Donor alloreactive T cells are required for tGVHD and damage the thymus in a dose-dependent manner.
Mature donor T cells in the BM allograft are the primary initiators of acute GVHD, and disease severity correlates with the dose of donor T cells. We therefore began by characterizing the numbers of donor alloreactive T cells in the allograft and their impact on systemic and tGVHD. We performed experiments in the well-defined MHC-disparate mouse model system C57BL/6 (B6, H-2b) → BALB/c (H-2d). BALB/c recipients received 8.5 Gy radiation and an allograft containing 5 × 106 B6 CD45.1 T cell–depleted BM (TCD-BM) cells and varying numbers of WT B6 CD45.2 T cells, which were insufficient to cause GVHD mortality. TCD-BM contained negligible numbers of contaminating T cells (0.1% cells; see Methods), and this process allowed for reliable titration of mature donor T cells in the allograft.
We assessed the sensitivity of the thymus to GVHD in experiments in which we titrated donor T cell numbers and assessed the effect on systemic and tGVHD. We first used very low doses of donor alloreactive T cells (0.25 × 105
, 0.5 × 105
, and 1 × 105
cells), which caused recipients to exhibit negligible weight loss (Figure A) or clinical GVHD (Figure B); by comparison, mice experiencing severe GVHD and resulting mortality can have weight loss of more than 50% and clinical scores of greater than 7 (25
). We then assessed thymic cellularity at week 4 after transplant and observed a striking dose-dependent decrease in donor BM-derived CD4+
thymocytes, with an approximately 50% loss, even with the addition of only 25,000 donor T cells (Figure C). We confirmed these results in additional experiments with 2.5 × 105
and 1 × 106
donor T cells, which also revealed an inverse relationship between thymocyte count and numbers of donor alloreactive T cells (data not shown). Upon monitoring recipients for survival, we noted that all mice receiving 0.25 × 106
or fewer donor T cells in the allograft had more than 95% survival up to day 28 after transplant. Survival for mice receiving 0.25 × 106
donor T cells is shown in Supplemental Figure 2 (supplemental material available online with this article; doi:
Clinical GVHD correlates with tGVHD and thymic function of B6 → BALB/c (8.5 Gy) mice.
We also tested our findings in the MHC-matched minor antigen–disparate model system B6 (H-2b) → LP (H-2b) and added different doses of B6 Thy1.1+ T cells (to distinguish between donor BM-derived cells, infused alloreactive T cells, and host cells) to induce varying degrees of GVHD.
In several experiments, we observed again dose-dependent decreases of donor CD4+CD8+ (DP) thymocytes at days 27 and 42 after transplant, in the absence of significant signs of clinical GVHD (including weight loss) (Supplemental Figure 3).
Although the kinetics of GVHD onset and the frequency of alloreactive T cells differ in this MHC-matched minor antigen–disparate model, as compared with the B6 → BALB/c model in which we performed most of our experiments in this report (described below), these observations confirm the sensitivity of the thymus to GVHD in clinically relevant model systems.
Recipients of low doses of donor alloreactive T cells exhibit tGVHD that is partially reversible at later time points.
To assess whether tGVHD is reversible, we repeated experiments in the model system B6 → BALB/c with TCD-BM with or without 0.5 × 105, 1 × 105, or 2.5 × 105 T cells and assessed thymic cellularity and composition at day 60 after transplant. We observed that total thymocyte numbers were significantly decreased (P < 0.05) in recipients of TCD-BM plus 2.5 × 105 T cells versus recipients of TCD-BM only (Supplemental Figure 4A). However, at lower donor T cell doses, we observed a recovery of thymus cellularity at day 60 (Supplemental Figure 4, A and B) compared with day 28 (Figure C). These data suggest that recipients of low doses of donor alloreactive T cells may have partially reversible GVHD. However, recipients of higher doses of T cells may have more prolonged thymic damage.
In addition to analyzing thymic cellularity and composition in recipients of TCD-BMT and T cell–replete allo-BMT, we tracked their clinical GVHD parameters and weight loss, to correlate tGVHD with systemic and clinical GVHD to day 60 after transplant (Supplemental Figure 4, C and D). These data show that recipients of T cell–replete allografts with up to 2.5 × 105 donor T cells developed partially reversible tGVHD, which was nonetheless correlated with low levels of sustained systemic GVHD.
These data suggest that recipients of low doses of donor alloreactive T cells may have partially reversible GVHD. However, recipients of higher doses of T cells may have more prolonged thymic damage.
GVHD damages thymic architecture, thymic output, and negatively influences peripheral T cell function.
We further studied the relationship between tGVHD and peripheral donor-derived T cell function in recipients of 5 × 106 TCD-BM with or without 0.25 × 105, 0.5 × 105, and 1 × 105 alloreactive T cells. This revealed an inverse relationship between numbers of peripheral BM-derived CD45.1+ T cells and numbers of donor alloreactive T cells that were infused (Figure D). Surprisingly, infusing greater numbers of donor CD45.2+ alloreactive T cells did not lead to increased numbers of CD45.2+ alloreactive T cells in the spleens at day 22 after transplant (Figure D). This may be due to the fact that after day 14 of an allo-BMT, the spleen assumes features of a GVHD target organ; decreased cellularity may therefore be interpreted as increased damage. Finally, we noted that all groups had 90% or greater total donor chimerism in the spleen, as measured by H-2b staining (data not shown).
As donor BM-derived and alloreactive T cells can both mediate antipathogen and antitumor activity, we studied the function of all donor-derived peripheral T cells by purifying them with CD5+ magnetic selection from the spleens of allo-BMT recipients on day 22 and testing the proliferation of these T cells with anti-CD3 and anti-CD28 stimulation. This revealed that donor-derived splenic T cells in recipients of TCD-BM only had significantly better proliferative responses upon anti-CD3 and anti-CD28 stimulation than recipients of TCD-BM and alloreactive donor T cells (Figure E).
To further study whether thymic cellularity was directly associated with thymic function (i.e., export) after T cell–replete allo-BMT, we transplanted irradiated BALB/c mice with 5 × 106 FVB background Rag2-EGFP TCD-BM only or Rag2-EGFP TCD-BM plus 0.1 × 106, 0.25 × 106, 0.5 × 106, or 1 × 106 WT FVB T cells. Recent thymic emigrants (RTEs) are Rag2+ and therefore EGFP+ in this model, thereby allowing for their identification in the periphery of the allo-BMT recipients. In addition to observing decreased thymic cellularity with increasing doses of donor T cells (data not shown), we also observed that, upon pooling data from all allo-BMT recipients transplanted with varying doses of donor T cells, including mice which received BM only (no GVHD) or mice which received 0.1 × 106, 0.25 × 106, 0.5 × 106, or 1 × 106 FVB T cells (varying degrees of tGVHD and thymic cellularity), absolute numbers of splenic RTEs correlated well with thymic cellularity (Figure F).
Additionally, we directly assessed the impact of increasing numbers of donor alloreactive T cells (thus increasing severity of tGVHD) on thymic export by transplanting irradiated B6D2F1 mice (13 Gy) with 105 Rag2-EGFP lineage– cells with or without 0.1 × 106, 0.25 × 106, 0.5 × 106, or 1 × 106 FVB T cells. We assessed thymic export and numbers of splenic RTEs (EGFP+ cells) on day 42 after transplant and noted that as few as 105 donor FVB T cells were sufficient to cause a significant decrease in thymic export (Figure G). Furthermore, this effect was dose-dependent, such that increasing numbers of donor FVB T cells in the allograft caused a corresponding decrease in RTE numbers (Figure G). Similar results were observed in an experiment using irradiated BALB/c recipients (data not shown). This suggests that increasing donor T cell numbers and tGVHD severity negatively impacts thymic function.
Finally, we assessed thymic architecture in allo-BMT recipients with GVHD. Recipients of TCD-BM only (no GVHD) had a thymic architecture with well-defined cortical and medullary areas (Figure H, left), similar to the normal thymus of a nontransplanted mouse. However, recipients of TCD-BM plus 0.25 × 106 B6 WT T cells (GVHD) had remarkable disruption of thymic architecture, including thinning of the cortex and loss of the corticomedullary junction (Figure H, right).
To assess the sensitivity of the thymus and its architecture to damage during tGVHD at even lower doses of donor T cells in the allograft, we transplanted BALB/c mice (8.5 Gy) with TCD-BM with or without 0.5 × 105, 1 × 105, or 2.5 × 105 WT T cells and quantified thymic cortical and medullary areas on day 28 after transplant in these recipients to assess in particular the loss of the thymic cortex and the loss of the corticomedullary junction. Recipients of as few as 1 × 105 donor T cells exhibited a statistically significant loss of thymic cortical area (Figure I and Table ) as well as a loss of the corticomedullary junction in some animals (Table ). These results again demonstrate the exquisite sensitivity of the thymus to tGVHD, which manifested as derangements in thymic cytoarchitecture, with even small numbers of donor alloreactive T cells.
Quantification of thymic cortical area
We conclude from these experiments that small numbers of donor alloreactive T cells cause dose-dependent thymic damage in allo-BMT recipients, which is associated with specific changes in thymic architecture and results in decreased export of T cells. Consequently, the thymus is exquisitely sensitive to GVHD.
Donor alloreactive T cells rapidly infiltrate the thymus after allo-BMT and become profoundly activated.
To assess the kinetics of donor alloreactive T cell infiltration of the thymus, we transplanted irradiated BALB/c mice with B6 TCD-BM and 0.25 × 106 B6 luciferase+ T cells, then tracked their migration and expansion with daily bioluminescent imaging studies. At this T cell dose, we began to detect a signal in the thymus by days 5 to 6 after transplant (Figure A). Additionally, in parallel allo-BMT experiments with a doses of 0.5 × 106, 1 × 106, or 10 × 106 B6 luciferase+ T cells, we were able to detect cells in the thymus as early as days 2 or 3 after transplant (Supplemental Figure 5), suggesting that a small percentage of donor alloreactive T cells traffic to the thymus with rapid kinetics.
Alloreactive T cells quickly infiltrate the thymus, undergo proliferation, and display an activated phenotype.
To further characterize thymus-infiltrating T cells, we transferred CFSE-labeled purified B6 CD45.1 splenic T cells into irradiated syngeneic (B6, 11 Gy) or allogeneic (BALB/c, 8.5 Gy) recipients. On day 6 after adoptive transfer, we observed that donor T cells comprised a significantly greater proportion of CD45+ hematopoietic cells in the thymus of allogeneic recipients as compared with syngeneic recipients and that the majority of donor T cells in allogeneic recipients were CFSEdim, indicating rapid proliferation and alloactivation (Figure B, left). In addition, the majority of donor T cells in the thymi of allo-BMT recipients were CD44hiCD62Llo, indicating a effector memory T cell phenotype, with a minority of CD44hiCD62Lhi central memory cells (Figure B, right). We conclude that alloactivated and proliferating donor T cells with an effector memory T cell phenotype infiltrate the thymus early after allo-BMT.
Donor alloreactive T cells have a characteristic peak at day 14 after transplant.
To assess the longer-term kinetics of T cell infiltration during tGVHD, we transplanted B6 and BALB/c mice with B6 Thy1.1 splenic T cells and B6 CD45.1+ TCD-BM. We observed a peak in donor alloactivated Thy 1.1+ T cells in the thymus on day 14 after allo-BMT, with a subsequent decline and low but sustained numbers at day 21 and day 28 after transplant (Figure C). We observed similar kinetics in the spleen, although there the decline in Thy1.1+ donor T cell numbers from day 14 to day 28 was more gradual (Figure C).
Donor alloreactive T cell–derived Tregs are dramatically decreased in the spleens but not thymi of allogeneic versus syngeneic BMT recipients. Tregs of donor and host origin have been demonstrated as important negative regulators of GVHD (26
). We therefore assessed donor FoxP3+
Tregs as a percentage of donor infused CD4 T cells in the spleens and thymi of BMT recipients. While the percentages of donor Tregs derived from the infused T cells were significantly reduced in the spleen of allo-BMT recipients versus syngeneic BMT recipients, the fraction of Tregs derived from the infused T cells in the thymi of allo-BMT recipients with GVHD versus syngeneic recipients was not significantly different (Figure D).
We also assessed the influence of tGVHD on the numbers of donor BM-derived Tregs in the spleen on day 28 after transplant. We observed similar percentages of donor CD45.1+CD4+FoxP3+ Tregs as a percentage of donor BM-derived CD45.1+CD4+ donor T cells in recipients of syngeneic and allogeneic BMT (Figure E). We interpret these data to signify that tGVHD does not disproportionately impact the reconstitution of donor BM-derived Tregs in the periphery.
Donor alloreactive T cells require FasL and TNF-related apoptosis-inducing ligand to mediate tGVHD.
Next, we assessed the T cell cytolytic molecules and pathways required for tGVHD. Comparing numbers of donor BM-derived thymocytes on day 28 in recipients of WT B6 versus recipients of B6.KO (deficient for cytolytic molecule) donor T cells, we observed that FasL and TNF-related apoptosis-inducing ligand (TRAIL) were both required for tGVHD, while TNF and perforin (Pfp
) were dispensable (Figure A). This agrees with our previous finding that alloactivated T cells can express FasL and TRAIL (28
). Interestingly, we also observed that donor T cell–derived IFN-γ was dispensable for tGVHD (Figure A).
Alloreactive T cells require FasL and TRAIL to mediate tGVHD.
Since recipients of generalized lymphoproliferative disease (gld, also known as Fasl) and Trail–/– T cells demonstrated significant increases in donor BM-derived CD45.1+CD4+CD8+ thymocytes, we asked whether interference with both these cytolytic pathways could further attenuate tGVHD. We therefore generated mice which were either deficient for both FasL and TRAIL (gld/Trail–/–) or mice additionally deficient for TNF (gld/Trail–/–Tnf–/–). Donor T cells from both of these mice were ineffective at mediating tGVHD (Figure A) or systemic GVHD (data not shown), and CD4+CD8+ thymocyte numbers on day 28 were similar to that of allo-BMT recipients of TCD-BM only (no GVHD). These results suggest that FasL and TRAIL are primary effector pathways by which donor alloreactive T cells damage the thymus.
We also assessed the requirements for tGVHD at higher doses of donor T cells (B6 → BALB/c with 0.5 × 106 T cells) and again confirmed that, even in this setting, the FasL and TRAIL pathways are required to mediate tGVHD, while the perforin and IFN-γ pathways were dispensable (Figure B). Moreover, recipients of gld/Trail–/– doubly deficient donor T cells exhibited additionally increased numbers of donor CD45.1+CD4+CD8+ thymocytes as compared with recipients of Trail–/– or gld singly deficient donor T cells (P < 0.05), suggesting a nonredundant role for these 2 pathways at this higher T cell dose (Figure B).
We further assessed the influence of donor alloreactive T cell FasL and TRAIL on thymic architecture and observed that while allo-BMT recipients of WT T cells had complete loss of distinction between the thymic cortical and medullary zones, recipients of Trail–/–, gld, and gld/Trail–/– T cells had intact thymic microarchitecture (Figure C).
Both CD4 and CD8 cells require FasL to mediate tGVHD, but TRAIL is only required for CD8 T cells to mediate tGVHD.
We further studied the importance of FasL and TRAIL on donor CD4+ and CD8+alloreactive T cells and performed transplants with 0.25 × 106 purified WT, gld, or Trail–/– donor CD4 or CD8 T cells. These experiments revealed that FasL is important on both CD4 and CD8 alloreactive T cells for mediating tGVHD, whereas TRAIL is only important on donor CD8 T cells (Figure D).
Donor alloreactive T cells damage host-derived thymic stroma via the FasL pathway to cause tGVHD.
To study whether donor alloreactive T cells mediated tGVHD against donor (syngeneic) BM-derived thymocytes or host tissues, we transplanted WT or lpr (Fas receptor–deficient) B6 mice with BALB/c TCD-BM and T cells and measured donor thymic cellularity on day 28 after transplant. These experiments revealed that while WT and lpr recipients of TCD-BM only had similar thymic cellularity, lpr recipients of T cell–replete allo-BMT had significantly increased numbers of donor BM-derived CD4+CD8+ thymocytes as compared with WT recipients, indicating that lpr allo-BMT recipients were resistant to tGVHD (Figure E). These results suggest that expression of the Fas receptor on host-derived thymic stroma is required for tGVHD.
In contrast, experiments in which we transplanted BALB/c mice with lpr BM versus those in which we transplanted BALB/c mice with B6 BM with or without WT T revealed no significant differences, indicating that Fas receptor on donor BM-derived thymocytes is not directly involved in sensitivity to tGVHD (Figure F).
Soluble DR5 agonists are sufficient to damage the thymus after TCD allo-BMT.
We further studied the role of TRAIL in mediating thymic damage after transplant by administering anti–mDR5-1 agonistic antibody to recipients of TCD allo-BMT, which did not receive any donor alloreactive T cells. The administration of a soluble TRAIL analog in this setting allowed us to clarify several aspects of TRAIL biology, which include the requirement for GVHD-associated inflammatory cytokines to enable TRAIL-mediated thymic damage in recipients of TCD allo-BMT and the requirement for other alloreactive T cell–mediated cytolytic pathways to enable TRAIL-mediated damage. Furthermore, we performed experiments in which we treated mice with anti-mouse DR5-1 agonistic antibody both immediately after transplant or with a delay in administration, which allowed us to probe the temporal sensitivity of the thymus to TRAIL/DR5-mediated killing.
In experiments in which we administered DR5-1 agonistic antibody “early,” during the peritransplant period (200 μg i.p. days –1, 1, 3, 5), and analyzed recipients on day 28 after transplant, we observed a highly significant decrease in total thymocyte numbers (Figure A) as well as donor CD45.1+CD4+CD8+ (DP) thymocyte numbers (Figure B) in recipients of this antibody as compared with recipients of hamster IgG control. We also analyzed the BM of recipients and found comparable total numbers of BM cells and early donor precursors (CD45.1+lineage–Sca-1+c-Kit+ [LSK] cells) in recipients of DR5-1 versus hamster IgG (Figure , C and D), suggesting that DR5-1 treatment did not directly influence the BM compartment. In addition, we found that recipients of DR5-1 had significantly decreased numbers of BM-derived T cells (Figure E), suggesting that DR5-1 antibody treatment impairs thymic output.
Soluble DR5 agonists are sufficient to damage the thymus after allo-BMT in 5×106 B6 CD45.1 + TCD-BM only → BALB/c (8.5 Gy) mice.
Next, to address the temporal sensitivity and effects of conditioning-regimen associated inflammation (e.g., cytokines) on the TRAIL-mediated thymic damage, we administered DR5-1 antibody or hamster IgG control (200 μg i.p.) on days 10, 12, 14, and 16 after transplant. On day 28, we again observed a decrease in total thymocyte numbers (Figure F) and donor CD45.1+CD4+CD8+ thymocyte numbers (Figure G) but no effect on total BM and donor LSK cellularity (Figure , H and I). Donor T cells in the spleen were again decreased in recipients of DR5-1 antibody (Figure J). These data suggest that conditioning-associated inflammation is not required for TRAIL/DR5-mediated thymic damage. Finally, we noted that donor CD45.1+ thymocytes expressed low levels of DR5, suggesting that DR5-1 does not directly act on donor thymocytes (Figure K).
Together, these experiments indicate that the thymus is sensitive to TRAIL/DR5-mediated damage after allo-BMT, in the absence of donor alloreactive T cells, their other cytolytic pathways, or the inflammatory cytokines associated with donor alloactivated T cells, the conditioning regimen, and GVHD. Additionally, the thymus appears to be sensitive to TRAIL/DR5-mediated damage throughout at least the early period of reconstitution (days 10–16).
Donor alloreactive T cells damage thymic medullary and cortical epithelium as well as thymic endothelium and fibroblasts.
Thymic stroma is a heterogenous population consisting of nonhematopoietic cells such as endothelium and cortical and medullary thymic epithelial cells (cTECs and mTECs) as well as fibroblasts. DCs and macrophages constitute the hematopoietic component of thymic stroma. As we were interested in host thymic stroma, we focused on nonhematopoietic cells and asked which populations were susceptible to tGVHD and damage by donor alloreactive T cells.
Upon digesting the thymi of allo-BMT recipients of TCD-BM with or without WT, gld, Trail–/–, and gld/Trail–/– T cells, we were surprised to note that all nonhematopoietic stromal cells, including endothelium, fibroblasts, cTECs, and mTECs, were increased in recipients of gld as compared with those of WT donor T cells (Figure A). Furthermore, recipients of Trail–/– T cells showed increased numbers of mTECs and cTECs but not fibroblasts and endothelium (Figure A). Recipients of gld/Trail–/– T cells had a phenotype similar to that of recipients of gld T cells. These observations suggested to us that donor alloreactive T cells use TRAIL to cause damage to mTECs and cTECs, whereas FasL is required for damage to all stromal cell subsets.
tGVHD causes damage to the thymic stroma, loss of thymic epithelial cells, and cortical thinning.
Donor alloreactive T cells and tGVHD cause cortical thinning in the thymus via the FasL and TRAIL pathways.
We further studied damage to the thymic stroma and thymic microarchitecture by staining sections from recipients of TCD-BM with or without WT T or gld/Trail–/– donor T cells. Analysis with the thymic cortical marker cytokeratin 8 (K8) and thymic medullary marker keratin 5 (K5) revealed that WT donor alloreactive T caused tGVHD and cortical thinning, whereas gld/Trail–/– donor alloreactive T cell did not (Figure B). In addition, a systemic quantitative analysis of cortical and medullary areas in recipients of T cell–depleted and T cell–replete allo-BMT revealed that recipients of WT T cells had a statistically significant loss of cortical area (P < 0.05) as compared with recipients of TCD-BM alone, whereas recipients of gld/Trail–/– T cells had similar thymic cortical area as recipients of TCD-BM only (Figure C). These observations further suggest that FasL and TRAIL are important for cortical thinning, a hallmark feature of tGVHD.
Thymic stromal cells upregulate Fas and DR5 and downregulate caspase-8–like inhibitory protein upon exposure to radiation.
Radiation both directly induces cellular apoptosis and sensitizes cells to other apoptotic stimuli. As radiation is an important part of many allo-BMT–conditioning regimens, we hypothesized that irradiation of the thymic stroma could be important for its sensitization to tGVHD and the death ligands FasL and TRAIL. We therefore irradiated nontransplanted BALB/c mice, and, in thymic stromal cells, we measured their cell-surface expression of Fas and DR5, which are receptors for FasL and TRAIL, respectively, as well as their expression of the intracellular protein caspase-8–like inhibitory protein (cFLIP), a negative regulator of Fas-mediated and DR5-mediated apoptosis.
These experiments revealed that cTECs and mTECs significantly upregulate DR5 by day 3 after irradiation and that thymic fibroblasts and cTECs upregulate Fas by day 3 after irradiation (Figure A). Furthermore, there is a transient decrease in cFLIP levels on day 1 after irradiation in mTECs and a similar trend in cTECs, fibroblasts, and endothelium (Figure B). This suggests that radiation injury indeed sensitizes thymic stromal cells to apoptosis via the FasL and TRAIL pathways.
Thymic stromal cells upregulate the death receptors Fas and DR5 and downregulate the antiapoptotic protein cFLIP, upon exposure to radiation.
Both canonical T cell thymus-trafficking molecules and T cell gut-trafficking molecules are implicated in tGVHD.
A number of T cell–trafficking molecules have been implicated in GVHD. We assessed a subset of these molecules, including CCR9, L selectin, αE and β7 integrin subunits, P selectin glycoprotein ligand-1 (PSGL-1), CCR2, and CXCR3, to evaluate their roles in mediating tGVHD. We performed experiments with B6.WT versus B6.KO (deficient for trafficking molecule) donor T cells, by transplanting B6.CD45.1+ TCD-BM into lethally irradiated BALB/c recipients (8.5 Gy) and assessing donor BM-derived CD45.1+CD4+CD8+ thymocyte numbers on day 28 after transplant (Figure A).
Alloreactive T cells require various homing molecules and costimulatory/inhibitory molecules to mediate tGVHD.
Recipients of donor alloreactive T cells deficient for molecules with documented functions in trafficking to the thymus (CCR9, L selectin, and PSGL-1; refs. 30
) accordingly exhibited increased numbers of BM-derived CD45.1+
thymocytes as compared with recipients of WT T cells, indicating a partial rescue of tGVHD (Figure A). However, to our surprise, alloreactive T cells deficient for trafficking molecules traditionally ascribed to gut trafficking (αE
integrin subunits, CCR2, CXCR3; refs. 35
) also mediated attenuated tGVHD (Figure A). We note however, that no deficiency in any single trafficking molecule tested was sufficient to completely abrogate the ability of donor alloreactive T cells to mediate tGVHD. This suggests both redundancy in the trafficking of donor alloreactive T cells to the thymus, and that many of the molecules we tested are partially but not completely required for thymic homing (Figure A).
WT CD4 T cells out-compete Itgb7–/– and Psgl1–/– CD4 T cells in early trafficking to spleen and thymus.
To distinguish whether the decrease in tGVHD was due to a specific, direct trafficking defect of (β7
donor T cells versus an indirect overall decrease in alloactivation of Itgb7–/–
donor T cells, we performed mixing experiments with Itgb7–/–
and WT donor T cells. We combined purified CD4 T cells of WT B6 (CD45.1) origin with either Itgb7–/–
(CD45.2) CD4 T cells or WT (CD45.2) T cells in a 1:1 ratio. Upon infusing CFSE-labeled cell mixtures into irradiated BALB/c mice and analyzing the spleens and thymus on day 6 after adoptive transfer, we noted similar proliferation kinetics (data not shown) but increased numbers of WT CD4 T cells in both the spleen and thymus of recipients of WT CD45.1+
mixtures (Figure B, top). Results of experiments performed with mixed CD8 T cells suggested that β7
integrin was not important for CD8 T cell trafficking (Supplemental Figure 6A). The observation that WT CD4 T cells out-compete Itgb7–/–
T cells in both spleen and thymus suggests that the β7
integrin subunit may be involved in trafficking of alloreactive donor T cells to the spleen and thymus. Similar experiments, in which whole WT versus Psgl1–/–
donor T cells were mixed, revealed also that WT T cells out-competed Psgl1–/–
T cells in the spleen and thymus (Figure B, bottom, and Supplemental Figure 6B), suggesting that PSGL-1 may have indirect effects in mediating tGVHD, despite its requirement for early thymic progenitors from the BM to seed the thymus (33
Ox40 and carcinoembryonic antigen-associated cell adhesion molecule 1 regulate the activation of alloreactive donor T cells during tGVHD.
We assessed a variety of costimulatory and coinhibitory molecules important for alloreactive T cell function during GVHD (39
) to determine their specific relevance in tGVHD. We found that inducible costimulator (ICOS) and glucocorticoid-induced TNF receptor (GITR) were dispensable for tGVHD (Figure C), whereas costimulatory molecule Ox40 was required for tGVHD (Figure C). Interestingly, both ablation and overexpression of carcinoembryonic antigen-associated cell adhesion molecule 1 (Ceacam1), a net negative regulator of T cell function, were able to attenuate tGVHD (Figure C).
We further assessed the role of the costimulatory molecule Ox40 for tGVHD because of its functions as an activator of both Teffs and Tregs. We performed experiments by mixing T effector cells (Teffs) and Tregs from WT and Ox40–/– FoxP3-GFP mice in a 1:4 ratio to assess the relative importance of Ox40 on Teff versus Treg for tGVHD. We observed that recipients of Ox40–/– Teffs have increased numbers of donor CD4+CD8+ (DP) thymocytes compared with recipients of WT Teffs (Figure D). These experiments suggest that while Ox40 expression on Teffs is important for donor T cells to mediate tGVHD, Ox40 expression on donor Tregs is dispensable.