Progression from Acute to Chronic Disease in a Murine Parent-into-F₁ Model of Graft-Versus-Host Disease

Mice

B6, DBA/2, B6D2F₁, BALB/c, and CB6F₁ female mice, 6–8 wk of age, were purchased from either Animal Production Area (Frederick, MD) or Charles River Breeding Laboratories (Wilmington, MA). Animals were cared for in accordance with the guidelines set up by the Institutional Animal Care and Use Committee. An animal protocol for studying GVHD was reviewed and approved by the National Cancer Institute Animal Care and Use Committee before starting experiments.

Preparation of murine cells

Single-cell suspensions were prepared from the spleens of mice in Dulbecco’s PBS (DPBS; Life Technologies, Grand Island, NY). Cell suspensions were filtered through a 70-μm sterile mesh screen (Becton Dickinson Labware, Franklin Lakes, NJ), and the cells were washed. For some applications, erythrocytes were lysed by using ACK Lysis Buffer (BioWhittaker, Walkersville, MD), or erythrocytes and dead cells were removed by using Lympholyte M (Accurate Chemical and Scientific, Westbury, NY). Cells were suspended in either DPBS or complete medium (CM) comprised of RPMI 1640 (Life Technologies) supplemented with 2 mM l-glutamine, penicillin, streptomycin, 5 × 10⁻⁵ M 2-ME (Life Technologies), 100 μM nonessential amino acids (Life Technologies), 5 μM HEPES (Life Technologies), and 10% heat-inactivated FBS (HyClone, Logan, UT) after the final wash, depending on use.

Induction of GVHD

Washed B6, DBA/2, BALB/c, B6D2F₁, and CB6F₁ splenocytes were suspended at 120 × 10⁶ viable cells/ml in DPBS. GVHD was induced by the injection of 60 × 10⁶ parental cells i.v. into CB6F₁ or B6D2F₁ mice. Control mice included uninjected age-matched mice and mice injected i.v. with 60 × 10⁶ syngeneic F₁ splenocytes. To maintain as much homogeneity between donor cell populations, both F₁ combinations were injected on the same day using cells processed simultaneously under the same conditions. Both CB6F₁ and B6D2F₁ mice were injected from the same pool of B6 splenocytes.

Flow cytometric analysis of parental cell engraftment

The spleens of GVHD mice were harvested at 1–4, 6, 8, and 12 wk postinoculation. Single-cell suspensions of splenocytes were prepared, and the number of cells per spleen was counted. Erythrocytes and dead cells were removed using Lympholyte M. Splenocytes were stained with FITC-conjugated anti-H-2^d (clone SF₁-1.1), PE-conjugated anti-H-2^b (clone AF6-88.5) to distinguish parental from F₁ cells in the presence of the FCγRIII clone 2.4G2 to block nonspecific staining. To further identify the splenocyte populations anti-CD3 (clone 145-2C11), anti-CD4 (clone RM4-5 or H129.19), anti-CD8α (clone 53-6.7), or anti-CD19 (clone 1D3) conjugated to CyChrome or biotin plus streptavidin-CyChrome were used. All mAbs were purchased from PharMingen (San Diego, CA). Data were collected on a FACScan flow cytometer and analyzed with CellQuest Software (Becton Dickinson, San Jose, CA).

Autoantibody detection

ELISAs of serum Abs recognizing ssDNA (20) and dsDNA (21, 22) were performed as described previously. Briefly, for ssDNA assays, 96-well Maxisorp plates (Nalge Nunc International, Roskilde, Denmark) were incubated with 10 μg/ml methylated BSA (Sigma, St. Louis, MO) for 90 min at room temperature, followed by incubation of the plates for 2 h with 10 μg/ml calf thymus DNA (Sigma) that had been heated to >80°C for 15 min. Plates were washed and blocked overnight with 5% FBS in DPBS. Mouse serum was incubated on the plates for 2 h in 5-fold serial dilutions from 1:10 to 1:1250. Bound serum Abs were detected using goat anti-mouse IgG conjugated to HRP (Cappel, Aurora, OH) and visualized using 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulfonate) (Kirkegaard & Perry Laboratories, Gaithersburg, MD) and measuring the OD at 405 nm. For dsDNA assays, 96-well Maxisorp plates (Nalge Nunc International) were incubated with 5 μg/ml poly-l-lysine for 2 h at room temperature followed by incubation with 5 μg/ml λ phage DNA (Boehringer Mannheim, Indianapolis, IN) for 2 h at room temperature, then 4°C overnight. Other components of this assay were performed as described above.

ELISAs to detect the presence of Ab to ENA were performed using plates from RELISA ENA single-well screening kits (Immuno Concepts, Sacramento, CA). Each microtiter well is coated with Sm, RNP, SS-A, SS-B, Scl-70, and Jo-1 autoantigens. Plates were blocked for 2 h at 37°C. Mouse serum was added to the plates in 5-fold serial dilutions from 1:10 to 1:1250. Bound serum Abs were detected with goat anti-mouse IgG-HRP (Cappel) and 3,3′,5,5′-tetramethylbenzidine (Kirkegaard & Perry Laboratories) stopped with 2 N H₂SO₄. OD was measured at 450 nm.

Detection of Ig complexes in kidney sections

Detection of Ig complexes in kidney sections were performed as previously described (23). Briefly, kidneys were removed from mice following euthanasia and snap frozen in liquid nitrogen. Six-micrometer sections were cut, air dried, and fixed with acetone. Ig deposits were detected using FITC-labeled goat anti-mouse IgG (Sigma) incubated on slides for 30 min and extensively washed with PBS. A fluorescence microscope was used to visualize specific staining. Immune complexes were evaluated according to density and strength of staining with scores ranging from (−) to (+++). Scores were converted to a numerical value and a mean and SD for each group of mice was determined.

CTL assays

Responding T cells from control and GVHD mice were prepared from pooled spleens, filtered, and washed with DPBS. Stimulator cells were splenocytes from normal F₁ mice treated with ACK lysing buffer and irradiated with 2000 cGy. Final cell concentrations were 2.5 × 10⁶ responding cells/ml and 1.25 × 10⁶ stimulators/ml in CM. Cultures were incubated at 37°C with 5% CO₂ for 5 days. Pooled effector cells were harvested and counted on day 5 for use in the CTL assays. Target cells were P815 (H-2^d) and EL-4 (H-2^b) cell lines labeled with 300 μCi ⁵¹Cr for 90 min at 37°C and washed extensively. Target and effector cells were incubated together at 37°C for 4 h at various E:T ratios and compared with target cells and CM for spontaneous release and target cells plus 3% Triton X-100 for maximum release. Supernatants were harvested using Skatron (Sterling, VA) harvesting filters and frames. The percent specific lysis equals [(experimental − spontaneous)/(maximum − spontaneous)] × 100.

Limiting dilution CTL assays

Single-cell suspensions were prepared from the spleens of B6, DBA/2, BALB/c, B6D2F₁, and CB6F₁ mice. Stimulator cells were CB6F₁ or B6D2F₁ splenocytes irradiated with 2000 cGy and used at 1 × 10⁶ cells/well in 96-well round-bottom plates (Costar, Corning, NY). CM was supplemented with recombinant murine IL-2 (BioSource International, Camarillo, CA) at a final concentration of 20 U/ml for these assays. Cells were cultured for 8 days at 37°C with 5% CO₂. Target cells were P815 (H-2^d) and EL-4 (H-2^b) cell lines labeled as above. Target cells were added to the limiting dilution wells at 3 × 10³ cells/well. After the addition of target cells, plates were briefly spun to collect cells at the bottom of wells. Plates were incubated for 4 h at 37°C.

Limiting dilution assays using B6 anti-CB6F₁ and B6 anti-B6D2F₁ were performed using 2-fold serial dilutions of responding cells in the range of 1.6 × 10⁴ to 125 cells/well. Assays using DBA/2 anti-B6D2F₁ and BALB/c anti-CB6F₁ were set up using 2-fold serial dilutions of responding cells in the range of 5 × 10⁴ to 781.25 cells/well. All cell concentrations were tested using 24 replicates. Spontaneous release of ⁵¹Cr was determined by incubating target cells for 4 h in wells that contained stimulator cells stimulated with CM and IL-2 for 8 days. All of the supernatant was harvested as above. Wells were scored positive for CTL activity if the cpm for a well was greater than the mean spontaneous release plus 3 SDs.

Both CTL precursor frequencies and Th precursor frequencies described below were determined by the least squares method using a computer program supplied by Dr. Charles Orostz (Ohio State University, Columbus, OH).

Limiting dilution proliferation assays

Single-cell suspensions of splenocytes from B6, B6D2F₁, and CB6F₁ mice were prepared as described above and used as responder cells. Stimulator cells were B6, B6D2F₁, or CB6F₁ splenocytes treated with 50 μg/ml mitomycin C (Sigma) per 50 × 10⁶ cells for 30 min at 37°C. Assays were conducted in flat-bottom 96-well plates, using 2.5 × 10⁵ stimulator cells/well and variable numbers of responder cells in CM. Responder cells were used in 0.75-fold serial dilutions from 1 × 10⁵ to 7508.5 cells/well in 48-well replicates. Four milliliters of responder cell suspension at 1 × 10⁶ cells/ml was added to 2 ml of medium in a fresh tube. The new cell suspension was mixed thoroughly, and 4 ml was removed to a fresh tube containing 2 ml of media. This process was continued until eight cell dilutions were ready for use in the assay. One hundred microliters of responder cells and stimulator cells were used per well. Plates were incubated for 4 days at 37°C, pulsed with 1 μCi/well [³H]thymidine, and incubated overnight. Plates were harvested using a Tomtec plate washer (Wallac, Gaithersburg, MD) and counted with a β plate reader (Wallac).

Parental cell engraftment in GVHD mice

It was previously demonstrated that injection of B6 splenocytes into immunocompetent B6D2F₁ mice resulted in high levels of parental cell engraftment and a short lethal disease course (2). Injection of DBA/2, the other parent, resulted in a chronic progressive disease with little parental cell engraftment (2). Typical engraftment profiles for acute and chronic GVHD obtained in this study are shown for this strain combination in Fig. 1⇓A. Injection of BALB/c splenocytes into CB6F₁ resulted in the engraftment pattern expected for chronic GVHD, with little engraftment of parental lymphocytes (Fig. 1⇓B). Injection of B6 splenocytes into CB6F₁ resulted in an initial burst of parental cell engraftment similar to that of B6-into-B6D2F₁. However, between the third and fourth week of GVHD, the percentage of parental cells unexpectedly began to decrease. This decline in parental B6 cells continued until week 12, at which time the disease mirrored chronic GVHD (Fig. 1⇓B). In contrast to the acute GVHD of B6-into-B6D2F₁ mice that die within 4–8 wk (Ref. 7 ; current study), we have observed B6-into-CB6F₁ GVHD mice surviving for >30 wk.

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FIGURE 1.

Parental cell engraftment profiles of GVHD. Flow cytometry was used to identify F₁ and parental cells in spleens from GVHD mice using H-2^d- and H-2^b-specific mAb. The percentage of parental cells in each GVHD model is plotted as a function of time after parental cell injection. A, DBA/2- and B6-into-B6D2F₁ mice. B, BALB/c- and B6-into-CB6F₁ mice. Results shown are averages (and SEM) of splenocytes from three different mice. Similar results were seen in two other separate experiments. ∗, Remaining mice in this group died before data could be collected at week 12.

B6-into-B6D2F₁ mice with acute GVHD exhibited a marked expansion of donor CD4⁺ and CD8⁺ lymphocytes with a concomitant decrease in total donor lymphocytes over the 8-wk disease course. By 8 wk, the spleens of acute GVHD consist of ∼88% (±1.9%) donor lymphocytes and 11.2% (±5.6%) host lymphocytes. The majority of donor cells in the affected spleens are CD4⁺, making up 37% of the total cell number (Fig. 2⇓A). B6-into-CB6F₁ mice exhibited a similar pattern of donor/host cell changes in the spleens of affected mice. Donor lymphocytes expanded over the first 3 wk of disease, with donor cells peaking at 62.2% (±11.7%). Donor CD4⁺ T cell numbers increased to 16.5% of the spleen and donor CD8⁺ T cell numbers increased to 10.6% of the spleen at week 3 (Fig. 2⇓B).

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FIGURE 2.

Detection of CD4⁺ and CD8⁺ cells from mice with B6-into-B6D2F₁ GVHD and B6-into-CB6F₁ GVHD. Splenocytes from mice with GVHD were stained with mAb to H-2^b and H-2^d to determine whether the cells were of host or donor origin. Abs recognizing either CD4 or CD8 were used simultaneously to identify the T cell subsets. A, Staining of CD4 and CD8 T cell subsets from B6-into-B6D2F₁ mice at weeks 1, 3, 6, and 12. B, Staining of CD4 and CD8 T cell subsets from B6-into-CB6F₁ mice at weeks 1, 3, 6, and 12. Similar results were seen in two other separate experiments.

Chronic GVHD has a distinctive pattern of donor cell engraftment in the spleen. The only cell population that engrafted was the donor CD4⁺ T cells at 2–7% of the total spleen cells. This population can be detected throughout the entire disease course and is responsible for the secretion of the cytokines that drive the affected mice to a chronic autoimmune disease (data not shown).

Cellular changes in GVHD mice

Although the percent changes in cells give an overview of the cell populations over time, they do not take into account the dynamics of changes in spleen size and cellularity during the course of disease. To obtain a more complete understanding of the changes within the spleens of the GVHD mice, the number of parental and F₁ splenocytes was determined by multiplying the percentage of each cell type in a given spleen by the total number of cells recovered from that spleen. In acute GVHD there was a drastic decrease in B6D2F₁ cell number by week 2 (Fig. 3⇓A). This dramatic decrease seen in host spleen cell numbers was due to the elimination of B6D2F₁ CD4⁺, CD8⁺, and CD19⁺ cells (Fig. 4⇓A). Concurrently, the B6 cells that were injected into the F₁ hosts expanded and gradually became the major cell population in the F₁ spleens. At 1 wk post-GVHD, there were 1.56 × 10⁷ B6 CD4⁺ T cells in spleens of B6-into-B6D2F₁ mice. This number decreased to 3.34 × 10⁶ cells at 4 wk and rebounded to 3.82 × 10⁷ cells at week 8 (Fig. 5⇓A). CD8⁺ T cells of B6 origin followed a similar pattern of expansion, regression, and expansion during acute GVHD (Fig. 5⇓A) with cell numbers ranging from 3.96 × 10⁶ to 1.21 × 10⁷.

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FIGURE 3.

Numbers of parental, F₁, and total cells in the spleens of F₁ mice as a function of time after parental cell injection. A, B6-into-B6D2F₁ (acute) GVHD; B, DBA/2-into-B6D2F₁ (chronic) GVHD; C, B6-into-CB6F₁ (sequential acute to chronic) GVHD; D, BALB/c-into-CB6F₁ (chronic) GVHD. Results shown are averages (and SEM) of splenocytes from three different mice. Similar results were seen in two other separate experiments.

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FIGURE 4.

Analysis of F₁ cell populations during GVHD. F₁ CD4⁺, CD8⁺, and CD19⁺ cell subsets in the spleens of GVHD mice are plotted as a function of time after parental cell injection. A, B6-into-B6D2F₁ (acute) GVHD; B, DBA/2-into-B6D2F₁ (chronic) GVHD; C, B6-into-CB6F₁ (sequential acute to chronic) GVHD; D, BALB/c-into-CB6F₁ (chronic) GVHD. Results shown are averages (and SEM) of splenocytes from three different mice. Similar results were seen in two other separate experiments.

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FIGURE 5.

Analysis of donor B6 T cell populations during GVHD. CD4⁺ and CD8⁺ cell subsets in the spleens of GVHD mice are plotted as a function of time after parental cell injection. A, B6-into-B6D2F₁ (acute) GVHD; B, B6-into-CB6F₁ mice (sequential acute to chronic) GVHD. Results shown are averages (and SEM) of splenocytes from three different mice. Similar results were seen in two other separate experiments.

Parental cell engraftment in chronic GVHD remained low throughout the disease course, ranging between 2 and 7% (see Fig. 1⇑, A and B). Converting this percentage to cell numbers resulted in a similar picture to that seen with percentages. Both DBA/2 and BALB/c parental cells were detected at low levels in their respective F₁ hosts throughout all the time points tested (Fig. 3⇑, B and D). The parental cells that were detected in the spleens of affected mice were predominantly CD4⁺ T cells (data not shown) as previously reported (1, 16). These CD4⁺ T cells have been suggested to provide help for driving host B cells and the autoimmune symptoms that are manifested later in the disease (16). F₁ CD4⁺ and CD8⁺ T cell levels remained relatively constant over the course of GVHD, whereas CD19⁺ B cells levels transiently increased during the initial phase of the disease, returned to normal levels, and then began to increase again at the later time points (Fig. 4⇑, B and D).

B6-into-CB6F₁ GVHD presents complex cellular interaction patterns not typical of either of the other two forms of GVHD. The number of CB6F₁ cells increased during the first 2 wk. A dramatic decrease in the number of CB6F₁ cells was observed between weeks 2 and 3, simultaneous with an increase in the number of parental B6 cells (Fig. 3⇑C). The loss of CB6F₁ cells included CD4⁺, CD8⁺, and CD19⁺ cells (Fig. 4⇑C), whereas the increase in B6 cells involved CD4⁺ and CD8⁺ T cells (Fig. 5⇑B). By week 4, these spleens had decreased in size from a peak at week 2 of 200 × 10⁶ to 60 × 10⁶ (Fig. 3⇑C). The continued decrease in spleen cell number between weeks 3 and 4 was due to a decrease in B6 cells (Fig. 5⇑B). B6-into-CB6F₁ GVHD continued to diverge from acute GVHD as disease progressed. The spleens reached a maximum size of 250 × 10⁶ cells by 6 wk postdisease induction. During this 2-wk period, massive expansion of CB6F₁ cells occurred. Both F₁ CD4⁺ and CD19⁺ cells expanded to greater than normal levels, whereas the F₁ CD8⁺ T cells returned to normal levels (Fig. 4⇑C). Concurrently, parental B6 cells underwent a small transitory increase in number (Fig. 5⇑B). By 12 wk postdisease induction, the spleens in B6-into-CB6F₁ GVHD mice exhibited a cellular consistency that was similar to that of chronic GVHD mice.

Limiting dilution CTL assays

It was important to compare the CTL precursor frequencies of B6 anti-B6D2F₁ and B6 anti-CB6F₁ because the B6-into-CB6F₁ GVHD resulted in the unexpected sequential acute to chronic pattern of GVHD (on the basis of host spleen repopulation and survival patterns), and acute vs chronic GVHD models were associated with differences in parent anti-F₁ CTL precursor frequencies (2). Therefore, we repeated and verified the earlier limiting dilution experiments indicating that the acute B6-into-B6D2F₁ GVHD vs the chronic DBA/2-into-B6D2F₁ GVHD could be accounted for by differences in parent anti-F₁ CTL precursor frequencies. Our frequencies were 1/2,295 for B6 anti-B6D2F₁ (compared with the earlier 1/1,450), and 1/19,510 for DBA/2 anti-B6D2F₁ (compared with 1/13,500) (2) (Fig. 6⇓). We also simultaneously compared the above parental anti-F₁ CTL precursor frequencies in the B6 anti-CB6F₁ (1/2,370) and BALB/c anti-CB6F₁ (1/19,230) CTL precursor frequencies (Fig. 6⇓). Despite the differences noted above in the GVHD profiles of B6-into-B6D2F₁ and B6-into-CB6F₁ ( Figs. 1–5⇑⇑⇑⇑⇑), the B6 anti-F₁ CTL precursor frequencies were indistinguishable. Furthermore, the CTL precursor frequencies for DBA/2 anti-B6D2F₁ and BALB/c anti-CB6F₁ were indistinguishable from each other, although they were 9-fold lower than the B6 anti-F₁ frequencies.

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FIGURE 6.

Limiting dilution CTL assays. Limiting dilution analysis for B6 anti-B6D2F₁, B6 anti-CB6F₁, DBA/2 anti-B6D2F₁, and BALB/c anti-CB6F₁ CTL responses. Similar results were seen in two other separate experiments.

Anti-H-2 CTL activity in GVHD mice

Acute GVHD is characterized by the expansion of parental CD8⁺ T cells and the ability to isolate anti-F₁ and anti-H-2^d cytotoxicity from the spleens of affected mice (2, 3, 5, 23). To further characterize the B6-into-CB6F₁ GVHD, spleens were removed at 2-wk intervals and tested for anti-H-2^d CTL activity. At 2 and 4 wk post-GVHD induction, similar anti-H-2^d CTL activity was detected in spleens from both acute and B6-into-CB6F₁ GVHD mice (Fig. 7⇓, A and B). This CTL activity could be detected at all time points tested in mice with acute GVHD (Fig. 7⇓, C and D). Anti-H-2^d CTL activity could be detected in B6-into-CB6F₁ GVHD during the first two, but not at the later two, time points (Fig. 7⇓, C and D). B6D2F₁ and CB6F₁ mice with chronic GVHD were also tested at all four time points for the presence of anti-H-2^b CTL activity. At no time during the 8 wk was anti-H-2^b activity detected (data not shown).

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FIGURE 7.

Anti-H-2^d CTL activity in the spleens of GVHD mice. B6D2F₁ and CB6F₁ mice injected with the B6 parental cells were tested for anti-H-2^d CTL activity at 2 (A), 4 (B), 6 (C), and 8 (D) wk after injection. The GVHD spleens were stimulated in vitro with irradiated F₁ spleen cells (B6-into-B6D2F₁ GVHD spleens stimulated with B6D2F₁ cells and B6-into-CB6F₁ GVHD spleens stimulated with CB6F₁ cells) and assayed on P815 (H-2^d) and EL-4 (H-2^b) target cells 5 days later. Data are shown for lysis of P815 targets. Lysis of EL-4 targets was <3.6% (data not shown) in all assays. Similar results were seen in one other independent experiment.

Autoimmune characteristics in GVHD mice

Chronic GVHD is associated with the production of Abs recognizing ssDNA (11, 12), dsDNA (11, 12, 13, 14, 15), and ENA (11, 15, 16). GVHD mice were bled at 16 wk post-GVHD induction, and the serum was tested for autoantibody production. B6D2F₁ mice injected with DBA/2 cells elicited Abs to all Ags tested (Fig. 8⇓, A–C). The BALB/c-into-CB6F₁ chronic GVHD mice and the B6-into-CB6F₁ GVHD mice resulted in Abs being produced to both ssDNA (Fig. 8⇓A) and dsDNA (Fig. 8⇓B) at higher titers than seen with DBA/2-into-B6D2F₁. At 16 wk post-GVHD induction, B6-into-CB6F₁ and both forms of chronic GVHD produced Abs recognizing ENA (Fig. 8⇓C). These Abs were not detected in syngeneic injected F₁ controls.

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FIGURE 8.

Autoantibody production in GVHD mice. Mice were screened at 16 wk following parental cell injection for production of Abs recognizing ssDNA (A–C), dsDNA (D–F), and ENA (G–I) in DBA/2-into-B6D2F₁ mice (A, D, and G), BALB/c-into-CB6F₁ mice (B, E, and H), and B6-into-CB6F₁ mice (C, F, and I). Results were confirmed at other time points in this experiment, and similar results were obtained in four additional independent experiments.

At 24 wk post-GVHD induction, the mice were euthanized and the kidneys tested for the presence of Ig deposits. Control animals injected with syngeneic F₁ cells had no detectable Ig deposits in the kidneys (B6D2F₁, Fig. 9⇓A; CB6F₁, Fig. 9⇓C). Both models of chronic GVHD, DBA/2-into-B6D2F₁ (Fig. 9⇓B) and BALB/c-into-CB6F₁ (Fig. 9⇓D), had significant Ig deposits. The B6-into-CB6F₁ GVHD also had significant numbers of Ig deposits within the kidneys (Fig. 9⇓E). Eight mice in each group were analyzed for Ig deposits, and numerical values were assigned to each score to compare staining between groups (Table I⇓). These results indicated that both models of chronic GVHD, DBA/2-into-B6D2F₁ and BALB/c-into-CB6F₁, resulted in very similar kidney-staining patterns and intensity. B6-into-CB6F₁ mice also had Ig deposits in the kidney, but the mean score of the group was lower than that seen in either of the chronic models of GVHD. Because the B6-into-CB6F₁ mice undergo a period of acute disease before progressing on to chronic disease, differences seen in kidney-staining patterns might be due to a delay in this group in developing renal pathology. Although the initiating events of chronic and B6-into-CB6F₁ GVHD are very different, the end result of these two pathways is an autoimmune disease similar to lupus.

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FIGURE 9.

Fluorescent Ab detection of murine Ig deposits in kidneys. Chronic and sequential acute-to-chronic GVHD mice were tested for the presence of Ig deposits within the kidneys as a parameter of disease. A representative mouse from each group is shown. Control B6D2F₁ injected with syngeneic splenocytes (A), DBA/2-into-B6D2F₁ mouse (B), control CB6F₁ injected with syngeneic splenocytes (C), BALB/c-into-CB6F₁ (D), and B6-into-CB6F₁ (E). Representative results are shown from a single mouse from each group (n ≥ 6 per group).

Role of Th in determining disease outcome

Because CTL precursor frequencies alone did not account for the differences between acute and B6-into-CB6F₁ GVHD, another mechanism must exist. Parental Th cells play a role in driving the F₁ B cell to hyperactivity in chronic GVHD and are presumed to play a supportive role for the CTL in acute GVHD (24). Therefore, it is also possible that differences in the parent anti-F₁ Th cell frequency contribute to determining the final disease outcome. Limiting dilution proliferation assays were performed to test for differences in the B6 response against mitomycin C-treated B6D2F₁ and CB6F₁. The Th cell limiting dilution curves for B6 anti-CB6F₁ and B6 anti-B6D2F₁ were identical and had a frequency of 1/79,120 (data not shown). It is possible that these proliferative Th cell frequencies are not different or that the stimulating alloantigens were limiting on F₁ stimulator cells, resulting in the Th frequencies appearing to be indistinguishable. Therefore, these limiting dilution proliferation assays were repeated using mitomycin C-treated homozygous BALB/c and DBA/2 stimulator cells. We observed a >3-fold difference in Th precursor frequency using allogeneic homozygous H-2^d stimulator cells (Fig. 10⇓). B6 anti-DBA/2 exhibited a Th precursor frequency of 1/19,420 spleen cells, whereas B6 anti-BALB/c had a Th precursor frequency of 1/65,900.

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FIGURE 10.

Limiting dilution Th assays. Limiting dilution proliferation assay analysis for B6 anti-DBA/2 and B6 anti-BALB/c Th responses. Results were confirmed by two additional independent experiments.