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The Journal of Immunology, 2006, 176: 3383-3390.
Copyright © 2006 by The American Association of Immunologists

Alloantigen Affinity and CD4 Help Determine Severity of Graft-versus-Host Disease Mediated by CD8 Donor T Cells1

Xue-Zhong Yu2,*,{dagger}, Michael H. Albert3,* and Claudio Anasetti4,*,{dagger}

* Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109; and {dagger} Department of Medicine, University of Washington, Seattle, WA 98195


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
TCR affinity dictates T cell selection in the thymus and also has a high impact on the fate of peripheral T cells. Graft-vs-host disease (GVHD) is a pathological process initiated by activation of donor T cells after adoptive transfer into an allogeneic recipient. How TCR affinity affects the potential of alloreactive T cells to induce GVHD is unclear. Using alloreactive CD4+ and CD8+ TCR transgenic (Tg) T cells, GVHD models are presented that allow for the visualization of how CD8+ alloreactive T cells behave in response to alloantigens with different TCR affinity in the absence or presence of CD4 help. In a nonmyeloablative transplant model where GVHD lethality is due to marrow aplasia, alloreactive CD8+ TCR Tg T cells induced significantly more severe GVHD in the recipients that express an intermediate-affinity alloantigen than in the recipients that express a high-affinity alloantigen. In a myeloablative transplant model where GVHD lethality is due to epithelium injury, CD8+ TCR Tg cells were also more pathogenic in the recipients with an intermediate-affinity alloantigen than in those with a high-affinity alloantigen. The presence of alloreactive CD4+ TCR Tg cells enhanced the potential of CD8+ TCR Tg cells to cause GVHD in recipients with an intermediate-, but not with a high-, affinity alloantigen. These findings underscore that alloantigen affinity and CD4 help control the fate and pathogenicity of alloreactive CD8+ T cells in vivo.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Graft-vs-host disease (GVHD)5 is a pathological process initiated by activation of donor T cells after adoptive transfer into an allogeneic recipient. The types and degree of histocompatibility disparities between the donor and recipient, host conditioning regimes, and ongoing inflammation control alloreactive T cell responses and GVHD manifestations (1). It is assumed that CD4 and CD8 T cell cooperation is required for the induction of lethal GVHD. Understanding the interactions between grafted CD4 and CD8 T cells is important for developing better strategies to prevent GVHD. However, donor T cells that recognize recipient alloantigens lack specific markers and are only present at a low frequency in vivo, making it difficult to monitor and characterize them directly in the host. The availability of an experimental model in which identifiable T cell populations with known alloantigen specificity cause GVHD would make it feasible to study the fate of T effector cells and the development of GVHD in vivo. With such a model, it would be possible to test strategies designed to eliminate or inactivate T cells responsible for GVHD while preserving other T cell populations that do not recognize recipient alloantigens thereby preserving anti-infectious or antitumor immunity.

The use of TCR transgenic (Tg) T cells has allowed the visualization of Ag-specific T cell population dynamics and function in vivo. Previous attempts to develop a clinical model of GVHD using alloreactive Tg T cells have met with limited success. Transfer of Ld (2, 3) or H-Y-specific (4) CD8+ Tg cells into Ag-expressing hosts results in a short burst of T cell expansion, followed by apoptosis and the development of anergy in the residual cells. Those Tg cells did not cause overt GVHD in these models. However, a polyclonal CD4+ T cell population, when transferred together, increased the severity of GVHD initiated by anti-Ld Tg T cells (5). Interaction of CD4 and CD8 T cells in the development of GVHD has been addressed by adoptive transfer of TCR Tg CD4+ and CD8+ cells into a recipient that expresses specific alloantigens for both T cell subsets (6). However, this study was limited to sublethal conditioning of the host, which does not adequately represent the clinical situation of GVHD because recipient death is caused by destruction of recipient hemopoiesis and not epithelial damage in typical GVHD target organs such as gut, skin, and liver.

To address these limitations, we developed a novel system in which CD4+ D10 and CD8+ 2C TCR Tg populations alone or together were transferred in combination with donor marrow cells into lethally irradiated recipients that express alloantigens to be recognized by either T cell population. The D10 TCR is positively selected by IAk in the thymus and recognizes the IAb alloantigen (7). The 2C TCR is positively selected by H-2Kb and negatively selected by H-2Ld in the thymus (8, 9). The 2C TCR binds to a natural peptide p2Ca presented by H-2Ld as an alloantigen with a high affinity (10). A naturally occurring mutant of H-2Kb, termed H-2Kbm3 (Asp77 to Ser, Lys89 to Ala) is also an alloantigen to 2C (11). The 2C TCR binds to a natural peptide dEV8 presented by H-2Kbm3 with an intermediate affinity and to the same peptide presented by H-2Kb with a low affinity, which is lower by factors of ~20 and 30, respectively, than the p2Ca/H-2Ld complex (12, 13, 14). Using the D10 and 2C TCR Tg systems, we were able to track the fate and pathogenicity of CD4+ or CD8+ effector cells alone or in combination in recipients that express specific alloantigens. Furthermore, we demonstrated that alloantigen affinity and CD4 help are the key factors that control GVHD development induced by CD8 alloreactive T cells.


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

C57BL/6J (B6), B6.SJL-Ly5a Ptprca Pep3b (B6.Ly5.1), C3H, B10.BR, B6.C-H2bm3/KhEgJ (B6.bm3), (B6 x C3H)F1, (BALB/c x B6)F1 (CB6F1) mice were purchased from The Jackson Laboratory. Founders of 2C TCR Tg mice were provided by D. Loh (Nippon Roche Research Center, Kamakurshi, Japan). Founders of D10 TCR-Tg mice were provided by D. Sant’Angelo (Memorial Sloan-Kettering Cancer Center, New York, NY) (7). (B6 x bm3)F1 mice were bred in our facility. Mice were housed in microisolator cages at the Fred Hutchinson Cancer Research Center (Seattle, WA). Experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee.

Isolation of T cells and bone marrow (BM) cells

CD4+ and CD8+ cells were purified from pooled spleen and lymph node cells by positive selection with a magnetic cell separation system (Miltenyi Biotec) as described previously (15). T cell purity ranged from 95 to 99%. BM was harvested from tibia and femurs, and T cells were depleted through complement lysis of Thy1.2+ cells.

Transplantation

In nonmyeloablative models, CB6F1, (B6 x bm3)F1, or (B6 x C3H)F1 mice were exposed to 750 cGy of irradiation at 20 cGy/min, and freshly isolated CD8+ cells or CD4+ cells at the doses indicated were injected via the tail vein to recipients within 24 h after irradiation. In myeloablative models, CB6F1, (B6 x bm3)F1, or (B6 x C3H)F1 mice were exposed to 1000–1200 cGy of irradiation at 20 cGy/min, and T cell-depleted (TCD)-BM cells alone or in combination with purified CD4 or/and CD8 cells from indicated donors were injected via the tail vein to recipients within 24 h after irradiation. Recipient mice were monitored every other day for mortality and clinical signs of GVHD, such as ruffled fur, hunched back, inactivity, or diarrhea. Body weight was measured two times a week. Animals judged to be moribund (i.e., unable to take food or water) were sacrificed and counted as GVHD lethality.

Immunofluorescence analysis

Peripheral blood samples or spleens were collected from recipients at the time points indicated. Cells were stained and analyzed using a FACScan flow cytometer and CellQuest software (BD Biosciences). Anti-CD4-FITC, anti-B220-PE, anti-H2b-biotin, anti-CD8-CyChrome, anti-CD4-CyChrome, streptavidin-PE, and streptavidin-CyChrome were purchased from BD Pharmingen. Biotin-labeled Ab specific for Ly5.1, 2C TCR (1B2), and D10 TCR (3D3) were prepared in our laboratory.

CTL assay

Cytotoxic activity of 2C cells was measured directly without in vitro restimulation as previously described (3). Briefly, spleen cells from each recipient were used as effectors against 51Cr-labeled P815 (Ld+) targets with a E:T at 40:1. Chromium release was measured after 4.5 h of incubation, and percent cytotoxicity was calculated as (experimental release – spontaneous release)/(maximal release – spontaneous release) x 100%.

Statistical analysis

The log-rank test was used to detect statistical differences in recipient survival in GVHD experiments. The Student t test was used to compare percentages or numbers of donor T cells and host B cells.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
The effect of alloantigen affinity on GVHD induced by TCR Tg CD8+ T cells

2C TCR Tg cells induce acute GVHD in alloantigen (Ld)-bearing CB6F1 recipients, where the target of GVHD is primarily host lymphoid tissues (3). However, 2C cells rapidly die shortly after their expansion in Ld+ recipients (2, 5). We hypothesized that the abortive response of 2C effector cells might be due to clonal deletion resulting from TCR engagement with a strong Ld alloantigen. To test this hypothesis, we compared the pathogenicity of 2C cells in CB6F1 and (B6 x bm3)F1 recipients that expressed a high- (Ld) and intermediate-affinity (Kbm3) ligand to the 2C TCR, respectively. After adoptive transfer, 2C cells engrafted, expanded, and eliminated host B cells and thymocytes in sublethally irradiated CB6F1 recipients (Ref.3 and data not shown), but most of those recipients survived long-term without clinical signs of GVHD (Fig. 1). 2C cells not only engrafted and expanded, but also caused death in the majority of (B6 x bm3)F1 recipients (Fig. 1). The GVHD lethality was significantly higher in (B6 x bm3)F1 than in CB6F1 recipients (p = 0.029).


Figure 1
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  		FIGURE 1. The fate and pathogenicity of 2C cells in allogeneic recipients. A, CB6F1 and (B6 x bm3)F1 mice were exposed to irradiation at 750 cGy, and then transferred with 5–8 x 106 purified CD8+ cells from 2C Tg mice. A group of irradiated F1 mice that were not transferred with donor T cells were used as controls without GVHD. Data were pooled from three separate experiments with the same setting. B, A total of 5 x 106 purified CD8+ cells from 2C Tg mice were transferred to sublethally irradiated CB6F1 mice. A group of CB6F1 mice were NK-depleted with 50 µg/mouse asialo GM1 Ab on days –1 and –2 before cell transfer. Irradiated F1 mice that were not transferred with donor T cells were used as controls without GVHD. Two weeks after cell transfer, blood samples were collected and blood cells were strained for the presence of 2C cells and host B cells. The percentage of 2C cells (1B2+) and host B cells (B220+) in total WBC are presented as the average ± 1 SD of each group (n = 6).

 
Because CB6F1 recipients express completely incompatible MHC Ags as compared with donor B6 mice, it is possible that the different fate and GVHD-inducing potential of 2C cells in the two different types of hosts resulted from hybrid resistance mediated by NK cells in the CB6F1 recipients. Considering that hybrid resistance can be overcome by high doses of donor cells (16), we tested whether higher doses of 2C T cells could induce the level of GVHD in CB6F1 recipients comparable to that in (B6 x bm3)F1 recipients. By titrating up the number of 2C cells, we found that increased cell doses (up to 15 x 106 cells/recipient) did not enhance engraftment and GVHD lethality in CB6F1 recipients (data not shown). To rule out the role of NK-mediated hybrid resistance against 2C cells in CB6F1 recipients, we depleted NK cells in the recipients before cell transfer with anti-NK cell Ab asialo GM1. We found that 2C cells were unable to cause GVHD lethality in CB6F1 mice regardless of NK depletion, and 2C expansion and host B cell elimination were comparable in both groups (Fig. 1B). Taken together with our previously published work that 2C cells have a higher level of expansion and a lower level of apoptosis in (B6 x bm3)F1 recipients than in CB6F1 recipients (17), the current data on recipient survival indicate that pathogenicity of CD8 effector cells activated by a high-affinity alloantigen is mitigated by rapid clonal deletion.

Pathogenicity of CD4+ TCR Tg cells in a nonmyeloablative model

We first evaluated the capability of CD4+ cells alone to induce GVHD. To visualize the CD4+ effector cells, we chose to test the response of D10 Tg cells in recipients that express a specific alloantigen (IAb) to the D10 TCR (7). We transplanted purified CD4+ D10 cells at various doses into sublethally irradiated syngeneic C3H or allogeneic (B6 x C3H)F1 mice. A group of irradiated (B6 x C3H)F1 mice were used as no transplant controls. All (B6 x C3H)F1 mice that received 0.1–2.5 x 106 D10 cells/mouse rapidly lost their body weights (data not shown) and died within 4 wk after transplantation (Table I). In contrast, with rare exception, the irradiation controls, syngeneic recipients, and allogeneic recipients of 0.02 x 106 or fewer donor cells survived long-term without significant signs of GVHD. Furthermore, survival increased after transplantation of 5 x 106 or more cells/mouse (Table I), which is consistent with prior data on transplantation of non-TCR Tg CD4+ T cells (18).


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  		Table I. TCR-Tg D10 cells induce lethal GVHD in (B6 x C3H)F1 recipientsa

 
To further evaluate GVHD in vivo, peripheral blood samples were obtained from each mouse at multiple time points to detect donor T cells and host B cells. The data indicate that D10 cells engrafted in both C3H and (B6 x C3H)F1 recipients (Fig. 2A). D10 cells expanded extensively at early time points and then rapidly disappeared 2 wk after transplantation (Fig. 2B, upper panel), and prevented host B cells from recovery (Fig. 2B, lower panel). In contrast, D10 cells expanded slowly but continuously in irradiated syngenic recipients without impairing host B cell reconstitution (Fig. 2B). The number of D10 cells in the recipient circulation was lower when 5 x 106 or more D10 cells/mouse were transferred into (B6 x C3H)F1 recipients (Fig. 2B), suggesting that fratricide of D10 cells occurred in allogeneic recipients at a high concentration of donor cells. These results are consistent with prior data on transplantation of non-TCR Tg CD4+ T cells.


Figure 2
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  		FIGURE 2. D10 cell expansion and host B cell recovery in syngeneic or allogeneic recipients. C3H (n = 5) or (B6 x C3H)F1 (n = 5) mice were exposed to irradiation at 750 cGy, and then transferred with purified CD4+ cells from D10 mice at the numbers indicated. A group of irradiated F1 mice (n = 5) not transferred with donor T cells were used as controls without GVHD. A, On day 15 after transplantation, a peripheral blood sample was collected from each mouse and stained for expression of CD4, D10 TCR (3D3), and B220. The numbers shown are percentage of CD4+/3D3+ T cells (left panels) or percentage of B220+ B cells (right panels) in total WBC. B, Peripheral blood samples were collected from each recipient on the days indicated. The absolute numbers of CD4+/3D3+ T cells (upper panel) or B220+ B cells (lower panel) in the blood were calculated from the WBC counts multiplied by the percentage of either population among WBC based on flow cytometric analysis.

 
Pathogenicity of CD4+ TCR Tg cells in a myeloablative model

We next studied the pathogenicity of D10 cells in a myeloablative model, which represents a close approximation of clinical acute GVHD. Lethally irradiated (B6 x C3H)F1 mice were transplanted with BM from D10 Tg– donors alone or plus CD4+ cells from D10 Tg+ donors, and clinical signs of GVHD and death were monitored. Recipients of BM alone or BM plus D10 cells survived long-term without significant clinical signs of GVHD (data not shown). To determine the fate and pathogenicity of D10 cells in vivo, we analyzed BM engraftment and D10 cell expansion in recipient splenocytes 15 days after transplantation. In the recipients of BM alone, B cells were predominantly of donor origin (H2b–) (Fig. 3, right panel), indicating the recipient was almost completely reconstituted with donor BM. As expected, there were no D10 cells detectable in the recipients of BM alone, but ~1 x 106 D10 cells could be found in the spleens of recipients of BM plus D10 cells (Fig. 3). There were significantly fewer donor B cells (B220+H2b–) (p < 0.0001) and CD4+ T cells (CD4+3D3–) (p = 0.001) in the recipients of BM plus D10 cells than in the recipients of BM alone (Fig. 3), suggesting that D10 cells prevented B cell reconstitution in the host. These data indicate that in this particular setting, TCR Tg CD4+ cells are unable to cause lethal GVHD, but do expand and impair B cell reconstitution in the recipients.


Figure 3
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  		FIGURE 3. D10 cell expansion and host B cell recovery in allogeneic recipients that were lethally irradiated. (B6 x C3H)F1 mice were exposed to irradiation at 1100 cGy, and then transferred with 10 x 106 TCD-BM alone or TCD-BM plus 2.5 x 106 purified D10 cells. On day 15 after transplantation, spleens were collected from all recipients and stained for expression of CD4, 3D3, B220, and H2b (the host MHC type). The absolute numbers of each population in the spleen were calculated from the total cell number multiplied by the percentage of each population among total spleen cells based on flow cytometric analysis. Data are presented as the average ± 1 SD of three to four mice in each group and the results are representative of two replicate experiments.

 
Cooperation of D10 and 2C cells in GVHD

We then studied the effects of TCR-Tg CD4 cells on GVHD mediated by 2C cells. Lethally irradiated CB6F1 mice were used as recipients, which express Ld to be recognized by 2C cells and express IAb to be recognized by D10 cells. CB6F1 mice that were transplanted with TCD-BM from B10.BR donors were used as controls without GVHD. B10.BR mice were used as BM donors in this myeloablative GVHD model, because neither D10 nor 2C cells react with the BM graft (H2k) whereas D10 cells react against B6 mice (H2b). In separate experiments where B6 mice were used as the source of donor BM, 2C cells alone also failed to induce GVHD lethality in CB6F1 recipients (data not shown), excluding a potential effect of MHC disparities between alloreactive T cells and the allograft on the development of GVHD.

Recipients of TCD-BM plus 2C cells survived long-term without obvious signs of GVHD. A subset of recipients of TCD-BM plus D10 cells or plus D10 and 2C cells died (Fig. 4), but the survival rate of either group was not significantly different from the group with BM alone (p > 0.05). The results indicate that 2C cells were unable to induce GVHD lethality in CB6F1 recipients even with CD4 help.


Figure 4
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  		FIGURE 4. Survival and weight changes of CB6F1 recipients that were transferred with 2C, D10, or both types of cells. CB6F1 mice were irradiated at 1200 cGy on day –1. Then each mouse was transferred with 8 x 106 TCD-BM cells alone, TCD-BM plus 2–3 x 106 CD4+ D10 cells, TCD-BM plus 5–7 x 106 2C CD8+ cells, or TCD-BM plus both. Survival (left panel) and average body weight changes in each group (right panel) are shown, and the data were pooled from two separate experiments with the same experimental setting.

 
To evaluate the fate and pathogenicity of TCR-Tg donor cells, we measured 2C, D10, and B cells in recipient spleens on day 15 after transplantation (Fig. 5A). The number of 2C cells was significantly higher in the recipients transplanted with 2C plus D10 cells than 2C cells alone (p = 0.005) (Fig. 5B, left panel), indicating that D10 cells facilitated 2C cell expansion. The number of D10 cells was also increased 2-fold when 2C cells were coadministered (Fig. 5B, middle panel, p = 0.1). A small proportion of host B cells (B220+H2d+) was still present in the recipients of TCD-BM alone, but not in the recipients of additional 2C or D10 cells (Fig. 5A, right panels), indicating that either type of donor T cells could eliminate host B cells.


Figure 5
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  		FIGURE 5. Engraftment and expansion of D10, 2C, and B cells in CB6F1 recipients that were lethally irradiated. CB6F1 mice were irradiated at 1000 cGy, and each of them was transferred with 10 x 106 TCD-BM cells alone (n = 5), TCD-BM plus 2.5 x 106 CD4+ D10 cells (n = 5), TCD-BM plus 7 x 106 CD8+ 2C cells (n = 4), or TCD-BM plus both (n = 5). A, On day 44, spleens were collected from each mouse and stained for expression of CD8, 1B2, CD4, 3D3, B220, and H2d. Percentages of CD8+/1B2+ (left panels), CD4+/3D3+ (middle panels), and B220+/H2d+ (right panels) in the spleen are shown from one representative mouse of each group. B, Absolute numbers of each population are calculated from the total splenocytes multiplied by the percentage of each population based on flow cytometric analysis. The data are presented as the average ± 1 SD.

 
The effect of alloantigen affinity on GVHD induced by 2C cells in the presence of CD4 help

The 2C TCR Tg cells induced a significantly higher rate of GVHD lethality in (B6 x bm3)F1 than in CB6F1 recipients in a nonmyeloablative model (Fig. 1). Therefore, we tested how alloantigen affinity affects the GVHD lethality mediated by 2C cells in the presence of CD4 help from D10 cells in a clinically more relevant myeloablative model. Lethally irradiated (B6 x bm3)F1 mice were used as recipients, which express H-2Kbm3 to be recognized by 2C cells and express IAb to be recognized by D10 cells. Irradiated F1 mice that were transplanted with TCD-BM from B10.BR donors were used as controls without GVHD. B10.BR mice were used as BM donors in this myeloablative GVHD model, as neither D10 nor 2C cells react with the BM graft (H2k) whereas D10 cells react against B6 mice (H2b).

The survival rate of recipients that were transplanted with TCD-BM plus D10 cells was not different from the recipients with TCD-BM alone (p = 0.174). In contrast, all the recipients that were transplanted with TCD-BM plus 2C cells died, significantly different from the recipients of TCD-BM alone (p < 0.001). Furthermore, GVHD was significantly more severe in the recipients with both 2C and D10 cells than those with 2C (p = 0.001) or D10 (p < 0.001) cells alone (Fig. 6). These results indicate that 2C cells but not D10 cells were able to induce GVHD lethality in (B6 x bm3)F1 recipients, but D10 cells provided help to enhance the pathogenicity 2C cells.


Figure 6
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  		FIGURE 6. Survival and weight changes of (B6 x bm3)F1 recipients that were transferred with 2C, D10, or both types of cells. (B6 x bm3)F1 mice were irradiated at 1200 cGy on day –1, and then each mouse was transferred with 10 x 106 TCD-BM cells from B10.BR donors alone, TCD-BM plus 2–3 x 106 CD4+ D10 cells (n = 11), TCD-BM plus 5–7 x 106 2C CD8+ cells, or TCD-BM plus both. Survival (left panel) and weight changes (right panel) are shown, and the data were pooled from three separate experiments with the same setting.

 
In comparing two types of recipients (Fig. 4 vs Fig. 6), D10 cells induced GVHD lethality similarly in CB6F1 and (B6 x bm3)F1 mice (p = 0.82). The results were expected because D10 cells recognize the same alloantigen (IAb) expressed on either recipient. In sharp contrast, 2C cells caused >70% lethality in (B6 x bm3)F1 recipients but 0% lethality in CBF1 recipients in the absence of CD4 help (p = 0.0002). In the presence of CD4 help, 2C cells also caused significantly more severe GVHD in (B6 x bm3)F1 recipients than in CB6F1 recipients (p < 0.0001).

To further analyze the cooperation between D10 and 2C cells in (B6 x bm3)F1 recipients, cytokine levels (TNF-{alpha}, IFN-{gamma}, and IL-5) in the serum as well as expansion of D10 and 2C cells and CTL activity of 2C effectors in the spleen were evaluated 7 days after transplantation. The levels of TNF-{alpha}, IFN-{gamma}, and IL-5 were comparable in the serum from recipients of D10 plus 2C cells and from recipients of 2C cells alone (p > 0.1), suggesting that cytokine production did not contribute to acute mortality of the recipients of D10 plus 2C cells. When D10 cells were coadministered, the expansion level of the 2C cells remained the same, but their CTL activity was increased (p = 0.07) (Fig. 7). Considering most of the activated CD8+ T cells presumably migrated into the target organs (i.e., gastrointestinal mucosa), this increase, although marginally significant, suggests that the D10 cells enhanced the CTL activity of 2C cells. Collectively, these data demonstrate that alloantigen affinity plays a key role in the development of GVHD and that CD4 help further enhances GVHD pathogenicity of alloreactive CD8+ T cells.


Figure 7
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  		FIGURE 7. Expansion and function of donor 2C and D10 T cells in (B6 x bm3)F1 recipients. (B6 x bm3)F1 mice were irradiated at 1200 cGy on day –1, and then each mouse was transferred with 10 x 106 TCD-BM cells alone, TCD-BM plus 2.5 x 106 CD4+ D10 cells (n = 11), TCD-BM plus 7 x 106 2C CD8+ cells, or TCD-BM plus both. Seven days after transplantation, spleen of each recipient was harvested and analyzed separately. A, Splenocytes were stained for expression of 2C TCR (1B2) and D10 TCR (3D3). The numbers in the legend showed the absolute cell count of 2C and D10 cells in the spleen, which were calculated from the total cell number multiplied by the percentage of each population among total spleen cells based on flow cytometric analysis. Data are presented as the average ± 1 SD (x104) of each group (n = 3–5). B, Splenocytes were used as effectors and their CTL activity was measured against P815 targets (E:T ratio at 40:1). The percentage of specific killing was shown in each recipient.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Transfer of 2C cells into Ld-bearing recipients results in a burst of expansion of 2C cells followed by apoptosis and anergy in the residual 2C cells, but no overt GVHD development in the recipient (2, 3, 5, 6). This study has shown that 2C cells induced GVHD lethality in Kbm3+ recipients with a significantly higher rate than in Ld+ recipients. The outcome was observed in sublethally (Fig. 1) as well as in lethally irradiated recipients (Figs. 4 and 6), and in the absence or presence of CD4 help (Figs. 4 and 6). These results indicate that alloantigen affinity plays a critical role in the fate and pathogenicity of donor CD8+ T cells.

Why do 2C cells have a limited expansion capacity and why are they unable to induce lethal GVHD in recipients that express high-affinity alloantigens? Lethal GVHD could be induced in Ld+ recipients by 2C cells that were deficient for IFN-{gamma} (19). Because IFN-{gamma} has been reported to play an important role in regulating the death of activated CD4+ and CD8+ cells (20, 21, 22, 23), the augmentation of GVHD in Ld+ recipients of IFN-{gamma}-deficient 2C cells is likely due to decreased cell death of those alloreactive CD8+ T cells. Furthermore, our recent work directly showed that 2C cells had a higher level of expansion, associated with a higher level of apoptosis and more severe injury of the lymphoid compartment in Ld+ recipients than in Kbm3+ recipients (17). Deletional tolerance of CD8+ T cells in response to high-dose or high-affinity TCR ligation is not restricted to 2C Tg cells. In an experimental autoimmune encephalomyelitis model with CD8+ Tg cells specific for myelin basic protein, immunization with wild-type Ag expanded the high-affinity T cells which was required to induce encephalomyelitis. In contrast, immunization with strongly antigenic analogs led to the elimination of T cells bearing high-affinity TCRs by apoptosis, thereby preventing disease development (24). In an autoimmune diabetes model with CD8+ Tg cells specific for OVA, a high dose of Ag led to tolerance of OVA-reactive T cells through deletion (25, 26). In the current study, we demonstrated that ligation with high-affinity alloantigens triggers death of alloreactive CD8+ T cells and thus facilitates transplantation tolerance.

The lack of CD4 help is another reasonable explanation for the inability of CD8+ T cells to mount an effective anti-host response. In support of this hypothesis, Gonzalez et al. (6) showed that the development of lethal GVHD occurred only when alloreactive CD8+ (2C) plus CD4+ (TEa) Tg T cells, but not CD8+ cells alone, were transferred into sublethally irradiated recipients. In this study, we found that CD4+ D10 cells enhanced 2C-mediated GVHD severity in lethally irradiated recipients that express either high- or intermediate-affinity alloantigens. These findings extend the existing knowledge about the role of CD4+ T cells in controlling the fate and function of CD8+ T cells in GVHD. The level of 2C expansion was significantly higher with CD4+ donor T cells than without (Fig. 5). Enhanced expansion of CD8+ T cells can result from an increased rate of cell division induced by cytokines secreted by CD4+ cells, and/or CD4 help may also prevent rapid deletion of CD8+ cells in those recipients (27). In addition, CD4 help can enhance the CTL activity of CD8+ cells, presumably through CD154-induced maturation of host dendritic cells (28, 29, 30).

2C cells cause rejection of Ld-expressing grafts in skin or heart transplantation (31, 32), but our work and others (19) showed that 2C cells fail to induce GVHD in Ld-expressed recipient. Rejection of skin or heart grafts is a hyperacute process (<14 days), while causing GVHD lethality takes longer. We surmise that 2C cells become tolerant before they can produce lethal GVHD. This assumption is supported by the observation that pre-exposure of Ld Ag to the recipient-bearing 2C cells results in a long-term Ld+ skin allograft acceptance (31).

In the current report, we also compared the fate and pathogenicity of CD4+ vs CD8+ donor T cells in GVHD. In nonmyeloablative models, either CD4+ (Table I) or CD8+ (Fig. 1) TCR Tg cells alone are able to induce lethal GVHD in the recipients that express the appropriate alloantigen (i.e., one with intermediate affinity). Under these circumstances, Tg CD4+ cells will not cause GVHD lethality when given in large doses (Table I). This phenomenon was also observed with non-Tg CD4+ cells and was termed as prozone (18). The prozone effect likely results from Fas-mediated fratricide of activated CD4+ T cells at high density in vivo. Such a phenomenon has not been observed for CD8+ cells, which might be due to the relative insensitivity of CD8+ cells to Fas-mediated killing.

Although Tg CD4+ cells were capable of inducing lethal GVHD in sublethally irradiated F1 recipients (Table I), those cells were not able to do so consistently in lethally irradiated F1 recipients (Figs. 4 and 6). We noticed that donor CD4+ cells were very capable of suppressing growth of lymphoid cells derived either from host or donor (Fig. 3), yet clinical GVHD was not overt in those recipients (data not shown). It is possible that cytotoxicity mediated by CD4 T cells may be sufficient to eliminate hemopoietic cells, but not sufficient to cause lethal injury in epithelial GVHD target organs.

The findings from this study underscore the importance of alloantigen affinity and CD4 help in controlling the fate of alloreactive CD8+ T cells in vivo. We developed a new system to visualize the fate and interactions of alloreactive CD4+ and CD8+ T cells in the pathogenesis of GVHD, facilitate studies of GVHD immunopathology, and assess potential strategies to prevent GVHD.


   Acknowledgments
 
We thank Drs. Paul Martin and Michael Bevan for helpful discussion of this project, and Sasha Mayer, Lisa Rapalus, Melissa Makris, Yaming Liang, and Kelli McIntyre for their technical assistance. The founder of D10 TCR Tg mice was kindly provided by Dr. Derek Sant’Angelo at the Memorial Sloan-Kettering Cancer Center.


   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 National Institutes of Health Grants CA 84132 (to X.-Z.Y.), CA 18029, AI 51693 (to C.A.), and by a grant from Deutsche Krebshilfe (to M.H.A.). Back

2 Address correspondence and reprint requests to Dr. Xue-Zhong Yu at the current address: Experimental Therapeutics Program, SRB-2, H. Lee Moffitt Cancer Center and Research Institute, University of South Florida, 12902 Magnolia Drive, Tampa, FL 33612. E-mail address: yuxz{at}moffitt.usf.edu Back

3 Current address: Dr. von Haunersches Children’s Hospital, Ludwig-Maximilians-University, Munich, Germany. Back

4 Current address: H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL 33612. Back

5 Abbreviations used in this paper: GVHD, graft-vs-host disease; Tg, transgenic; BM, bone marrow; TCD, T cell depleted. Back

Received for publication June 7, 2005. Accepted for publication January 6, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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