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The Journal of Immunology, 2005, 175: 69-81.
Copyright © 2005 by The American Association of Immunologists

Autoimmune CD4+ T Cell Memory: Lifelong Persistence of Encephalitogenic T Cell Clones in Healthy Immune Repertoires 1

Naoto Kawakami*, Francesca Odoardi*, Tjalf Ziemssen*, Monika Bradl{dagger}, Thomas Ritter{ddagger}, Oliver Neuhaus*, Hans Lassmann{dagger}, Hartmut Wekerle* and Alexander Flügel2,*

* Department of Neuroimmunology, Max-Planck-Institute for Neurobiology, Martinsried, Germany; {dagger} Neurological Institute, University of Vienna, Vienna, Austria; and {ddagger} Institute of Medical Immunology, Charité, Humboldt-University, Berlin, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
We embedded green fluorescent CD4+ T cells specific for myelin basic protein (MBP) (TMBP-GFP cells) in the immune system of syngeneic neonatal rats. These cells persisted in the animals for the entire observation period spanning >2 years without affecting the health of the hosts. They maintained a memory phenotype with low levels of L-selectin and CD45RC, but high CD44. Although persisting in low numbers (0.01–0.1% of lymph node cells) they were sufficient to raise susceptibility toward clinical autoimmune disease. Immunization with MBP in IFA induced CNS inflammation and overt clinical disease in animals carrying neonatally transferred TMBP-GFP cells, but not in controls. The onset of the clinical disease coincided with mass infiltration of TMBP-GFP cells into the CNS. In the periphery, following the amplification phase a rapid contraction of the T cell population was observed. However, elevated numbers of fully reactive TMBP-GFP cells remained in the peripheral immune system after acute experimental autoimmune encephalomyelitis mediating reimmunization-induced disease relapses.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Human autoimmune diseases, such as multiple sclerosis and rheumatoid arthritis, commonly take chronic, disabling courses. In some patients disease progresses steadily, while in others, relapses alter with remissions (1). According to present understanding, disease is caused and controlled by autoimmune T cells, which attack their target tissues, and thus determine activity and clinical character (2, 3). However, the nature of the autoimmune T cells over time remains largely unclear. In multiple sclerosis, for example, it is important to know whether different clones are recruited and activated from relapse to relapse. Newly recruited T cells could recognize distinct autoantigenic epitopes on the same autoantigen, or even be specific for different autoantigens, termed intra- and intermolecular determinant spreading, respectively (4, 5, 6, 7). As a less popular alternative, sequential disease episodes could be driven by members of the initial pathogenic clone(s) throughout the entire course of disease. Such a pathogenesis would invoke formation and persistence of autoimmune memory T cells (8).

Mechanisms of disease chronicity are difficult to study in model systems. This holds especially true for models induced by autoimmunization, because these either result in monophasic disease episodes with full and definite recovery, or, depending on large-scale irreversible tissue damage, take a chronic course. In this study, we established a model system which enabled us to study the fate of individual bona fide autoimmune memory cell clones in the Lewis rat. We transferred myelin basic protein (MBP) 3-reactive CD4+ T cells retrovirally engineered to express the gene of GFP (9, 10) into neonatal recipient animals. Using this technique we tracked and functionally characterized defined (autoreactive) memory T cell populations in vivo over time. The T cells integrated into the immune repertoire of immune-competent recipients leaving the endogenous immune system unaffected. They conferred T and B cellular reactivity to the recipient animals including specific T cell proliferation, proinflammatory cytokine release, and accelerated and increased autoantibody production. Importantly, the persisting autoreactive memory T cells raised the susceptibility toward autoimmune CNS inflammation and disease. After recovery from acute disease, they remained functionally fully intact in the peripheral immune system and, upon activation, evoked repeated disease episodes.

Thus, the data point toward long-term persistence of an autoimmunologic T cell memory as a critical factor in chronic autoimmune disease.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals, CD4+ TGFP cells, and neonatal transfer of TGFP cells

Lewis rats were obtained from the animal breeding facilities of the Max-Planck-Institute for Neurobiology (Martinsried, Germany) and were kept under standardized conditions. Ag-specific T cell clones used in the study were specific for guinea pig MBP and OVA. MBP was purified from guinea pig brains as described (11). Hen egg OVA was obtained from Sigma-Aldrich. All animal experiments were performed with the license of the Regierung von Oberbayern (No. 209.1/211-2531-56/99). CD4+ TGFP cells have been generated and tested for their phenotype, cytokine profile, and Ag specificity as described before (9). The lines consisted of oligoclonal CD4+ {alpha}{beta}TCR+ Th1 cells and were highly specific for their respective Ags (see Table I, see Fig. 7D). Intraperitoneal transfer of TGFP cells (2 x 106 cells, in 0.5 ml/animal in Eagle’s HEPES medium) into newborns was performed under hypothermia within 48 h after birth. After T cell transfer, the newborn rats were kept under a 30°C humid atmosphere until fully recovered, then returned to their mother. Four different TMBP-GFP and three different TOVA-GFP cell lines were used for neonatal transfer. The activation state of the T cells did not influence the capacity of T cells to become memory cells. In most of the experiments, the T cells were transferred 4–5 days after restimulation with Ag.


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  		Table I. Characterization of Ag-specific T cell lines

 


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  		FIGURE 7. Repeated immunization of memory animals with MBP/IFA. EAE in 6-mo-old memory-rats and age-matched non-memory control rats after reimmunization with MBP/IFA 3 mo after the first immunization round. Clinical scores (left axis, bars) and weight loss (right axis, lines). A, Double-immunized memory animals (black), first time-immunized memory rats (white). Means ± SD of four animals per group. B, First time-immunized non-memory control rats neither developed disease, nor weight loss (gray). Reimmunization of such control animals (black) produced mild clinical signs of disease. C–E, Reactivity of Tmemory cells after repeated immunizations. C, GFP+ Tmemory cells in LN cultures of double-immunized memory animals respond to specific Ag. LN cells isolated from first time-immunized 8-mo-old memory animals (triangles, blue) and double-immunized 18-mo-old memory animals (squares, red) exposed to MBP (dark color) or OVA (bright color). Relative cell numbers of GFP+ cells (days 2, 3, 4, or 5) divided by cell numbers at day 0. Triplicate values ± SD (never exceeded 5% of the original value). D, Cytokine profile of Tmemory cells (real-time PCR). GFP+ Tmemory cell isolated from LNs after 2-fold immunization ({blacksquare}). The cells were enriched in vitro by G418. mRNA was isolated from T cell blasts (>3 x 106 cells) 2 days after restimulation. Cultured counterparts of the same descendent T cell line ({cjs2108}). E, IFN-{gamma} secretion (ELISPOT). Numbers of IFN-{gamma}-producing cells in LNs of 2-fold-immunized 18-mo-old memory rats ({blacksquare}) and 12-mo-old non-memory control rats ({cjs2108}) after exposure to Ag or PMA/ionomycin. Quadruplicates from 106 LN cells/well (MBP/OVA) or 105 LN cells/well (PMA/iono).

 
Cell isolation, cytofluorometry, and FACS

Single-cell suspensions from organs were obtained as described previously (9, 10, 12). For cytofluorometric analysis, FACSCalibur operated by CellQuest software (BD Biosciences) was used (9, 10). The following mAbs were used for surface membrane analysis: W3/25 (anti-CD4; Serotec), R73 ({alpha}{beta}TCR), OX-6 (rat MHC class II), OX-40 Ag (CD134), OX-39 (CD25, IL-2R{alpha} chain), OX-22 (CD45RC), H-CAM (CD44), L-selectin (CD62L), V{beta} 8.2, 8.5, 10, 16 (all BD Biosciences). RPE-Cy-5-labeled anti-mouse Ab (DAKO) was used as secondary Ab.

Induction of adoptive transfer of experimental autoimmune encephalomyelitis (tEAE) and animal preparation

Adoptive transfer of the encephalitogenic T cell lines was performed by i.v. injection. The dose of T cells injected was adjusted to 5 x 106 T cell blasts/animal. Animals were monitored daily by measuring weight and examining disease scores (0, no disease; 1, flaccid tail; 2, gait disturbance; 3, complete hind limb paralysis; 4, tetraparesis; and 5, death).

Organ distribution of neonatally transferred TMBP-GFP and TOVA-GFP cells

Numbers of persisting TMBP-GFP following neonatal transfer were evaluated cytofluorometrically. The organs were carefully dissected and the relative numbers of GFP per organ cells were measured.

Histology

Histological analysis was performed as described (10). Briefly, after perfusion with 4% paraformaldehyde (PFA), lymph nodes (LNs) were prepared and postfixed in 4% PFA overnight. The frozen organs were cut into 20-µm sections. Following fixation for 30 min at room temperature with 4% PFA the sections were incubated with OX-33 (CD45RA, B cell marker, dilution 1/200; BD Biosciences) overnight at 4°C. Cy3-labeled anti-mouse antiserum (Dianova) was used as secondary Ab. The sections were counterstained with biotinylated anti-TCR{alpha}{beta} Ab (R73, dilution 1/300), detected by Cy5-labeled streptavidin (Molecular Probes). Fluorescence analysis was performed with confocal laser-scanning microscopy (Leica). Immunohistochemical staining for GFP and analysis with a Zeiss EM10 transmission electron microscope was performed as described (13). For quantification of GFP+ cells, organs were treated as described (14). The inflammatory index in spinal cord lesions was determined after quantification of T cells (W3/13, dilution 1/50; Harlan Seralab) and monocytes/macrophages (ED1, dilution 1/1000; Serotec) on five randomly selected complete spinal cord cross-sections per animal. The section area was determined using a morphometrical grid. The values represent means (±SD) of two individual animals per group.

Ex vivo reactivity assays

LN cells were cultured in 96-well plates (in DMEM 1% rat serum) without Ag, with ConA (2.5 µg/ml), or in the presence of specific Ag (10 µg/ml MBP or OVA, respectively), or with the irrelevant Ag purified protein derivative (10 µg/ml), respectively. [3H]dT (2 Ci/mmol; Amersham-Buchler) was added to the cultures after 48 h. The radioactive label present in the different cultures was determined as described (10).

Amplification of ex vivo-isolated GFP+ memory T cells was measured by cytofluorometry. Their numbers were determined in relation to a known absolute number of added PE-labeled plastic beads (BD Biosciences). The amplification rate was calculated in relation to the GFP+ T cell numbers at day 0.

Intracellular IFN-{gamma} staining, IFN-{gamma} ELISPOT

Intracellular IFN-{gamma} staining was performed with anti-mouse/rat IFN-{gamma} Ab (clone DB-1; BD Biosciences) as described (15). Control IgG (mouse IgG MOPC31) was obtained from Sigma-Aldrich. IFN-{gamma}-ELISPOT analyses were performed as described (15) using polyclonal goat anti-rat-IFN-{gamma} and biotinylated goat anti-rat-IFN-{gamma} antiserum (R&D Systems), and an automated imaging system equipped with appropriate computer software (KS ELISPOT Automated Image Analysis System; Zeiss).

Quantitative PCR

TaqMan analysis was performed as reported (10) using the ABI Prim 7700 Sequence Detector TaqMan (Applied Biosystems). For quantification of cytokine mRNAs, the expression of the cytokine mRNA was set in relation to a housekeeping gene ({beta}-actin). All PCR data were obtained by two independent measurements. The cycle threshold (CT) value of the measurements did not differ >0.5 amplification cycles.

Ab measurements

ELISA. Serial blood samples were obtained from tails of the rats. After clotting at 4°C, the sera were collected by centrifugation and stored at –20°C. ELISA was performed according to the protocol of Stefferl et al. (16). The rat sera were diluted 1/1000. Mouse mAbs specific for rat Ig and isotypes IgM, IgG1, IgG2a, IgG2b, and IgE were obtained from Serotec. Secondary goat anti-mouse peroxidase conjugate was purchased from Dianova (dilution 1/8000). O-phenylenediamine dihydrochloride (Sigma-Aldrich) was used as substrate. The reaction was stopped with 3 M HCl, and OD was determined at 490 nm.

ELISPOT. Plasma cells were determined by reversing the present sandwich method (17). Briefly, ELISPOT plates (MAHA N45; Millipore) were coated with goat anti-rat IgG (H+L) Ab (10 µg/ml, 100 µl/well in carbonate buffer, pH 9.3; Jackson ImmunoResearch Laboratories). After blocking (5% BSA-PBS), cells were added in a serial dilution (100 µl/well) in triplicates and incubated for 24 h. After removal of cells, plates were incubated for 2 h with biotinylated Ag (MBP or OVA, respectively), or biotinylated goat anti-rat IgG (H+L) Ab (0.5 µg/ml, 100 µl/well in 0.5% BSA-PBS; Jackson ImmunoResearch Laboratories). Spots onto the ELISPOT plates were visualized using streptavidin-alkaline phosphatase with 5-bromo-4-chloro-3-indolyl phosphate/NBT as substrate (Sigma-Aldrich), and analyzed with an automated ELISPOT reader system (KS ELISPOT; Zeiss, Jena, Germany). Total Ig-spots were considered total numbers of plasma cells, MBP/OVA-spots represented MBP/OVA-specific plasma cells. No second biotinylated detector was used as negative control.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Introduction of GFP-expressing CD4+ T cells into the immune repertoire by neonatal transfer

GFP-transduced CD4+ T cells (9) specific for MBP or OVA (OVA, TMBP-GFP, or TOVA-GFP, respectively), were injected i.p. into syngeneic Lewis rat pups (2 x 106 cells, within 48 h of life). Both TOVA-GFP and TMBP-GFP cells consisted of highly specific oligoclonal CD4+ T cell populations which displayed a Th1-like cytokine profile with high levels of IFN-{gamma} and TNF-{alpha}, but no IL-4 (Table I) (10). TMBP-GFP cells transferred clinical disease to adult animals, which in course and severity was indistinguishable from EAE transferred by their non-manipulated counterparts. However, neither of the MBP-specific T cells attacked newborn recipients (18, 19).

We traced the transferred, Ag-experienced T cells in the recipients over periods of >2 years. The cells settled throughout the immune system including LNs, spleen, thymus, and bone marrow (Fig. 1A), and they were also found in some non-immune organs including lung, liver, and gut. Importantly, however, they never spontaneously invaded the CNS (Fig. 1A). In LNs, the T cells populated the paracortical T cell areas and the medulla of the LNs. A few scattered green T cells were seen within lymph follicles (Fig. 1B). In the spleen, the persisting GFP+ T cells sat both in the periarteriolar sheaths of the white pulp and in the red pulp (data not shown). Within the thymus, most, if not all of the cells were located in the medulla (data not shown). The persisting T cells (Tmemory-MBP cells) did not demonstrably influence the host’s immune cell composition and immune competence (data not shown).



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  		FIGURE 1. Persistence and distribution of Tmemory cells. A, Persistence of Tmemory cells. TMBP-GFP cells were counted by cytofluorometry in the indicated tissues of 2-, 8-, 12-, and 25-mo-old rats. Relative numbers (10–3 x GFP+ cells/organ cells). Means ± SD of three individual animals. Representative data of at least two independent experiments. B, GFP+ Tmemory-MBP cells in parathymic LNs of 10-wk- (left panel) or 1-year- (right two panels) old memory rats. Tmemory-GFP+ cells: green, arrows. Left panel, TCR stain in red; yellow, autofluorescence. LF, Lymph follicle; M, medulla; PC, paracortex. Right panels, Double staining for TCR (blue) and B cell marker OX-33 (red). Lower panel, Inset of the indicated area of the upper figure. Bars, 10 µm.

 
The numbers of GFP-labeled T cells remained remarkably stable over time (Fig. 1A). Notably, after transfer of oligoclonal (instead of monoclonal) populations, the proportions of the clonal components were maintained (Table II).


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  		Table II. TCR V{beta} usage (%) of CD4+ T cellsa

 
Previous studies had shown that encephalitogenic T cells undergo profound phenotypic changes before and during clinical tEAE. During the 3–4 days preceding onset of disease, most of the T cells reside in the peripheral immune organs, where they down-regulate activation markers. Instead, they express other sets of genes, including chemokine receptors, required for their migratory activity. We termed T cells in this prodromal stage migratory T cells. Upon arrival in the CNS by days 3–4, the Tmigratory cells become maximally reactivated, assuming the full-blown effector phenotype, a process directly related to development of clinical disease (10).

We examined the membrane phenotype of memory T cells by comparing Tmemory cells, which had persisted in their host for 8 mo, with T cells directly from culture (Tculture cells), and with T cells that had been isolated from spleens of syngeneic adult animals 4 days after transfer, right before migration into the CNS and onset of EAE (Tmigratory cells) (Fig. 2A). Importantly, T cells from all stages were progeny of the same original GFP-transduced T cell line. Tmemory cells had low levels of activation markers IL2-R and OX-40. In addition, Tmemory and Tmigratory cells stained for MHC class II, which was missing in Tculture cells.



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  		FIGURE 2. Membrane phenotype of Tmemory cells. A, Cytofluorometric characterization of ex vitro TMBP-GFP cells (6 days after restimulation, red filled histogram, culture), of migratory TMBP-GFP cells isolated ex vivo 4 days posttransfer (p.t.) (blue overlay histograms, migratory), or of Tmemory-GFP cells from LNs of 8-mo-old memory rats (green overlay histograms, memory). Yellow histograms in A and B: membrane Ags of GFP-negative CD4+ T cells in LNs of memory animals. Orange histograms in A and B: membrane Ags of CD4+ T cells in LNs of non-memory animals. B, Memory phenotype of Tmemory-GFP cells 18 mo after neonatal transfer (violet histograms).

 
There was no evidence of Tmemory cells reverting back to a marker profile of naive T cells. Even after >1.5 years, Tmemory cells maintained their memory phenotype with high CD44 and low CD45RC (OX-22) and L-selectin (CD62L) expression (Fig. 2B). OVA-specific Tmemory cells behaved in all respects like their MBP-specific counterparts (data not shown).

Ag reactivity of Tmemory cells ex vivo

Immunological memory involves an accelerated and enhanced response against repeated Ag encounter. We compared the specific proliferation of LN cells from animals harboring Tmemory-MBP cells for 9 wk, 8 mo, or 1 year (MBP-memory animals). LN cells from all three groups of memory animals responded by specific proliferation, while their unmanipulated counterparts did not (Table III). The increased reaction of these memory LN cells was largely due to expansion of GFP-expressing Tmemory cells. Cytofluorometry demonstrated a massive increase of GFP+ T cells (~400-fold) within 5 days following exposure to the specific Ag (Fig. 3A). This response was seen in MBP as well as in OVA-specific T cells and was maintained even by memory cells residing in their host for 1 year. The multiplication rate of Tmemory cells exceeded the one of their in vitro cultured counterparts >10-fold (Fig. 3B).


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  		Table III. Memory rats respond to specific Aga

 


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  		FIGURE 3. Ag reactivity of long-term Tmemory cells. A and B, Proliferation of Tmemory-GFP cells (cytofluorometry). LN cultures were analyzed 0, 2, 3, 4, and 5 days following exposure to specific or control Ag. Amplification rate: NGFP cells days 2–5/NGFP cells day 0. A, GFP+ Tmemory cells of 12-mo-old OVA- (squares) or MBP-(circles) memory rats. Dark and bright blue squares: OVA-memory T cells exposed to OVA and MBP, respectively. Red and orange circles: MBP-memory cells exposed to MBP and OVA, respectively. Triplicate values ± SD (never exceeded >5% of the original value). Representative data of four independent experiments. B, Proliferative potential of Tmemory cells. Proliferation rates of cultured MBP-specific (red circles) or OVA-specific (green squares) T cells upon stimulation with their cognate Ag compared with their counterparts isolated from memory animals. Tmemory-MBP cells: magenta unfilled circles; Tmemory-OVA cells: blue unfilled squares. Tculture cells exposed to control Ags did not proliferate (data not shown). C, IFN-{gamma} secretion by LN cells from memory animals (ELISPOT). LN cells from 12-mo-old OVA- or MBP-memory rats (gray and black, respectively) and age-matched non-memory control rats (white) exposed to Ag or phorbol myristate ester plus ionomycin (PMA/iono). A total of 106 LN cells/well (MBP/OVA) or 105 LN cells/well (PMA/iono). Quadruplicate measurements of three pooled animals. D, Tmemory cells produce IFN-{gamma}. Intracellular IFN-{gamma} staining of GFP+ cells in LN cultures of 12-mo-old MBP-memory animals. The LN cells were exposed with MBP (left panel, MBP) or PMA/ionomycin (right panel). Filled histograms: isotype control. Red overlays: IFN-{gamma} staining. E, Th1 phenotype of the specific immune response in memory animals. LN cells from 12-mo-old MBP- or OVA-memory ({blacksquare} and {cjs2108}, respectively) or age matched non-memory control rats ({square}) were exposed to MBP (upper graphs) or OVA (lower graphs). Specific mRNA for IFN-{gamma} (left), IL-4 (middle), or IL-10 (right) in relation to {beta}-actin mRNA was determined by quantitative PCR. F, Encephalitogenic potential of GFP+ Tmemory cells. Transfer EAE of GFP+ Tmemory cells isolated from LNs of 6- and 12-mo- (data not shown) old MBP-memory rats (MBP, clinical score: black bars, weight loss: black squares and lines). Control animals (control, white) received activated TOVA-GFP blasts. Means ± SD of three rats per group.

 
Tmemory cells maintained the Th1-like cytokine phenotype. We used intracellular IFN-{gamma} staining, IFN-{gamma} ELISPOT assays, and real-time PCR for IFN-{gamma}, IL-4, and IL-10 to monitor the cytokine responses of LN cells isolated from rats harboring Tmemory cells for 12 mo. Tmemory-OVA cells containing LN cells exposed to OVA developed a high number of IFN-{gamma}-producing cells. Exposure of the same populations to MBP did not evoke any significant response. Conversely, LN cells from 12-mo-old MBP-memory rats mounted a specific IFN-{gamma} response toward exposure to MBP, but not to OVA (Fig. 3C). Unmanipulated control rats responded neither to OVA nor to MBP, while nonspecific stimulation with PMA/ionomycin induced high numbers of IFN-{gamma}-producing spots in all cultures including controls (Fig. 3C). The ELISPOT results were confirmed by intracellular IFN-{gamma} staining and cytofluorometry, which revealed that ~50% of ex vivo-isolated GFP+ Tmemory-MBP cells produced IFN-{gamma} upon exposure to the specific Ag. After maximal stimulation with PMA/ionomycin >80% were positive (Fig. 3D). Quantitative PCR confirmed a strong and specific IFN-{gamma}-dominated response within the memory LNs (Fig. 3E).

Tmemory-MBP cells also maintained their encephalitogenic potential. GFP+ TMBP cells were isolated from LNs of MBP-memory animals 6 and 12 mo after neonatal transfer and selectively expanded in vitro under negative selection with neomycin-derivative G418. The purity of GFP+ MBP-specific T cells exceeded 98% (data not shown). Adoptive transfer of these activated TMBP blasts (5 x 106/animal) induced severe adoptive transfer EAE (disease scores of 3, Fig. 3F).

Active induction of EAE in MBP-memory rats

We never noted spontaneous EAE in rats harboring Tmemory-MBP cells. To examine susceptibility of these animals to classical actively induced EAE, we first immunized memory rats with MBP in CFA (MBP/CFA, Fig. 4A). Surprisingly, MBP-memory animals showed disease responses similar to naive age-matched control rats. Although EAE started slightly earlier in memory animals (day 5 or 6 in MBP-memory and day 11 or 12 in control animals), disease severity was milder than in control rats.



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  		FIGURE 4. Active EAE induction in memory rats. A, EAE induction by MBP/CFA. Clinical signs of disease (left y-axis, bars) and weight changes (right y-axis, curves) of 12-mo-old memory animals (black) and age matched non-memory control rats (white). Means ± SD of six animals/group. B, EAE induction by MBP/IFA. Immunization of 6-mo-old memory rats (black) and age matched non-memory controls (white). Representative data of three independent experiments. Means ± SD of four animals/group. C, GFP+ cells in CNS infiltrates of MBP/IFA induced EAE lesions (cytofluorometry). Absolute numbers of TGFP-MBP cells (right axis, white) and their percentage within all infiltrating CD4+ T cells (left axis, gray bars). Means ± SD of three independent measurements. D, Distribution of GFP+ T cells in EAE lesions. GFP+ T cells (brown) within meninges (upper panel, closed arrows, open arrows: submeningeal parenchyme), perivascular areas (left lower panel, closed arrows, open arrows: parenchyme), and the parenchyme of the white matter (WM, open arrows) and gray matter (GM: closed arrows, right lower panel). Magnification bars, 10 µm. E, Cytokine pattern in EAE lesions. Real-time PCR from spinal cord tissue 8 day p.i. of 6-mo-old MBP-memory rats immunized with MBP/IFA ({blacksquare}) or OVA/IFA ({cjs2108}). {square}, Age-matched non-memory control rats immunized with MBP/IFA. Means ± SD of three animals/group.

 
In marked contrast, MBP-memory rats showed a superior response against MBP in IFA (MBP/IFA), a regimen, which fails to induce EAE in naive Lewis rats. MBP/IFA-treated MBP-memory animals developed clinical disease with clinical scores of 1–2 (Fig. 4B). Clinical signs started at day 7, and peaked by days 10–12, and paralleled the development of inflammatory CNS infiltrates (Fig. 4, C and D; Table IV). Control animals showed neither clinical nor histological changes during the acute phase. Very mild and clinically silent CNS inflammation was observed in control rats at day 21 postimmunization (p.i.) (inflammatory index 0.19, data not shown).


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  		Table IV. Distribution of TMBP-GFP cells after actively induced EAEa

 
The CNS lesions of MBP/IFA-treated memory animals were indistinguishable from classical EAE, and were dominated by T cells and activated monocytes/macrophages (data not shown). Importantly, in the early EAE lesions, up to 50% of all CD4+ cells were GFP+ Tmemory-MBP cells, a proportion that decreased to <5% at day 14 p.i. (Fig. 4C). Real-time PCR of CNS tissue 8 days p.i. with MBP/IFA documented a clear elevation of Th-1 cytokines in the infiltrates of memory animals but not in controls (Fig. 4E).

Kinetics and reactivity of Tmemory cells in the course of EAE

After active immunization with MBP/IFA GFP+ T cells in the draining LNs, the control LNs and the spleen were quantified by serial cytofluorometry. In the draining LNs, the number of Tmemory-MBP cells increased steeply (up to 20-fold) to reach a peak 8 days postimmunization (Fig. 5A). Immunofluorescence of the LNs showed numerous reactivated T cells with increased size and up-regulated production of GFP. Quite commonly, in blastoid cells the transgenic GFP was not restricted to the cytoplasm, but permeated into the nucleus, indicating disintegration of the nuclear membrane in association with cell division (Fig. 5C). Accordingly, numerous mitotic TGFP cells could be identified corresponding to the massively amplified number of GFP+ cells (Fig. 5, A and C). Proliferation of Tmemory-MBP cells in spleens and non-draining LNs was markedly lower (5- and 3-fold, respectively), and was noted after a delay of 2 days (Fig. 5A). Within 24 h p.i., GFP+ T cells in the draining LNs showed clear signs of reactivation. IL-2R, OX-40 Ag, and transferrin receptor (OX-26) came up and peaked by day 8 p.i., while the {alpha}{beta} TCR (TCR{alpha}{beta}) complex was down-modulated (Fig. 5D). By day 21 p.i. Tmemory cells had returned to their resting state (Fig. 5D). However, the numbers of the autoreactive GFP+ Tmemory cells in LNs remained elevated at least for the observation period (60 days p.i., Fig. 5A). After immunization with OVA, we did not observe a significant increase of Tmemory-MBP cells in the peripheral immune organs nor did Tmemory-MBP cells infiltrate into the CNS (Fig. 5B).



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  		FIGURE 5. Reactivation of Tmemory cells in vivo by Ag/IFA. A, Amplification of Tmemory cells in vivo (cytofluorometry). GFP+ cells in draining LNs, nondraining LNs, and spleens of 6-mo-old MBP-memory animals 1, 4, 6, 8, 14, 21, and 60 days p.i. with MBP/IFA. Absolute numbers of GFP+ cells/106 organ cells. Triplicate measurements of three pooled animals. Representative data of two independent experiments. B, Kinetics of MBP-specific Tmemory cells after immunization with control Ag. Numbers of MBP-specific Tmemory cells in draining LNs, non-draining LNs, spleens, and CNS of 6-mo-old MBP-memory animals 4, 8, and 14 days p.i. with OVA/IFA. Absolute numbers of GFP+ cells/106 organ cells determined by cytofluorometry. Triplicate measurements of three pooled animals. C, GFP+ Tmemory cells in vivo. Upper panel, Tmemory cells (arrows, brown diaminobenzidine (DAB) stain for GFP) in the draining (popliteal) LNs (paracortical area) of 3-mo-old memory animals 4 days p.i. Middle panels, Confocal microscopy with Topro staining (red; Molecular Probes) labeling nuclei of LN cells. Green: GFP. Left panel, Tmemory cells (arrows) in LNs before immunization. Right panel, Tmemory cells 4 days p.i. Lower four panels, Immunoelectron microscopy of Tmemory cells within LNs before immunization (upper left panel) and in different phases of the cell division 4 days p.i. (IFA). The electron dense DAB stain of GFP imposes as dark grains. Note the enlargement of the cytoplasm and the chromatolysis of the nucleus (upper right panel) followed by disintegration of the nuclear membrane (lower left panel). Lower right panel, A mitotic Tmemory cell. Magnification bars, 10 µm. D, Activation of GFP+ Tmemory cells. Draining LNs (popliteal and inguinal LNs, red line histograms) overlaid on non-draining LNs (cervical LNs, filled histograms) on days 0, 1, 4, and 21 p.i. with MBP/IFA. GFP+ cells were examined for the membrane markers as indicated. E, IFN-{gamma}-producing cells (IFN-{gamma}-ELISPOT assay) in draining LNs 4, 6, 8, 14, 21 days p.i. with MBP/IFA of MBP-memory rats (black) and age matched non-memory control rats (gray). Absolute numbers of IFN-{gamma}+ dots/106 organ cells after addition of MBP (left y-axis, bars) or without stimulation (right y-axis, lines). Means of quadruplicate measurements ± SD. LN cells of three individual rats per time point were pooled.

 
Tmemory cell-amplification after immunization with MBP/IFA tightly correlated with the number of IFN-{gamma}-producing cells in LNs displayed by ELISPOT assays (Fig. 5E). In contrast, MBP/IFA-immunized non-memory control rats did not raise a significant IFN-{gamma} response (Fig. 5E).

B cell support by Tmemory cells

One crucial effector function of CD4+ T cells is to support specific Ab generation. To detect a possible effect of Tmemory cells on B cell responses, we studied memory and, in parallel, age-matched naive rats immunized with Ag in IFA. MBP-memory animals responded to MBP faster and reached higher titers of specific anti-MBP IgG and IgM Abs than did naive control rats (Fig. 6, B–D). This response was specific, because the reaction of MBP-memory rats against control Ag OVA/IFA did not differ from the one of the naive animals (Fig. 6A).



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  		FIGURE 6. Tmemory cells accelerate specific Ab response. A, Ab response (ELISA) to control Ag. Anti-OVA IgG Ab response of 3-mo-old MBP-memory animals (black) and age matched non-memory control rats (white) immunized against OVA/IFA after 0, 4, 6, 8, and 12 days p.i. Means ± SD of four rats per group. Titers measured in quadruplicate assays. B, Ab response to memory Ag. Three-month-old MBP-memory animals (black) developed earlier and higher titers of specific IgG p.i. with MBP/IFA than age-matched non-memory control rats (white, p < 0.001, *). Representative data of at least three independent experiments. C, Ab response is age independent. Anti-MBP Ab response in 12-mo-old memory animals (black) and age-matched non-memory control rats (white, p < 0.001, *). Means ± SD of four rats per group. D, Acceleration and enhancement of specific IgM responses. Anti-MBP IgM titers of 12-mo-old memory rats (black) and age matched non-memory control rats (white, p < 0.001, *). Means ± SD of four rats per group. E, Elevated numbers of specific Ab-forming B cells. Numbers of anti-MBP Ig-forming B cells determined by ELISPOT in 12-mo-old MBP-memory animals (black) and age matched non-memory control rats (white). LN cells of three individual rats per time point were pooled. Mean values ± SD of quadruplicates (p < 0.001, *). F, Isotypes of anti-MBP Ig in memory rats (memory) and age-matched non-memory control rats (control). Ig isotypes (ELISA) in 12-mo-old memory and control rats at the peak of Ab response (8 days p.i. in memory animals and 12 days p.i. in controls). Means ± SD of four rats per group.

 
This accelerated and enhanced Ab response against specific memory Ag was noted throughout the life of memory rats. Thus, the Ab response was similar in 3- and 12-mo-old memory animals (Fig. 6, B and C). The kinetics of Ab titers in memory animals were paralleled by an increase of specific Ab-secreting B cells in the draining LNs as determined by ELISPOT analyses (Fig. 6E). Unexpectedly, however, the anti-MBP response did not follow a clear Th1-like pattern. The majority of anti-MBP Abs in both memory and control animals were of the IgG1 and IgG2a subclass, with a slight dominance of IgG2a levels in memory rats, and IgG1 in control rats (Fig. 6F). Similar data were obtained with OVA-memory animals (data not shown).

Tmemory-MBP cells survive actively induced EAE episodes and remain functionally responsive

We previously showed that in tEAE, most of the transferred effector T cells are lost both from the CNS and from the peripheral immune system (10). We now observed that Tmemory-MBP cells behave very differently. Although they were effectively cleared from the CNS following an acute EAE bout (Fig. 4C), they persisted in the periphery even in elevated numbers (Fig. 5A). Furthermore, these cells remained functionally intact. Reimmunization of memory rats with MBP/IFA 3 mo after a first EAE attack led again to clinical disease starting 7 days p.i., and this second EAE episode became more severe (weight loss, clinical score 2–3: hind limb pareses) than the preceding one (Fig. 7A). Control animals undergoing repeated immunization with MBP/IFA also developed EAE though with mild intensity (no weight loss, clinical score 0.5: partial tail paresis) indicating that the animals had raised and maintained autoreactive memory T cells after the first immunization (Fig. 7B).

We determined the numbers and the reactivity of Tmemory cells after repeated immunizations. Tmemory cells 2 mo after the first and 14 mo after the second immunization (>18 mo after neonatal transfer) persisted in frequencies of 0.17% (±0.02%) and 0.02% (±0.001%) of LN cells, respectively. Upon Ag exposure the Tmemory cells massively amplified (Fig. 7C, >1500-fold). Real-time analysis of the cells revealed that they maintained their Th-1 cytokine profile (Fig. 7, D and E). Further, they remained encephalitogenic: after expansion in vitro and transfer to healthy recipients, the Tmemory cells induced severe clinical EAE (maximal clinical score 3, data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Formation of memory responses is a hallmark of the adaptive immune system. A first encounter of a particular Ag conditions the immune system to respond faster and more vigorously when exposed repeatedly to Ag. The memory response is due to the expansion and differentiation of Ag-specific memory cells which are more sensitive to the Ag and are able to produce effector molecules more efficiently than their naive progenitors (20).

Immunological memory has evolved to warrant rapid and efficient elimination of microbial agents that repeatedly enter the organism. As a rule, immunological memory builds up, following successful elimination or neutralization of the Ag from the organism. In contrast, persistence of Ag, like in chronic infectious diseases, often leads to the exhaustion of the immune response (21). In autoimmune responses, the target autoantigen is not eliminated, but persists throughout life. Thus, would encephalitogenic T cells build up memory against autoantigens, and if so, do memory cells play a role in the course of chronic or relapsing autoimmune disease? The long-lasting persistence of autoimmune T cell clones in human blood (22, 23, 24), and the memory phenotype displayed by at least some of these cells (25, 26, 27) may argue in favor of autoimmune memory. The opposite prediction would come from EAE studies, where priming of rats with MBP in CFA or IFA confers protection from subsequent EAE induction, but does not prime for enhanced inducibility (28).

A model to study autoimmune memory is required to allow identification and functional characterization of autoreactive memory T cell populations in an intact immune repertoire over extended periods of time. We describe here an experimental system which seems to satisfy these requirements. The model is based on the introduction of labeled autoreactive T cells into the neonatal, immature immune system, and their analysis in adulthood. The retrovirally transduced, GFP-expressing T cell lines lent themselves for our investigations, because they replenish their fluorescent label over years both in vivo and in vitro. Furthermore, as described before, retroviral manipulation does not interfere with the programmed function of the cells (9, 10). Although the GFP-labeled T cells produce a foreign protein which would cause their eventual rejection in adults, the cells are tolerated lifelong after transfer into neonatal hosts. Our model is based on previous work which had established that encephalitogenic T cells transferred into neonatal hosts neither induce EAE nor activate counterregulatory T cell loops (18, 19). Instead, the inoculated T cells persist in the recipients’ immune organs throughout life (29). The late formation of CNS myelin (30) may be one factor responsible for the resistance to transferred disease. A deficit in T cell responsiveness and susceptibility to tolerance induction of the young immune system may be another factor (31).

Neonatal tolerance of foreign cells is not a simple phenomenon of passive acceptance, but relies on several mechanisms. These may include deletion of the grafted T cells from the host’s repertoire, the activation of regulatory T cells, and the formation of anti-inflammatory milieus (32). Obviously, any of these mechanisms could influence the function of the myelin autoreactive T cells. However, this was not the case in our model. The GFP-labeled MBP-specific T cells maintained their functional properties throughout their persistence in the hosts’ immune tissues. Their responsiveness against Ag remained unimpaired and they maintained their full encephalitogenic potential.

Transfer of mature T cells into neonatal organisms may be a less artificial situation, as it may appear on first glance. It should be kept in mind that the introduction of foreign, i.e., maternal immune cells into a neonatal organism is a natural event. T cells enter the fetus via placenta, and the neonate via colostral milk (33). Maternal immune cells, which in humans preferentially display memory properties, are demonstrable for long periods of time (34). They may well influence reactivity of the maturing immune system for better or worse (35).

The phenotypic stability of in vivo persisting memory T cells was remarkable. Even after periods of >1.5 years, the T cells maintained their "memory phenotype", with high levels of CD44, but low levels of CD45RC and L-selectin (Fig. 2B). The cells never showed any reversion toward a "naive" phenotype, as has been described in murine models (36, 37). Furthermore, the memory T cells retained a stable cytokine response pattern. When isolated from adult rats, GFP-labeled MBP-specific T cells responded to specific Ag by prompt production of Th1-like cytokines, i.e., IFN-{gamma}, TNF-{alpha}, and IL2. Th1-like responses were also triggered in vivo. This phenotypic stability is in contrast with memory T cells transferred into adult RAG mutant mice, which turned either Th1 or Th2 depending on the nature of the antigenic stimulus applied (38). Did the autoimmune T cells persist as "central" or "effector" memory cells, categories that had been invoked in human (39) and mouse (40) studies? Although we did not study chemokine receptors, the low L-selectin levels and their functional properties would be compatible with the "effector memory" option.

Although our memory rats contained considerable numbers of highly autoreactive T cells, none of them ever developed spontaneous EAE bouts. In contrast to naive rats, however, memory rats were susceptible to EAE induction by MBP in IFA, and such treated rats responded with accelerated formation of anti-MBP Abs, especially of the IgG2a isotype. But, unexpectedly, the reaction of memory rats to MBP/CFA was barely enhanced. An MBP/CFA stimulus triggered a slightly accelerated EAE response (though without increased severity). The memory T cells, which respond to autoantigen so promptly in vitro, either are shielded from activating signals in vivo, or they are under the tight control of down-regulatory mechanisms.

One regulatory loop was established by the work of Cohen and colleagues (41) who showed that intact or attenuated encephalitogenic T cell lines protect adult Lewis rats from subsequent attempts to induce EAE. The vaccination is due to the activation of CD8+ regulatory T cells, which suppress encephalitogenic T cells in vivo and destroy them in vitro (42, 43). In the mouse, CD8+ regulatory T cells recognize clonotypic structures of the target CD4+ T cells in the context of atypical MHC class Ib proteins (44, 45). This mechanism is not effective in our system, because transfer of encephalitogenic T cells into neonatal recipients fails to recruit protective CD8+ T cells (19).

Another regulatory loop which has resurfaced recently is CD4+CD25+ regulatory T cells, which down-regulate the pathogenic potential of autoreactive cells in vivo and in vitro via membrane-dependent contacts (46, 47). Control by regulatory cells, however, is less likely in our system, as demonstrated by strong proliferation and cytokine production of the GFP+ memory T cells in vitro as well as in vivo.

Autoimmune T cells undergo radical changes on their way into the target organ. They must be maximally activated to become pathogenic (48). But when introduced into a naive recipient, the T cells first settle in peripheral immune organs, where they assume a "migratory phenotype", which involves a profound reorganization of the membrane protein pattern including down-regulation of activation markers IL-2R and OX-40 and induction of chemokine receptors. Upon arrival in the CNS, the autoimmune T cells become reactivated again (10). The memory T cells described here resemble resting T cells with regard to their membrane phenotype, but functionally, they are distinct. Even after repeated immunizations and persistence for >1.5 years, the proliferative response of memory T cells to Ag by far exceeded the one of resting cultured T line cells or ex vivo isolated migratory T cells (Figs. 3 and 7). Memory T cells amplified several 100-fold, in contrast to the other T cell stages with maximally 20–50 amplification rounds. One possible explanation could lie in an intrinsic property of the proliferative machinery of the memory T cells allowing higher cell division rates. Alternatively, lower levels of apoptosis, e.g., based on elevated levels of antiapoptotic molecules might enable the cells to proliferate even under suboptimal stimulation conditions (49, 50).

Which are the factors that keep MBP-specific memory T cells in vivo alive over years? The roles of Ag-dependent signals and cytokines of the local milieu for persistence of memory CD4 T cells are matters of debate. Cytokines, such as IL-7 and IL-15, are considered as potential candidates as well for CD8+ and CD4+ memory T cells (51, 52). Several transfer studies using recipients lacking either relevant Ag or MHC products argue against a major role of continuous antigenic signaling (53, 54, 55). However, other work indicates that contact with Ag helps to maintain Ag reactivity of memory cells over time (56). It is not established whether MBP-specific memory T cells would find their nominal Ag presented by APCs in the peripheral lymphoid tissues, where they persist. Definitely, MBP-related genes are expressed in cells of the lymphoid system (57, 58), but to date immunogenic presentation of these proteins has not been shown with certainty.

The present memory model allows investigations of the fate of autoimmune T cells beyond a disease episode, a clinically important question, which so far has eluded detailed investigation. In previous studies we analyzed the migratory behavior of GFP-labeled encephalitogenic T cells on their way to and within the CNS tissue (9, 10, 15). However, due to antigenicity of the GFP protein, it was impossible to reliably trace the cells for periods longer than 1 wk. Our results now show that memory T cells integrated in a naive host’s immune system can be activated in vivo to contribute to a transient EAE attack. Most important, while numerous effector cells are deleted in the lesion, another substantial proportion of the cells remains within the lymphoid system, where it can be reactivated at a later time point.

In conclusion we show here that autoreactive memory T cells can persist in a healthy organism essentially throughout life. Under "regular" circumstances, they do not spontaneously produce disease, but they clearly participate in induced autoimmune attacks. It is therefore reasonable to conclude that autoimmune memory T cells may participate in human chronic autoimmune disease, such as in multiple sclerosis, and as we hope, animal models of autoimmune memory may contribute to better understanding of these long-lasting disorders.


   Acknowledgments
 
We thank Simone Bauer, Ingeborg Haarmann, Katrin Vogt, Dietmute Büringer, and Karin Brückner for excellent technical assistance. We thank Drs. Klaus Dornmair, Edgar Meinl, and David Johnson for critically reading the manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 The work was supported by the Deutsche Forschungsgemeinschaft (SFB 455) and the European Community (Mechanisms of Brain Inflammation: QLG3-CT-2002-00712). Back

2 Address correspondence and reprint requests to Dr. Alexander Flügel, Max-Planck-Institute of Neurobiology, Department of Neuroimmunology, Am Klopferspitz 18, 82152 Martinsried, Germany. E-mail address: Fluegel{at}neuro.mpg.de Back

3 Abbreviations used in this paper: MBP, myelin basic protein; p.i., postimmunization; EAE, experimental autoimmune encephalomyelitis; tEAE, adoptive transfer of EAE; PFA, paraformaldehyde; LN, lymph node. Back

Received for publication December 30, 2004. Accepted for publication April 6, 2005.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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