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The Journal of Immunology, 2007, 179, 3305 -3314
Copyright © 2007 by The American Association of Immunologists, Inc.
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*Substance via MeSH
Medline Plus Health Information
*Stem Cells

CD4+ T Cells Generated De Novo from Donor Hemopoietic Stem Cells Mediate the Evolution from Acute to Chronic Graft-versus-Host Disease1

Yi Zhang*, Elizabeth Hexner*, Dale Frank{dagger} and Stephen G. Emerson2,*

* Department of Medicine and Pediatrics and {dagger} Department of Pathology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Acute and chronic graft-versus-host disease (GVHD) remain the major complications limiting the efficacy of allogeneic hemopoietic stem cell transplantation. Chronic GVHD can evolve from acute GVHD, or in some cases may overlap with acute GVHD, but how acute GVHD evolves to chronic GVHD is unknown. In this study, in a classical CD8+ T cell-dependent mouse model, we found that pathogenic donor CD4+ T cells developed from engrafted hemopoietic stem cells (HSCs) in C57BL/6SJL(B6/SJL, H-2b) mice suffering from acute GVHD after receiving donor CD8+ T cells and HSCs from C3H.SW mice (H-2b). These CD4+ T cells were activated, infiltrated into GVHD target tissues, and produced high levels of IFN-{gamma}. These in vivo-generated CD4+ T cells caused lesions characteristic of chronic GVHD when adoptively transferred into secondary allogeneic recipients and also caused GVHD when administered into autologous C3H.SW recipients. The in vivo generation of pathogenic CD4+ T cells from engrafted donor HSCs was thymopoiesis dependent. Keratinocyte growth factor treatment improved the reconstitution of recipient thymic dendritic cells in CD8+ T cell-repleted allogeneic hemopoietic stem cell transplantation and prevented the development of pathogenic donor CD4+ T cells. These results suggest that de novo-generated donor CD4+ T cells, arising during acute graft-versus-host reactions, are key contributors to the evolution from acute to chronic GVHD. Preventing or limiting thymic damage may directly ameliorate chronic GVHD.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Graft-versus-host disease (GVHD)3 and its related complications remain the major barriers limiting the efficacy of allogeneic hemopoietic stem cell transplantation (allo-HSCT) (1, 2, 3, 4, 5, 6). GVHD is caused by donor T cells that recognize and react to host histocompatibility differences between the donor and host, and occurs in two distinct syndromes, acute and chronic GVHD (1, 2, 3, 4, 5, 6). Although acute and chronic GVHD have historically been defined by whether signs occur before or after 100 days after HSCT, this arbitrary distinction is now recognized as inappropriate, especially in the era of nonmyeloablative HSCT (7). Instead, the two syndromes are recognized as having overlapping but distinct clinical presentations. Acute GVHD is characterized by the cytotoxic destruction of recipient skin, liver, and gastrointestinal tract, whereas chronic GVHD has diverse clinical manifestations with chronic inflammation affecting multiple tissues, including lymphoid organs, and has many clinical and laboratory features reminiscent of chronic autoimmune disorders (1, 3, 4, 5, 6, 8, 9, 10). In most patients, chronic GVHD develops in the setting of preexisting acute GVHD, and in some patients chronic GVHD begins before acute GVHD resolves (5, 11, 12). These observations suggest that chronic GVHD may be caused as a by-product of acute graft-versus-host (GVH) reactions, but the pathologic mechanism whereby acute GVHD proceeds to chronic GVHD has yet to be defined.

Using an experimental mouse model of CD8+ T cell-dependent GVHD directed against minor histocompatibility Ags (miHAs), we now demonstrated that donor CD4+ T cells generated from engrafted donor HSCs mediate chronic GVHD. These pathogenic donor CD4+ T cells develop in vivo in impaired and stressed thymi during acute GVHD. Keratinocyte growth factor (KGF) treatment improves the restoration of thymic dendritic cells (DCs) and prevents the de novo generation of pathogenic CD4+ T cells causing chronic GVHD. Thus, in vivo development of pathogenic CD4+ T cells is a critical step in the evolution from acute GVHD into chronic GVHD.


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

B6/SJL (H-2Db, CD45.1+) and C3H.SW (H-2Db, CD45.2+, and Ly9.1+) mice were purchased from The Jackson Laboratory. Thymectomized B6/SJL mice were purchased from Taconic Farms. Drinking water of bone marrow transplant (BMT) recipients was supplemented with neomycin sulfate and polymyxin B (Sigma-Aldrich) as previously described (13, 14, 15).

Abs, cytokines, and flow cytometry analysis

Biotinylated anti-mouse CD44 Ab and all other Abs used for immunofluorescence staining were obtained from BD Pharmingen. Microbead-conjugated Abs and streptavidin were purchased from Miltenyi Biotec. Immunofluorescence analyses of cell surface phenotypes and intracellular cytokines were performed by FACScan (BD Biosciences) as previously described (14, 15, 16).

Cell preparations

Bone marrow (BM) cells were prepared from donor mice as previously described (13, 14, 15). T cell-depleted (TCD) BM cells were isolated with anti-CD4- and anti-CD8 Ab-conjugated Dynal beads (Dynal). In some experiments, c-kit+ hemopoietic stem cells (HSCs) were magnetically sorted from the TCD BM to prepare TCD c-kit+ HSCs. CD8+ T cells from spleens and lymph nodes of mice were incubated with microbead-conjugated Ab to CD8, followed by positive selection using magnetic cell sorting (MiniMACS; Miltenyi Biotec). CD4+ T cells were magnetically separated from the spleens and lymph nodes using microbead-conjugated Ab to CD4. CD11c+ cells were magnetically removed by microbead-conjugated Ab to CD11c before purifying donor T cells. To assure stringent purification of CD44lowCD8+, purified CD8+ T cells were stained with allophycocyanin-conjugated anti-CD44 Ab and PE-conjugated anti-CD4 Ab and sorted into CD4–CD44lowCD8+ T cells by FACS (MoFlow; DakoCytomation). The purity of the sorted T cell subset was consistently >98%. CFSE labeling of donor CD8+ T cells was performed as previously described (13, 14, 15).

Mature DCs were prepared from B6/SJL BM as previously described (15, 16, 17). Briefly, B6/SJL BM were stained with biotinylated anti-c-kit Ab followed by streptavidin-conjugated microbeads (Miltenyi Biotec). The c-kit+ hemopoietic progenitor cells were magnetically sorted and incubated for 6 days in IMDM (Invitrogen Life Technologies) supplemented with 10% FBS in the presence of GM-CSF, stem cell factor, and IL-4. CD11c+ immature DCs were magnetically sorted from this 6-day culture using anti-CD11c Ab-conjugated microbeads and stimulated with GM-CSF plus TNF-{alpha} for an additional 2 days to induce their maturation.

GVHD induction

Mice underwent allo-HSCT as previously described (14, 15). Briefly, for C3H.SW anti-B6 mouse GVHD, B6/SJL recipients were irradiated with 10.0 Gy administered in two fractions from a 137Cs source. C3H.SW TCD BM (5 x 106), mixed with or without C3H.SW CD8+ T cells, were transplanted into B6/SJL recipients via tail vein injection (four to eight mice per group per experiment) immediately after irradiation. Recipient mice were weighed twice weekly and monitored for the clinical signs of GVHD and survival. Established clinical grading criteria for the cutaneous inflammation and clinical score of acute GVHD were followed (18). Mice were sacrificed and specimens of liver, skin, and intestine were taken for histopathologic assessment of GVHD as previously described (19, 20). Chronic GVHD was defined as leichenoid skin inflammation, epidermal hyperplasia and collagen I deposition, and chronic inflammation in other organs including the liver, intestine, and thymus (5, 8, 10, 11).

KGF administration

Recombinant human KGF was administered at 5 mg/kg per day s.c. from day –3 to day +1 after allo-HSCT, with mice receiving PBS injection as controls (21).

Pathologic examination of tissues

Mice were sacrificed, and specimens of liver, skin, and intestine were taken for histopathologic analysis. All samples were placed in 10% neutral-buffered formalin (Sigma-Aldrich), embedded in paraffin, sectioned, and stained with H&E for histopathologic assessment.

Immunohistochemical staining of tissues

Immunohistochemical staining of liver and skin was performed as previously described (14, 15). Sections of liver were stained with rat anti-CD8 Ab or anti-CD4 Ab (BD Pharmingen), followed by HRP-conjugated goat anti-rat IgG (Fab')2 (Vector Laboratories). Sections of skin were stained with rabbit anti-collagen I (Sigma-Aldrich) followed by HRP-conjugated goat anti-rabbit IgG (Fab')2 (Vector Laboratories) and were developed with a diaminobenzidine substrate kit (Vector Laboratories). Isotype-matched rabbit IgG (Sigma-Aldrich) was used as a negative control.

Ex vivo stimulation of CD4+ T cells

Donor CD4+ T cells from normal B6/SJL mice or donor CD4+ T cells recovered from B6 mice receiving C3H.SW TCD c-kit+ HSCs plus CD8+ T cells were cultured in IMDM containing 10% FBS in 96-well plates, with or without addition of B6 BM-derived mature DCs at a DC:T cell ratio of 1:3.

Statistical analysis

Survival data were analyzed by life table methods using the Mantel-Peto-Cox summary of {chi}2. All values are expressed as mean ± SD. Experimental data were analyzed by the nonparametric unpaired Mann-Whitney U test. Values of p < 0.05 were to be considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
In vivo generation of host-reactive donor CD4+ T cells in GVHD recipients

We first examined donor CD4+ T cells arising in recipients during the evolution of acute GVHD in vivo. Donor CD44lowCD8+ T cells (2 x 106) and TCD BM (5 x 106) from C3H.SW mice (H-2b) were transplanted into lethally irradiated B6/SJL mice (H-2b) to induce GVHD. B6/SJL mice receiving C3H.SW TCD BM, with or without donor naive CD4+ T cells, served as controls. As expected, B6/SJL mice receiving C3H.SW CD8+ T cells plus TCD BM, but not donor TCD BM alone or donor TCD BM plus naive CD4+ T cells, developed clinical GVHD (Fig. 1A). Although donor grafts contained only TCD BM plus CD8+ T cells, donor-derived CD4+ T cells were detected in the spleens and livers of GVHD B6/SJL mice by day 14 after transplantation and persisted throughout the clinical course of these mice (Fig. 1B). By days 35–42 after transplantation, these recipients had 3.5- and 4.9-fold more donor CD4+ T cells in their livers than did control mice receiving TCD BM and control mice receiving TCD BM plus naive CD4+ T cells, respectively, but 4- to 6-fold less donor CD4+ T cells in their spleens than the controls (Table I). Histologic examination of the liver demonstrated dense infiltrates of both donor-derived CD8+ T cells and CD4+ T cells in portal tracts (Fig. 1C). Unlike control naive T cells from B6/SJL mice, CD4+ cells isolated from the liver of B6/SJL mice with ongoing GVHD produced high levels of IFN-{gamma} upon ex vivo restimulation (Fig. 1D). These results suggest that donor CD4+ T cells are generated in vivo in hosts with ongoing acute GVHD and possess the ability to traffic to GVHD target organs.


Figure 1
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  		FIGURE 1. In vivo generation of CD4+ T cells from engrafted donor BM. Donor C3H.SW TCD BM transplanted with or without donor C3H.SW CD44lowCD8+ T cells, or C3H.SW CD4+ T cells, into lethally irradiated B6/SJL recipients. A, The survival of animals following allo-BMT. {blacktriangleup}, TCD BM (n = 4); {diamondsuit}, TCD BM + CD4+ T cells (n = 4); {blacksquare}, TCD BM + CD44lowCD8+ T cells (n = 8). B, The percentage of donor CD4+ T cells in the spleens and livers of B6/SJL mice receiving donor C3H.SW CD44lowCD8+ T cells + TCD at day 14 (n = 6), day 28 (n = 12), and days 35–42 (n = 16) after transplantation. C, Histologic examination shown represents the livers harvested from B6/SJL mice receiving C3H.SW TCD BM (n = 6), B6/SJL mice receiving donor C3H.SW CD44lowCD8+ T cells + TCD BM (n = 8), and B6/SJL mice receiving donor C3H.SW CD4+ T cells + TCD BM (n = 4) at day 42 after transplantation by routine H&E staining and immunohistochemistry with Ab to CD8 and CD4, respectively. Original magnification: upper, x200; lower, x400. D, Dot plots show percentage of in vivo-generated donor CD4+ T cells recovered from the livers of B6/SJL recipients with GVHD 42 days after transplantation and the percentage of IFN-{gamma}+ T cells of gated subsets in gated CD45.2+ CD4+ T cells stimulated for 16 h in vitro (mean ± SD). Results shown are from one of four independent experiments.

 

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  		Table I. In vivo-generated donor HSC-derived CD4 T cells after allo-HSCT

 
We next characterized the phenotype, proliferative capacity, and immune specificity of donor CD4+ T cells generated in vivo in the context of CD8+ T cell-mediated GVHD. Donor CD4+ T cells were recovered at days 35–42 after transplantation from GVHD B6/SJL mice receiving donor CD8+ T cells plus TCD BM (termed GVHD-CD4+) and non-GVHD B6/SJL mice receiving donor TCD BM alone (termed non-GVHD-CD4+), respectively. Flow cytometry analysis showed that ~70% of non-GVHD-CD4+ T cells were CD44lowCD62Lhigh naive-like T cells, whereas 82% of GVHD-CD4+ T cells expressed the effector memory phenotype CD44highCD62Llow along with increased levels of CD69 and CD25 (Fig. 2A). Five days after ex vivo stimulation by B6/SJL DCs, we recovered 4-fold more donor CD4+ T cells from the culture of GVHD-CD4+ T cells than that of non-GVHD-CD4+ T cells and 25-fold more than that of syngenic B6 CD4+ naive T cells (Fig. 2B). Cultured GVHD-CD4+ T cells, but not non-GVHD-CD4+ T cells, produced high levels of IFN-{gamma} (Fig. 2C). Taken together, these results suggest that in vivo-generated donor GVHD-CD4+ T cells are recently activated cells and have the ability to rapidly proliferate and generate effector cells when reactivated by host DCs.


Figure 2
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  		FIGURE 2. In vivo-generated donor CD4+ T cells are host-reactive cells. At day 42 after the transplantation of C3H.SW TCD BM with or without CD44lowCD8+ T cells, cells were recovered from the spleens and livers of these B6/SJL recipients: GVHD-CD4 T cells derived from GVHD recipients (n = 4) of donor C3HSW TCD BM plus CD44lowCD8+ T cells and non-GVHD-CD4 T cells derived from non-GVHD recipients (n = 4) of donor C3HSW TCD BM alone. A, Dot plots show the percentage of each cell fraction in the gated in vivo-generated donor CD45.2+CD4+ T cell population. Cells were pooled from four mice in each group, which represents three independent experiments. B and C, Donor C3HSW GVHD-CD4+ and non-GVHD-CD4 T cells were magnetically isolated from the spleens and livers of B6/SJL mice, respectively, labeled with CFSE, and cultured in the presence of B6/SJL BM-derived DCs at a ratio of 2 x 105 CD4 T cells and 0.6 x 105 DCs/well in 96-well plates. Naive CD4+ T cells from normal B6/SJL mice were cultured as controls. Five days later, cells were collected, numerated, and stained with Ab to CD4 for flow cytometry. Each group contained three mice, which represents one of two experiments. B, The number of recovered CD4+ T cells in each well at day 5 after ex vivo stimulation. *, p < 0.01. C, Dot plots show the production of IFN-{gamma} and dilution of CFSE in gated CD4+ T cells. Results shown are from one of two independent experiments.

 
In vivo-generated donor GVHD-CD4+ T cells cause chronic GVHD

We then asked whether these in vivo-generated donor GVHD-CD4+ T cells mediated inflammatory reactions in vivo. Donor GVHD-CD4+ T cells were recovered from the spleens and livers of GVHD B6/SJL between days 35 and 42 after transplantation and were adoptively transferred into lethally irradiated secondary B6/SJL recipients, with B6/SJL mice receiving C3H.SW TCD BM with or without non-GVHD-CD4+ T cells as controls. We found that control B6/SJL mice receiving C3H.SW TCD BM with or without non-GVHD-CD4+ T cells did not show any clinical signs of GVHD (Fig. 3, A and B). However, animals receiving non-GVHD CD4+ T cells plus TCD BM did not gain as much weight as those receiving TCD-BM (Fig. 3, A and B), without evident histologic change (data not shown). In contrast, in vivo administration of GVHD-CD4+ T cells caused clinical GVHD, as characterized by weight loss, cutaneous inflammation, and diarrhea (Fig. 3, A and B) as well as a marked reduction in double-positive (DP) thymocytes (Fig. 3C). Histologic examination showed inflammation in several target tissues including thymus, liver, intestine, and skin (Fig. 3, D and E). Damage to the thymus was severe, with effacement of thymic architecture and the infiltration of numerous tingible-body macrophages (Fig. 3E). Biopsies of skin lesions in these B6/SJL mice receiving GVHD-CD4+ T cells showed epidermal hyperplasia and large amounts of collagen I deposition, consistent with chronic GVHD (8, 9, 10, 11) (Fig. 3, E and F). These results show that in vivo-generated donor GVHD-CD4+ T cells are pathogenic T cells with the capacity to induce chronic GVHD.


Figure 3
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  		FIGURE 3. In vivo-generated donor BM-derived CD4 T cells induce chronic GVHD. GVHD was induced in primary B6/SJL mice receiving C3H.SW TCD BM + CD44lowCD8+ T cells. At day 35 after transplantation, donor C3HSW GVHD-CD4+ T cells (3 x 105) were magnetically isolated from these B6/SJL recipients with ongoing GVHD and adoptively transferred along with donor C3H.SW TCD BM (5 x 105) into lethally irradiated secondary B6/SJL mice (n = 21). B6/SJL mice (n = 12) receiving C3H.SW TCD BM and B6/SJL mice (n = 8) receiving C3H.SW TCD BM + non-GVHD-CD4+ T cells were used as controls. Weight changes (A) and clinical scores (B) of GVHD. C, Dot plots show each thymocyte fraction in gated donor CD45.2+ cells (n = 3 for each group, which represents one of two experiments). D, The scores of histologic examination (H&E) of the skin, liver, and intestine from secondary B6/SJL mice receiving TCD-BM (n = 4), non-GVHD-CD4 (n = 4), and GVHD-CD4 (n = 9), respectively. *, p < 0.05. E, Histologic examination of the thymus, liver, intestine, and skin from one of B6/SJL mice receiving C3H.SW TCD BM (n = 4) and B6/SJL mice receiving donor C3H.SW TCD BM + donor GVHD-CD4+ T cells (n = 9), respectively. Original magnification: thymus, x50; liver, intestine, and skin, x200. Arrow in the intestine section indicates the apoptotic crypt epithelial cell. F, Immunohistochemistry staining of collagen I in the skin of secondary recipients. Original magnification, x200. Data shown represent one of four mice for each group.

 
We next tested the ability of these in vivo-generated C3HSW GVHD-CD4+ T cells reactive to B6/SJL to induce GVHD in C3H.SW mice. B6-reactive C3H.SW GVHD-CD4+ T cells were adoptively transferred along with C3H.SW TCD BM into lethally irradiated C3H.SW mice, with C3H.SW mice receiving C3H.SW TCD BM with or without non-GVHD-CD4+ T cells as controls. In vivo administration of donor GVHD-CD4+ cells caused GVHD in syngeneic recipients, manifested as weight loss, diarrhea, and mortality (Fig. 4A). Histologic examination showed hepatic and intestinal inflammation, although cutaneous inflammation was not observed in these syngenic C3H.SW recipients of GVHD-CD4+ T cells (Fig. 4B and data not shown). By contrast, mice receiving non-GVHD-CD4+ T cells did not develop any clinical or histologic signs of GVHD (Fig. 4, A and B). Thus, donor GVHD-CD4+ T cells generated in GVHD recipients are also autoreactive.


Figure 4
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  		FIGURE 4. In vivo-generated donor BM-derived CD4+ T cells induce autologous GVHD. GVHD was induced in primary B6/SJL mice receiving C3H.SW TCD BM plus CD44lowCD8+ T cells. At day 35 after transplantation, magnetically isolated donor GVHD-CD4+ T cells (3 x 105) from these GVHD B6/SJL recipients were adoptively transferred along with donor C3H.SW TCD BM (5 x 105) into lethally irradiated secondary C3H.SW mice. Irradiated C3H.SW mice receiving C3H.SW TCD BM and C3H.SW mice receiving C3H.SW TCD BM plus non-GVHD-CD4+ T cells derived from non-GVHD B6/SJL recipients were used as controls (n = 6 for each group). Survival of animals (A) and histologic examination (B) of the liver and intestine of C3H.SW mice receiving GVHD-CD4 and non-GVHD-CD4 T cells, respectively. Arrows show both scattered portal and lobular inflammatory infiltrates in the liver and chronic inflammation in colonic mucosa and submucosa. Original magnification, x200. Data are representative of two independent experiments.

 
Donor GVHD-CD4+ T cells develop in impaired thymus

To ask whether host-reactive donor CD4+ T cells develop via thymopoiesis vs extrathymic maturation, we looked for de novo thymopoiesis in situ and measured in vivo CD4+ T cell production. To rule out the possibility that residual donor CD4+ T cells in the grafts might contribute to the in vivo generation of CD4+ T cells, we rigorously depleted CD4+ T cells from donor C3H.SW CD44lowCD8+ T cells and positively selected c-kit+ HSCs from donor TCD BM. Flow cytometry analysis showed that residual mature CD4+ T cells were not detected in either the CD8+ T cells or HSC grafts (data not shown). We found that thymic cellularity was dramatically reduced in GVHD B6/SJL mice receiving donor CD44lowCD8+ T cells plus TCD c-kit+ HSCs as compared with non-GVHD B6 mice receiving donor TCD c-kit+ HSCs and normal C3H.SW mice at day 35 after transplantation. However, donor HSC-derived thymocytes were clearly detected and 7.6% of them were donor-derived CD4+ T cells. The percentage of single-positive (SP) CD4+ T cells and double positive (DP) CD4+CD8+ T cells was reversed in B6/SJL mice receiving donor CD44lowCD8+ T cells plus TCD c-kit+ HSCs (55.0 ± 3.0% vs 9.5 ± 4.8%) (Fig. 5A). To further define the role of the thymus, we infused donor CD44lowCD8+ T cells plus TCD c-kit+ HSCs into lethally irradiated thymectomized B6/SJL mice, with euthymic B6/SJL mice as controls. Thymectomized B6/SJL recipients of CD44lowCD8+ T cells developed GVHD with similar kinetics to control euthymic B6/SJL mice (Fig. 5B). Twenty-eight days after transplantation, we found that these thymectomized recipient mice had 52- and 8.5-fold fewer donor CD4+ T cells in the spleens and peripheral blood, respectively, than did control euthymic recipients (0.24 ± 0.06 x 104 vs 12.8 ± 8.9 x 104 for the spleen, and 1.5 ± 0.5 x 104/ml vs 12.7 ± 4.5 x 104/ml for the peripheral blood, respectively) (Fig. 5C). Thus, donor anti-host CD4+ T cells generated from engrafted donor HSCs during acute GVHD are primarily produced in the thymus, under the stress of impaired thymopoiesis produced by CD8+ T cell-mediated thymic damage.


Figure 5
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  		FIGURE 5. Thymus-dependent generation of donor HSC-derived CD4+ T cells. Donor C3H.SW TCD c-kit+ HSCs were transplanted with or without donor C3H.SW CD44lowCD8+ T cells depleted of CD4+ T cells into lethally irradiated euthymic or thymectomized B6/SJL mice, respectively. At day 35 after transplantation, thymocytes were recovered from these mice, numerated, and stained with Ab to CD45.2, CD4, and CD8 for flow cytometry analysis. Thymocytes from normal C3H.SW mice were used as controls. A, The number of thymocytes recovered from euthymic B6/SJL recipient mice. Dot plots show the percentage of donor-derived CD4+ T cells and thymocyte subsets in each fraction (n = 3 for each group). *, p < 0.05. B, Clinical scores of GVHD developed in euthymic B6/SJL mice receiving donor C3H.SW TCD BM (n = 3) or TCD BM + CD8+ T cells (n = 6) and in thymectomized B6/SJL mice receiving donor C3H.SW TCD BM (n = 3) or TCD BM + CD8+ cells (n = 6). C, At day 28 after transplantation, cells were recovered from the spleen and peripheral blood of these recipients and stained with Ab to CD45.2, CD4, and CD8 for flow cytometry. Dot plots show the percentage of each cell subset in euthymic B6/SJL mice receiving donor C3H.SW TCD BM + CD8+ T cells (n = 6) and in thymectomized B6/SJL recipients of donor C3H.SW TCD BM + CD8+ T cells (n = 6).

 
To assess when thymopoiesis recovers following allo-HSCT, donor C3HSW CD44lowCD8+ T cells (1 x 106 cells/mouse) were transplanted in combination with TCD BM (0.5 x 106 cells/mouse) derived from normal B6/SJL mice into lethally irradiated B6/SJL mice as we previously described (22). This allowed us to distinguish BM-derived T cells from those cells derived from infused mature T cells in the thymus. We found that thymic cellularity was decreased to the nadir by day 7 after transplantation (0.05 ± 0.02 x 106 cells) in these recipients, but dramatically increased by 238-fold to 11.9 ± 2.1 x 106 by day 14. Both donor HSC-derived SP CD4+ and DP CD4+CD8+ T cells clearly developed in these recipient mice at day 14 after transplantation. In parallel, alloreactive CD8+ T cells did not invade the thymus until day 14 after transplantation (data not shown). Thus, despite the presence of alloreactive GVH reactions, thymopoiesis indeed occurs as early as 14 days after transplantation.

KGF treatment prevents in vivo generation of pathogenic CD4+ T cells

We next asked whether protection of the thymus prevents the in vivo generation of pathogenic CD4+ T cells. KGF was administered to B6/SJL mice receiving C3H.SW TCD BM plus CD44lowCD8+ T cells at day –3, –2, –1, 0, and +1 after transplantation as previously described (21, 23, 24, 25, 26, 27, 28). Normal saline was administered to B6/SJL mice that received C3H.SW TCD BM alone or C3H.SW TCD BM plus CD44lowCD8+ T cells as controls. KGF-treated B6 recipients of C3H.SW TCD BM plus CD44lowCD8+ T cells developed less severe GVHD than did untreated control B6/SJL mice receiving donor TCD BM plus CD8+ T cells (Fig. 6A), and generated 10-fold more thymocytes than did control B6/SJL mice with GVHD (Fig. 6B). The percentages of DP and SP CD4+ thymocytes in KGF-treated GVHD B6/SJL mice were similar to those of untreated control B6/SJL mice receiving donor TCD BM alone by day 35 after transplantation (79.5 vs 76.4% and 14.8 vs 14.7%, respectively) (Fig. 6C). Donor CD4+ SP thymocytes generated in KGF-treated B6/SJL mice expressed higher levels of CD69 than those of KGF-untreated GVHD B6/SJL recipients of donor CD8+ T cells plus TCD BM, suggesting enhanced TCR activation of these CD4+ SP cells (Fig. 6C). In parallel to the improved thymopoiesis, thymic CD11c+ DCs were clearly reconstituted in KGF-treated B6/SJL mice receiving donor CD44lowCD8+ T cells plus TCD BM (0.17%) and control B6 recipients of donor TCD BM alone (0.14%), whereas thymic DCs were not detected in KGF-untreated control GVHD B6 mice receiving donor CD44lowCD8+ T plus TCD BM (Fig. 6D). In contrast to GVHD-CD4+ T cells recovered from saline-treated control mice, GVHD-CD4+ T cells derived from KGF-treated B6 mice did not cause any clinical signs of GVHD when adoptively transferred into secondary allogeneic B6/SJL recipients (Fig. 7). These results indicate that in vivo administration of KGF enhances the restoration of thymic DCs and thymopoiesis and prevents the thymic development of pathogenic CD4+ T cells mediating chronic GVHD.


Figure 6
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  		FIGURE 6. KGF treatment enhances the in vivo generation of donor CD4+ T cells and thymic DCs. GVHD was induced in B6/SJL mice receiving donor C3H.SW TCD BM + CD44lowCD8+ T cells, with B6/SJL mice (n = 6) receiving donor TCD BM alone as controls. B6/SJL mice that received donor C3H.SW TCD BM + CD44lowCD8+ T cells were treated with or without KGF from days –3 to +1 after transplantation (n = 8 for each group), as indicated KGF(+) and KGF(–), respectively. A, Clinical scores. B, The number of thymocytes recovered from B6/SJL recipient mice (n = 3 for each group). *, p < 0.05; ***, p = 0.36. C, Dot plots show the percentage of donor-derived CD4+ and CD8+ T cells, and histograms show the expression of CD69 on gated SP CD4+ T cells (n = 3 for each group). D, Dot plots show the percentage of thymic CD11c+ DCs and the number of thymic DCs recovered from B6/SJL recipient mice (n = 3 for each group). *, p < 0.05; **, p = 0.27.

 

Figure 7
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  		FIGURE 7. KGF treatment prevents the in vivo generation of pathogenic donor CD4+ T cells causing chronic GVHD. GVHD was induced in B6/SJL mice receiving donor C3H.SW TCD BM plus CD44lowCD8+ T cells, with B6/SJL receiving donor TCD BM alone as controls. B6/SJL mice that received donor C3H.SW TCD BM plus CD44lowCD8+ T cells were treated with or without KGF from days –3 to +1 after transplantation, as indicated KGF(+) and KGF(–), respectively. At day 35 after transplantation, magnetically isolated donor GVHD-CD4+ T cells (3 x 105) from these B6/SJL recipients were adoptively transferred along with donor C3H.SW TCD BM (5 x 105) into lethally irradiated secondary B6/SJL mice. B6/SJL mice receiving C3H.SW TCD BM plus non-GVHD-CD4+ T cells derived from non-GVHD B6/SJL recipients of TCD BM alone were transplanted as controls. Weight changes (A), clinical scores (B), and survival animals (C; n = 5 for each group).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
These results demonstrate that stem cell-derived donor CD4+ T cells are pathogenic in hosts with CD8+ T cell-induced acute GVHD. We found that these host-reactive donor CD4+ T cells developed via thymopoiesis despite thymic dysfunction during acute GVH reactions. Like alloreactive CD8+ T cells, these donor HSC-derived CD4+ T cells were activated, infiltrated into GVHD target organs, and were able to vigorously proliferate and produce IFN-{gamma} upon re-encounter with host DCs. Secondary adoptive transfer of these pathogenic donor CD4+ T cells caused chronic GVHD, primarily targeting the thymus and skin, and to a lesser extent the liver and intestine. KGF treatment improved thymopoiesis and normal immune reconstitution and prevented the in vivo generation of pathogenic donor CD4+ T cells capable of causing chronic GVHD. We observed a concomitant increase in the number of donor-derived mature thymic DCs in hosts receiving CD8+ T cell-replete allo-HSCT and KGF. Thus, thymic-derived host-reactive donor CD4+ T cells generated during alloreactive T cell-mediated acute GVH reactions are key contributors to the evolution from acute to chronic GVHD.

The pathophysiology of chronic GVHD is complex (5, 6, 11, 12) and has been illuminated by several animal models (8, 9, 10, 29, 30, 31, 32). Transplantation of parental lymphocytes into nonirradiated F1 recipients induces lupus-like autoimmune diseases mediated by host-reactive B cells (31), whereas infusion of MHC-matched donor T cells causes chronic GVHD characterized by CD4+ T cell-mediated sclerodermatous skin damage and collagen I deposition (e.g., B10D2->irradiated BALB/c and DBA/2 ->irradiated BALB/c) (29, 30, 32). Similarly, murine models that more closely mimic clinical BMTs suggest that donor CD4+ T cells also play an important role in mediating chronic GVHD. After allo-HSCT chronic GVHD occurs later than acute GVHD primarily mediated by CD8+ T cells and is characterized by noncytotoxic CD4+ T cell-induced host tissue injury (8, 9, 10, 32). In addition to the effect of infused donor T cells on mediating chronic GVHD, data from other studies indicate that alloreactive T cells may attack the recipient thymus during acute GVHD, leading to the aberrant immune recovery and the subsequent in vivo generation of host-reactive T cells (33, 34, 35, 36, 37). However, although these models show that host-reactive T cells play a role in chronic GVHD, none clarify the cellular pathophysiology of how acute GVHD evolves into chronic GVHD.

Previous studies have demonstrated that both acute and chronic GVHD occur in irradiated B6 recipients of MHC-identical donor LP/J mouse lymphocytes that recognize and react to host miHAs (8, 9, 10). In this mouse model, although clinical chronic GVHD occurs later following allo-HSCT, host-reactive CD4+ T cells causing chronic GVHD developed during acute GVHD initiated by donor CD8+ T cells (8, 9, 10). In a similar MHC-identical but miHA-mismatched GVHD model, we recently found that while infusion of naive CD4+ T cells derived from normal donor mice did not induce GVHD in recipients, activated donor CD4+ T cells that were derived from GVHD mice receiving cotransfer of donor naive CD4+ and CD8+ T cells were potent inducers of chronic-like GVHD (38). Collectively, these data suggest that host tissue injury mediated by alloreactive CD8+ T cells during acute GVH reactions is critical to the generation of host-reactive CD4+ T cells (8, 10, 38). In the present study, we found that a substantial proportion of these host-reactive donor CD4+ T cells were generated via thymic-dependent pathways in recipients with donor CD8+ T cell-mediated acute GVHD. Because the thymus is not only the organ where new T cells are generated, but is also the place where central immune tolerance develops (39), it is likely that damage to the thymus mediated by alloreactive CD8+ T cells results in the aberrant recovery of immune function and dysregulation of central tolerance, leading to the development of pathogenic, GVHD-inducing CD4+ T cells.

Our findings that GVHD-CD4+ T cells cause GVHD when adoptively transferred into autologous, as well as allogeneic hosts, further support this conclusion. The disruption of thymic architecture by alloreactive T cells is an important component of murine GVHD. It has been shown that GVHD induces thymic dysplasia, disruption of thymic epithelial cells and DCs, and loss of Hassall’s corpuscles, which collectively result in a failure of both the positive and negative selection of newly developing T cells, and a subsequent period of prolonged immune deficiency (1, 3, 23, 26, 27, 33, 34, 35, 36, 37, 40). Impaired thymic-negative selection results in the generation of autoreactive T cells, leading to the development of autoimmune diseases. An autoimmune syndrome resembling GVHD that develops in hosts after autologous or syngeneic BMTs has been termed autologous or syngeneic GVHD (41, 42, 43, 44). T cells from K14 mice that express MHC class II (MHC-II) only on thymic cortical epithelium are also able to induce autologous GVHD, suggesting that the thymic expression of MHC-II in hemopoietic APCs and medulla cells is critical to eliminating host-reactive T cells (43, 44). This has been confirmed in HSCT models that inactivation of MHC-II on thymic hemopoietic APCs results in donor T cells with the ability to induce autoimmune colitis (45), autologous GVHD in normal syngeneic mice (42), and chronic GVHD in allogeneic mice with MHC differences (46). In the present study, we found that inflammatory cells caused severe damage to the thymic architecture of mice receiving donor CD8+ T cells. Compared with thymi from normal mice and from mice receiving donor TCD BM alone, the percentage of donor HSC-derived SP CD4+ cell populations was dramatically increased in euthymic mice receiving donor CD8+ T cells plus TCD BM, but not in control thymectomized recipients. Thus, CD8+ T cell-dependent acute GVHD leads to a failure of negative thymic selection and subsequent generation of donor HSC-derived host-reactive CD4+ T cells causing chronic GVHD that may coexist with acute GVHD.

Impaired survival or function of thymic DCs may account for the in vivo generation of host-reactive CD4+ T cells seen in our studies. Studies from other groups (42, 43, 44, 46) have demonstrated that T cells with a strong affinity for thymic-peptide complexes are deleted in the thymic medullary via negative selection, mediated primarily by DCs and, less efficiently, thymic medullary epithelial cells. We have found that in vivo-generated GVHD-CD4+ T cells proliferate, expand, and produce high levels of IFN-{gamma} upon re-encounter with host DCs and induce severe thymic injury. These results demonstrate that host DC-reactive donor CD4+ T cells derived from donor stem cells are not eliminated during thymopoiesis. Indeed, we found that thymic DCs were not detected in mice with acute GVHD after receiving allogeneic CD8+ T cells, demonstrating that the thymic DC compartment fails to reconstitute in mice with ongoing GVHD. The present results support and extend previous observations that inactivation of MHC-II expression on thymic DCs results in the failure to negatively select autoreactive CD4+ T cells (42, 43, 44, 46, 47, 48). Furthermore, recent studies (49) have demonstrated that Hassall’s corpuscle-instructed DCs are essential for the natural development of functional regulatory T cells (Treg) in the thymus. Treg have a potent suppressive effect on immune responses to self-Ags (18, 50) and can decrease the incidence and severity of GVHD in T cell-replete allo-HSCT recipients (51). It is possible that impaired thymic DCs may also lead to the failure of the positive selection of Treg cells in the thymus and that this failure may contribute to the subsequent development of chronic GVHD.

Given the essential role of thymic DCs in eliminating host-reactive T cells, one can then ask whether restoring functional thymic DCs can restore appropriate negative selection and prevent the in vivo generation of pathogenic T cells. Data from recent studies indicate that in vivo administration of Flt3 ligand (Flt3L) enhances murine thymopoiesis and increases the numbers of splenic DCs, but exacerbates GVHD in mice receiving T cell-replete allo-HSCT (52). This prevents the potential application of Flt3L for improving the reconstitution of thymic DCs. In contrast to Flt3L, we found that KGF treatment restored the recovery of donor-derived thymic DCs in recipient mice receiving CD8+ T cell-replete allo-HSCT to levels similar to those of non-GVHD mice receiving TCD BM alone. This improved donor reconstitution of thymic DCs in mice receiving T cell-replete allo-HSCT was accompanied by inhibited generation of pathogenic donor CD4+ T cells causing chronic GVHD, although acute GVHD in peripheral tissues was not completely inhibited in response to this treatment with KGF.

The cellular and molecular mechanisms whereby KGF treatment restores the reconstitution of thymic DCs remain to be further determined. KGF was first described as a growth factor for epithelial cells (7, 53, 54, 55, 56, 57, 58, 59). KGF treatment before T cell-replete allo-HSCT decreased damage to epithelial cells such as those of the skin and gastrointestinal mucosa, leading to reduced morbidity and mortality (21, 23, 24, 25, 26, 27, 60, 61, 62, 63). It has been shown that KGF treatment essentially preserves both cortical and medullary epithelial cells in the thymus of nonconditioned F1 recipients despite GVHD in peripheral tissues (27). Data from recent studies further demonstrated that KGF is required for postnatal thymic regeneration (21). Although KGF gene-deficient mice showed defects in thymopoiesis after sublethal irradiation, KGF treatment enhanced the thymic reconstitution in irradiated and immunosuppressive drug-treated mice (21). Because the KGF receptor expression is detected on thymic epithelial cells (7, 54, 56, 57, 58), it is likely that KGF treatment protects thymic epithelial cells against alloreactive T cell-mediated thymic GVHD, leading to improved restoration of thymic DCs. Palifermin (Kepivance; Amgen), a recombinant human KGF, has demonstrated protection against chemotherapeutic or irradiation injury as well as aiding the healing process of various epithelia (21, 59, 64, 65) and is generally safe in allo-HSCT (60). Our findings suggest that palifermin may improve the reconstitution of thymic DCs and the subsequent immune recovery in allo-HSCT patients and suggests the utility of studies of palifermin to prevent the development of chronic GVHD in the clinic.

Mature T cells can undergo homeostatic expansion in vivo in lymphopenic hosts (40, 66). We found that thymectomized recipients with ongoing GVHD had 52-fold fewer donor CD4+ T cells in their spleens than did control euthymic recipients. Thus, it seems unlikely that the expansion of residual donor CD4+ T cells are essential to mediating chronic GVHD in our study. However, because data from previous studies from other laboratories and our own indicate that donor CD4+ T cells are able to induce GVHD under certain circumstances (5, 9, 10, 38, 42, 45, 46, 67), we do not rule out the possibility that mature CD4+ T cells the in graft may contribute to chronic GVHD.

In summary, we believe that in vivo-generated donor HSC-derived CD4+ T cells are the "Hidden Dragon" of CD8+ T cell-mediated GVHD. These de novo-generated donor CD4+ T cells are alloreactive and autoreactive and key contributors to the progression from acute GVHD to chronic GVHD. Donor anti-host CD4+ T cells may provide key insight into mechanisms involved in chronic GVHD and represent a potential therapeutic target for preventing or treating chronic GVHD. Furthermore, investigating how KGF improves the reconstitution of thymic DCs and their interaction with pathogenic CD4+ T cells will allow for the development of new approaches for restoring normal immune function following allo-HSCT.


   Acknowledgment
 
We greatly appreciate the technical support given by Shuqin Wang (Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA).


   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 This work was supported by a grant of the Specialized Center for Research from the Leukemia and Lymphoma Society of America and Grant R01 CA102464-01 from the National Institutes of Health. Back

2 Address correspondence and reprint requests to Dr. Stephen G. Emerson, Department of Medicine, Division of Hematology and Oncology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104. E-mail address: emersons{at}mail.med.upenn.edu Back

3 Abbreviations used in this paper: GVHD, graft-versus-host disease; allo-HSCT, allogeneic hemopoietic stem cell transplantation; HSC, hemopoietic stem cell; GVH, graft-versus-host; miHA, minor histocompatibility Ag; KGF, keratinocyte growth factor; DC, dendritic cell; BMT, bone marrow transplant; BM, bone marrow; TCD, T cell depleted; DP, double positive; SP, single positive; MHC-II, MHC class II; Treg, regulatory T cell; Flt3L, Flt3 ligand. Back

Received for publication February 1, 2007. Accepted for publication June 10, 2007.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
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R. Kapur, S. Ebeling, and A. Hagenbeek
B-cell involvement in chronic graft-versus-host disease
Haematologica, November 1, 2008; 93(11): 1702 - 1711.
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