Encephalitogenic T lymphocytes develop from SJL/J hematopoietic cells transplanted into severe combined immimodeficient ( SCID) mice

Journal of Neuroimmunology ELSEVIER

Journal of Neuroimmunology 57 (1995) 155-164

Encephalitogenic T lymphocytes develop from SJL/J hematopoietic cells transplanted into severe combined immunodeficient (SCID) mice Richard E. Jones a,bj*, Ruth H. Whitham a,b, Tim Sullivan a, Michele Mass b, Dennis N. Bourdette a,b a Research Service, 151-D, VA Medical Center, 3710 S. U! US Veterans Hospital Rd., Portland, OR 97207, USA h Department of Neurology, Oregon Health Sciences Uniclersity, Portland, OR 97201, USA

Received 8 July 1994; revised 1 November 1994; accepted 1 November 1994

Abstract Previously, we constructed chimeras by injecting hematopoietic cells from experimental autoimmune encephalomyelitis (EAE)-susceptible SJL (H-2S) strain mice into severe combined immunodeficient (SCID) C.B-17scid/scid (H-2d> mice. These SCID mouse-SJL mouse hematopoietic cell chimeras developed passive EAE following adoptive transfer of PLP S139-151specific SJL T lymphocyte line cells, but were resistant to active EAE induced by primary immunization with PLP S139-1.51. In order to gain an understanding of the encephalitogenic potential of transplanted hematopoietic progenitors in SCID mouse-SJL mouse chimeras, we attempted to induce EAE in hematopoietic chimeras constructed with or without an additional SJL fetal thymus implant. Chimeras with the thymus implant were susceptible to passive and active EAE while chimeras without the thymus implant were susceptible to passive but not active EAE. Encephalitogenic, CD4+, TCR+ T lymphocytes were selected in vitro from PLP S139-151-immunized, thymus-implanted chimeras. These results showed that hematopoietic SJL progenitors developed into antigen-presenting accessory cells and immunocompetent encephalitogenic T lymphocytes following transplantation into SCID mice. The development of primary immune reactivity depended on a fetal thymus implant for expression in SCID mouse-SJL mouse chimeras. Keywords:

Experimental mice; SJL mice

autoimmune

encephalomyelitis;

Hematopoietic

1. Introduction

Immune responses mediated by T or B lymphocytes do not occur in SCID mice due to an absence of these cell types (Bosma et al., 1983; Schuler et al., 1986). Transplant rejection, autoimmune disease and specific immune responses to antigenic challenge are each absent in SCID mice (Bosma and Carroll, 1991). This combination of characteristics in SCID mice has provided a unique environment for studying the regulation and development of immune system functions arising from transplanted lymphatic and hematopoietic cells and tissues.

* Corresponding

author. Phone (503) 273 5177; Fax (503) 273 5351

0165.5728/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0165-5728(94)00179-O

chimeras;

Severe

combined

immunodeficient

(SCID)

The conditions required for development, survival and function of immunocompetent T cells in SCID mouse chimeras have remained uncertain. a/p TCRexpressing human T cells have been detected in SCID mouse-human hematopoietic cell chimeras constructed with a human thymus implant but not in chimeras constructed without a thymus implant (McCune et al., 1988), indicating that the human but not the SCID thymic environment was necessary for the development of LX//~ TCR-expressing T cells from transplanted human hematopoietic cells. In contrast, the presence of (Y/P TCR-expressing rat T cells in SCID mouse-rat hematopoietic cell chimeras constructed without a thymus implant has been reported (Surh and Sprent, 1991) and neonatal but not adult SCID mice were reconstituted with T lymphocytes after transplantation of T-depleted allogeneic mouse

156

R.E. Jones et al./Journal of Neuroimmunology57 (1995) 155-164

bone marrow cells (Cowing and Gilmore, 1992). Thus, T lymphocytes have been identified in some, but not all hematopoietic chimeras in SCID mice. However, conclusions regarding requirements for development of cellular immune functions in SCID chimeras have been limited because the development of meaningful antigen-specific, T cell-mediated immune responses from transplanted hematopoietic progenitors in SCID mice has not been described. Inbred SJL-strain mice (H-2S) are susceptible to passive experimental autoimmune encephalomyelitis (EAE) induced by the adoptive transfer of PLP S139151-specific SJL T lymphocytes and immunization with PLP S139-151 in complete CFA induces active EAE in this strain (Tuohy et al., 1988; Whitham et al., 1991a). SCID mice (H-2d), previously reconstituted with hematopoietic fetal liver tissue from SJL mouse donors, also developed passive EAE following adoptive transfer of encephalitogenic, PLP S139-151-specific SJL T lymphocytes but identical SCID mouse-SJL mouse hematopoietic cell chimeras were resistant to EAE induced by active immunization with PLP S139-151 (Jones et al., 1993). SCID mouse-Lewis rat hematopoietic cell chimeras constructed with a Lewis rat fetal thymus implant were susceptible to active EAE induced by PLP S139-151. Together, these studies raised the possibility that a thymus implant might be required for the development of encephalitogenic T lymphocytes from transplanted hematopoietic progenitors in certain SCID mouse-hematopoietic cell chimeras. Transplanted hematopoietic tissue-derived cells (e.g. macrophages, microglial cells) residing in the CNS and CNS parenchymal cells (astrocytes, endothelial cells) have each been shown to function as antigen-presenting cells (APC) in MHC-restricted, inflammatory immune reactions directed against neural antigens (Traugott et al., 1985; McCarron et al., 1986; Hinrichs et al., 1987; Massa et al., 1987; Hickey and Kimura, 1988; Myers et al., 1993). In SCID mouse-SJL mouse chimeras, transplanted SJL hematopoietic tissue-derived APC were present in the CNS at sufficient levels for the induction of passive EAE by encephalitogenic, H-2s-restricted, SJL T lymphocytes. Thus, resistance to the induction of active EAE in chimeras was not due to an absence of hematopoietic-derived, SJL APC in the CNS. For the present study, groups of SCID mouseSJL mouse chimeras were constructed by transplanting SJL hematopoietic tissue into SCID mice with or without an SJL fetal thymus implant. Chimeras, with or without the thymus implant, were compared for their ability to develop active or passive EAE. Thymus implanted chimeras were susceptible to both active and passive EAE and served as donors of CD4+, encephalitogenic T lymphocytes following immunization with encephalitogenic peptide. These results demonstrated conditions permitting the development and selection of

encephalitogenic T lymphocytes from hematopoietic progenitors within chimeric SCID mice.

2. Materials

and methods

2.1. Animals

Female SCID C.B-17scid/scid mice, 8-10 weeks old, were obtained from the SCID mouse breeding colony at the Portland Veterans Affairs Medical Center Animal Research Facility. Female SJL/J mice (6-8 weeks of age) and male breeder SJL/J mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Timed-pregnant (16 days of gestation) SJL mouse fetal-tissue donors were bred at the Portland Veterans Affairs Medical Center Animal Research Facility. 2.2. Ag The 43-64 and 139-151 peptides of myelin proteolipid protein (PLP) were synthesized by the Merrifield solid phase method as described (Hashim et al., 1986) and provided by Dr. George Hashim, St. Luke’sRoosevelt Center, New York, NY. The sequences of PLP peptides were as follows: PLP S43-64, (C)EKLIETYFSKNYQDYEYLINVI(G); PLP S139151, HCLGKWLGHPDKF (Whitham et al., 1991b). PLP residues were numbered according to the published sequence of bovine PLP (Laursen et al., 1984). Mouse BP was extracted from whole mouse brains and purified by ion exchange chromatography as described (Eylar et al., 1971). 2.3. Chimera construction SCID mouse-SJL mouse chimeras were constructed by the i.v. injection into SCID mice of 2 x 10’ fetal liver hematopoietic cells, obtained from 16-day gestational age SJL mouse fetal donors, prepared as a single-cell suspension in 0.3 ml buffered saline. In some hematopoietic cell chimera constructs, SJL mouse fetal thymus tissue (16-day gestational age) was surgically implanted beneath the SCID mouse renal capsule at the caudal pole of the left kidney (Fig. 1). Passive or active EAE was induced in SCID mouse-SJL mouse chimeras 5-7 weeks after chimera construction. 2.4. Induction of EAE Immunization of mice with PLP S139-151

peptide

Animals were immunized by S.C. injection in the flanks, at four sites, with 150 pg peptide in a total volume of 0.2 ml of a water-in-oil emulsion of CFA containing 200 pg heat-inactivated Mycobacterium tuberculosis (strain H37Ra) (Difco). Actively immunized

R.E. Jones et al. /Journal

of Neuroimmunology 57 (1995) 155-164

157

ated thymic accessory cells as a source of APC. After successive cycles of Ag stimulation and IL-2 expansion, cells were phenotyped, stimulated for passive transfer or assayed for mRNA expression.

SJL Mouse

Hematopoletic Liver

2.4. Spinal cord histology

Fig. 1. Chimera construction method. All chimeras in this study were constructed in SCID mice with transplanted SJL hematopoietic fetal liver cells @CID mouse-SJL mouse chimeras). Some chimeras (SCID mouse-SJL mouse+ thymus chimeras) also received an SJL fetal thymus implanted beneath the left kidney capsule.

mice were followed daily for appearance of clinical EAE or were used as donors for cell line selection. Transfer of SJL PLP S139-151-specific

T cells

Lymph node cells from peptide-immunized SJL mouse donors were selected in vitro with PLP S139-151 (5 pg/ml) through successive cycles of antigen stimulation and expansion in interleukin (IL)-2-containing medium, as described (Whitham et al., 1991a). The indicated number of peptide-stimulated T lymphoblasts were transferred by i.p. injection into naive SJL mouse, SCID mouse and SCID mouse-SJL mouse chimera recipients. Passive recipients were followed daily for appearance of clinical EAE. EAE severity was scored by assessing ascending paralysis on a scale of O-4, as follows: 0, no signs; 1, partial tail paralysis; 2, single hindlimb weakness; 3, single hindlimb paralysis; 4, single forelimb paralysis. The ‘ +’ symbol following the disease score signifies complete tail paralysis or signifies a clinical abnormality (weakness or paralysis) affecting both limbs. Results are expressed as the disease severity for each animal and the day of onset of disease for each animal. 2.5. Selection of antigen-specific cells from chimeras

Splenic mononuclear cells from actively immunized chimeras were prepared as a single-cell suspension and were stimulated in vitro for 3 days (10 X lo6 cells/ml) with PLP S139-151 (10 pg/ml) in stimulation medium (RPM1 1640; 2 mM L-glutamine; 1 mM sodium pyruvate (GIBCO); 5 X lop5 M 2-ME (Sigma); 1% (v/v) syngeneic mouse serum>. After stimulation, cells were expanded for 5-7 days in stimulation medium containing rIL-2 (Hazelton Laboratories) before being restimulated with peptide and syngeneic SJL mouse irradi-

Spinal columns were removed from mice during acute disease and immersion fixed in 10% buffered formalin. Fixed spinal cords were dissected from the surrounding tissue, and sections l-2 mm in length were embedded in paraffin. Four to five paraffin sections (10 pm) per cord were stained with LFB-periodic acid Schiff reagent-Hematoxylin, Hematoxylin and Eosin, or silver (Garvey et al., 1987). 2.7. Proliferation assay After expansion in IL-2-containing medium, 2 x lo4 selected line cells and 1 X lo6 irradiated syngeneic SJL mouse thymocytes per well in 0.2 ml stimulation medium were cultured for 3 days in a 96-well microtiter plate (Falcon) with medium alone (control), the T cell mitogen concanavalin A (ConA) (Sigma Chemical) (1.25 pg/ml), the PPD of M. tuberculosis (Statens Seruminstitut, Copenhagen) (2.5 pg/ml), mouse BP (5, 20 or 50 pg/ml), or the synthetic peptides PLP S139-151 or PLP S43-64 (5,20 or 50 pg/ml). Cells were labeled with [3H]thymidine (0.5 pCi/well) for the last 18 h of culture. After 3 days, cells were harvested onto glass fiber filters and [3H]thymidine uptake was measured by liquid scintillation counting. Results are expressed as the mean of triplicate wells. 2.8. Phenotype assay The established SCID mouse-SJL mouse chimeraderived cell line was phenotyped after expansion in IL-2. The following monoclonal antibodies specific for cellular differentiation markers were used as staining reagents with the appropriate FITC-conjugated second antibody: T cells, Thy 1.2; CD8, Lyt 2.2; CD4, L3T4 (Accurate Chemical). Monoclonal antibodies directed against mouse TCR VP chains were as follows: B20.6, Vp2; KT410, Vp4; F23.1, V/?S; KJ23, VP17a (Whitham et al., 19931. 10 X lo6 cultured hybridoma cells per mouse were injected into SCID mice primed 7 days previously with 0.1 ml pristane. Ascites fluid was harvested from SCID mice 8-14 days after cell injection. Affinity-purified anti-TCR antibodies were eluted from protein A (F23.1 and KJ23) or protein G (B20.6 and KT410) with 0.1 M glycine buffer (pH 3) and dialyzed against normal saline. Antibodies were used at 1 pg/106 cells with the appropriate FITC-conjugated second antibody. FITC-stained cells were analyzed on a Becton-Dickinson FACSCAN.

R. E. Jones et al. /Journal

158

of Neuroimmunology 57 (1995) 155-164

2.9. TCR VP mRNA detection

Table 1 Passive EAE in SJL, SCID and SCID mouse-SJL

Total cellular RNA was isolated from 2 X lo6 cells by phenol-chloroform extraction (Chomczynski and Sacchi, 1987). cDNA was synthesized from isolated mRNA using M-MLV reverse transcriptase (GIBCO, BRL) and the TCR Cp DNA primer, M3CB4(CP) (S-GCAATCTCTGCTTTTGATGGCTC-3’) in a total volume of 50 ~1. TCR VP-specific mouse sequences were amplified by PCR from 0.25~~1 aliquots of thawed cDNA using a single V/?-specific primer and the C/3-specific primer, M3CB4 (Cp> (0.285 PM each) in 35 ~1 of buffer (100 mM KCL, 100 Mm Tris, pH 8.3, 10 mM MgCl,) containing 2.5 U Taq polymerase (GIBCO-BRL), 350 PM dNTPs and 8.5% glycerol. The V/I primers used were: VP 1, 5’-TAAACAGTTGATTCGAAATGAGAC-3’; V/32,5’-CTGTTCACTCTGCGGAGTCCT-3’; Vp3, 5’-CAAGAAGTTCTTCAGCAAATAGAC-3’; Vp4, 5’-AGAGTTCATGTTTTCCTACAGCTA-3’; Vp5, 5’-CCCAGCAGATTCTCAGTCCAAC-3’; Vp6, 5’-GAGACTGATCTACTATTCAATAAC-3’; VP 7, 5’-CCTGGTCTGGGGCTACAGCT-3’; VPS, 5’CATGGGCTGAGGCTGATCCATT-3’; V/39, 5’TGATAAGATMTGAACAGGGAAGC-3’; V/310, 5’GCTACAATAATAAGCAACTCATTG-3’; VP 11, 5’CAAGCTCCTATAGATGATICAGG-3’; Vp12, 5’TTCCCCCTTATGGAAGATGGTG-3’; Vp13, 5’CCTAAAGGAACTAACTCCACTCT-3’; Vp14, 5’CCAGGTAGAGTCGGTGGTGC-3’, Vpl5, 5’-TTTCTACTGTGAACTCAGCAATC-3’; Vp16, 5’-

Recipient

EAE Incidence

Onset

SJL SCID SCID-SJL SCID-SJL+

5/5 o/5

88888 , >1,

3,3,3 + ,3 + ,4

00000 , ,1>

00000 ,, 2 2

4/5 5/5

0,21,23,24,32 16,22,24,21,27

0,1,2+,2+,3 1+,1+,2+,3+,3+

100

60

3

;

60

; 5 $

40

20

n

0 Control

Con A

PPD

Sl39151 Antigen

543-64

MoBP

Fig. 2. In vitro proliferation of PLP S139-151~selected SJL T cell line. Pooled lymph node cells from SJL mice immunized previously with PLP S139-151 in CFA were selected in vitro. Line cells were assayed in triplicate microtiter wells with no stimulus (control), ConA, PPD, and PLP S139-151, PLP S43-64 and mouse BP at 50, 20, or 5 pg/ml.

thymus

’

mouse

chimeras

a

Severity

a SCID mouse-SJL mouse chimeras (SCID-SJL) were constructed by injecting into 6-week-old SCID mice 2 X 10’ SJL mouse fetal liver cells alone @CID-SJL) or received also an SJL fetal thymus implant beneath the left kidney capsule (SCID-SJL+ thymus). 5 weeks after chimera construction, chimeras and age-matched SCID and SJL mice received by i.p. injection 12X lo6 encephalitogenic SJL mouse PLP S139-151specific T cells. Animals were scored daily for disease signs (see Materials and methods). b Cells were transferred on day 0. Onset was the 1st day on which disease signs appeared.

CAGATGGAGTTICTGGGTAC-3’; VP 17, STGGTCAAGAAGAGATTCTCAGCT-3’. PCR products were amplified through 30 cycles using a thermocycler (Perkin-Elmer 9600, Norfolk, CT). Each cycle included denaturation for 20 s at 94.5”C, annealing for 60 s at 60°C and extension for 60 s at 72°C. VP-specific PCR amplification products were detected in reaction mixtures by HPLC using a modification of a published method (Katz and Dong, 1990). PCR amplified reaction mixtures (35 ~1) were applied to a low volume, TSK DEAE-NPR, 2.5 pm diameter, non-porous resin, anion exchange column (Perkin Elmer) equilibrated with binding buffer consisting of 30% buffer A (25 mM Tris, 1.0 M NaCl, pH 9.0) and 70% buffer B (25 mM Tris, pH 9.0) using an automated gradient controller (Millipore-Waters 600E pump, UK6 sample injector, system control software). PCR amplification products were eluted from the column with increasing NaCl using a linear solvent gradient from 30 to 70% buffer A. PCR product peaks were detected by UV absorbance (260 nm, Millipore-Waters 990 diode array detector) and quantitated by automatic integration of the eluent absorbance profiles using a single, uniform set of peak recognition criteria (Waters 990+ software, NEC powermate 2 computer). The mean + standard deviation for each VP-specific PCR product amount was calculated using results from three separate assays of a single cDNA preparation. PCR reaction mixture tubes containing all reaction ingredients except the VP primer were used as negative controls and these produced no detectable PCR reaction products. Unamplified VP2-specific reaction tubes also produced no detectable PCR product. Furthermore, since V/35, 8, 9, 11, 12 and 13 are not expressed by SJL (Behlke et al., 19861, these VP reaction mixtures may be viewed as internal negative controls. Statistical analysis was accomplished by Student-New-

R. E. Jones et al. /Journal

of Neuroimmunology 57 (1995) 155-l 64

Fig. 3. SCID mouse-SJL mouse chimera spinal cord. Transverse section from the spinal cord of a SCID mouse-SJL mouse chimera (constructed with a fetal thymus implant) which received encephalitogenic T cells and developed EAE. This LFB-PAS-Hematoxylinstained section revealed prominent myelin loss (absence of the lipophilic Luxol fast blue pigment) in the posterior white matter. This view of the cord also showed a white matter cellular infiltrate with some lipid-laden cells. Magnification X 500.

man-Keuls multiple comparisons test between possible combination of paired reaction tubes.

every

3. Results 3.1.

Passive

EAE in SCID mouse-SJL

mouse chimeras

Lymph node cells from PLP S139-151-immunized SJL mice were selected in vitro with PLP S139-151 peptide. The resulting cell line proliferated upon in vitro stimulation with ConA or PLP S139-151. The cell line did not respond to the PPD of M. tuberculosis, a

Fig. 5. SCID mouse-SJL mouse chimera spinal cord. Higher magnification of adjacent Hematoxylin-Eosin-stained transverse section from same mouse as Fig. 3 revealed meningeal lymphocytic infiltrates and lateral white matter inflammatory lesions composed predominantly of darkly stained mononuclear cells and few polymorphonuclear leukocytes. Magnification x 1000.

component of the immunogen, CFA, or to the myelin antigens, PLP S43-64 or mouse BP (Fig. 2). 12 X 10h SJL PLP S139-151 line cells were transferred by i.p. injection into SJL and SCID mice and into SCID mouse-SJL mouse chimeras constructed with either SJL fetal liver cells alone or fetal liver cells and thymus (Table 1). As expected, the SJL PLP S139-151 specific T cells were encephalitogenic in SJL but not SCID recipients. SCID mouse-SJL mouse chimeras constructed with SJL fetal liver, and chimeras constructed with fetal liver and thymus also developed passive EAE. EAE in chimeras had a delayed onset compared with EAE in SJL recipients.

,-

Fig. 4. SCID mouse-SJL mouse chimera spinal cord. Adjacent transverse section from same mouse as Fig. 3 stained with silver revealed regions of axonal sparing and prominent axonal loss throughout areas where myelin loss occurred. Magnification X 250.

159

.-

.

,

\_‘

‘.

Fig. 6. SCID mouse-SJL mouse chimera spinal cord. Transverse section from the spinal cord of a SCID mouse-SJL mouse chimera (constructed with a fetal thymus implant) which was immunized with PLP S139-151 in CFA and developed EAE. This LFB-PASHematoxylin-stained section revealed prominent myelin loss in the posterior white matter and a white matter cellular infiltrate with some lipid-laden cells. Magnification x 500.

R.E. Jones et al./Journal

160

Table 2 Active EAE in SJL, SCID and SCID mouse-SJL

of Neuroimmunology 57 (1995) 155-164

mouse chimeras a

100

r

EAE

Recipient

Incidence

Onset b

Severity

5/5 o/5 o/5 4/4

11,11,12,12,12

2+,2+,3,3,3+

00000 t 9,) 00000 1, I >

00000 > I1 > o,o,o,o,o

17,31,31,31

1+,1+,1+,3

60

SJL SCID SCID-SJL SCID-SJL+ thymus

a SCID mouse-SJL mouse chimeras (SCID-SJL) were constructed by injecting into 6-week-old SCID mice 2 X 10’ SJL mouse fetal liver cells alone (SCID-SJL) or received also an SJL fetal thymus implant beneath the left kidney capsule (SCID-SJL + thymus). 7 weeks after chimera construction, chimeras received by S.C.injection 0.2 ml CFA containing 150 pg PLP S139-151 peptide and 200 pg Mycobac-

20

terium tuberculosis. b Immunization occurred on day 0. Onset was the 1st day on which

disease signs appeared. Ocmbol

T cells

CD8

CD4

‘AS

‘h4

VbB

vi17

Phenotype

Chimeras with passive EAE had multiple foci of histopathology in the lateral and dorsal spinal cord. Demyelination (Fig. 3), axonal loss (Fig. 41, and inflammation (Fig. 5) were present. 3.2. Active EAE in SCID mouse-SJL

mouse chimeras

Active immunization with PLP S139-151 peptide induced active EAE in SJL but not SCID mice (Table 2). SCID mouse-SJL mouse chimeras constructed without the thymus implant failed to develop active EAF whereas chimeras constructed with SJL fetal liver and thymus developed active EAE. Thymus implanted chimeras with active EAE had pathological changes

Fig. 8. Expression of differentiation antigens and TCR detected by monoclonal antibody staining. The PLP S139-151-specific cell line selected from SCID mouse-SJL mouse donors expressed phenotypic markers recognized by several monoclonal antibodies. The following monoclonal antibodies were used to detect specific differentiation molecules: Thy 1.2, pan T cell marker; Lyt 2.2, CD8; L3T4, CD4; B20.6, VP2 TCR; KT410, VP4 TCR; F23.1, VP8 TCR; KJ23, Vj317 TCR.

similar to those of chimeras with passively transferred EAE (Fig. 6). SCID mouse-SJL mouse chimeras which resisted active EAF had normal spinal cord histology. 3.3. T cell line selection from SCID mouse-SJL chimeras

mouse

Spleen cells from PLP S139-151 peptide immunized, thymus-implanted SCID mouse-SJL mouse chimeras with active EAE were selected in vitro with

Table 3 Passive EAE from SCID mouse-SJL SCID mice a

3

% 60-

Recipient

$ 5 Z 4o

SJL SCID

20 -

Control

PPD

Con A Antigen

Fig. 7. In vitro proliferation of SCID mouse-SJL mouse chimera-derived cell line. Pooled spleen cells from SCID mouse-SJL mouse thymus implanted chimeras immunized previously with PLP S139-151 in CFA were selected in vitro. Line cells were assayed in triplicate microtiter wells with no stimulus (control), ConA, PPD, or PLP S139-151 peptide at 50, 20, or 5 pg/ml.

mouse chimeras to SJL and

EAE Incidence

Onset b

Severity

5/5 O/5

11,12,13,13,14 00000 7, , ,

2,2 + ,2 + ,3,3

00000 , , t ,

a SCID mouse-SJL mouse chimera donors (SCID-SJL+ thymus) were constructed by injecting into 6-week-old SCID mice 2 X 10’ SJL mouse fetal liver cells and implanting an SJL fetal thymus beneath the left kidney capsule. 7 weeks after chimera construction, chimeras were immunized with PLP S139-151 peptide in CFA and developed EAE on days 17-31. On day 60, three chimeras with EAE were sacrificed and a pooled donor spleen cell culture was selected in vitro with PLP S139-151. After the second restimulation with antigen, 20x lo6 SCID-SJL line cells were injected i.p. into naive recipients. Recipient SJL and SCID mice were scored daily for disease signs (see Materials and methods). b Cells were transferred on day 0. Onset was the 1st day on which disease signs appeared.

R.E. Jones et al. /Journal

of Neuroimmunology 57 (1995) 155-164

I61

Table 4 Analysis PRIMER

of VP-specific ’

mRNA

expression

Experiment VP1 VP2 VP3 VP4 VP5 VP6 VP7 VP8 VP9 VP10 VP11 vp12 vp13 vp14 VP15 VP16 vp17 CP

by detection

of VP-specific

PCR products

B

Experiment

3

(AU x 10-6)*MIN

1763 8338 0 4701 0 1261 386 0 0 1224 0 0 0 3812 1026 1914 815 0

1

Experiment

2

649 5838 0 3970 0 613 0 0 0 1230 0 0 0 1522 584 274 0 0

0 7326 0 2949 0 2064 0 0 0 492 0 0 0 663 0 300 0 0

Mean k S.D.

P value

804 + 728 7167 f 1027 Ok0 3873 + 719 Ok0 1313 + 593 129 + 182 o*o O&O 982 f 346 o+o o+o Ok0 1999 & 1329 537 + 420 829 + 767 272 f 384 Ok0

> < > i > > > > > > > > > < > > > >

0.05 0.001 c 0.05 0.001 c 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 c 0.05 0.05 0.05 0.05

a Cellular mRNA was extracted from encephalitogenic SCID mouse-SJL mouse cell line, and used to make a cDNA preparation. cDNA was stored at -20°C until used in the V&specific PCR assay, as described in Materials and methods. Experiments 1, 2 and 3 represent three separate PCR assays of a single cDNA preparation. Assays using a different cDNA preparation from the same cell line gave similar results (not shown). (AU x 10m6) * min equals the integrated area under the PCR product elution absorbance peak at 260 nm wavelength. b All tubes contained identical reagents including the Cp primer and either one of the Vp primers (as indicated) or no Vp primer (Cp tube). ’ Significant response based on the maximal P value obtained by Student-Neuman-Keuls comparison against each of the other means. Thus. VP2 and VP4 were significantly different from all other VP PCR products (P < 0.001 for each comparison). VP14 was significantly different from all other PCR products (PC 0.05 for each comparison except for P 0.05 for each comparison except against Vp2, VP4 and Vp14).

PLP S139-151 peptide. The resulting cell line proliferated upon stimulation with ConA or PLP S139-151 (Fig. 7) but was unresponsive to the PPD of M. tuberculosis. This T helper cell line was positive for cell

Fig. 9. SJL mouse spinal cord. LFB-PAS transverse section from the spinal cord of an SJL mouse which received encephalitogenic T cells selected from a SCID mouse-SJL mouse chimera donor. The LFBPAS-Hematoxylin-stained sections from this donor had meningeal lymphocytic infiltrates and white matter mononuclear inflammation and demyelination and some polymorphonuclear leukocytes. Arrowheads are directed towards areas of myelin loss in the white matter. Magnification x 250.

surface expression of Thy 1.2 (88%), CD4 (99%), VP2 (26%) and V/34 (22%) TCR. The cell line did not express CD8 (5%), VP8 (1%) or V/317 (3%) TCR at significant levels above control, second antibody staining (2%) (Fig. 8). 20 X lo6 stimulated SCID mouse-SJL mouse line cells transferred clinical and histological EAE with paralysis (Table 3) and inflammation and demyelination (Fig. 9) into naive SJL recipients but not into SCID mice. mRNA isolated from this cell line contained sequences specific for VP2 and VP4 which were detected at highly significant levels. VP14 mRNA was also detected at less significant levels than VP2 and Vp4. All other VP mRNA sequences were present at insignificant levels, using the PCR method (Table 4). Spleen cells from actively immunized chimeras constructed without an SJL thymus implant did not respond to specific antigen in vitro and attempts to select a T cell line from actively immunized chimeras constructed without a thymus implant were unsuccessful, here and previously (Jones et al., 1993).

4. Discussion There has been considerable interest in the use of chimeras constructed in SCID mice for the study of autoimmune diseases (Duchosal et al., 1990; Tighe et

162

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al., 1990;Bosma and Carroll, 1991; Saeki et al., 1992; Martin0 et al., 1993). However, there remains uncertainty about how to optimally construct SCID chimeras, particularly for studying the development of diseasecausing T cells. Previously, SCID mice reconstituted with hematopoietic cells from allogeneic, EAE-susceptible, SJL donors resisted active EAE following immunization with encephalitogenic myelin peptide. The present study extends the previous observations by demonstrating conditions enabling the development of encephalitogenic T lymphocytes from transplanted hematopoietic progenitors within chimeras constructed in the SCID mouse. These findings provide useful information on the transplant requirements for constructing SCID chimeras that are susceptible to T cell-mediated autoimmune diseases. The development of encephalitogenic SJL T lymphocytes within thymus-implanted chimeras was demonstrated in two ways: (i) immunization with PLP S139-151 induced active EAE and; (ii) it was possible to select encephalitogenic, CD4+, TCR+, PLP S139151-specific SJL T lymphocytes from peptide-immunized chimera donors. Thus, transplanted SJL hematopoietic tissue retained the potential for T lymphocyte development in SCID hosts and expression of this developmental potential required an SJL thymus implant. Previous studies showed that adoptive transfer of cell-mediated immune responses and EAE required MHC compatibility between recipient APC and transferred antigen-specific T cells (Standage et al., 1985; Hinrichs et al., 1987; Myers et al., 1993). In the CNS of SCID mouse-SJL mouse hematopoietic cell chimeras, the only possible source of donor H-2s-compatible APC was the transplanted SJL hematopoietic cells. Spleens removed from SCID mouse-SJL mouse hematopoietic cell chimeras with passive EAE had sufficient SJL APC activity to induce in vitro, antigen-dependent proliferation of previously transferred SJL PLP S139151-specific cells in the absence of freshly added SJL APC (Jones et al., 1993), and it was possible here to select peptide-specific SJL T lymphocytes from peptide-immunized, thymus-implanted SCID mouse-SJL mouse chimeras. Therefore, the molecular and cellular interactions required for development of an immunologically functional APC population from transplanted SJL hematopoietic cells occurred within the SCID host and these transplant-derived APC repopulated multiple tissues within chimeras, including spleen and CNS. Consequently, chimeric SCID mice reconstituted with a source of APC, such as transplanted hematopoietic fetal liver or bone marrow cells, appear susceptible to autoimmune diseases induced by antigen-specific T cells that are histocompatible with the transplant-derived APC and histocompatibility with non-hematopoietic cells is not required.

The SCID mouse, C.B-17scid/scid strain is derived from a C57BL IgH-gene congenic BALB/c H-2d inbred strain (Bosma et al., 1983). Rodent strains such as BALB/c have genetic resistance to active EAE (Hughes and Stedronska, 1973; Singer et al., 1981; Ben-Nun et al., 1982). Genetically EAE-resistant rodent strains have been made susceptible to active EAE by transplantation of hematopoietic cells from genetically susceptible strains (Singer et al., 1981; Korngold et al., 1986; Matsumoto et al., 1990), but this was not sufficient for induction of active EAE in SCID mouseSJL mouse chimeras without the SJL thymus implant. Although susceptibility to active EAE may be controlled, in part, by the genotype of the hematopoieticderived cells, the present results suggest that development of PLP S139-151-specific, encephalitogenic SJL T lymphocytes depends on the genotype of the thymus in SCID mouse-SJL mouse chimeras. However, the architecture and cellularity of the SCID mouse thymic microenvironment is abnormal because the structure of the thymic medulla depends on hematopoietic-derived cells absent from the SCID thymus (Van Ewijk, 1991). Thus, in the absence of a thymus implant, the abnormal morphology of the SCID thymus, rather than genetic factors, may impede the development of encephalitogenic T lymphocytes from transplanted SJL hematopoietic cells. The participation of MHC class II in adoptive EAE and in vitro proliferation to BP has been demonstrated by inhibition with antibodies directed against MHC class II molecules (Sriram and Steinman, 1983; Offner et al., 1986) and rodent strain-specific resistance or susceptibility to active EAE has been correlated with depressed or elevated MHC class II gene expression by astrocytes in the resistant BALB/c and susceptible SJL strains, respectively (Massa et al., 1987). In the SCID mouse-SJL mouse chimeras constructed here, non-hematopoietic-derived cells (e.g. SCID, H-2d astrocytes) expressed the H-2d allotype, not the H-2” allotype. Thus, expression of the H-2” allotype by nonhematopoietic-derived cells in the CNS was not required for the development of an inflammatory response directed by H-2”-restricted, SJL T cells against target SCID, H-2d neural tissue components. Consequently, in chimeras, differential expression of MHC class II by non-hematopoietic cells (e.g. astrocytes) could not have influenced EAE susceptibility solely through an influence on MHC class II-restricted, antigen-presentation to encephalitogenic T lymphocytes. Encephalitogenic, CD4+, SJL T cell clones specific for PLP S139-151 express TCR V@2, Vp4, Vp6, VP10 or V/317a (Kuchroo et al., 1992; Whitham et al., 1993) and cells obtained from SJL EAE spinal cords expressed Vp2, VplO, VP16 and VP17 (Whitham et al., 1993). Two of the previously identified encephalitogenie, PLP S139-151-specific SJL VP TCR, VP2 and

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VP4 were expressed by SJL T lymphocytes selected from peptide-immunized, thymus-implanted SCID mouse-SJL mouse chimeras. Although V/?lCspecific PCR products were detected in the mRNA expression assay, it is not known whether this particular VP is also important in the encephalitogenic, SJL PLP S139-151 response. Thus, the T cell response to PLP S139-151 of SCID mouse-SJL mouse chimeras was similar to that of SJL mice, particularly with regards to encephalitogenicity and expression of the TCR VP2 and VP4 genes, and conditions within chimeras allowed the development of a typical SJL T cell response to antigenic challenge. The use of SCID mouse-human chimeras for the study of human autoimmune and infectious diseases has been described previously (Duchosal et al., 1990; Tighe et al., 1990; Bosma and Carroll, 1991; Saeki et al., 1992; Martin0 et al., 1993). The results here suggest that SCID mice reconstituted with hematopoietic precursors only, such as mature human T cell-depleted bone marrow cells, will resist active immunization due to an absence of detectable, functional antigen-specific T lymphocytes. The development of functional, immunocompetent T lymphocytes in SCID mouse-human hematopoietic chimeras will probably require special conditions for T lymphocyte development, such as a human thymus implant. However, SCID mouse-human hematopoietic chimeras without an immunocompetent T lymphocyte population still may be susceptible to passively transferred autoimmune diseases using human antigen-specific T cells. SCID mouse-human chimeras then may be useful for testing the ability of in vitro selected, human myelin-reactive T cells to induce demyelinating diseases.

Acknowledgements

The authors thank Verna Russell for preparing histology slides and Carolyn Steuer for expert animal care. This work was supported by the US Department of Veterans Affairs and the Medical Research Foundation of Oregon.

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