CD8-de” cient SJL mice display enhanced susceptibility to ...5)/409-420.pdf · infected¯2M-de” cient SJL mice and wild-type SJL controls. TMEV-IDD was induced in all mice using

Journal of NeuroVirology, 7: 409± 420, 2001c° 2001 Taylor & Francis ISSN 1355± 0284/01 $12.00+.00

CD8-de�cient SJL mice display enhancedsusceptibility to Theiler’s virus infectionand increased demyelinating pathology

Wendy Smith Begolka, Lia M Haynes, Julie K Olson, Josette Padilla, Katherine L Neville, Mauro Dal Canto,Joann Palma, Byung S Kim, and Stephen D Miller

Department of Microbiology-Immunology and Interdepartmental Immunobiology Center, Northwestern UniversityMedical School, Chicago, Illinois, USA

Theiler’s murine encephalomyelitis virus (TMEV) infection of the central ner-vous system (CNS) induces a chronic, progressivedemyelinating disease in sus-ceptible mouse strains characterized by in�ammatory mononuclear in�ltratesand spastic hind limb paralysis. Our lab has previously demonstrated a criticalrole for TMEV- and myelin-speci�c CD4+ T cells in initiating and perpetuatingthis pathology. It has however, also been shown that the MHC class I loci areassociated with susceptibility/resistance to TMEV infection and persistence.For this reason, we investigated the contribution of CD8+ T cells to the TMEV-induced demyelinating pathology in the highly susceptible SJL/J mouse strain.Here we show that ¯2M-de�cient SJL mice have similar disease incidencerates to wild-type controls, however ¯2M-de�cient mice demonstrated earlieronset of clinical disease, elevated in vitro responses to TMEV and myelin prote-olipid (PLP) epitopes, and signi�cantly higher levels of CNS demyelination andmacrophage in�ltration at 50 days post-infection. ¯2M-de�cient mice also dis-played a signi�cant elevation in persisting viral titers, as well as an increasein macrophage-derived pro-in�ammatory cytokine mRNA expression in thespinal cord at this same time point. Taken together, these results indicate thatCD8+ T cells are not required for clinical or histologic disease initiation orprogression in TMEV-infected SJL mice. Rather, these data stress the criticalrole of CD4+ T cells in this capacity and further emphasize the potential forCD8+ T cells to contribute to protection from TMEV-induced demyelination.Journal of NeuroVirology (2001) 7, 409–420.

Keywords: ¯2-microglobulin; CD8; Theiler’s virus; demyelination; multiplesclerosis

Address correspondence to Stephen D Miller, PhD, Departmentof Microbiology-Immunology, Northwestern University MedicalSchool, 303 E. Chicago Avenue, Chicago, IL 60611, USA. E-mail:[email protected]

Received 5 April 2001; revised 7 May 2001; accepted 29 May2001.

1Abbreviations: CNS, central nervous system; CTL, cytolytic/cytotoxic T lymphocyte; DTH, delayed-type hypersensitivity; KO,knock-out; MS, multiple sclerosis; OVA, ovalbumin; p.i., postin-fection; PLP, proteolipid protein; TMEV-IDD, Theiler’s murine en-cephalomyelitis virus-induced demyelinating disease; ¯2M, ¯2-microglobulin.

Introduction

Although the etiology of multiple sclerosis (MS),1

an immune-mediated demyelinating disease of thecentral nervous system (CNS) in humans, remainslargely unknown, several lines of evidence suggestthe potential for a virus as the initial causative agent(Kurtzke, 1993; Waksman, 1995). Theiler’s murineencephalomyelitis virus (TMEV) is a member ofthe Picornaviridae family that induces a chronicprogressive demyelinating pathology that closely re-sembles MS in susceptible strains of mice following

Severe TMEV-IDD pathology in CD8-de® cient SJL mice

410 WS Begolka et al

intracerebral inoculation. TMEV-induced demyelin-ating disease (TMEV-IDD) is characterized clinicallyby an abnormal waddling gait resulting from an as-cending hind limb spastic paralysis, and histologi-cally by the presence of perivascular in�ammatorymononuclear in�ltrates within the CNS that result inprimary demyelination (Lipton and Dal Canto, 1979;Miller and Karpus, 1994).

It is believed that the mechanisms involved in thedevelopment of demyelination involve both directand bystander immune-mediated processes as evi-denced by the presence of in�ammatory CD4C andCD8C T cells and macrophages within the target or-gan (Lipton et al, 1984; Pope et al, 1996), and toa lesser extent, direct viral damage of oligodendro-cytes and microglia due to persistent viral infection(Rodriguez et al, 1983; Graves et al, 1986; Rodriguezet al, 1988; Ohara et al, 1990). However, the exactnature of these immune effector responses and theircontribution to disease pathogenesis is still a matterof some controversy. There is considerable evidencefrom our own lab, as well as others, that indicatesa critical role for CD4C Th1 cells in mediating boththe initial and chronic pathogenesis of TMEV-IDDin the susceptible SJL/J strain, as demonstrated byantibody depletion studies (Rodriguez et al, 1986a;Friedmann et al, 1987; Welsh et al, 1987), the cor-relation of delayed-type hypersensitivity (DTH) re-sponses with the severity of disease (Miller et al,1997), and the increasing amounts of CD4C Th1-derived pro-in�ammatory cytokines expressed in theCNS throughout disease progression (Begolka et al,1998). Despite these signi�cant �ndings, there alsoexist numerous lines of often contradictory evidencefrom similar studies conducted in resistant C57BL/6mice with gene deletions to additionally support therole of CD8C T cells, not only in viral clearanceand protection but also in promoting the demyelinat-ing process (Rodriguez et al, 1986a; Rodriguez andSriram, 1988; Fiette et al, 1993; Pullen et al, 1993;Njenga et al, 1996; Murray et al, 1998).

Therefore, in this study we sought to de�nitivelyestablish the effector role of CD8C T cells in the highlysusceptible SJL/J mouse, by utilizing mice whichare de�cient in the expression of ¯2-microglobulin(¯2M), which fail to positively select any CD8C

T cells (Koller et al, 1990; Zijlstra et al, 1990 ). Ourresults clearly indicate, in correlation with our pre-vious data in this system, that CD8C T cells fail toprovide a signi�cant and demonstrable effector con-tribution to either the initiation or progression ofTMEV-IDD clinical pathology or demyelination. Inaddition, our data also convincingly demonstratesthat the absence of CD8C T cells leads to an accel-erated and more severe disease phenotype, as mea-sured clinically, histologically, and by several in vitroparameters, leading to the conclusion that the role ofCD8C T cells is primarily for protection and/or regula-tion in this system. These data are consistent with ourongoing model for the SJL/J mouse wherein persistent

CNS infection with TMEV leads to the continual ac-tivation and clonal expansion of TMEV-speci�c, andeventually neuroantigen-speci�c, CD4C Th1 effectorcells capable of secreting proin�ammatory cytokinesand chemokines. These proin�ammatory moleculesin turn lead to the recruitment and eventual accu-mulation of activated macrophages/microglia withinthe CNS resulting in demyelination via a nonspeci�cbystander response.

Results

Accelerated onset and increased clinical sever-ity of TMEV-IDD in ¯2M-de�cient mice Wild-typeSJL and ¯2M-de�cient mice were monitored forthe development of clinical signs of demyelinationfollowing infection with TMEV (Figure 1). Althoughthe incidence rate for developing TMEV-IDD was notstatistically distinct between the two groups (93.1%vs 75.8%), ¯2M-de�cient mice developed a height-ened disease phenotype as measured by several pa-rameters. ¯2M-de�cient mice had an accelerated dis-ease onset when compared to control mice (MDO D29.89 vs 35.08; P D :038), and a rapidly progressingchronic disease that was dramatically increased inseverity over controls from day 32 postinfection (p.i.)throughout the measured clinical course.

The increaseddisease severity in the ¯2M-de�cientmice was also evident from gross CNS histology atday 50 p.i. (Figure 2), showing large numbers ofperivascular and parenchymal in�ammatory in�l-trates, which appear to be comprised primarily of

Figure 1 Comparison of clinical disease course between TMEV-infected ¯2M-de�cient SJL mice and wild-type SJL controls.TMEV-IDD was induced in all mice using the BeAn strain of virusand animals were graded for clinical signs as described in Materi-als and Methods. Results are expressed as mean clinical score ofaffected animals vs days p.i. from three pooled experiments. Dis-ease scores at 32, 38, and 48 days p.i. are signi�cantly increasedin ¯2M-de�cient vs controls as measured by the Mann-Whitneytest (*P < .006). Mean day of clinical onset (MDO) is also acceler-ated in ¯2M-de�cient mice (**P D :038). No signi�cant differenceis observed in the disease incidence rate between ¯2M-de�cientmice (22/29—75.9%) and the wild-type controls (27/29—93.1%)as measured by Â 2 using Fisher’s exact test (P D :1443).


WS Begolka et al 411

Figure 2 Spinal cord histology of TMEV-infected ¯2M-de�cient SJL and wild-type SJL control mice. Two mice from each group weresacri�ced by perfusion on day 50 p.i., and 10 1-¹m-thick sections from the lumbar region of the spinal cord were stained with toluidineblue and examined by light microscopy for evidence of in�ammatory in�ltrate and demyelination. Pathologic changes were gradedas follows: , no disease; C/ , meningeal in�ammation; C, focal parenchymal in�ammation with demyelination; CC, multiple areasof parenchymal in�ammation and demyelination; CCC, extensive in�ammation and demyelination with con�uent lesions. Shown isrepresentative section for each group of mice, with the wild-type SJL receiving an overall score of C, and the ¯2M-de�cient SJL receivingan overall score of CCC.

myelin debris-containing macrophages, as well asextensive white matter demyelination, compared tothe milder perivascular CNS damage in wild-typecontrols. To address if there were qualitative and/orquantitative changes in the nature of the in�amma-tory in�ltrate between the two groups, we also per-formed immunohistochemistry for CD4, CD8, andF4/80 in�ltrates. The increased CNS in�ltrates in the¯2M-de�cient mice were largely the result of accu-mulation of F4/80C macrophages/microglia, ratherthan CD4C effector cells which were fairly equiva-lent between wild-type and ¯2M-de�cient mice (Fig-ure 3). As expected, staining for CD8C T cells wasdetected in the wild-type infected CNS, albeit at lowlevels, yet completely absent in ¯2M-de�cient mice,con�rming their phenotype. Isotype-matched con-trols for each antibody as well as staining of CNStissue from na¨õ ve mice revealed no positive cells forany of the three tested markers (data not shown).

Increased viral persistence in the CNS of TMEV-infected ¯2M-de�cient mice While the clinical andhistological disease was exacerbated in the ¯2M-de�cient mice, it was also of interest to determineif the severe disease pathology could be attributed toalterations in viral clearance within the CNS due tothe lack of cytolytic CD8C T cells. Viral plaque assaysof brain and spinal cord tissue from TMEV-infected¯2M-de�cient mice at day 50 p.i. revealed a 2–2.5-fold increase in viral titer from both the brain andspinal cord as comparedto the wild-type control mice(Figure 4A). Overall, higher levels of virus were iso-lated from the spinal cord in both groups correlatingwith the previous observation that TMEV persists

to a higher level in this CNS tissue at later timepoints in disease (Lipton et al, 1984). As expected,ho-mogenized kidney samples from both TMEV-infectedgroups failed to yield any detectable plaques (data notshown). Semiquantitative RT-PCR from spinal cordsamples at this same time point also demonstratedan increase in mRNA levels for the TMEV capsid pro-teins VP1 and VP3 as seen in the plaque assays (Fig-ure 4B). This data illustrates the critical role of theCD8C T cell in the highly susceptible SJL mouse inregulating levels of infectious virus, and not in con-tributing to the TMEV-IDD demyelinating pathology.

T cell responses to both TMEV and myelin epi-topes are increased in ¯2M-de�cient mice The datato this point indicate that the clinical presentation ofTMEV-IDD differs dramatically in the presence or ab-sence of CD8C T cells in the SJL host, and that in ¯2M-de�cient mice the exacerbated disease severity maybe attributed to the pronounced in�ammatory in�l-trate and increasedviral titer. We next sought to deter-mine if the pattern of peripheral immune reactivityto TMEV and myelin proteins, as measured by bothin vivo and in vitro assays was altered between the¯2M-de�cient and control mice, and thereby provideadditional evidence of a contributing mechanism forthe observed accelerated disease. Delayed-type hy-persensitivity (DTH) analysis of TMEV-infected ¯2M-de�cient at day 50 p.i. demonstrated no signi�cantdifference in the level of in vivo reactivity to eitherUV-inactivated TMEV, the immunodominant VP270-86 capsid protein peptide, or the immunodomi-nant myelin epitope PLP139-151 when compared tothe wild-type infected controls (Figure 5). This does



Figure 3 Immunohistochemicalstaining for CD4C and CD8C T cells, and F4/80C macrophages/microglia in the spinal cords of wild-typeand ¯2M-de�cient SJL mice with TMEV-IDD. Spinal cord sections from mice from day 50 p.i. were stained for expression of CD4, CD8,and F4/80 as described in Materials and Methods. Shown is a representative section from each group of mice for each cell surface marker.In the top panels F4/80 staining is shown in red; in the middle panels, CD4 staining is shown in green with a blue DAPI counterstain; and,in the bottom panels, CD8 staining is shown in blue with red propidium iodide counterstain. Magni�cation is 100£. Isotype-matchedcontrol antibody did not stain spinal cords from affected mice, nor was any positive staining revealed in the spinal cords of na¨õ ve mice(data not shown).



Figure 4 Increased viral titers and mRNA for viral proteins are found in the brain and spinal cord of TMEV-infected ¯2M-de�cient SJLmice. In panel A, three mice at day 60 p.i. from each group were sacri�ced, and brain, spinal cord, and kidney (not shown) were isolated,pooled, and processed for a viral plaque assay as described in Materials and Methods. Viral plaques in a BHK monolayer were countedover a series of four dilutions of tissue lysate, with the data shown as the mean PFU (plaque forming unit) per mg of brain and spinalcord starting tissue. Control kidney lysate did not yield any viral plaques. These results are representative of two separate experiments.In panel B, spinal cord-derived RNA from mice at day 60 p.i. was used as a template for semi-quantitative RT-PCR for the TMEV viralcapsid proteins VP1 (495bp) and VP3 (445bp).

indicate, however, that ¯2M-de�cient mice are fullycompetent in developing and sustaining a CD4C Th1-mediated response.

Although this observation is interesting, it is alsopossible that we may have missed an accelerated acti-vation and expansion of PLP139-151-speci�c T cells

Figure 5 ¯2M-de�cient mice display signi�cant DTH responsesto whole TMEV and myelin epitopes. DTH responses to TMEV,PLP139-151, and OVA323-339 were assessed at day 50 p.i. fromTMEV-infected ¯2M-de�cient and wild-type control mice (n D5). The results are expressed as the 24-h ear swelling in units of10 4 inches § SEM in response to ear challenge with 5 ¹g UV-inactivatedTMEV, 10 ¹g PLP139-151,or 10 ¹g OVA323-339.Theseresults are representative of two separate experiments. *DTH re-sponses were signi�cantly greater than uninfectedcontrols as mea-sured by the Student’s t-test (P < .001).

at the day-50 time point, given that the majority of¯2M-de�cient mice have already progressed to a se-vere clinical disease, and may now be displaying en-hanced reactivity to additional myelin epitopes nottested. We therefore performed DTH at an earlier timepoint in disease (day 35 p.i.), and despite yieldingsimilar results for TMEV and VP2 70-86, the ¯2M-de�cient mice now displayed a trend (bordering onsigni�cance) toward increased responses to PLP139-151 compared to controls, indicative of acceleratedepitope spreading (data not shown).

This trend toward increased neuroantigen reac-tivity was also observed in the in vitro splenicproliferative responses to both viral and myelin epi-topes. ¯2M-de�cient mice demonstrated compara-ble to slightly higher responses, as measured bystimulation indexes, to intact TMEV and VP2 70-86 (Figure 6A), yet dramatically increased reactiv-ity to PLP139-151 (SI D 10.1 vs 3.2) (Figure 6B). Re-sponses to the irrelevant antigen OVA 323-339 werenot detected in any experimental group. Given thatwe have previously demonstrated the ability to detectonly low levels of proliferation to this immunodom-inant epitope at this time point from wild-type ani-mals (Miller et al, 1997), collectively these data ad-ditionally support the immunological phenomenonof epitope spreading as a contributing factor in theaccelerated disease phenotype observed in the ¯2M-de�cient mice.

Increased levels of macrophage-derived pro-in�ammatory mediators in the CNS of ¯2M-de�cientmice With evidence of increased cellular in�ltrate,viral titer and cellular responses, we were also



Figure 6 Proliferative responses to TMEV and PLP139-151 are in-creased in ¯2M-de�cient mice. Splenic cells isolated from TMEV-infected ¯2M-de�cient or wild-type SJL mice 50 days p.i. werecultured with UV-inactivated TMEV and VP2 70-86 (panel A), andPLP139-151 and OVA323-339 (panel B). Data represent the mean3[H]-thymidine uptake of triplicate cultures stimulated for a totalof 96 h with antigen (shown is 5 ¹g for TMEV and 10 ¹g for pep-tides) and is plotted as 1 CPM (no antigen controls subtracted)§ SEM. Shown above certain results in parentheses are also theproliferation data expressed as the stimulation index (SI D CPM ofantigen-containing wells/CPM of control wells). These results arerepresentative of two separate experiments.

interested in the resulting cytokine pro�le duringTMEV-IDD within the CNS of infected animals, aswe have previously demonstrated that the levels ofmRNA for pro- and anti-in�ammatory cytokines con-tinue to increase throughout the disease course in thewild-type SJL mouse (Begolka et al, 1998). Any in-creases in the levels of these cytokines in the ¯2M-de�cient mouse as compared to wild-type would alsobe indicative of, and contributing to, the observed ac-celerated disease. Total RNA isolated from mice atday 50 p.i. revealed similar results as the immuno-histochemical analysis in Figure 3, demonstrating a2-fold increase in message for the F4/80 cell surfacemarker in ¯2M-de�cient mice, but not for CD4, anda concomitant increase in macrophage-derived pro-in�ammatory cytokines such as TNF-®, IL-10, iNOS,and IL-12, without a corresponding increase in com-

parable cytokines produced by in�ammatory CD4C Tcells, as measured by semiquantitative RT-PCR (Fig-ure 7A–C). A notable exception to this for the CD4C

population is the readily detectable increase in IL-4mRNA, which may be increased as an attempt by thehost at intrinsic regulation of a more severe diseasepathology.

Discussion

A large body of research has attempted to ascertainwhether CD4C or CD8C T cells are involved in theinitiation, progression and/or regulation of TMEV-IDD. The evidence for CD4C T cells as mediators ofTMEV pathology in susceptible SJL mice is signi�-cant in that: 1) monoclonal antibodies against CD4,MHC Class II, or IL-12 either prevented or dimin-ished clinical signs (Friedmann et al, 1987; Welshet al, 1987; Inoue et al, 1998); 2) �ow cytometric andhistological data from infected SJL/J mice revealedan increased level of CD4C T cells and their pro-in�ammatory cytokines during clinical disease pro-gression, and transfer of a CD4C VP2-speci�c T cellline leads to increased incidence and accelerated on-set of disease (Gerety et al, 1994; Pope et al, 1996;Begolka et al, 1998); 3) the development of virus andmyelin antigen-speci�c DTH corresponds with onsetand chronicity of demyelination (Clatch et al, 1986;Miller et al, 1997); 4) epitopes of TMEV that elicita Th1 response and DTH can accelerate the patho-genesis of TMEV upon active immunization (Yauchet al, 1998); and 5) tolerance via ECDI-coupled cellswith TMEV, known to reduce IL-2 and IFN-° fromTh1 cells, reduces the severity of disease (Petersonet al, 1993; Karpus et al, 1995).

Although these data seem to rigorously supportthe role of CD4C Th1 cells in both initiation andprogression of TMEV-IDD, there are also a numberof published reports suggesting the same is true forCD8C T cells (Rodriguez et al, 1986a; Rodriguez andSriram, 1988; Matloubian et al, 1994; Njenga et al,1996; Murray et al, 1998). However, as the majorityof these studies were conducted using mice on a re-sistant genetic background (C57BL/6 and 129/J), inthis study we sought to determine the outcome of theTMEV-IDD disease phenotype in the absence of CD8C

T cells in the highly susceptible SJL/J mice that werede�cient in the expression of ¯2M. Previous reportsexamining the effects of this mutation or the MHCclass I knockout counterpart have provided evidencethat the absence of CD8C T cells on the resistant back-ground leads to the establishmentof chronic demyeli-nation with or without clinical signs, extensive CNSin�ltrate, and high viral titers, leading one to con-clude that the primary role of these cells is in pro-tection and regulation of disease (Fiette et al, 1993;Pullen et al, 1993; Rodriguez et al, 1993; Rivera-Quinones et al, 1998; Lavi et al, 1999). The currentresults support this central role for CD8C T cells, with



Figure 7 Semiquantitative RT-PCR analysis of cytokine mRNA levels from the spinal cords of TMEV-infected ¯2M-de�cient and wild-type control SJL mice. At 50 days p.i., three mice per group were sacri�ced by perfusion and spinal cord RNA was isolated. This total RNAserved as the template for RT-PCR as detailed in Materials and Methods. Shown in panel A are the gel images of the ampli�ed productsfor the control transcript HPRT, the macrophage/microglia cell surface marker F4/80, and the macrophage-derived proin�ammatorymediators, TNF-®, IL-12, IL-10, and iNOS. Shown in panel B are the ampli�ed products of the effector T cell marker CD4 and the Tcell-derived cytokines IFN-° , IL-4, and TNF-¯ (lymphotoxin). Shown in panel C are the densitometry values for the ampli�ed cytokineproducts in panels A/B following normalization to the housekeeping gene, HPRT. The data are expressed as a percentage of normalizedHPRT expression. Results are representative of two separate experiments.



the secondary conclusion that CD4C effector T cellsare suf�cient to mediate disease pathogenesis.

First, ¯2M-de�cient mice are highly susceptible toTMEV-IDD, as there was no difference in the inci-dence rate when comparing them to control mice;however, their progression of disease was remarkablewhen observing several severity parameters. ¯2M-de�cient mice displayed an accelerated onset of dis-ease, as well as a more severe clinical progression asmeasured by mean clinical score over the durationof the time course observed. In addition, in one ex-periment where the mice were allowed to progressto beyond 55 days p.i., 5 out of 11 ¯2M-de�cientmice progressed to a clinical score of 4, with sev-eral mice eventually succumbing to the disease byday 60, an observation rarely seen with the wild-type SJL mouse (data not shown). Second, the se-vere clinical disease in the ¯2M-de�cient mice alsocorrelated with the presence of large numbers of in-�ammatory in�ltrates comprised mainly of activatedmacrophages/microglia in conjunction with exten-sive white matter demyelination as compared to thecontrol mice. Expression of cytokine mRNA from theCNS of infected mice additionally con�rms the in-crease in activated macrophage/microglia in�ltrateas well as their secreted pro-in�ammatory mediatorsknown to promote the CD4C Th1 environment. Third,¯2M-de�cient mice were effectively able to mount aspeci�c immune response to both virus and myelinepitopes that was at a minimum equivalent to, yet of-ten exceeded, that of wild-type controls. Last, ¯2M-de�cient mice were signi�cantly hampered in theirability to control viral persistence in both the brainand spinal cord.

Impressively, the current data extend the priorwork done with resistant animals to the susceptibleSJL/J mouse and con�rm the phenotype previouslyobserved. This correlation between outcomes regard-less of the genetic background lends strong supportto the distinct roles of CD4C and CD8C T cells in theTMEV model. Although data indicating the pivotalrole of CD4C T cells in the pathogenesis of TMEV-IDD have been previously reported, an equally com-pelling body of work also demonstrates the criticalrole CD8C T cells play in the control of disease sus-ceptibility. It has been clearly shown that : 1) sus-ceptibility/resistance to TMEV maps to Class I H-2D (Clatch et al, 1985; Rodriguez and David, 1985;Rodriguez et al, 1986b; Kappel et al, 1991); 2) mono-clonal antibody depletion of CD8C T cells leads toearlier onset and increased disease severity (Larsson-Sciard et al, 1997); 3) virus-speci�c CTLs are foundat a higher frequency in the CNS of resistant micethan susceptible mice, and in vivo IL-2 therapy caneffectively boost CTL responses in susceptible strainsand diminish viral persistence (Lindsley et al, 1991;Pena Rossi et al, 1991; Lin et al, 1995; Dethlefs et al,1997); 4) perforin KO mice on the normally resistantC57BL/6 background are able to be persistently in-

fected with TMEV (Pena-Rossi et al, 1998); and 5)in the BALB/c system, transfer of a CD8C popula-tion from the spleen of TMEV-infected donors pro-tects recipients from disease induction (Nicholsonet al, 1996; Haynes et al, 2000). Taken as a whole,our data in the susceptible SJL/J mouse supplementand are consistent with these previously documentedobservations and further support a critical role forCD8C T cells in regulating disease susceptibility andseverity.

Our results are, however, in direct contrast withdata suggesting CD8C T cells contribute to the dis-ease pathology of TMEV-IDD (Rivera-Quinones et al,1998; Rodriguez and Sriram, 1988). There are severalpossible explanations for these observed differences.With regard to the absence of CD4C T cells leadingto an exacerbated disease, the ability to provide cog-nate B cell to help production of antibody to controlthe peripheral and CNS viremia is critically impaired(Borrow et al, 1993). This is supported by the factthat the anti-TMEV humoral response in suscepti-ble animals is unable to completely clear the virusbut is capable of dramatically limiting the spreadearly in the infection (Lipton and Gonzalez-Scarano,1978). To that end, CD4-de�cient animals die of anoverwhelming virally mediated encephalitis within25 days p.i. (Rodriguez and Sriram, 1988). The deple-tion of CD4C T cells in the chronic stages of TMEV-IDD has not been directly addressed in the literatureto this point, however, given the compelling evidencefor a pathologic role of myelin-speci�c autoimmunityin chronic disease progression (Miller et al, 1997),it is strongly believed that in�ltrating CD4C T cellscontribute more signi�cantly to disease progressionthan its regulation. Our lab has recently addressedthis hypothesis through the use of tolerance to a panelof myelin autoepitopes at later time during chronicdisease with the observed outcome of diminishedclinical score and decreased demyelinating pathol-ogy (manuscript submitted). In addition, as it hasbeen demonstrated that susceptible mice are capableof mounting virus-speci�c CTL responses (Lindsleyet al, 1991), it is interesting to note that CD4C T cellshave been reported to be required to perpetuate long-term CTL reactivity when infections become persis-tent, as in the case of LCMV (Matloubian et al, 1994).

The observation that the absence of CD8C T cellsleads to a less severe disease is more dif�cult to rec-oncile with our data (Rivera-Quinones et al, 1998).It is possible that differences related to the strain ofvirus used (DA vs BeAn) may be a contributing factor,as it has not been completely determined what typeof CD8C responses are elicited during active infec-tion with each strain of virus. It is well documentedin both human and mouse that na¨õ ve CD8C T cellsare capable of differentiating into Tc1 or Tc2 popu-lations, analogous to Th1 and Th2 cells, dependingon the cytokine environment present during initialstimulation (Croft et al, 1994; Sad et al, 1995; Sad



and Mosmann, 1995; Seder and Le Gros, 1995; Carterand Dutton, 1996). Tc1 development is promoted byIL-18, IL-12, and IFN-° leading to more of a cytolyticresponse, whereas Tc2 development is promoted byIL-4 leading to a predominant regulatory response (Liet al, 1997). It is possible, then, that in the resistantC57BL/6 strain, where virus is cleared rapidly, thatthe predominant response is Tc1 in nature. This ideais supported by data indicating that C57BL/6-perforinKO mice are able to be persistently infected withTMEV (Pena-Rossi et al, 1998), and that the absenceof CD8C T cells leads to viral titers that are dramat-ically increased (Figure 4) (Fiette et al, 1993; Pullenet al, 1993; Rodriguez et al, 1993; Rivera-Quinoneset al, 1998; Lavi et al, 1999). This would be in contrastto the susceptible SJL/J strain where the predominantresponse may be Tc2 in nature, and/or minimally aweak Tc1, and the initial viremia is controlled, butnot cleared. These mice, although capable of mount-ing a cytolytic response during active infection, mustbe dependent on additional mechanisms to supportviral clearance, with the most likely candidate be-ing antibody-dependent responses. This is supportedby our observation that the majority of ¯2M-de�cientmice, while displaying an accelerated disease onsetand severity of TMEV-IDD, do not succumb to thepathology until after 60 days p.i. Their rapid progres-sion of disease is then most likely a combination ofincreased viral titer leading to enhanced immune in-�ltrate and demyelination rather than a virally medi-ated encephalitis, indicative of a signi�cant regula-tory response that is now absent.

Collectively, the current data lend additional sup-port to our model of TMEV-IDD in the suscepti-ble SJL/J mouse wherein persistent CNS infectionwith TMEV leads to the continual activation andclonal expansion of TMEV-speci�c, and eventuallyneuroantigen-speci�c, CD4C Th1 effector cells ca-pable of secreting pro-in�ammatory cytokines andchemokines. These pro-in�ammatory molecules con-tinue to promote an environment favoring Th1 ac-tivation, and in turn lead to the additional re-cruitment and eventual accumulation of activatedmacrophages/microglia within the CNS resulting indemyelination via a nonspeci�c bystander response.The role of CD8C T cells in this model appears tobe a combination of both Tc1 cytolytic and Tc2 reg-ulatory responses, but overall they do not appear tocontribute signi�cantly to either the demyelinatingpathology or the onset of neurological de�cit.

Materials and methods

Mice Female SJL/J mice, 6–7 weeks old, were pur-chased from Harlan Laboratories (Indianapolis, IN).¯2M-de�cient SJL/J mice were purchased from theJackson Labs (Bar Harbor, ME). All mice were housedin the Northwestern animal care facility and main-

tained on standard laboratory chow and water ad li-bitum. Severely paralyzed mice were afforded easieraccess to food and water.

Peptides PLP139-151(HSLGKWLGHPDKF), OVA323-339 (ISQAVHAAHAEINEAGR), and VP2 70-86(WTTSQEAFSHIRIPLP) were purchased from thePeptides International (Louisville, KY). Amino acidcomposition was veri�ed by laser desorption massspectroscopy and purity (>98%) was assessed byHPLC.

Induction and clinical evaluation of TMEV-IDD Mice were anesthetized with methoxy�urane(Mallinckrodt Veterinary, Mundelein, IL) and inocu-lated in the right cerebralhemisphere with (2.9 £ 106)plaque-forming units of TMEV, strain BeAn 8386,in 30 ¹l DMEM. Mice were examined in a single-blinded fashion 2–3 times per week for the devel-opment of chronic gait abnormalities and spasticparalysis indicative of demyelination, and assigned aclinical score of 0 to 6 as follows: 0 D asymptomatic,1 D mild waddling gait, 2 D severe waddling gait,intact righting re�ex, 3 D severe waddling gait, spas-tic hind limb paralysis, impaired righting re�ex, 4 Dsevere waddling gait, spastic hind limb paralysis, im-paired righting re�ex, mild dehydration, and/or mal-nutrition, 5 D total hind limb paralysis, severe dehy-dration, and/or malnutrition, 6 D death. The data areplotted as the mean clinical score for animals fromthree pooled experiments.

In vitro T cell proliferation assays T cell prolif-erative responses were assessed by incorporation of[3H]-thymidine. A total of 106 viable splenocytes re-covered from TMEV infected mice at day 50 p.i. werecultured in triplicate in 96-well, �at-bottom platesin 0.2 ml HL-1 media (Biowhittaker, Walkersville,MD) supplemented with 5% FBS (Sigma, St. Louis,MO), 2 mM glutamine, 100 ¹g/ml streptomycin, and100 U/ml penicillin (Gibco BRL, Gaithersburg, MD).A range of VP2 70-86, PLP139-151, and OVA323-339peptide antigen concentrations (0.1–100 ¹M �nal),and 5 ¹g of UV-inactivated intact TMEV were tested.Plates were pulsed with 1 ¹Ci [3H]-thymidine after72 h of culture and harvested for scintillation count-ing on a Packard TopCount plate reader 24 h later.The results are expressed as mean CPM of antigencontaining cultures.

Delayed-type hypersensitivity (DTH) DTH re-sponses were quantitated using a standard 24-h ear-swelling assay. Prechallenge ear thickness was de-termined using a Mitutoyo model 7326 engineer’smicrometer (Schlesinger Tools, Brooklyn, NY). DTHresponses were elicited by injected 10 ¹g of peptide(in 10 ¹l of PBS), or 5 ¹g of UV-inactivated TMEV intothe dorsal surface of the ear using a 100-¹l Hamiltonsyringe �tted with a 30-gauge needle. Twenty-fourh after ear challenge, the increase in ear thicknessover pre-challenge measurements was determined.Results are expressed in units of 10 4 in § SEM. Ear-swelling responses were the result of mononuclear



cell in�ltration and showed typical DTH kinetics (i.e.,minimal swelling at 4 h, maximal swelling 24–48 h).

CNS histology At day 50 p.i., 2 mice from eachgroup were anesthetized and sacri�ced by perfusionthrough the left ventricle with 10 ml of PBS, fol-lowed by 100 ml of chilled 3% glutaraldehyde inPBS, pH 7.3. Spinal cords were dissected from thevertebral canal, sectioned at 1-mm intervals, post-�xed in 1% osmic acid for 1 h, and processed forEpon embedding. Ten 1-¹m-thick sections from eachspinal cord were stained with toluidine blue and ex-amined by light microscopy. Pathologic changes weregraded in a blinded manner as follows: , no disease;C/ , meningeal in�ammation; C, focal parenchymalin�ammation with demyelination; CC, multiple ar-eas of parenchymal in�ammation and demyelina-tion; CCC, extensive in�ammation and demyelina-tion with con�uent lesions.

Immunohistochemistry for CD4, CD8, and F4/80At day 50 p.i., 2 mice from each group were anes-thetized and sacri�ced by perfusion through the leftventricle with 30 ml of ice-cold PBS. Spinal cordswere removed by dissection, and 2–3-mm spinalblocks were immediately frozen in OCT (Miles Labo-ratories, Elkhart IN) in liquid nitrogen. The blockswere stored at 80±C in plastic bags to preventdehydration. Then, 5–6-¹m-thick sections from thelumbar region (approximately L2-L3) were cut ona Reichert-Jung Cryocut 1800 cryotome (Leica In-struments, Deer�eld, IL), mounted on SuperfrostPlus electrostatically charges slides (Fisher Scien-ti�c, Hanover Park, IL), air dried, and stored at 80±C.

Slides were stained using Tyramide Signal am-pli�cation (TSA) Direct Kit (NEN Life ScienceProducts, Boston, MA) according to the manu-facturer’s instructions. Sections from each groupwere thawed, air-dried, �xed in acetone at roomtemperature, and rehydrated in 1£ PBS. Nonspe-ci�c staining was blocked using anti CD16/CD32(Fc° R II/III-2.4G2, Pharmingen, San Diego, CA),and an avidin/biotin blocking kit (Vector Laborato-ries, Burlingame, CA) in addition to the blockingreagent provided by the TSA kit. Slides were stainedwith the following biotin-conjugated antibodies: anti-macrophage (F4/80) (Caltag, Burlingame, CA); anti-CD4 (H129.19), and anti-CD8a (53-6.7) (Pharmingen).Sections were counterstained with DAPI (Sigma) orpropidium iodide (Molecular Probes, Eugene, OR),and then coverslipped with Vectashield mountingmedium (Vector). Slides were then examined byepi�uorescence using a chroma triple-band �lter(Chroma Technology Corporation, Brattleboro, VT).Four serial sections from each sample per group wereanalyzed at 100£ and 400£ magni�cation through-out the entire spinal cord section, which includedgray and white matter, and dorsal, ventral, and lat-eral regions.

Plaque assay for viral titer At day 50 p.i., 3–4mice per group were sacri�ced by anesthesia. Thebrain, spinal cord, and kidney were removed and ho-

mogenized in PBS, and diluted in serum-free DMEMto obtain at least three serial dilutions for the assay.BHK-21 cells seeded 2 days prior in 60 £ 15-cm tissueculture plates, and grown to con�uency, were washed1£ with serum-free DMEM prior to the addition of ho-mogenizedtissue dilutions. Infection of the BHK cellsis accomplished by incubation of the serial dilutionsof homogenate with the cells at room temperature for1 h with occasional swirling. A sterile 2% top agar so-lution is added in equal volume to 2£ DMEM supple-mented with 2% FBS, 5 mM L-glutamine, 200 ¹g/mlpenicillin, and 200 ¹g/ml streptomycin. After the1-h incubation, the DMEM/agar mixture is added tothe plate and the BHK cells are incubated at 34±Cfor 6 days in a humidi�ed environment. Followingthe 6-day incubation, the agar layer is removed andthe BHK cells are �xed with methanol. The cells arethen stained with a crystal violet solution (6 g crys-tal violet, 100 ml ethanol, and 400 ml H2O), and theplaques are visualized on each plate. The number ofplaques counted on each plate are multiplied by thehomogenate dilution and the amount of homogenateadded to the plate to determine the plaque formingunits (PFU) per ml. The weight of the original start-ing tissue (mg) per ml of homogenate is then used tocalculate the PFU/mg.

Isolation of RNA At day 50 following inductionof TMEV-IDD, two mice from each group were anes-thetized and perfused through the left ventricle with50 ml of PBS. Spinal cords were extruded by �ush-ing the vertebral canal with PBS and then rinsed inPBS. Tissues were forced through a 100-mesh stain-less steel screen to give a single cell suspension andpelleted by centrifugation at 1200 RPM for 5 minat 4±C. The pellets were vigorously resuspended in16 ml of 4 M guanidinium isothiocyanate/50 mMTris-Cl (pH 7.5)/25 mM EDTA (Gibco BRL); 1% 2-mercaptoethanol. Shearing of DNA was facilitated byforcing the resulting suspension repeatedly througha 23-gauge needle. Total RNA was isolated by high-speed gradient centrifugation (27,000 RPM) of 8 mllysate through a 3-ml 5.7 M CsCl pad using a SW41swinging bucket rotor for 20 h at 4±C. The resultingRNA pellet was resuspended to a �nal concentrationof 1 ¹g/¹l with diethylpyrocarbonate (DEPC)-treatedwater and stored in aliquots at 70±C.

First strand cDNA synthesis and RT-PCR Firststrand cDNA was generated from 2 ¹g total RNAusing Advantage-RT Kit (Clontech, Palo Alto, CA)using 20 pmole oligo-dT primer as per the manufac-turer’s provided protocol in a total volume of 20 ¹l.Following �rst strand synthesis, each cDNA samplewas brought to a �nal volume of 100 ¹l with distilledwater. Final PCR conditions included 50 mM KCl;10 mM Tris-Cl (pH 8.3); 2.5–5.0 mM MgCl2 (empiri-cally determined for each primer pair); 2 mM dNTPs;100 pmoles of each 50 and 30 gene-speci�c primer;1 U Taq polymerase (Qiagen, Chatsworth, CA) and 5–10 ¹l diluted cDNA. Cycling conditions were 94±C,40 s; 60±C, 20 s; 72±C, 40 s, for a total of 30 cycles;



linked to a �nal 72±C extension program for 3 min andthen to a �nal 4±C soak program. PCR products wererun on an ethidium-bromide-containing 2% agarosegel and illuminated using an ultraviolet light source,then photographed using Polaroid type 667 �lm. Gelimages were then scanned into Adobe Photoshop us-ing an Epson 1200-C scanner and imported as TIFF�les into Kodak 1D Digital Science for densitometry.The sum intensity and band area were determined for

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CD8-de” cient SJL mice display enhanced susceptibility to ...5)/409-420.pdf · infected¯2M-de” cient SJL mice and wild-type SJL controls. TMEV-IDD was induced in all mice using