Allogeneic donor T cells play a pivotal role in beneficial graft-versus-tumor (GVT) activity and detrimental graft-versus-host disease (GVHD) after HSCT. Although allo-antigens are omnipresent in the recipient, GVHD is nonetheless limited to a few select organs such as the liver, intestine, and skin. To date, this organ selectivity remains poorly understood.
We hypothesized that the homing properties of alloreactive T cells, as determined by their expression patterns of adhesion molecules, such as selectins ligands, integrins, and chemokine receptors, as well as the expression of cognate integrins, selectins, and chemokine ligands in GVHD target tissues, would be important in the development of specific GVHD target organ pathology.
Based upon our preliminary data, which included a systematic microarray-based kinetic analysis of the expression of chemokines, chemokine receptors, and other adhesion molecules in GVHD target organs and the expression of selectins and integrins on alloreactive T cells in situ, we postulated that the expression of the chemokine receptors CCR2 and CCR9 and the integrin α4β7 (LPAM-1) on alloreactive T cells play a critical role in intestinal GVHD, while the expression of CCR4 and CCR10 and the selectin ligands PSGL-1 and CLA on T cells are important for skin GVHD.
We are also interested in the cognate ligands for the chemokine receptors (e.g., CCL3, 4, and 5 for CCR5, CCL25 for CCR9) and adhesion molecules (e.g., MAdCAM for LPAM, P-selectin for PSGL-1).
We are using mice deficient for CCR2 and CCR9, β7 integrin, PSGL-1, T cell selection strategies, small molecule inhibitors, and other blockade strategies, such as neutralizing antibodies, in order to probe the physiology of these molecules in effector cell homing, systemic GVHD, cutaneous and intestinal GVHD, and GVT in clinically relevant mouse models for HSCT.
As an example of our studies, our studies with β7 integrin have revealed exciting results: when T cells deficient for this integrin are infused into hosts, a significant decrease in the severity of gut and liver GVHD and overall mortality is observed as demonstrated by histopathology analysis and T cell infiltrates into target organs.
In a GVT model, we found no increased mortality from tumor in recipients of β7 deficient donor T cells. In fact, autopsy data and in vivo imaging, using luciferase transfected P815, suggest that â7-deficient donor T cells may have improved efficacy at eradicating residual tumor.
Recipients of α4β7- T cells Develop Less GVHD Mortality and Morbidity.
Lethally irradiated (1300 cGy) CBA/J (A) and (1100-1200 cGy) C57BL/6J (B) recipients were transplanted with B10.BR TCD-BM (5x106) and splenic T cells (0.5-1 x 106). T cells were included in the allograft as unsorted and/or á4â7+ CD3+ and α4β7- CD3+ sorted populations from B10.BR donors.
Kaplan-Meier survival and clinical GVHD score (±SEM) curves are shown representing 4 (BM only), 8-11 (BM + T cells) and 13-16 (BM + LPAM+ or - T cells) recipients per group from 2 combined experiments.
Statistical analysis: A) Left - vs. P=0.007, vs. P<0.0001; Right - vs. P<0.01, vs. P<0.01. B) Left - vs. P=0.0179; Right - vs. P<0.02.
GVT Activity is Preserved in Recipients of á4â7- T cells.
C57BL/6 mice were transplanted as described. Recipients were given 1 x 105 EL-4 murine leukemia/lymphoma cells as a separate intravenous injection at the time of transplantation.
Survival is depicted as a Kaplan-Meier curve representing mice that received BM only (n=4) or BM + á4â7+ or - T cells (n=12). The cause of death (GVHD vs. tumor) for all recipients that succumbed during the course of the experiment is shown in the Table.
Statistical analysis: vs. P=0.025, vs. P=0.0013.
â7-/- Donor T cells Cause Less GVHD Organ Damage.
Tissues were harvested on day 7 and 14, and analyzed by semi-quantitative histology. â7-deficient T cells have statistically significant decreases in organ damage in the small bowel, large bowel, and liver.
Another homing project we are actively pursuing is the study of the chemokine CCR2.
In a series of experiments using Affymetrix DNA microarrays and ELISA analysis, we found that monocyte chemoattractant protein-1 (MCP-1, also called CCL2), is upregulated in GVHD target organs in the first 2 weeks after HSCT. CC chemokine receptor 2 (CCR2) is the primary receptor for CCL2 and is expressed on a variety of hematopoietic cells including monocytes, macrophages, and memory T cells.
The relevance of CCR2 for homing of monocytes and macrophages has been demonstrated in a variety of disease models, but data on the relevance of CCR2 for T cell homing are limited. Therefore, we analyzed the effects of CCR2-deficiency on the homing capacity of donor T cells in clinically relevant murine GVHD models.
We first demonstrated that proliferation, alloactivation, IFN-ã production, and cytotoxicity of B6.CCR2-/- T cells were intact. However, preliminary studies with a donor T cell inoculum, consisting of 50 percent B6.WT and 50 percent B6.CCR2-/- T-cells, showed a reduced infiltration of B6.CCR2-/- donor CD8+ T cells into the intestinal epithelium of C3FeB6F1 recipients. Homing to other organs and homing of CD4+ T cells was not impaired.
In accordance with these findings, we could demonstrate a reduction of GVHD morbidity and mortality in recipients of B6.CCR2-/- CD8+ T cells while preliminary data showed no major difference for CD4+ T cells. Recipients of B6.CCR2-/- CD8+ T cells had a significant reduction in large intestine histopathology scores and showed a trend toward lower scores for the small intestine.
Moreover, the graft-versus-tumor (GVT) activity of B6.CCR2-/- CD8+ T cells against P815 mastocytoma in B6D2F1 recipients was intact, as demonstrated by in vivo bioluminescent imaging. Again GVHD mortality was reduced for B6D2F1 recipients of B6.CCR2-/- CD8+ T cells.
In conclusion, our data suggest that the MCP-1-CCR2 pathway is involved in intestinal homing of alloreactive T cells during GVHD but is not essential for GVT activity.
We expect these studies and studies of other homing-associated molecules to contribute to a better mechanistic understanding of organ-selective GVHD and to provide new therapeutic targets and strategies to treat or prevent organ-specific GVHD.
We expect our studies to also reveal novel means for the separation of GVT from GVHD activity based on control of effector cell trafficking patterns.