Memory cells specific for myelin oligodendrocyte glycoprotein (MOG) govern the transfer of experimental autoimmune encephalomyelitis

Journal of Neuroimmunology 234 (2011) 84–92

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Journal of Neuroimmunology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j n e u r o i m

Memory cells specific for myelin oligodendrocyte glycoprotein (MOG) govern the transfer of experimental autoimmune encephalomyelitis☆ Jessica L. Williams, Aaron P. Kithcart, Kristen M. Smith, Todd Shawler, Gina M. Cox, Caroline C. Whitacre ⁎ The Ohio State University, Department of Molecular Virology, Immunology, and Medical Genetics, 760 Biomedical Research Tower, 460 W 12th Avenue, Columbus, OH 43210, United States

a r t i c l e

i n f o

Article history: Received 29 September 2010 Received in revised form 3 January 2011 Accepted 9 February 2011 Keywords: Experimental autoimmune encephalomyelitis Multiple sclerosis Myelin oligodendrocyte glycoprotein Adoptive transfer Memory T cell

a b s t r a c t Multiple sclerosis (MS) is an inflammatory disease of the CNS mediated by CD4+ T cells directed against myelin antigens. Experimental autoimmune encephalomyelitis (EAE) is induced by immunization with myelin antigens like myelin oligodendrocyte glycoprotein (MOG). We have explored the transfer of EAE using MOG35-55-specific TCR transgenic (2D2) T cells. Unsorted 2D2 Th1 cells reliably transferred EAE. Further, we found that CD44hiCD62Llo effector/memory CD4+ T cells are likely responsible for the disease transfer due to the up-regulation of CD44. Given the importance of MOG in MS pathogenesis, mechanistic insights into adoptively transferred EAE by MOG-specific Th1 cells could prove valuable in MS research. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Myelin oligodendrocyte glycoprotein (MOG), located on the outermost lamellae of the myelin sheath and on the surface of oligodendrocytes, is highly conserved among species (Johns and Bernard, 1999). Although MOG is a quantitatively minor component of CNS myelin, there is strong evidence that MOG serves as an autoantigen and plays an important role in the pathogenesis of multiple sclerosis (MS) (Sun et al., 1991; Kerlero de Rosbo et al., 1997; Egg et al., 2001). T cells from MS patients readily proliferate and secrete IFN-γ in response to MOG peptides relative to other myelin antigens such as myelin basic protein (Kerlero de Rosbo et al., 1997). In addition, anti-MOG IgG-secreting B cells are more abundant in both the peripheral blood and cerebrospinal fluid of MS patients compared to controls (Sun et al., 1991), suggesting a role for MOG in the pathogenesis of MS. Experimental autoimmune encephalomyelitis (EAE) is the most frequently studied animal model of multiple sclerosis (MS) and can be credited for the development of several currently approved therapies (Zamvil and Steinman, 2003). EAE shares many clinical and histopathological features with MS and is mediated by myelinAbbreviations: MOG, myelin oligodendrocyte glycoprotein; MS, multiple sclerosis; EAE, experimental autoimmune encephalomyelitis; 2D2, MOG35-55-specific TCR transgenic mice. ☆ These studies were supported by National Institutes of Health Grant AI43376 and National Multiple Sclerosis Society Grant RG 3272. ⁎ Corresponding author at: Department of Molecular Virology, Immunology, and Medical Genetics, The Ohio State University, 208 Bricker Hall, 190 N Oval Mall, Columbus, OH 43210, United States. Tel.: + 1 614 247 8356; fax: + 1 614 292 6602. E-mail address: [email protected] (C.C. Whitacre). 0165-5728/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2011.02.008

reactive CD4+ T cells. EAE is induced in genetically susceptible mouse strains by immunization with myelin antigens including myelin basic protein (Zamvil et al., 1986), proteolipid protein (Tuohy et al., 1989), and MOG (Mendel et al., 1995). MOG35-55 is the major immunodominant epitope of MOG and elicits neurological impairment following immunization with MOG35-55 and adjuvants (Mendel et al., 1995). Although the active induction of EAE is an important tool in MS research, it introduces many confounding factors such as adjuvants, which are not present in the human disease. To circumvent the drawbacks of active immunization, models of passive immunization have been developed and used for decades (Pettinelli and McFarlin, 1981). In adoptively transferred EAE, lymphoid cells are harvested from the myelin antigen immunized mice, stimulated ex vivo and transferred, without the use of adjuvants, to the recipient mice. In 2003, a MOG35-55-specific TCR transgenic (2D2) mouse was reported in which transgenic T cells were not deleted or tolerized, and were found to be fully competent. The unique feature of this mouse strain was that more than 30% of mice spontaneously developed optic neuritis without clinical or histological evidence of EAE. Only 4% of 2D2 mice were reported to develop spontaneous EAE (Bettelli et al., 2003). Since 2003, substantial difficulties have been encountered in achieving successful transfer of EAE using 2D2 donor cells. In 2009, Jäger et al. reported that naïve (CD4+CD62L+) 2D2 T cells, following primary differentiation in Th1 or Th17 culture conditions, were unable to transfer disease reliably. However, when those cells were restimulated, both Th1 and Th17 cells were able to transfer EAE indicating that secondary T cell stimulation is a critical event in establishing encephalitogenicity (Jäger et al., 2009). Given the importance of MOG in EAE and MS, using 2D2 T cells to induce EAE could provide insight into the development and pathogenesis of MS.

J.L. Williams et al. / Journal of Neuroimmunology 234 (2011) 84–92

The progression of MS and EAE has been attributed to several properties of T cells. Both IFN-γ-producing Th1 and IL-17-secreting Th17 cells are thought to contribute to the advancement of CNS pathology in EAE and MS (Stromnes et al., 2008). More recently, the Th1 transcription factor, T-bet has been shown to be essential for the encephalitogenicity of T cells (Yang et al., 2009). T-bet expression is restricted to Th1 cells and controls the induction of IFN-γ, repressing IL4, creating a Th1-biased environment for the perpetuation of an inflammatory response (Szabo et al., 2000). Myelin-reactive T cells from MS patients more readily proliferate in response to antigen in the absence of costimulation compared to healthy controls, suggesting that T cells from MS patients have previously been activated and have a memory phenotype (Lovett-Racke et al., 1998). In addition to accelerated activation, memory cells are able to more readily extravasate across endothelial barriers as they express high amounts of surface CD44 (DeGrendele et al., 1996). This suggests that memory cells are well poised to access the CNS parenchyma and initiate inflammation. We examined the adoptive transfer of EAE using Th1- and Th17differentiated 2D2 cells. We demonstrate that IFN-γ-producing, T-bet+ Th1 2D2 cells are capable of inducing EAE. Further, we show that memory T cells are necessary, and likely responsible, for the transfer of disease following Th1 differentiation. While our results are consistent with those of Jäger et al. (2009), we provide here a more direct method of transferring EAE using 2D2 Th1 cells. Additionally, we observed that Th1-differentiated 2D2 cells from male mice transfer more severe disease into sex-matched WT recipients than their female counterparts, corresponding to a difference in the number of memory cells between the sexes. 2. Materials and methods 2.1. Mice C57Bl/6 mice were purchased from The Jackson Laboratory. 2D2 mice were a kind gift from Dr. Vijay Kuchroo (Center for Neurologic Disease, Brigham and Women's Hospital, Harvard Medical School, Boston, MA). Mice were housed in a specific pathogen-free animal facility at The Ohio State University on a 12-h light/dark cycle and given food and water ad libitum. All 2D2 mice were screened using flow cytometric analysis of PBMCs using specific antibodies to Vα3.2 and Vβ11 (BD Biosciences). Mice used in experiments were 6–9 weeks of age at the initiation of the studies. All animal procedures were performed in accordance with approved university protocols.

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moribund or death. Brains and spinal cords were removed 14 days post immunization and 18 days following adoptive transfer, fixed, and processed for H&E staining. Stained tissue sections were examined for mononuclear cell infiltration. 2.3. Differentiation and transfer of naïve and memory 2D2 T cells Spleens from 6–10 week-old male 2D2 mice were harvested and dissociated into a single cell suspension. After erythrocyte lysis, CD4+CD62L+ T cells were isolated using the CD4+CD62L+ T Cell Isolation Kit II for mouse (Miltenyi Biotec) according to manufacturer's instructions. Briefly, for the depletion of non-CD4+ cells, biotin-conjugated antibodies and anti-biotin conjugated microbeads were used in conjunction with a magnetic column. Positive selection of naïve CD4+CD62L+ cells was achieved using microbeads conjugated to monoclonal anti-mouse CD62L Ab. To isolate memory T cells, the MagCellect Mouse Memory CD4+ T Cell Isolation Kit (R&D Systems) was used according to manufacturer's instructions. CD4+ populations that were N95% CD62L+ and N90% CD44+ were used for naïve and memory T cell cultures, respectively. Isolated naïve and memory T cells were cultured with WT APCs in 24-well plates in the presence of 20 μg/ml MOG35-55 and 0.5 ng/ml IL-12. Following a 48-h incubation, 5 × 106 purified naïve and unsorted cells were washed in PBS and injected i.p. into WT recipients. 2.4. Intracellular staining and flow cytometry Flow cytometric analysis was performed to evaluate intracellular cytokine and T-bet expression. Following culture, cells were suspended in medium containing BD Golgi Plug (brefeldin A) (BD Pharmingen) for 3.5 h. Cell surfaces were then stained with PerCP-conjugated anti-CD4 mAb. After cells were fixed and permeabilized (Cytofix/Cytoperm Kit; BD Biosciences), cells were stained with PE-conjugated anti-IL-17, allophycocyanin (APC)-conjugated anti-IFN-γ (BD Biosciences), and FITC-conjugated anti-T-bet (Santa Cruz) mAb. All samples were analyzed by flow cytometry (FACSCanto II; BD Biosciences). 2.5. Analysis of secreted cytokines by ELISA Supernatants were collected following cell culture. Supernatant IFNγ and IL-17 concentrations were determined using the BD OptEIA mouse IFN-γ ELISA set (BD Biosciences) and the DuoSet ELISA kit for mouse IL-17 (R&D Systems) according to manufacturer's instructions.

2.2. Induction and assessment of EAE 2.6. Statistical analysis C57Bl/6 and 2D2 mice were immunized s.c. with 100 μl of an emulsion containing 200 μg of MOG35-55 (Princeton Biomolecules) and CFA, containing 200 μg of heat-killed Mycobacterium tuberculosis Jamaica strain, over the 4 flanks. Pertussis toxin (250 ng) (List Biological Laboratories) in 0.2 ml PBS was given i.p. on the day of immunization and 48 h later. EAE was also induced via adoptive transfer. Spleens from 6–8 weekold 2D2 mice were dissociated into single cell suspensions and cultured in RPMI 1640 containing 10% FBS, 25 mM HEPES, 2 mM L-glutamine, 50 U/ml penicillin, and 50 μg/ml streptomycin. Cells were activated with 20 μg/ml MOG35-55 unless otherwise specified and a combination of cytokines and neutralizing antibodies for the development of Th1 or Th17 cells. Cytokine and antibody concentrations were as follows: 0.5 ng/ml IL-12, 25 ng/ml IL-6, 5 ng/ml TGF-β, 2 μg/ml anti-IFN-γ, 1 μg/ ml anti-IL-4, and 0.65 μg/ml anti-IL-12 unless otherwise indicated. After 48 h, cells were washed in PBS and injected i.p. into naïve, sex-matched C57Bl/6 mice. Mice were monitored daily for clinical signs of disease and were scored as follows: 0, no signs; 1, limp tail; 2, limp tail and ataxia; 3, paralysis of one hind limb; 4, complete hind limb paralysis; and 5,

A two-tailed student's t test was used to determine statistical differences when comparing cumulative disease index, day of onset, and maximum clinical score between groups. The Mann–Whitney U test was performed to determine differences when assessing clinical EAE using GraphPad Prism software (GraphPad). 3. Results 3.1. 2D2 T cells are able to infiltrate the CNS and induce severe EAE 2D2 transgenic mice, with an over-representation of T cells specific for MOG35-55, were first described by Bettelli et al. (2003). A high proportion of these mice demonstrated optic neuritis, but only 4% of 2D2 mice were reported to develop spontaneous EAE (Bettelli et al., 2003). To examine the ability of 2D2 cells to initiate EAE, WT and 2D2 mice were immunized with MOG35-55 and adjuvants. Following immunization, 2D2 mice developed more severe clinical signs of EAE and earlier onset compared to WT controls, resulting in 60% mortality (Fig. 1A, Table 1). To assess the ability of 2D2 cells to migrate to the CNS, we

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for the generation of Th1- and Th17-differentiated 2D2 cells to establish a reliable adoptive transfer system. Several strategies for generating an encephalitogenic 2D2 T cell population were attempted including culture with antigen, antigen with Th1-inducing cytokines, antigen with Th17-inducing cytokines in several different combinations as well as anti-CD3/anti-CD28 stimulation. Moreover, variation in culture length, concentration of antigen, and number of cells transferred as well as inhibition of specific cytokine action and dilution of T cells with irradiated WT splenocytes were carried out (Table 2). Despite significant T cell proliferation (data not shown) and expression and secretion of IFN-γ and IL-17, 2D2 cells were unable to transfer EAE to WT recipients in many circumstances (Table 2). Successful transfer was achieved by culturing unsorted splenocytes with MOG35-55 and IL-12 for 48 h, and transferring 107 cells either i.v. or i.p. to unmanipulated recipients (Table 2). 3.3. Following initial stimulation, Th1-differentiated 2D2 cells exhibit an encephalitogenic phenotype

Fig. 1. 2D2 mice exhibit early onset EAE following immunization with MOG35–55 compared to WT mice. A, 2D2 and WT mice were immunized with MOG35–55 in CFA, given pertussis toxin, and monitored for clinical signs of EAE. B and D, Brains and; C and E, spinal cords were harvested 14 days following immunization and stained with H&E. Arrows designate focal areas of mononuclear cell infiltration (WT n = 3; 2D2 n = 5).

analyzed histological sections of the brain and spinal cord 14 days post immunization. Significant perivascular cuffing and parenchymal mononuclear cell infiltration were evident in the cerebellum and spinal cords of immunized 2D2 mice (Fig. 1D and E). These data indicate that 2D2 T cells are primed in the periphery and are able to traffic to the CNS and initiate tissue damage that results in clinically severe EAE.

3.2. Specific culture conditions are required for reliable transfer of EAE by 2D2 T cells Both Th1 and Th17 cells are implicated in the pathogenesis of MS and EAE (Langrish et al., 2005; Kebir et al., 2007). While IFN-γ-producing Th1 cells have been defined as the primary mediators of disease, Th17 cells are critical mediators of inflammation and cell recruitment to the CNS, contributing to lesion formation (Stromnes et al., 2008; Bielekova et al., 2000). Therefore, we investigated a variety of culture conditions

Table 1 2D2 mice are susceptible to and develop severe, early onset EAE via active immunizationa. Incidence WT 2D2

3/3 (100%) 5/5 (100%)

Mortality 0/3 (0%) 3/5 (60%)

Day of onset ± SEM c

11.0 ± 0.6 8.2 ± 0.7

Yang et al. (2009) described several culture conditions for promoting Th1 and Th17 cells. A T-bet-dependent pathway was found to be responsible for the generation of an encephalitogenic T cell population able to adoptively transfer EAE. We first sought to characterize the pre-culture population of CD4+ T cells from 2D2 mice, defining the CD44lo/CD62Lhi naïve cells, CD44hi/CD62Llo effector memory cells, and CD44hi/CD62Lhi central memory cells (Fig. 2A). To activate T cells prior to transfer, splenocytes from male 2D2 mice were cultured in the presence of Th1 or Th17 culture conditions. In Th1 cultures, MOG and IL-12 were added; and in Th17 cultures, MOG, IL-6, anti-IFN-γ, anti-IL-4, and anti-IL-12 were added for 48 h (Table 2, conditions 7 and 8, respectively). Following Th1 differentiation, 2D2 CD4+ T cells expressed and secreted a substantial amount of IFN-γ, a low level of IL-17, and were T-bet+ (Fig. 2B–D). Conversely, cells driven towards a Th17 phenotype expressed and secreted only IL-17, and did not express T-bet (Fig. 2B, C and D). These results suggest that Th1-driven 2D2 cells are an encephalitogenic T cell population able to transfer EAE and induce CNS inflammation. 3.4. Th1-differentiated 2D2 cells transfer EAE and infiltrate the CNS To determine the pathogenic capacity of Th1- and Th17-differentiated 2D2 cells, we injected the activated cells described in Fig. 2 i.p. into naïve, syngeneic WT recipients without the administration of additional adjuvants. Recipients were then observed for clinical signs of EAE. While Th17 cell recipients failed to develop appreciable signs of EAE, Th1 cell recipients exhibited signs of disease beginning 9 days following transfer (Fig. 3A and Table 3). Th1 and Th17 cells have been shown to traffic to varying areas of the CNS depending on culture condition during EAE (Stromnes et al., 2008). To establish the pattern of infiltration of activated 2D2 cells within the CNS, brains and spinal cords were taken 18 days post transfer and stained using H&E. In the cerebellum of Th1 but not Th17 recipients, significant perivascular cuffing is evident (Fig. 3B and E). In addition, parenchymal mononuclear cell infiltrates were found within the cerebellum of mice that received Th17 but not Th1 cells (data not shown). As expected, in the white matter of the spinal cord of Th1 cell recipients, heavy cell infiltration is apparent (Fig. 3 C–D). Thus, following initial stimulation, Th1-differentiated, T-bet+ 2D2 cells are able to infiltrate the CNS and initiate damage resulting in clinical EAE.

CDIb ± SEM 53.8 ± 2.9c 97.0 ± 17.5

a Wt and 2D2 mice were immunized with MOG35–55 + CFA and pertussis toxin and monitored daily for signs of EAE. b Cumulative disease index (mean sum of clinical scores). c p b 0.05 by Student's t test.

3.5. Memory 2D2 T cells generated in vivo transfer EAE Jäger et al. (2009) describe an adoptive transfer model using purified naive CD4+CD62L+ 2D2 T cells differentiated into effector Th1 cells. Following the primary stimulation of naïve cells, the recipients of Th1 cells did not develop EAE; however, following secondary

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Table 2 A variety of culture conditions do not reliably transfer EAE using 2D2 cells. Culture conditionsa

Length of IFN-γ+b(%) culture

IL-17+b(%) [IFN-γ] (pg/ml)

[IL-17] (pg/ml) Cells transferred

1 MOG

72 h

NAf

NA

3965.0

NA

2 3 4 5 6

72 h 72 h 1h 72 h 72 h

NA NA NA 6.1 33.6

NA NA NA 18.2 3.7

4063.3 NA NA 402.4 NDf

NA NA NA 1015.3 133.1

48 h 48 h 72 h

20.3 ± 2.9e 0.5 ± 0.2e 19.0

1.3 ± 0.3e 2.7 ± 1.3e 1.5

161921 ± 95823e ND NA

163.5 ± 19.6e 318.8 ± 59.9e NA

MOG, IL-12 (1.0 ng/ml) 1:5 cell dilutionc, MOG, IL-12 MOG MOG (60 μg/ml), IL-6 (100 ng/ml), IL-23 (10 ng/ml), TGF-β Anti-CD3d (5 μg/ml), Anti-CD28 (5 μg/ml), IL-6 (10 ng/ml), TGF-β, Anti-mIFN-γ (10 μg/ml), Anti-mIL-4 (10 μg/ml), Anti-mIL-2 (10 μg/ml) 7 MOG (20 μg/ml), IL-12 (0.5 ng/ml) 8 MOG (10 μg/ml), IL-6, Anti-IFN-γ, Anti-IL-4, Anti-IL-12 9 MOG (2 μg/ml), IL-12, 1:3 cell dilutionc

a b c d e f

10, 20, 30, 40 × 106, i. v. 10, 20, 30 × 106, i.v. 5, 10, 20 × 106, i.v. 5, 10 × 106, i.v. 30 × 106, i.v. 10 × 106, i.v.

EAE induction − − − − – −

10 × 106, i.v., i.p. +++ 10, 20 × 106, i.p. + 6 5 × 10 , i.p. (adapted − from Yang et al. (2009)

Unsorted splenocytes (lysed RBC) were used for all culture conditions. Cells positive for intracellular cytokines were gated on CD4+ cells. 2D2 cells were diluted with irradiated WT splenocytes. Anti-CD3 was bound to culture flask. Values are representative of three independent experiments (± SEM). NA denotes values that were not done, ND denotes values that were not detectable.

stimulation, Th1 cells were able to transfer disease (Jäger et al., 2009). We found that unsorted splenocytes, following an initial 48-h culture under Th1 differentiating conditions, are able to transfer EAE, suggesting that an inherent memory T cell population in 2D2 mice is responsible for

the transfer of EAE as opposed to a naïve T cell population. Thus, we assessed the ability of purified naïve (CD4+CD62L+) T cells to transfer EAE following a primary stimulation in our Th1-differentiating conditions.

Fig. 2. Following differentiating culture conditions, 2D2 cells exhibit Th1 and Th17 phenotypes. A, CD4+ splenocytes from male 2D2 mice were analyzed by flow cytometry for the presence of CD62L and CD44 prior to culture. Cells were then incubated in the presence of 20 μg/ml MOG35–55 and 0.5 ng/ml rIL-12 or in Th17 differentiating conditions with 10 μg/ml MOG35–55, 25 ng/ml IL-6, 2 μg/ml anti-IFN-γ, 1 μg/ml anti-IL-4, and 0.65 μg/ml anti-IL-12. Following a 48 h incubation, B, Th1 and Th17 cells were analyzed for the expression of IL-17 and IFN-γ and C, T-bet; cell populations were gated on CD4+ cells. D, Supernatants were also analyzed for the presence of secreted IFN-γ and IL-17; error bars represent SEM. Data are representative of 3 independent experiments.

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Fig. 3. Th1- but not Th17-differentiated 2D2 cells initiate CNS inflammation and cause EAE following initial stimulation. Male 2D2 splenocytes were cultured in Th1- or Th17differentiating conditions. After 48 h, 10 × 106 were injected i.p. into naïve male WT recipients. A, Th1 and Th17 cell recipients were monitored for the development of EAE; error bars represent SEM (⁎⁎⁎p b 0.0001). Brain and spinal cords were taken from transfer recipients 18 days post transfer and stained with H&E. B and E, Brain sections were visualized at 10× and spinal cord sections were visualized at 10× (for panels C and F), and 20× (for panels D and G). Data represent 3 combined experiments (Th1 cell recipients n = 19; Th17 cell recipients n = 11).

Prior to culture, 77% of unsorted 2D2 CD4+ cells were also CD62L+ and 9.4% were CD62L−, while 96% of 2D2 CD4+ cells were CD62L+ following the enrichment of naïve cells (data not shown). Following a 48 h-incubation in the presence of MOG35-55 and IL-12, CD4+ T cells from both unsorted and naïve 2D2 cultures exhibited a Th1 phenotype expressing a significant amount of IFN-γ and T-bet (Fig. 4A–B). Cells were washed and 5 × 106 were injected i.p. into naïve WT recipients. Although both unsorted and naïve T cells had a strong Th1 phenotype when transferred, only unsorted cultures that contained memory CD4+ T cells, were able to induce EAE in recipient mice (Fig. 4C), indicating that the memory cell population is critical for disease transfer. Similarly, when purified CD4+CD44+CD62L− memory T cells were cultured in Th1-driving conditions, they express intracellular IFN-γ and T-bet similar to that of the unsorted cells (Fig. 4D). However, following

Table 3 Th1-differentiated 2D2 cells transfer EAE following initial stimulation. Incidence Th1 Th17 a b

15/19 (79%) 1/11 (9%)

Day of onset ± SEM 11.9 ± 0.5 19.0 ± 0

Mean peak score ± SEM b

2.2 ± 0.3 0.1 ± 0.1

Cumulative disease index (mean sum of clinical scores). p b 0.05 by Student's t test.

CDIa ± SEM 15.2 ± 2.6b 0.2 ± 0.2

differentiation, memory 2D2 T cells have greater expression of the adhesion molecule, CD44 (Fig. 4E). On average, 4% of the unsorted splenocytes in female 2D2 mice were effector memory T cells; thus, following a Th1 differentiation, 0.4× 106 purified memory T cells were washed in PBS and injected i.p. into female WT recipients. The transfer of that number of differentiated memory T cells did not result in the transfer of EAE. Taken together, these data suggest that CD44 expression on CD4+ T cells is important for the adoptive transfer of EAE using 2D2 T cells and highlights the CD44hiCD62Llo T cell population.

3.6. Male 2D2 T cells transfer EAE with greater proficiency than female 2D2 T cells In the course of our experiments, we observed that male 2D2 mice had an expanded CD44hiCD62Llo memory/effector CD4+ T cell population compared to female 2D2 mice (Fig. 5A–C). We hypothesized that because the memory cell population is greater in male 2D2 mice, unsorted Th1-differentiated male 2D2 splenocytes would transfer EAE to a greater extent relative to female 2D2 Th1 cells. To compare the ability of male versus female 2D2 T cells to transfer disease, we cultured the unsorted splenocytes in the presence of MOG35-55 and IL-12 for 48 h. Following culture, 10 × 106 cells were

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Fig. 4. Intrinsic memory T cells are required for Th1-differentiated 2D2 cells to transfer EAE following initial stimulation. Unsorted 2D2 T cells, purified naïve (CD4+CD62L+) cells, and purified memory (CD4+CD44+) cells were cultured with WT APCs in 24-well plates in the presence of 20 μg/ml MOG35–55 and 0.5 ng/ml IL-12 for 48 h. A, CD4+ cells were then analyzed for the expression of intracellular IL-17, IFN-γ, and B, T-bet. C, Unsorted and naïve T cells (5 × 106) were injected i.p. into WT recipients and monitored for the development of EAE (⁎p b 0.05) (unsorted T cell recipients n = 6; naïve T cell recipients n = 5). D, Unsorted and purified memory cells were analyzed for the expression of intracellular IFN-γ and Tbet and E, cell surface-associated CD44 following culture.

injected i.p. into sex-matched WT recipients. As expected, female 2D2 Th1 cells transferred EAE to a significantly lesser extent than male 2D2 T cells despite robust intracellular IFN-γ (41%) and T-bet (62%) expression in CD4+ T cells following culture (Fig. 5D, Table 4). To further demonstrate the importance of memory cells in the transfer of EAE, we normalized the number of CD44+CD62L− memory 2D2 Th1 cells transferred to male and female WT recipients. When the absolute number of effector memory 2D2 splenocytes transferred was the same between males and females, the effect of gender on disease course was abrogated (Fig. 5E). This further suggests that the expansion of the effector/memory T cell population upon initial stimulation accounts for the ability of unsorted 2D2 Th1 cells to transfer EAE to syngeneic WT recipients. 4. Discussion Multiple sclerosis is a complex neuroimmune disorder perpetuated by the targeting of myelin antigens by self-reactive CD4+ T cells. Although myelin reactive T cells have been found in healthy individuals and MS patients, those found in MS patients have an activated, memory phenotype (Lovett-Racke et al., 1998). This suggests that effector/memory

T cells may be a fundamental component in MS pathogenesis. MOG is a prominent neuroantigen in MS pathogenesis and by using a MOG35-55specific TCR transgenic mouse (2D2) with an inherent memory T cell population, the effects of these cells on disease in an experimental model can be explored. Our results demonstrate a role for 2D2 Th1-differentiated effector/ memory CD44hiCD62Llo T cells in the initiation of EAE. We have shown that purified naïve T cells are unable to transfer EAE following an initial stimulation (Fig. 4C). Further, we have demonstrated that male 2D2 mice have a larger CD44hiCD62Llo memory population relative to female 2D2 mice and thus transfer EAE with greater proficiency following similar culture conditions (Fig. 5A–D). Importantly, this sex difference was abrogated when the absolute number of CD44hiCD62Llo memory T cells transferred to sex-matched recipients was normalized (Fig. 5E). Our studies have demonstrated a requirement for a population of previously primed memory 2D2 T cells for EAE transfer regardless of IFN-γ or T-bet expression (Fig. 4) suggesting education of the T cell, and not cytokine expression, is a critical factor in determining encephalitogenicity. The cultures of 2D2 cells in this study included memory T cells; therefore, we were able to transfer EAE following a single stimulation.

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Fig. 5. Male 2D2 mice have a greater number of effector/memory T cells resulting in enhanced EAE following adoptive transfer. Unsorted splenocytes from male and female 2D2 mice were cultured in the presence of 20 μg/ml MOG35–55 and 0.5 ng/ml IL-12 for 48 h. A, Male and B, female 2D2 CD4+ splenocytes were analyzed for effector/memory (CD44hiCD62Llo), central memory (CD44hiCD62Lhi), and naïve (CD44loCD62Lhi) cell populations. C, Central and effector memory populations from male and female 2D2 mice were quantified (⁎p b 0.05). Plots shown are representative of data collected from 4 2D2 males and 4 2D2 females, from which the quantification in panel C was derived. Cells were washed following culture and 10 × 106 cells were injected i.p. into naïve WT sex-matched recipients. D, Transfer recipients were monitored for the development of EAE. Data represent 3 separate experiments (⁎⁎p = 0.01) (female recipients n = 15; male recipients n = 9). E, Unsorted male and female splenocytes were normalized for the absolute number of memory T cells such that the number of memory cells transferred was the same between males and females. Following culture, 8–10 × 106 2D2 cells were injected i.p. into naïve WT sex-matched recipients and monitored for EAE (female recipients n = 5; male recipients n = 4).

In a report by Jäger et al. (2009), effector memory T cells were eliminated from the culture conditions (through the selection for CD4 + CD62L+ cells) and thus EAE was not transferred following an initial stimulation. Only after a second stimulation, which was necessary to convert naïve 2D2 T cells to effector memory cells, was the transfer of EAE possible (Jäger et al., 2009). Taken together, these data further confirm that the encephalitogenic potential of a T cell is dependent on its education and activation state. Several factors are thought to be responsible for the encephalitogenic properties of T cells in EAE and MS. Increased frequency of myelin-specific Th1 cells expressing IFN-γ has been correlated with exacerbations in MS suggesting a vital role for Th1 cells in CNS inflammation (Ferber et al., 1996). Although myelin-specific CD4+ Th1 cells alone are sufficient to induce EAE, IFN-γ-deficient mice develop severe EAE (Lock et al., 2002). Similarly, the transfer of myelin-specific IL-17-secreting Th17 cells results in EAE; and, microarray analysis demonstrates the presence of IL-17 within MS lesions (Haak et al., 2009). However, the loss of both IL-17A and IL-17F had little impact on EAE development (van Oosten et al., 1997). The

Table 4 Male Th1-differentiated 2D2 cells transfer more severe EAE. Incidence Male Female a b

8/9 (89%) 7/15 (47%)

Day of onset ± SEM b

11.3 ± 0.6 13.2 ± 0.3

Mean peak score ± SEM b

2.1 ± 0.4 0.8 ± 0.3

Cumulative disease index (mean sum of clinical scores). p b 0.05 by Student's t test.

CDIa ± SEM 9.7 ± 2.2b 2.7 ± 0.9

perpetuation and even initiation of EAE and MS have long been thought to be the product of CD4+ T cell targeting of selfneuroantigens. In a 1997 randomized, double-blind, phase II clinical trial, relapsing–remitting and secondary progressive MS patients were treated with a monoclonal anti-CD4 depleting antibody (cMT412). Surprisingly, following a marked reduction in the total number of circulating CD4+ cells, there were no significant changes in the primary outcome measure, cumulative number of active gadolinium enhancing lesions over the 9-month treatment period, when CD4-depleted MS patients were compared to those that were placebotreated (Rep et al., 1997). In a follow-up study, it was observed that naïve, CD45RApos/ROneg T cells were more sensitive to mAbmediated depletion than primed, CD45Rneg/ROpos T cells. Further, it was found that the previously primed (memory), IFN-γproducing Th1-type cell population remained relatively stable following CD4 depletion. Thus, the authors concluded that the lack of effect of the CD4 depletion on MS disease progression was a consequence of the depletion-resistant, previously primed Th1-type, IFN-γ-producing cells that remained in the circulation following treatment (Rep et al., 1997). These data highlight the importance of CD4+ effector/memory populations in MS pathogenesis, since a relatively small population of these cells was able to perpetuate CNS inflammation. We found that following Th1 differentiation, purified 2D2 CD44hiCD62Llo memory cells have an enriched population of CD44hi cells compared to unsorted splenocytes despite similar expression of IFN-γ and T-bet (Fig. 4D–E). We transferred 0.4 × 106 female Th1differentated purified memory cells to female WT recipients, as this

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was the average proportion of T cells that were of a memory phenotype in female unsorted splenocyte adoptive transfers. Surprisingly, the transfer of purified memory Th1 cells did not result in observable signs of EAE in female WT recipients. The lack of disease transfer by purified memory Th1 cells is predicted to be a consequence of the low number of cells transferred. We hypothesize that memory Th1 cells can readily access the CNS and are capable of inducing inflammation due to the expression of intracellular IFN-γ and T-bet and the high expression of surface CD44. CD44 is a key determinant of the efficiency by which effector memory T cells navigate inflammation. CD44 binds to E-selectin and hyaluronic acid on the surface of endothelial cells and facilitates adhesion via the integrin VLA-4 (Siegelman et al., 2000). The CD44/VLA-4 complex is dependent on CD44 for enabling activated T cells to halt rolling and engage in firm adhesion to the endothelium in preparation for diapedesis into tissues (Nandi et al., 2004). T cells expressing CD44 and integrin α4 are able to enter the CNS independent of their antigen-specificity and blocking mAbs specific for CD44 and integrin α4 prevent primed MBP-specific T cells from entering the CNS and transferring EAE (Brocke et al., 1999). Thus, the population of CD44hiCD62L− memory cells we observe following Th1 differentiation is likely critical for the initiation of CNS inflammation during adoptively transferred EAE using 2D2 T cells. Survival of long-term memory T cells requires TCR engagement provided by self-peptide/MHC complexes. This process of positive selection has proven to be important not only in the selection of naïve T cells in the thymus, but also sufficient for the maintenance of memory T cell populations in the periphery (Markiewicz et al., 1998). Further, there is a sexual dimorphism in the frequency of oligodendrocytes such that the density in males is 20–40% greater than that of females in three separate areas of the CNS including the ventral columns of the spinal cord (Cerghet et al., 2006). Thus, a possible explanation for the increase in memory T cells in 2D2 males is that there is a greater probability of positive selection of T cells due to the higher density of myelin-producing oligodendrocytes. Another possible explanation for sex differences seen in effector/ memory T cell populations in males versus females is hormonal variation. In EAE, Bebo et al. (1999) have shown that naïve T cells in the presence of sex hormones have a Th1 to Th2 shift in cytokine production and that this effect is persistent upon further stimulation of T cells even in the absence of additional hormone treatment. This suggests that sex hormones have significant influences on the development of effector T cell responses. In CD4+CD25+ regulatory T cell populations, estrogen (E2) has been shown to enhance the expression of the T regulatory cell (Treg) transcription factor, FoxP3. This estrogen-mediated increase in FoxP3 expression correlated with augmented suppressive activity of CD4+CD25+ Treg cells (Polanczyk et al., 2004, 2005). It is well documented that FoxP3+ Tregs are a source of TGF-β and it has been shown that Treg-derived TGF-β is required for the inhibition of Th1 differentiation (Li et al., 2007). In addition, a majority of naïve CD4+CD62Lhi T cells become FoxP3+ in the presence of TGF-β (Bettelli et al., 2006). In the female milieu, where estrogen is present in the microenvironment in which T cells differentiate, it is possible that there is a greater tendency for naïve T cells to evade an effector phenotype. Interestingly, we found that while there is a difference in effector memory T cell populations between sexes, a similar population of central memory T cells exists between male and female 2D2 mice (Fig. 5C). Effector and central memory cells are distinct subsets of T cells and have distinguishable trafficking patterns and effector functions. As opposed to effector memory T cells, central memory T cells express both CD62L and CCR7, an adhesion molecule and chemokine receptor important for cell retention in secondary lymphoid organs (Sallusto et al., 1999). Additionally, unlike central memory cells, effector memory T cells secrete large amounts of IFN-γ following polyclonal stimulation, suggesting they have a more

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prominent role in modulating Th1-mediated diseases like MS and EAE (Sallusto et al., 1999). T cell subsets such as Th1, Th17, Treg, and memory/effector cells have been implicated as contributing to disease outcome in MS and EAE. The adoptive transfer of EAE using MOG-specific T cells, without the use of adjuvants or previous T cell differentiation in immunized animals, can help elucidate the roles of various T cell populations in disease pathogenesis. Similar to the findings in MS and EAE, we have also demonstrated a role for MOG-specific memory cells in neuroimmune inflammation and have described a sex dimorphism present in 2D2 mice. Taken together, the data presented here demonstrate the pathogenic potential of effector/memory MOG-specific T cells in EAE. Disclosure The authors have no financial conflicts of interest. Acknowledgements We thank Dr. Vijay K. Kuchroo for his generous gift of 2D2 mice. In addition, we would like to thank Drs. Amy E. Lovett-Racke and Michael Racke for their helpful scientific discussion and Ingrid Gienapp for her outstanding technical support. References Bebo Jr., B.F., Schuster, J.C., Vandenbark, A.A., Offner, H., 1999. Androgens alter the cytokine profile and reduce encephalogenicity of myelin-reactive T cells. J. Immunol. 162, 35–40. Bettelli, E., Carrier, Y., Gao, W., Korn, T., Strom, T.B., Oukka, M., Weiner, H.L., Kuchroo, V.K., 2006. Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature 441, 235–238. Bettelli, E., Pagany, M., Weiner, H.L., Linington, C., Sobel, R.A., Kuchroo, V.K., 2003. Myelin oligodendrocyte glycoprotein-specific T cell receptor transgenic mice develop spontaneous autoimmune optic neuritis. J. Exp. Med. 197, 1073–1081. Bielekova, B., Goodwin, B., Richert, N., Cortese, I., Kondo, T., Afshar, G., Gran, B., Eaton, J., Antel, J., Frank, J.A., McFarland, H.F., Martin, R., 2000. Encephalitogenic potential of the myelin basic protein peptide (amino acids 83–99) in multiple sclerosis: results of a phase II clinical trial with an altered peptide ligand. Nat. Med. 6, 1167–1175. Brocke, S., Piercy, C., Steinman, L., Weissman, I.L., Veromaa, T., 1999. Antibodies to CD44 and integrin α4, but not L-selectin, prevent central nervous system inflammation and experimental autoimmune encephalomyelitis by blocking secondary leukocyte recruitment. Proc. Natl. Acad. Sci. USA 96, 6896–6901. Cerghet, M., Skoff, R.P., Bessert, D., Zhang, Z., Mullins, C., Ghandour, M.S., 2006. Proliferation and death of oligodendrocytes and myelin proteins are differentially regulated in male and female rodents. J. Neurosci. 26, 1439–1447. DeGrendele, H.C., Estess, P., Picker, L.J., Siegelman, M.H., 1996. CD44 and its ligand hyaluronate mediate rolling under physiologic flow: a novel lymphocyteendothelial cell primary adhesion pathway. J. Exp. Med. 183, 1119–1130. Egg, R., Reindl, M., Deisenhammer, F., Linington, C., Berger, T., 2001. Anti-MOG and antiMBP antibody subclasses in multiple sclerosis. Mult. Scler. 7, 285–289. Ferber, I.A., Brocke, S., Taylor-Edwards, C., Ridgway, W., Dinisco, C., Steinman, L., Dalton, D., Fathman, C.G., 1996. Mice with disrupted IFN-γ gene are susceptible to the induction of experimental autoimmune encephalomyelitis (EAE). J. Immunol. 156, 5–7. Haak, S., Croxford, A.L., Kreymborg, K., Heppner, F.L., Pouly, S., Becher, B., Waisman, A., 2009. IL-17A and IL-17F do not contribute vitally to autoimmune neuroinflammation in mice. J. Clin. Invest. 119, 61–69. Jäger, A., Dardalhon, V., Sobel, R.A., Bettelli, E., Kuchroo, V.K., 2009. Th1, Th17, Th9 effector cells induce experimental autoimmune encephalomyelitis with different pathological phenotypes. J. Immunol. 183, 7169–7177. Johns, J.G., Bernard, C.C.A., 1999. The structure and function of myelin oligodendrocyte glycoprotein. J. Neurochem. 72, 1–9. Kebir, H., Kreymborg, K., Ifergan, I., Dodelet-Devillers, A., Cayrol, R., Bernard, M., Giuliani, F., Arbour, N., Becher, B., Prat, A., 2007. Human TH17 lymphocytes promote blood-brain barrier disruption and central nervous system inflammation. Nat. Med. 13, 1173–1175. Kerlero de Rosbo, N., Hoffman, M., Mendel, I., Yust, I., Kaye, J., Bakimer, R., Flechter, S., Abramsky, O., Milo, R., Karni, A., Ben-Nun, A., 1997. Predominance of the autoimmune response to myelin oligodendrocyte glycoprotein (MOG) in multiple sclerosis: reactivity to the extracellular domain of MOG is directed against three main regions. Eur. J. Immunol. 27, 3059–3069. Langrish, C.L., Chen, Y., Blumenschein, W.M., Mattson, J., Basham, B., Sedgwick, J.D., McClanahan, T., Kastelein, R.A., Cua, D.J., 2005. IL-23 drives a pathogenic T cell population that induces autoimmune inflammation. J. Exp. Med. 201, 233–240. Li, M.O., Wan, Y.Y., Flavell, R.A., 2007. T cell-produced transforming growth factor-beta1 controls T cell tolerance and regulates Th1- and Th17-cell differentiation. Immunity 26, 579–591.

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