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The Journal of Immunology, 1998, 161: 5321-5330.
Copyright © 1998 by The American Association of Immunologists

The Nephritogenic T Cell Response in Murine Chronic Graft-Versus-Host Disease1

Catherine M. Meyers2,*, John E. Tomaszewski{dagger}, Joan D. Glass* and Clarice W. Chen*

* Department of Medicine, Renal-Electrolyte and Hypertension Division, and {dagger} Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To investigate mechanisms of cell-mediated events in chronic glomerulonephritis, T cell clones were isolated from kidneys of animals with murine chronic graft-vs-host disease. This systemic disorder is induced in normal (C57BL/6 x DBA/2)F1 recipients (H-2b/d) following transfer of parental (DBA/2) T cells (H-2d). These studies demonstrate that mouse renal (MR) T cells isolated from nephritic kidneys of diseased recipients are host-derived CD4+ {alpha}/ß+ T cells. Adoptive transfer of a panel of MR clones to naive (C57BL/6 x DBA/2)F1 recipients reveals distinct functional subsets. One subset does not transfer renal disease, and one induces severe renal inflammation and damage. In vitro proliferative responses of nephritogenic MR clones reveal predominant reactivity toward autologous class II MHC (I-Ed/I-Ad) determinants, and selected nephritogenic MR clones preferentially recognize renal Ag preparations derived from normal (C57BL/6 x DBA/2)F1 kidneys. In addition, cytokine profile analysis of MR clones indicates a Th2 pattern with IL-4 and IL-10 expression, although nephritogenic T cell clones also express IFN-{gamma}. These data suggest that the nephritogenic T cell response in chronic graft-vs-host disease is autoreactive in nature and may be restricted by determinants shared by both graft and host (Iad).


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Murine chronic graft-vs-host disease (cGVHD)3 is induced in genetically susceptible animals by adoptive transfer of parental CD4+ T cells to an MHC-incompatible F1 recipient (1). This systemic autoimmune syndrome develops in normal (C57BL/6 x DBA/2)F1 (B6D2 F1) mice (H-2b/d) following injection of parental DBA/2 (H-2d) T cells (1, 2, 3). A multisystem disorder, cGVHD generally comprises a spectrum of abnormalities, such as vasculitis, polyarthritis, and mononuclear cell infiltration of multiple organs (4, 5). In addition, excess collagen deposition and fibrosis typically develop in affected organs, particularly the kidney (6). B6D2 F1 recipient animals with cGVHD develop nephritis within 2–4 wk following transfer of parental T cells (2, 5). Previous reports demonstrate that animals develop an immune complex glomerulonephritis that progresses to global glomerulosclerosis and renal failure (6).

Previous in vitro studies conducted in cGVHD have demonstrated differential abnormalities in host B cells as well as CD4+ and CD8+ T cells within 2 wk of parental DBA/2 T cell transfer to B6D2 F1 recipients (7, 8, 9). A variety of circulating and deposited autoantibodies are detected in affected animals, including anti-DNA, anti-histone, and anti-laminin Abs, that are produced by host B cells (2, 10, 11, 12, 13). Models of early immunologic events in cGVHD suggest that recognition of allogeneic H-2b on B6D2 F1 class II MHC-expressing cells by DBA/2 CD4+ T cells provides help to host B cells, with subsequent activation and expansion of autoreactive B cells (7, 14). Abnormalities in host T cells have also been described (8, 9). A selective deficiency in host CD4+ Th function occurs in the setting of marked decreases in IL-2 production, while CD8+ T cell function appears intact (8, 9). The potential pathogenic roles of alloreactive CD4+ donor T cells or autoreactive host T cell subsets in cell-mediated manifestations of cGVHD, however, have not been clearly elucidated.

As the renal pathology observed in cGVHD is characterized by mononuclear cell (T cell) infiltrates as well as immune complex deposition, we investigated mechanisms of cell-mediated injury in this renal disease. For this purpose, we isolated T cell lines and clones from the kidney of diseased B6D2 F1 recipients with cGVHD. Our analyses reveal that mouse renal (MR) cells are host-derived CD4+ T cells that express {alpha}/ß heterodimeric TCRs. Adoptive transfer studies demonstrate that most of these renally derived T cell clones induce a severe pattern of interstitial inflammation in syngeneic B6D2 F1 recipients. In vitro proliferative responses of renally derived MR clones reveal predominant reactivity toward autologous class II MHC (I-Ed/I-Ad) determinants, and selected nephritogenic MR clones preferentially recognize renal Ag preparations derived from normal B6D2 F1 kidneys. Further analysis of MR clones with cytokine studies reveals a Th2 pattern of IL-4 and IL-10 expression, with differential IFN-{gamma} expression in clones that transfer disease. These studies describe an interesting T cell subset that should facilitate our understanding of the autoreactive nephritogenic T cell response in cGVHD.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

C57BL/6 (H-2b), DBA/2 (H-2d), B6D2 F1 (H-2b/d), B10.HTG, B10.A, and C3H.LG were purchased from The Jackson Laboratory (Bar Harbor, ME).

Induction of cGVHD and isolation of T cells

Disease was induced in 6- to 8-wk-old female B6D2 F1 mice following transfer of two sequential injections of 50 x 106 T cells derived from spleen, lymph node, and thymus preparations of 6- to 8-wk-old female DBA/2 mice (2). The second injection was administered 7 days following the first inoculum. Control B6D2 F1 animals were injected with similarly prepared T cells derived from other 6- to 8-wk-old B6D2 F1 mice. Following induction of disease with parental DBA/2 T cells, animals were evaluated every 2 wk for urinary protein excretion using the Bio-Rad assay (Bio-Rad, Richmond, CA) with a BSA standard.

MR cells were isolated from saline-perfused nephrotic kidneys of animals at 14 wk (MR1), 11 wk (MR2), and 7 wk (MR3) following disease induction. Isolated kidneys were finely minced into small pieces, gently pressed through a mesh sieving screen, and washed with ice-cold PBS. T cells were separated by Lympholyte M (Cedarlane, Hornby, Ontario, Canada) extraction at room temperature. The interface cell layer containing viable T cells was washed with PBS and plated at a density of 0.5 x 106/well of a 24-well tissue culture plate. MR T cells were propagated by weekly passage with 10% MLA-144 supernatants as a source of IL-2 and other growth factors, renal Ag preparation (10 µg/ml) derived from collagenase-digests of normal B6D2 F1 kidneys, and 5 x 106 irradiated (2500 rad) syngeneic B6D2 F1 spleen cells (15, 16). Mouse spleen (MS) cells were isolated and propagated from spleens of diseased animals (at 14 wk) in a similar fashion. HEL cells (CD4+, {alpha}/ß TCR+) were isolated from the draining lymph nodes of HEL-immunized B6D2 F1 mice and were propagated by weekly stimulation with IL-2, HEL (100 µg/ml), and irradiated APC (17). MR, MS, and HEL cell lines were cloned by limiting dilution (17). T cells were plated at a density of 0.3 cells/well in 96-well microtiter plates. Wells with evidence of T cell growth were then transferred to 24-well plates and further expanded by weekly stimulation. T cell culture medium consisted of RPMI 1640 (Life Technologies, Gaithersburg, MD) supplemented with glutamine, antibiotics (penicillin, streptomycin, and gentamicin), 10% decomplemented FCS, 5% NCTC-109 (BioWhittaker, Walkersville, MD), and 2 x 10-5 M 2-ME. Typically, wells became confluent at 5–7 days and were carried every 10–14 days. All T cells were cultured at 37°C in a 5% CO2 incubator.

Flow cytometry

Flow cytometry was performed on T cells harvested from day 10 cultures. Cells were washed in PBS, and aliquots of 1 x 106 cells were resuspended in 50 µl of PBS with 0.1% BSA (staining buffer). Conjugated Abs, (FITC: anti-TCR {alpha}ß, anti-TCR {gamma}{delta}, anti-CD4, anti-CD8, anti-H-2kb, anti-H-2kd; PE: anti-TCR {alpha}ß; PharMingen, San Diego, CA), were incubated with samples on ice for 20 min, and the samples were washed three times with staining buffer. For two-color analysis, a second incubation on ice was conducted with PE-conjugated {alpha}/ß TCR Ab, and samples were again washed with staining buffer. Stained cells were then fixed in 500 µl of 4% paraformaldehyde in PBS. Fluorescence was recorded on a FACScan cytofluorograph (Becton Dickinson, Mountain View, CA), and the data were analyzed with LYSYS II software. In each run at least 10,000 live gated cells were analyzed.

Proliferation assays

MR T cell clones (1 x 105) harvested from day 7–9 cultures were added to irradiated splenocytes (5 x 105) derived from either syngeneic animals or recombinant strains with 200 µl of T cell medium. For some of these studies 10 µg/ml of anti-class I MHC or anti-class II MHC Abs (PharMingen) were added. Different Ag preparations (10 µg/ml) derived from collagenase digests of normal B6D2 F1 organs (kidney and liver) were also added to subsets of proliferation assays with MR T cell clones. For all studies, cells were pulsed with [3H]TdR at 48 h and then harvested at 72 h for scintillation counting. Values represent the mean of triplicate samples ± SD.

Adoptive transfer of disease

Renal subcapsular transfers were performed with naive 6- to 12-wk-old B6D2 F1 mice, as previously described (17). In brief, T cells were harvested at end passage (days 10–14) and washed with PBS, and aliquots of 10 x 106 in approximately 75 µl of PBS were injected under the kidney capsule with a 30-gauge needle. This volume uniformly lifted the capsule off most of the parenchyma without bleeding. Seven to 18 days later the kidneys were harvested and longitudinally sectioned with preservation of the subcapsular cell layer. Sections of kidney tissue for immunofluorescence were embedded in OCT compound (Miles, Elkhart, IN), snap-frozen in liquid nitrogen, and stored at -70°C. Other kidney sections were fixed in 10% buffered formalin and paraffin-embedded for staining (hematoxylin-eosin and periodic acid-Schiff) and examined for histologic evidence of disease.

Assessment of renal disease

Semiquantitative methods assessed renal disease in affected kidneys. Sections of paraffin-embedded tissue (4 µm) were stained with hematoxylin and eosin and periodic acid-Schiff, and examined for cellular infiltrates and tubular atrophy by a pathologist blinded to the experimental protocol (J.E.T.). The area of the mononuclear cell infiltrate was measured in two dimensions. The density of the infiltrate was graded on a scale of 0–4, where 0 = no inflammatory cells found in the area of abnormal appearing tubular cells, 1 = 1–10% of cells in the histologically abnormal areas were mononuclear cells, 2 = 11–25% inflammatory cells in histologically abnormal areas, 3 = 26–50% inflammatory cells in the histologically abnormal areas, and 4 = >=51% inflammatory cells in histologically abnormal areas. Parameters of tubular cell damage noted on histologic sections included marked basophilia of tubular epithelium, increased nuclear/cytoplasmic ratio, microvacuolar cytoplasmic changes, and decreased cytoplasmic volume. Tubular cell damage in the area of the infiltrate was also graded on a scale of 0–4 with 0 = no tubular cells in the area of infiltrate demonstrating evidence of damage, 1 = 1–10%, 2 = 11–25%, 3 = 26–50%, and 4 = >=51% of tubular cells in the area of infiltrate showing evidence of tubular damage. The data from both methods were expressed as a mean for each group ± SEM.

Immunofluorescence

Cryostat sections of frozen kidney tissue, 4 µm in thickness, were fixed in ether-ethanol (1/1) and 100% ethanol, and then stained with FITC-conjugated Abs for 45 min at room temperature in a humidified chamber (18). Abs for these studies included FITC-rat anti-mouse IgM, FITC-rat anti-mouse IgG, FITC-goat anti-rat Ig, and normal rat IgG (Boehringer Mannheim, Indianapolis, IN). Following the final wash, each mount was covered with Aquamount (Fisher Scientific, Medford, MA) and examined with a fluorescence microscope (Zeiss, Thornwood, NY).

Immunohistochemistry

Frozen kidney sections were stained with a panel of mAbs to cell surface markers using the avidin-biotin-peroxidase complex technique (Vector, Burlingame, CA). Frozen sections were fixed in chilled (-20°C) acetone, air-dried, rehydrated in PBS, and then incubated with the avidin-biotin blocking reagent (Vector) for 60 min. Appropriate (1/50 to 1/200) dilutions of the primary Abs were applied, and the slides were incubated for 60 min at room temperature. The slides were then washed in PBS and incubated with biotinylated anti-rat IgG (Vector) for 60 min. Following another PBS wash, slides were incubated with the Vectastain ABC reagent (Vector) for an additional 60 min. Slides were then incubated with diaminobenzidine (Vector) and counterstained with hematoxylin (Fisher Scientific, Medford, MA). Abs used for these studies were rat anti-mouse preparations anti-CD4 and anti-CD8 (PharMingen). Control sections consisted of staining without primary Ab (PBS) and staining with an irrelevant primary Ab (normal rat IgG).

RNA extraction

Total cellular RNA was isolated from end-passage T cell clones (1–2 x 106 cells/ml) 24 h after stimulation with 1 µg/ml Con A (Sigma, St. Louis, MO) by the single-step method of acid guanidium thiocyanate-phenol-chloroform extraction (19). After assessing the purity of the final products by OD ratios at 260/280 nm, which were typically >1.7, the RNA samples were used for Northern blot analyses.

Northern blot hybridization

Total RNA isolated from 6 x 106 T cells was fractionated on a 1.5% agarose-formaldehyde gel and then transferred to a Zetabind membrane (Cuno Laboratory Products, Meriden, CT). Blots were hybridized to 32P-labeled (Amersham, Arlington Heights, IL) murine probes (sp. act., 2.0 x 109 dpm/µg) for 16 h at 55°C. Probes for IL-2 (20), IL-4 (21), IL-10 (22), IFN-{gamma} (23), and ß-actin (24) were used in these studies. After hybridization the blots were washed three times with 0.1x SSC/0.1% SDS at 65°C for 30 min. Autoradiography was performed with intensifying screens at -70°C. Exposed films were scanned with a laser densitometer (Hoefer, San Francisco, CA), and cytokine mRNA levels were calculated relative to those of ß-actin.

Secreted cytokine measurements

The production of IL-2, IL-4, IL-10, and IFN-{gamma} by T cell clones was measured in cell-free culture supernatants by cytokine-specific ELISA (25). Supernatants were obtained from end-passage T cell clones (1–2 x 106/ml) plated for 24 h with fresh T cell medium in the presence or the absence of Con A (1 µg/ml). Purified anti-cytokine capture mAb (PharMingen) was diluted to 1.0 µg/ml in 0.1 M NaHCO3, pH 8.2, and 50-µl aliquots were added to wells of an ELISA plate. Following overnight incubation at 4°C and washing with PBS/0.05% Tween, wells were blocked with 200 µl of PBS/10% FCS at room temperature for 2 h, washed again with PBS/0.05% Tween, and then coated with cytokine standards and T cell clone supernatants diluted in PBS/10% FCS in duplicate. Plates were incubated overnight at 4°C, washed with PBS/0.05% Tween, and coated with 100-µl aliquots of biotinylated anti-cytokine detecting Ab (PharMingen) diluted to 1 µg/ml in PBS/10% FCS. Following a 45-min incubation at room temperature, the plates were washed with PBS/0.05% Tween, coated with 100-µl aliquots of avidin-peroxidase (Sigma) diluted in PBS/10% FCS, and incubated again at room temperature for 30 min. The plates were then washed with PBS/0.05% Tween, and 100-µl aliquots of peroxidase substrate reagent (Bio-Rad) were added to each well, allowed to develop at room temperature for 5–10 min, and read on an ELISA plate reader at 405 nm. Sensitivity limits for the ELISA were: IL-2, 15 pg/ml; IL-4, 30 pg/ml; IL-10, 30 pg/ml; and IFN-{gamma}, 0.3 U/ml.

Southern hybridization studies

Liver and T cell clonal DNA were isolated with standard methods (25). Each sample containing 10 µg of genomic DNA was digested to completion with a restriction enzyme (HindIII, EcoRI, or BamHI; Boehringer Mannheim), separated on a 0.7% agarose gel, and transferred to a Zetabind membrane (Cuno Laboratory Products). The blots were prehybridized and hybridized at 65°C in 0.5 M NaPO4 buffer, 7% SDS, 1% BSA, 1 mM EDTA, 50 mg/ml poly(A) (Boehringer Mannheim), and 50 µg/ml ssDNA. The blots were hybridized to a 32P-labeled (random primed) murine TCR Cß probe (sp. act., 2.0 x 109 dpm/mg) for 16 h, and then washed three times for 30 min each time in 0.1x SSC/0.1% SDS at 65°C. Autoradiograph exposures were obtained with intensifying screens at -70°C.

Statistical analysis

Differences between experimental groups were determined by Student’s t test where appropriate (26). Statistical significance between groups of adoptively transferred renal lesions was determined by Kruskal-Wallis one-way analysis of variance by ranks and the Mann-Whitney U test (26).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Isolation and phenotypic characterization of MR T cell clones

Chronic GVHD was induced in a cohort (n = 42) of B6D2 F1 recipients by transferring two sequential inocula of 50 x 106 parental DBA/2 T cells. The progression of renal disease in B6D2 F1 mice with cGVHD was assessed by evaluating both the level of proteinuria and histologic evidence of renal disease in B6D2 F1 recipients. Histologic findings in kidneys obtained from animals with cGVHD at relevant time intervals following initial transfer of parental DBA/2 T cells are illustrated in Fig. 1Go and are similar to previously reported pathology in (C57BL/10 x DBA/2)F1 mice (2, 5). Glomerular changes were apparent 2–7 wk following induction of disease and consisted of mesangial matrix expansion with glomerular capillary basement membrane thickening (Fig. 1GoA). Proliferative glomerular lesions progressed over subsequent weeks with evidence of focal and segmental sclerosis appearing at 6–12 wk following induction. Evidence of perivascular and tubulointerstitial inflammation was also observed in all kidneys harvested within 2–6 wk following disease induction. These inflammatory changes were characterized by a preponderance of mononuclear cell infiltrates, largely CD4+ T cells, associated with tubular dilatation and atrophy (14 wk; Fig. 1Go, B and D). Tubulointerstitial injury, like glomerular pathology, progressed over several weeks in all B6D2 F1-recipient animals and ultimately resulted in an end-stage renal lesion with marked tubular dilatation, persistent interstitial mononuclear infiltration, and renal fibrosis. Kidneys harvested from some animals also revealed a number of glomerular crescents as well as tubulointerstitial inflammation (Fig. 1GoC; 7 wk).



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  		FIGURE 1. Renal pathology associated with murine cGVHD. A, Glomerular lesions are apparent within 2 wk following induction of disease and typically exhibit mesangial matrix expansion with capillary basement membrane thickening (periodic acid-Schiff; x313). B, In addition to glomerular changes, perivascular and tubulointerstitial mononuclear cell infiltration is generally seen following induction of cGVHD. Such changes progress over subsequent weeks and are associated with tubular dilatation and atrophy (14 wk; hematoxylin-eosin; x200). C, Glomerular crescent formation observed at 7 wk after induction of cGVHD (periodic acid-Schiff; x500). D, Representative photograph of infiltrating mononuclear cells in cGVHD (from B; 14 wk), that demonstrates a preponderance of CD4+ T cells (immunoperoxidase with hematoxylin counterstain; x313).

 
In view of histologic data suggesting a preponderance of T cells in this progressive autoimmune glomerulonephritis, MR T cell lines and clones were isolated from diseased kidneys of nephritic B6D2 F1 recipients. For these studies, we isolated MR1 at 14 wk (Fig. 1GoB), MR2 at 11 wk, and MR3 at 7 wk (Fig. 1GoC) after induction of cGVHD. FACS analysis of MR clones derived from these cell lines determined their cell surface phenotype and origin (parental or B6D2 F1). As shown in Fig. 2GoA, MR1.3 and MR1.12 are CD4+ {alpha}/ß+ T cell clones. As cGVHD is induced by transfer of immunocompetent parental DBA/2 T cells (H-2d) to B6D2 F1 recipients (H-2b/d), we also evaluated the origin of T cell clones cultured from diseased B6D2 F1 kidneys. Fig. 2GoB reveals a representative two-color FACS analysis of MR clones, MR1.3, MR1.6, and MR1.12, and demonstrates that these clones express H-2Kb, a host-specific class I MHC determinant. Similar FACS analysis of MR2.5, MR2.12, and MR3.3 (data not shown) indicate that they are all host-derived CD4+ {alpha}/ß+ T cell clones.



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  		FIGURE 2. FACS analysis of day 10 MR clones. A, Single-color analysis of MR1.3 and MR1.12 incubated with FITC-conjugated anti-CD4/CD8 Abs and FITC-conjugated anti-TCR Abs. B, Two-color FACS analysis of MR clones, MR1.3 and MR1.12, incubated with PE-conjugated anti-TCR and FITC-conjugated anti-class I MHC (H-2Kb) Abs. Horizontal and vertical axes were placed to yield <1% positive cells with control Abs, and the percentages of respective cell populations are noted in each quadrant. Horizontal and vertical scales are log10 fluorescence intensity.

 
Nephritogenic potential of T cells isolated from diseased kidneys

We examined the in vivo function of MR T cells by investigating their ability to induce renal injury in naive B6D2 F1 recipients. In previous studies we have found that renal subcapsular transfer of immunocompetent T cells is a sensitive and reproducible means of determining the ability of discrete T cell subsets to elicit inflammation in renal parenchyma (17, 27, 28). We therefore used this technique to compare the nephritogenic potential of MR cells in this model of cGVHD. To assess induced patterns of renal injury, eight groups of kidneys were evaluated for evidence of renal pathology. Adoptive transfers were performed with six different MR clones, MS4 (CD4+ {alpha}/ß+ T cell lines isolated from spleens of diseased B6D2 F1 mice) and HEL.4 (CD4+ {alpha}/ß+ HEL-reactive T cell clone). Fig. 3Go (A and C–F) shows representative histology of lesions induced by nephritogenic T cell clones MR1.3, MR1.12, MR2.5, MR2.12, and MR3.3. Affected kidneys exhibited cortical inflammation with focal mononuclear cell infiltrates as well as evidence of tubular dilatation and atrophy. All the renally derived clones were not nephritogenic, however, as tubulointerstitial inflammation was not observed following transfer of T cell clone MR1.6 to naive B6D2 F1 kidneys (Fig. 3GoB). Similarly, the diseased spleen-derived T cell line (MS4) and HEL.4 did not induce renal inflammation or injury on transfer to naive mice (Fig. 3Go, G and H). Fig. 4Go compares the immunofluorescence staining pattern of renal IgG deposition in kidneys obtained form cGVHD mice (Fig. 4Go, A and B) and those from adoptively transferred lesions (Fig. 4GoD). Immune deposits (IgG/IgM) were not detected in any of the T cell-induced renal lesions despite severe inflammation. Immune deposits were also not detected in kidneys that received nonnephritogenic T cells (data not shown).



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  		FIGURE 3. Adoptive transfer studies of cultured T cells in cGVHD. Cultured T cells were injected under the kidney capsule of naive syngeneic mice. After 7–18 days the kidneys were harvested and sectioned for histologic analysis. Representative photos of histologic changes observed following transfer of MR1.3 (A), MR1.6 (B), MR1.12 (C), MR2.5 (D), MR2.12 (E), MR3.3 (F), MS4 (G), and HEL.4 (H). Affected cortical areas (A and C–F) contain mononuclear cell infiltrates with evidence of tubular cell damage and atrophy (hematoxylin-eosin; x200). Similar transfer of MR1.6, MS4, and HEL.4 T cells did not result in interstitial inflammation (B, G, and H), as the tubulointerstitial architecture remained normal (hematoxylin-eosin; x200).

 


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  		FIGURE 4. Immunofluorescence studies of adoptive transfers. A, Direct immunofluorescence of renal tissue isolated at 2 wk following induction of disease demonstrates IgG deposition along the glomerular capillary wall (x500). B, Direct immunofluorescence of kidney sections obtained at 6 wk reveals a more granular pattern of IgG distribution along the glomerular basement membrane (x640). C, Direct immunofluorescence of kidney sections isolated from a sex- and age-matched control for B demonstrates the lack of IgG deposition (x500). D, Similarly performed direct immunofluorescence of kidney section isolated 2 wk after adoptive transfer of MR1.3 to a normal B6D2 F1 recipient (x500). Immunofluorescence staining for IgM in MR-induced lesions revealed similar findings (data not shown).

 
As delineated in Table IGo, several renally derived T cell clones induced tubulointerstitial inflammation and injury within 7–18 days after transfer. Table IGo compares infiltrate area, inflammatory cell density, and patterns of tubular epithelial cell damage induced in eight experimental groups of mice. Five nephritogenic MR clones (MR1.3, MR1.12, MR2.5, MR2.12, and MR3.3) induced similar patterns of injury by histologic scores. Tubulointerstitial infiltrates or damage were not detected after transfer of MR1.6, MS4, or HEL.4. In these experimental groups the T cell inocula remained in the renal subcapsular space following transfer and did not infiltrate the kidney. These analyses indicate that approximately two-thirds of all T cell clones isolated from diseased B6D2 F1 animals are nephritogenic to naive syngeneic recipients.


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  		Table I. Nephritogenic potential of cultured T cells

 
Proliferative responses of MR T cell clones

As adoptive transfers suggested that a distinct subset of MR clones induced renal disease, further in vitro analysis evaluated patterns of nephritogenic clonal recognition. In the first set of proliferation assays, we observed preferential recognition of renally derived Ag by selected MR clones. The studies in Fig. 5GoA were conducted with MR clones, irradiated syngeneic APC, and an Ag preparation (10 µg/ml) obtained through collagenase digests of normal B6D2 F1 kidney or liver (15). MR1.3, MR1.6, MR2.5, and MR1.12, which proliferate to B6D2 F1 APC in the absence of Ag, are not affected by exogenous peptides at this concentration or at 1 and 100 µg/ml Ag concentrations (data not shown). Examination of MR2.12 and MR3.3 reactivity, however, reveals preferential recognition of renal Ag, an effect not elicited by similarly digested liver preparations.



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  		FIGURE 5. Proliferative responses of MR clones. Reported findings are derived from a single experiment that is representative of three separate experiments. A, MR clones (1 x 105) were added to irradiated syngeneic splenocytes (5 x 105) and various Ag preparations (10 µg/ml). For all studies, cells were pulsed with [3H]TdR at 48 h and then harvested at 72 h for scintillation counting. *, p < 0.001 vs no Ag control wells. B, MR clones (1 x 105) were added to irradiated splenocytes (5 x 105) derived from the recombinant strains listed above with 200 µl of T cell medium. *, p < 0.001 vs B6D2F1 wells. C, MR clones (1 x 105) were added to irradiated syngeneic splenocytes (5 x 105) and various blocking Abs (10 µg/ml). Studies with T cell clones MR2.12 and MR3.3 were also conducted in the presence of B6D2 F1 renal Ag (10 µg/ml). *, p < 0.001 vs no Ab control wells. Values represent the mean of triplicate samples ± SD.

 
Proliferation assays were then conducted with MR clones and APC derived from a panel of intra-MHC recombinant strains to delineate relevant MHC determinants recognized by these T cell clones. As shown in Fig. 5GoB, three distinct patterns of recognition are apparent from these studies. MR1.3, MR1.12, and MR2.5 respond to autologous APC (H-2b/d), and the responses to other recombinant strains in Fig. 5GoB also suggest that these nephritogenic clones demonstrate selective reactivity toward autologous class II MHC (Iad) determinants. By contrast, nephritogenic clones MR2.12 and MR3.3 do not respond to autologous APC (H-2b/d) or other stimulating strains in the absence of exogenous antigen. In addition, the nonnephritogenic T cell clone MR1.6 recognizes only autologous (B6D2 F1) APC and none of the other stimulating strain APC.

Further blocking Ab studies were then performed with MR clones and syngeneic APC. Fig. 5GoC illustrates MR clonal responses to B6D2 F1 APC with a panel of blocking Abs (anti-H-2d, anti-I-Ad, and anti-I-Ed Abs). Anti-I-Ed Ab inhibited MR1.6, MR1.12, MR2.5, and MR3.3 (in the presence of renal Ag) proliferation to B6D2 F1 APC, whereas anti-H-2Kd and anti-I-Ad Abs had no significant effect on this response. Similarly, anti-I-Ad Ab inhibited MR1.3 and MR2.12 (in the presence of renal Ag) proliferation to B6D2 F1 APC, responses not blocked by either anti-class I MHC or anti-I-Ed Abs. These blocking Ab studies therefore suggest that nephritogenic MR clone recognition is restricted to Iad, determinants expressed in both graft and host in this model of cGVHD. The nonnephritogenic clone MR1.6, however, recognized only B6D2F1 APC and exhibited no reactivity against H-2d determinants.

Analysis of cytokine expression in MR T cells

Numerous studies have demonstrated that subsets of murine CD4+ T cell clones express unique cytokine profiles that directly correlate with function (29, 30). To determine the cytokine profile of renally derived T cells, nephritogenic and nonnephritogenic clones were compared by Northern analysis. Nephritogenic clones, MR1.3, MR1.12, MR2.5, MR2.12, and MR3.3, expressed IL-4, IL-10, and IFN-{gamma}, but not IL-2, following Con A stimulation (Fig. 6Go). By contrast, nonnephritogenic clones, MR1.6, MS3.1, MS4.5, and HEL.4, expressed IL-4 and IL-10 but not IL-2 or IFN-{gamma} under similar conditions (Fig. 6Go). These analyses illustrate the predominant Th2 cytokine profile (IL-4 and IL-10) of all T cell clones tested.



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  		FIGURE 6. Expression of cytokine transcripts in MR clones. Total RNA preparations from Con A-stimulated T cell clones were isolated and hybridized with radiolabeled DNA cytokine probes. Relative cytokine mRNA production in a panel of MR, MS, and HEL T cell clones, determined by densitometric analysis of autoradiograms, is expressed as a percentage of control ß-actin mRNA expression.

 
Analysis of secreted cytokines in supernatants of MR and MS T cells corroborated initial Northern hybridization studies. Quantitative assessment of secreted cytokines was performed by ELISA on a panel of Con A-stimulated T cell clones. As shown in Table IIGo, supernatants from nephritogenic clones MR1.3, MR1.12, MR2.5, MR2.12, and MR3.3 contained significant amounts of IL-4 and IL-10. Consistent with our Northern hybridization data, nephritogenic MR clone supernatants also contained low levels of IFN-{gamma} (8–42 U/ml) and did not contain IL-2. Although the intensity of the IFN-{gamma} hybridization signal for MR1.12 (Fig. 6Go) appeared greater than that observed with other MR clones, we did not detect a significant difference in secreted cytokine at 24 h in these assays. Supernatants from MR1.6, which did not induce renal inflammation, contained IL-4 and IL-10, but not IL-2 or IFN-{gamma}. Similarly, other nonnephritogenic clones, MS3.1, MS4.5, and HEL.4, expressed IL-4 and IL-10 but not IL-2 or IFN-{gamma}.


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  		Table II. Analysis of secreted cytokines

 
MR clonal TCR gene rearrangement

Given the common functional properties of nephritogenic MR T cells, we evaluated these cells for TCR-ß gene rearrangement to verify that they are distinct clones. The Southern blot analysis in Fig. 7Go displays B6D2 F1 liver and MR clonal DNA digested with HindIII and hybridized to a labeled Cß probe. As shown in Fig. 7Go, HindIII digestion of B6D2 F1 liver DNA resulted in a 9.4-kb Cß1 fragment and a 3.0-kb Cß2 fragment. Similar analysis of clonal DNA revealed the germline 3.0-kb Cß2 fragment in all T cells, although clones MR1.3, MR1.6, MR1.12, MR2.5, MR2.12, and MR3.3 exhibited Cß1 fragments that differed from germline configuration. Additional restriction enzyme digestion with EcoRI and BamHI further demonstrated that these MR clones (MR1.3 and MR2.12, MR2.5 and MR3.3) are distinct (data not shown).



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  		FIGURE 7. Southern blot analysis of MR clones. Ten micrograms of DNA from MR clones and control DNA from B6D2 F1 liver was digested with HindIII, electrophoresed, blotted onto a nylon membrane, and hybridized to a Cß probe. The germline configuration (lane 1) from liver DNA reveals the 9.4 Cß1 and 3.0 Cß2 fragments. Digestion of six MR clones, MR1.3 (lane 2), MR1.6 (lane 3), MR1.12 (lane 4), MR2.5 (lane 5), MR2.12 (lane 6), and MR3.3 (lane 7) demonstrates Cß fragments that differ from germline configuration. The migration distance in kilobases of m.w. standards is indicated on the left.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The studies presented in this paper suggest a pathogenic role for autoreactive T cells in renal parenchymal injury observed in cGVHD. These studies demonstrate that selected renally derived CD4+ {alpha}/ß+ T cell clones of host origin adoptively transfer tubulointerstitial inflammation to kidneys of normal syngeneic mice. Proliferation assays reveal heterogeneous patterns of nephritogenic MR clonal recognition, restricted by autologous class II MHC determinants (I-Ad/I-Ed). Cytokine analysis of MR T cell clones indicate a predominant Th2 profile with IL-4 and IL-10 expression, but nephritogenic T cells also express IFN-{gamma}. Although events that initiate the cascade of immune reactivity in animals with cGVHD are probably induced by early allogeneic responses of the parental DBA/2 T cell inocula, isolation of multiple host-derived T cell clones from nephritic kidneys suggests that elements of parenchymal organ damage in this disease are mediated by autoreactive T cells.

As mechanisms that initiate or regulate mononuclear cell infiltration and their correlation with glomerular disease in the renal lesion of cGVHD have not been extensively characterized, we examined the role of T cells in this pattern of renal inflammation with lymphocytes cultured from diseased kidneys, when renal mononuclear cell infiltration was most abundant. Their pathogenic role is suggested by the ability to transfer inflammatory renal disease to normal B6D2 F1 recipients. Nephritogenic MR clones predictably induced severe lesions characterized by tubulointerstitial inflammation and damage in naive hosts. The lack of Ab deposition in these transferred lesions suggests that some forms of renal injury in this model may indeed occur independent of parenchymal immune complex formation. Previous adoptive transfer studies in other models of autoimmune renal disease have revealed similar observations of distinct T cell-mediated patterns of renal injury (31). Distinct nephritogenic T cell clones in anti-tubular basement membrane disease, for example, elicited qualitatively different renal lesions in susceptible animals (31). Such findings may be relevant to observations of progressive renal injury with varied phenotypic expression of both tubulointerstitial and glomerular inflammation and damage generally apparent in histologic samples derived from diseased kidneys (31, 32).

Lymphocyte transfer studies in models of autoimmune renal disease have revealed only a few examples of renal damage induced by T cells derived from diseased animals (17, 33, 34, 35, 36). In the context of a systemic autoimmune disease, it is noteworthy that the renally derived MR T cell clones elicited primarily tubulointerstitial injury, particularly in view of data from models of glomerular injury, demonstrating that progressive renal disease is closely correlated with both chronicity and severity of tubulointerstitial changes (37, 38, 39). Subsequent analyses have also demonstrated that the MR T cell line induces glomerular inflammation (C. M. Meyers, unpublished observation). Indeed, multiple studies examining renal inflammation and damage in the MRL/lpr model of systemic autoimmunity indicate a pivotal role of cell-mediated events in progressive disease (40, 41, 42, 43). Depleting Ab studies (anti-Thy1.2, anti-CD4, and anti-CD3) as well as CD4+ T cell deficiency induced by deleting MHC class II expression have successfully inhibited autoantibody production and renal disease in susceptible animals (40, 41, 42, 43). Recent production of MRL/lpr mice congenitally deficient in {alpha}ß T cells also suggests a critical role for cell-mediated mechanisms in progressive renal damage (44). TCR- MRL/lpr mice in reported analyses develop hypergammaglobulinemia and evidence of both circulating and deposited (renal) autoantibodies (44). Although the TCR- MRL/lpr mice develop renal immune deposits, they do not develop the typical pattern of renal inflammation and damage or the characteristic, rapidly progressive renal disease of TCR+ MRL/lpr mice (44).

Investigations examining immune responses in this model of cGVHD have traditionally focused on humorally mediated mechanisms of disease (7, 14). Early reports of this form of cGVHD observed persistent lymphoid hyperplasia, hypergammaglobulinemia, pathogenic autoantibodies, and lupus-like histopathologic lesions (7, 14). Models for these immunostimulatory B cell events emphasize cGVHD-induced activation of autoreactive B6D2 F1 B cells by so-called abnormal T-B cell cooperation (7, 14). In such T-B cell cooperation, F1 B cells receive relevant helper signals from alloreactive donor DBA/2 (H-2d) CD4+ T cells in response to H-2b determinants expressed in B6D2 F1 recipients that ultimately effect the production of a variety of IgG autoantibodies (1, 2, 3).

In addition to autoreactive B cell stimulation, however, our current studies suggest chronic activation of autoreactive T cells in this model of cGVHD. Predominant Iad reactivity of nephritogenic MR clones is noteworthy, as this systemic autoimmune syndrome is induced by transfer of DBA/2 (H-2d) T cells to B6D2 F1 (H-2b/d) recipients. The nonnephritogenic clone MR1.6 displays a distinct pattern, however, with a response only to B6D2 F1 APC and that is inhibited by anti-I-Ed blocking Abs. This response may indicate reactivity to hybrid I-E(b/d) molecules, or perhaps H-2b determinants presented in the context of I-Ed, in the F1 recipient. In aggregate, our observations suggest that the nephritogenic autoimmune T cell response in this disorder targets cell surface determinants common to both graft and host. Pathogenic autoreactive T cells isolated from diseased kidneys appear restricted to the initial cGVHD-inducing parental H-2d determinants.

Recent studies in models of systemic autoimmunity induced by allogeneic interactions or chemicals have suggested that preferential activation of Th2 cells in diseased animals associated with an increase in IL-4 and IL-10 production plays an integral role in disease pathogenesis (45, 46, 47). Our cytokine analyses of autoreactive T cell clones in cGVHD several weeks after disease induction that reveal IL-4 and IL-10 expression are consistent with a Th2 effect in this disease. We were interested in the differential IFN-{gamma} expression in the Th2-like MR clones, however, in view of the proscriptive influence of IL-10 on its synthesis in CD4+ T cell clones (48). Although culture conditions may effect uncharacteristic changes in cytokine gene expression in cloned T cell lines, similar Th2-like observations in other models of autoimmunity suggest that this particular CD4+ T cell phenotype may be relevant to disease expression (49, 50, 51). Such observations include autoreactive T cell clones isolated from MRL/lpr mouse kidneys and gold salt-injected Brown Norway rat lymph nodes as well as allergen-specific human Th2 cells (49, 50, 51). These analyses indicate that IFN-{gamma} secretion by a Th2-like CD4+ T cell may be of functional significance in vivo (49, 50, 51).

Along these lines, as IFN-{gamma} expression was detected only in MR clones that transfer disease, its expression in nephritogenic T cell clones may be one mechanism of eliciting and propagating renal inflammatory responses. Other investigators have also suggested that T cell-derived IFN-{gamma} is an important mediator of inflammatory reactions elicited by CD4+ T cell clones (52). Moreover, IFN-{gamma}-induced up-regulation of class II MHC expression on renal epithelial cells in vivo may be a crucial event in local trafficking of autoimmune CD4+ T cells and may facilitate the adoptively transferred MR renal lesions observed in this report (53, 54, 55). Recent studies conducted in our laboratory support this hypothesis (manuscript in preparation), and current analyses are examining renal expression of costimulatory molecules relevant to nephritogenic clonal activation. Explication of relevant mechanisms of MR T cell-mediated responses will provide further insight into both the activation and the target recognition of pathogenic T cells in this model of cGVHD.


   Acknowledgments
 
We thank Michael Madaio and Larry Turka for their comments and critical review of the manuscript, and Carolyn Kelly for her support.


   Footnotes
 
1 This work was performed during the tenure of a Grant-in-Aid Award from the American Heart Association (95012790; to C.M.M.). It was also supported in part (to C.M.M.) by the Thomas B. and Jeannette E. Laws McCabe Fund; the Margaret Q. Landenberger Research Foundation; the National Kidney Foundation of the Delaware Valley; the Lupus Foundation of Philadelphia, Inc.; the Measey Foundation of the University of Pennsylvania; National Institutes of Health Grants DK-07006, DK-49724, and DK-44513 (to J.E.T.); and administrative/educational funds from the Dialysis Clinic, Inc., Renal Education and Development Fund. Back

2 Address correspondence and reprint requests to Dr. Catherine M. Meyers, Renal-Electrolyte and Hypertension Division, University of Pennsylvania, 700 Clinical Research Building, 415 Curie Blvd., Philadelphia, PA 19104. Back

3 Abbreviations used in this paper: cGVHD, chronic graft-versus-host disease; B6D2 F1, (C57BL/6 x DBA/2)F1; MR, mouse renal-derived T cells; MS, mouse spleen-derived T cells, HEL, hen egg lysozyme; PE, phycoerythrin. Back

Received for publication June 23, 1997. Accepted for publication July 14, 1998.


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 Introduction
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 Results
 Discussion
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