, we analyzed PLP multimer staining in conjunction with TCR expression on CD4+ T cells in the CLN and CNS of PLP-immunized mice. We found a higher proportion of PLP139–151 multimer+ cells in the TCRlo CD4+ T cell population in the CLN during both pre-clinical (day 3) and clinical (day 14) stages of EAE (Fig. 3). At day 3 post-immunization, the percentage of PLP multimer+ TCRlo CD4+ T cells (7.3%) in the CLN is significantly higher (N3-fold) as compared to total multimer+(2.2% of TCRlo/hi) CD4+ T cells. At day 14 post-immunization, a higher proportion of multimer+ cells reside in the TCRlo

132

M. Mohindru et al. / Journal of Neuroimmunology 155 (2004) 127–135

Fig. 3. Correlation of PLP multimer staining with the activation phenotype of cervical or CNS CD4+ T cells. (A) T cells were isolated from the cervical LN and the CNS of PLP-immunized mice during pre-clinical (day 3) or clinical EAE (day14) and examined for cell surface TCR expression. CD4+ T cells expressing different levels of TCR were analyzed for PLP multimer staining. The top and bottom panels show the percentage of multimer+ CD4+ T cells in total (TCRhi/lo) and TCRlo (highly activated) populations, respectively. (B) Activation phenotype of CD4+ T cells infiltrating the CNS was analyzed by flow cytometry. The expression of CD45RB, CD44, CD69 and CD25 molecules on the surface of CD4+ T cells infiltrating the CNS at 14 days post-immunization was determined in conjunction with the expression levels of TCR. The proportions of CD45RBhi, CD69hi and CD25hi are drastically different between TCRlo and TCRhi CD4+ T cell population. A representative of three separate experiments is shown here.

CD4+ T cell population in the CNS (6.6% in TCRlo vs. 3.7% in TCRlo/hi). It is possible that anti-TCR antibody might inhibit multimer staining. If this is the case, multimer binding will be preferentially blocked on T cells with low TCR density, an opposite of our finding. In addition, the overall levels of multimer binding in the presence or absence of anti-TCR antibody are very similar, i.e., 3.8% vs. 3.1%, respectively. 3.5. Distinct activation markers on TCRlo and TCRhi populations in the CNS of PLP139–151-immunized mice The identification of a distinct population of CD4+ T cells in the CNS of PLP139–151-immunized SJL/J mice (i.e TCRlo and TCRhi CD4+ T cells; Fig. 3A) led us to further characterize the CNS-infiltrating T cell population by comparing the expression of various activation makers (Fig. 3B). We analyzed the levels of CD45RB, CD44, CD69 and CD25 molecules on the surface of CNS-infiltrating TCRlo and TCRhi CD4+ T cell populations at 14 days postimmunization. The expression of activation makers was drastically different between TCRlo and TCRhi populations. Although the level of CD44hi expression (99–100%) was

similar, levels of CD45RB, CD69 and CD25 were dramatically different between these populations. The presence of CD44hi T cells in both populations is consistent with the previous observation that expression of CD44 molecule is required for the extravasation of T cells into the site of inflammation (Brennan et al., 1999; DeGrendele et al., 1997). In particular, 93% of TCRlo CD4+ T cell population displayed CD45RBhi as compared to only 9% of TCRhi CD4+ population. Similarly, the levels of CD69hi (17% vs. 64%) and CD25hi (11% vs. 48%, respectively) populations in TCRlo CD4+ T cells were different from those in TCRhi CD4+ T cells. The expression pattern of the activation markers on the TCRlo CD4+ T cell population in the CNS suggests that this is a unique T cell population in the CNS of mice with EAE and perhaps represents a pathogenic effector CD4+ T cell population. 3.6. IFN-g production by PLP139–151-specific CD4+ T cells in the CNS upon in vitro stimulation In order to further correlate PLP139–151 multimer+ CD4+ T cells with T cell function, we analyzed IFN-g production by CD4+ T cells from the CLN and CNS after brief in vitro

M. Mohindru et al. / Journal of Neuroimmunology 155 (2004) 127–135

133

Fig. 4. Antigen-specific IFN-g production by cervical or CNS T cells during the course of EAE. (A) Intracellular cytokine staining was performed on T cells isolated from cervical lymph nodes or CNS at various times during the course of EAE as described in Materials and methods. This figure shows the percentage of IFN-g-producing PLP139–151-specific or non-specific (plate-bound anti-CD3 responding) CD4+ T cells in the cervical lymph nodes and CNS of PLPimmunized mice. (B) IFN-g production by CNS-infiltrating TCRlo (first row), TCRhi (second row) and TCRhi/lo (third row) CD4+ T cells during clinical EAE (day 14 post-immunization) upon stimulation with no peptide, PLP peptide or anti-CD3 antibody. The numbers in the plots represent the percentage of IFN-gproducing CD4+ T cells. The meanFstandard error of the mean from three separate experiments is shown here. *pb0.05 and **pb0.01 between CD4+ T cells in the CNS and in the CLN of PLP-CFA-immunized mice based on unpaired (two-tailed) t-test.

stimulation with PLP139–151 peptide or anti-CD3 antibody on various days after immunization. Very few CLN T cells produce IFN-g upon stimulation with either PLP139–151 peptide or anti-CD3 antibody throughout the entire course (Fig. 4A), although the efficiency of the T cells to produce IFN-g is gradually increased at the onset of disease. In contrast, a significant proportion of CD4+ T cells infiltrating the CNS produce IFN-g as early as day 9 post-immunization, coinciding with the start of expansion/accumulation of PLP multimer+ cells in the CNS (Fig. 2). The IFN-gproducing CD4+ T cells continue to increase in the CNS at day 14 post-immunization, when mice are clinically affected by EAE. These results indicate that cytokine-producing effector CD4+ T cells specific to PLP139–151 appear in the CNS only by day 9 and continue to persist as well as

increase in numbers through the clinical phase of EAE. Thus, functional maturity of IFN-g-producing effector CD4+ T cells appears to occur preferentially in the CNS. Our findings are consistent with recent observations by ELISPOT assays that CNS-infiltrating T cells produce IFNg upon antigen stimulation (Di Rosa et al., 2000; Flugel et al., 2001; Targoni et al., 2001). Because a higher proportion of PLP139–151 multimer+ T cells in the CNS were present in the TCRlo population, we also compared IFN-g production by TCRlo and TCRhi CD4+ T cells in the CNS (Fig. 4B). In agreement with our multimer staining data, a higher proportion of TCRlo CD4+ T cells produce IFN-g (12.2% of TCRlo CD4+ T cells) as compared to TCRhi CD4+ T cells (4.3%). Although a higher number of CNS-infiltrating CD4+ T cells produce IFN-g upon stimula-

134

M. Mohindru et al. / Journal of Neuroimmunology 155 (2004) 127–135

tion with anti-CD3 compared to PLP-stimulated CD4+ T cells, N80% CD4+ T cells in the CNS could not be stimulated with anti-CD3 antibody treatment (Fig. 4B). These results suggest that a vast majority of CNS-infiltrating CD4+ T cells may not be able to produce inflammatory cytokines in response to TCR-mediated stimulation.

4. Discussion In this report, we have made significant improvements in the isolation of expressed MHC class II (I-As) molecules as well as in the efficiency of peptide loading onto soluble MHC molecules. To improve the isolation procedure, the His tag was introduced to one of the class II chains in order to facilitate easy purification using nickel-column (Fig. 1). Moreover, to increase the efficiency of peptide loading, pre-assembled multimers were used to load the peptide in a small reaction volume. Many previously reported multimers including PLP139-151/I-As have been prepared with covalently linked epitope peptides. However, this requires generating individual multimer constructs for each epitope whereas our multimers can be pre-made and loaded various epitope peptides. Furthermore, a free-loaded multimer might have specificity for the natural, diverse T cell repertoire while a covalently ligated peptide multimer might be more restricted. Hence externally loaded peptides may be able to bind MHC class II molecules in multiple forms of several overlapping epitopes within the peptide region that are recognized by bulk T ell populations (Kim et al., 1999; Pu et al., 2002). The majority of previous studies analyzing autoreactive CD4+ T cells in mice with EAE are based on assessment of proliferative responses and/or cytokine production of peripheral T cells rather than CNS-infiltrating T cells (Smeltz and Swanborg, 1998). Our results indicate that very few CD4+ T cells from CLN produce IFN-g through the entire course of EAE, whereas a significant proportion of CD4+ T cells infiltrating the CNS produce IFN-g (Fig. 4). Our observation that CNS-infiltrating T cells produce IFN-g is consistent with other recent publications (Di Rosa et al., 2000; Flugel et al., 2001; Targoni et al., 2001). Since a relatively high level of PLP multimer+ CD4+ T cells are detected in the CLN despite the lack of IFN-g-producing cells, functional maturity of IFN-g-producing effector CD4+ T cells appears to occur preferentially in the CNS. However, the majority of (N80%) CD4+ T cells in the CNS do not produce IFN-g following stimulation with either PLP peptide or anti-CD3 antibody. It is conceivable that these non-responsive, CNS-infiltrating CD4+ T cells may represent autoreactive T cells, which may be in a state of anergy in the CNS as shown previously (Brabb et al., 2000). Our results utilizing the PLP multimer suggest that PLP139–151-specific T cells accumulate first in the CLN and then CNS (Fig. 2). The presence of low CNS-autoantigen reactive T cells in the periphery of animals with EAE is

consistent with the previous study tracing GFP-tagged MBPspecific cells in rats (Flugel et al., 2001). A higher proportion of PLP139–151 multimer+ cells are present in the TCRlo CD4+ T cell population of the CLN and CNS during both preclinical and clinical stages of EAE, suggesting that these cells are activated (Fig. 3). A similar observation has also been reported with human gliadin specific T cells following in vitro antigenic stimulation (Quarsten et al., 2001). Therefore, staining with peptide multimers may underestimate the numbers of highly activated epitope-specific T cells in the CNS of mice with autoimmune demyelinating disease. The overall CNS-infiltrating CD4+ T cells during clinical disease show various T cell activation markers on the surface, which are consistent with the activated status (Fig. 3). However, the expression of CD45RB is uniquely high on the majority of TCRlo CD4+ T cells and the expression of CD69 and CD25 is low as compared to TCRhi populations. It was previously suggested that CD45RBlo CD4+ T cells may be involved in the pathogenesis of EAE (Jensen et al., 1992; Renno et al., 1994). In addition, treatment of animals with anti-CD45RB antibodies was able to prevent EAE (Schiffenbauer et al., 1998). Taken together, our results suggest that the autoreactive CD4+ T cells accumulate first in the CLN and then in the CNS. In the CNS, these T cells further differentiate into potentially pathogenic effector Th1 cells that are capable of producing IFN-g . It is conceivable that these T cells expand in the CLN after encountering the autoantigen from CNS and get bprimedQ before migrating to the CNS following a gradient of autoantigens from the CNS to CLN (de Vos et al., 2002; Fontana et al., 1996; Phillips et al., 1997). However, many of these activated T cells appear to downregulate their TCRs and yet produce IFN-g, suggesting that autoreactive effector CD4+ T cells in the CNS are highly sensitive to functional stimulation. Further characterization and understanding the nature of these CD4+ T cells in the CNS will ultimately provide important information critical for immune intervention of such diseases.

Acknowledgements This work was supported by grants RO1 NS 28752 and RO1 NS 33008 from USPHS. We thank Drs. Ellis Reinherz and H.-C. Chang (Dana Farber Cancer Institute, Boston, MA) for LZ-containing plasmids, pCRII acid and pCRII base, Dr. Goldstein (Howard Hughes Medical Institute, La Jolla, CA) for insect cell expression vector, pRmHa-3 and Dr. V.K. Kuchroo (Harvard Medical School, Boston, MA) for 5B6 hybridoma.

References Alcover, A., Alarcon, B., 2000. Internalization and intracellular fate of TCR–CD3 complexes. Crit. Rev. Immunol. 20, 325 – 346.

M. Mohindru et al. / Journal of Neuroimmunology 155 (2004) 127–135 Altman, J.D., Moss, P.A., Goulder, P.J., Barouch, D.H., McHeyzerWilliams, M.G., Bell, J.I., McMichael, A.J., Davis, M.M., 1996. Phenotypic analysis of antigen-specific T lymphocytes. Science 274, 94 – 96. Anderson, A.C., Nicholson, L.B., Legge, K.L., Turchin, V., Zaghouani, H., Kuchroo, V.K., 2000. High frequency of autoreactive myelin proteolipid protein-specific T cells in the periphery of naive mice: mechanisms of selection of the self-reactive repertoire. J. Exp. Med. 191, 761 – 770. Brabb, T., von Dassow, P., Ordonez, N., Schnabel, B., Duke, B., Goverman, J., 2000. In situ tolerance within the central nervous system as a mechanism for preventing autoimmunity. J. Exp. Med. 192, 871 – 880. Brennan, F.R., O’Neill, J.K., Allen, S.J., Butter, C., Nuki, G., Baker, D., 1999. CD44 is involved in selective leucocyte extravasation during inflammatory central nervous system disease. Immunology 98, 427 – 435. Cameron, T.O., Norris, P.J., Patel, A., Moulon, C., Rosenberg, E.S., Mellins, E.D., Wedderburn, L.R., Stern, L.J., 2002. Labeling antigenspecific CD4(+) T cells with class II MHC oligomers. J. Immunol. Methods 268, 51 – 69. Crawford, F., Kozono, H., White, J., Marrack, P., Kappler, J., 1998. Detection of antigen-specific T cells with multivalent soluble class II MHC covalent peptide complexes. Immunity 8, 675 – 682. de Vos, A.F., van Meurs, M., Brok, H.P., Boven, L.A., Hintzen, R.Q., van der Valk, P., Ravid, R., Rensing, S., Boon, L., t Hart, B.A., Laman, J.D., 2002. Transfer of central nervous system autoantigens and presentation in secondary lymphoid organs. J. Immunol. 169, 5415 – 5423. DeGrendele, H.C., Estess, P., Siegelman, M.H., 1997. Requirement for CD44 in activated T cell extravasation into an inflammatory site. Science 278, 672 – 675. Di Rosa, F., Serafini, B., Scognamiglio, P., Di Virgilio, A., Finocchi, L., Aloisi, F., Barnaba, V., 2000. Short-lived immunization site inflammation in self-limited active experimental allergic encephalomyelitis. Int. Immunol. 12, 711 – 719. Flugel, A., Berkowicz, T., Ritter, T., Labeur, M., Jenne, D.E., Li, Z., Ellwart, J.W., Willem, M., Lassmann, H., Wekerle, H., 2001. Migratory activity and functional changes of green fluorescent effector cells before and during experimental autoimmune encephalomyelitis. Immunity 14, 547 – 560. Fontana, A., Constam, D., Frei, K., Koedel, U., Pfister, W., Weller, M., 1996. Cytokines and defense against CNS infection. In: Ransohoff, R.M., Benveniste, E.N. (Eds.), Cytokines and the CNS. CRC Press, Boca Raton, FL, USA, pp. 188 – 219. Hackett, C.J., Sharma, O.K., 2002. Frontiers in peptide–MHC class II multimer technology. Nat. Immunol. 3, 887 – 889. Homann, D., Teyton, L., Oldstone, M.B., 2001. Differential regulation of antiviral T-cell immunity results in stable CD8+ but declining CD4+ Tcell memory. Nat. Med. 7, 913 – 919. Horton, R.M., Cai, Z.L., Ho, S.N., Pease, L.R., 1990. Gene splicing by overlap extension: tailor-made genes using the polymerase chain reaction. BioTechniques 8, 528 – 535. Jensen, M.A., Arnason, B.G., Toscas, A., Noronha, A., 1992. Preferential increase of IL-2R+ CD4+ T cells and CD45RB CD4+ T cells in the central nervous system in experimental allergic encephalomyelitis. J. Neuroimmunol. 38, 255 – 261. Kang, B.S., Lyman, M.A., Kim, B.S., 2002. The majority of infiltrating CD8+ T cells in the central nervous system of susceptible SJL/J mice infected with Theiler’s virus are virus specific and fully functional. J. Virol. 76, 6577 – 6585. Kim, B.S., Bahk, Y.Y., Kang, H.K., Yauch, R.L., Kang, J.A., Park, M.J., Ponzio, N.M., 1999. Diverse fine specificity and receptor repertoire of T cells reactive to the major VP1 epitope (VP1230-250) of Theiler’s virus:

135

V beta restriction correlates with T cell recognition of the c-terminal residue. J. Immunol. 162, 7049 – 7057. Klein, L., Klugmann, M., Nave, K.A., Tuohy, V.K., Kyewski, B., 2000. Shaping of the autoreactive T-cell repertoire by a splice variant of self protein expressed in thymic epithelial cells. Nat. Med. 6, 56 – 61. Kwok, W.W., 2003. Challenges in staining T cells using HLA class II tetramers. Clin. Immunol. 106, 23 – 28. Kwok, W.W., Liu, A.W., Novak, E.J., Gebe, J.A., Ettinger, R.A., Nepom, G.T., Reymond, S.N., Koelle, D.M., 2000. HLA-DQ tetramers identify epitope-specific T cells in peripheral blood of herpes simplex virus type 2-infected individuals: direct detection of immunodominant antigenresponsive cells. J. Immunol. 164, 4244 – 4249. Lider, O., Beraud, E., Reshef, T., Friedman, A., Cohen, I.R., 1989. Vaccination against experimental autoimmune encephalomyelitis using a subencephalitogenic dose of autoimmune effector T cells: 2. Induction of a protective anti-idiotypic response. J. Autoimmun. 2, 87 – 99. Mallet-Designe, V.I., Stratmann, T., Homann, D., Carbone, F., Oldstone, M.B., Teyton, L., 2003. Detection of low-avidity CD4+ T cells using recombinant artificial APC: following the antiovalbumin immune response. J. Immunol. 170, 123 – 131. Martin, R., McFarland, H.F., McFarlin, D.E., 1992. Immunological aspects of demyelinating diseases. Annu. Rev. Immunol. 10, 153 – 187. McRae, B.L., Kennedy, M.K., Tan, L.J., Dal Canto, M.C., Picha, K.S., Miller, S.D., 1992. Induction of active and adoptive relapsing experimental autoimmune encephalomyelitis (EAE) using an encephalitogenic epitope of proteolipid protein. J. Neuroimmunol. 38, 229 – 240. Novak, E.J., Liu, A.W., Nepom, G.T., Kwok, W.W., 1999. MHC class II tetramers identify peptide-specific human CD4(+) T cells proliferating in response to influenza A antigen. J. Clin. Invest. 104, R63 – R67. Phillips, M.J., Needham, M., Weller, R.O., 1997. Role of cervical lymph nodes in autoimmune encephalomyelitis in the Lewis rat. J. Pathol. 182, 457 – 464. Pu, Z., Carrero, J.A., Unanue, E.R., 2002. Distinct recognition by two subsets of T cells of an MHC class II–peptide complex. Proc. Natl. Acad. Sci. U. S. A. 99, 8844 – 8849. Quarsten, H., McAdam, S.N., Jensen, T., Arentz-Hansen, H., Molberg, O., Lundin, K.E., Sollid, L.M., 2001. Staining of celiac disease-relevant T cells by peptide–DQ2 multimers. J. Immunol. 167, 4861 – 4868. Radu, C.G., Anderton, S.M., Firan, M., Wraith, D.C., Ward, E.S., 2000. Detection of autoreactive T cells in H-2u mice using peptide–MHC multimers. Int. Immunol. 12, 1553 – 1560. Reddy, J., Bettelli, E., Nicholson, L., Waldner, H., Jang, M.H., Wucherpfennig, K.W., Kuchroo, V.K., 2003. Detection of autoreactive myelin proteolipid protein 139–151-specific T cells by using MHC II (IAs) tetramers. J. Immunol. 170, 870 – 877. Renno, T., Zeine, R., Girard, J.M., Gillani, S., Dodelet, V., Owens, T., 1994. Selective enrichment of Th1 CD45RBlow CD4+ T cells in autoimmune infiltrates in experimental allergic encephalomyelitis. Int. Immunol. 6, 347 – 354. Schiffenbauer, J., Butfiloski, E., Hanley, G., Sobel, E.S., Streit, W.J., Lazarovits, A., 1998. Prevention of experimental allergic encephalomyelitis by an antibody to CD45RB. Cell. Immunol. 190, 173 – 182. Smeltz, R.B., Swanborg, R.H., 1998. Concordance and contradiction concerning cytokines and chemokines in experimental demyelinating disease. J. Neurosci. Res. 51, 147 – 153. Targoni, O.S., Baus, J., Hofstetter, H.H., Hesse, M.D., Karulin, A.Y., Boehm, B.O., Forsthuber, T.G., Lehmann, P.V., 2001. Frequencies of neuroantigen-specific T cells in the central nervous system versus the immune periphery during the course of experimental allergic encephalomyelitis. J. Immunol. 166, 4757 – 4764.