A Role For MMP-3 In The Modulation Of Early T Cell Responses In Gvhd

During graft-versus-host disease (GVHD), activated alloreactive T cells recirculate from the secondary lymphoid organs and extravasate via the blood vessels into target tissues, causing pathology which if severe enough can ultimately lead to mortality. Extensive work from our laboratory has shown that lethal GVHD can be induced between MHC-matched murine strains expressing multiple minor histocompatibility antigen (miHA) differences. In the C57BL/6y (B6)–>BALB.B model, both CD4⁺ and CD8⁺ donor T cells can mediate lethal GVHD, whereas in the B6 –>CXB-2 model, only CD8⁺ T cells are lethal. CXB-2 mice are a recombinant inbred strain which expresses a subset of the same miHA as BALB.B mice, and B6 CD4⁺ T cells do initially respond by proliferation in both recipients, yet tissue pathology, particularly in the intestinal tract is much more limited in CXB-2 mice. We therefore hypothesized that differences at the level of the host issue may have an influence on GVHD development. In order for alloreactive T cells to infiltrate target tissues, they must traverse the extracellular matrix (ECM), which makes up the majority of the intercellular space in solid tissues. While the role of various T cell subsets has been studied extensively in GVHD, the involvement of ECM structural and regulatory proteins, specifically matrix metalloproteinases (MMP), is much less defined. MMP not only participate in ECM turnover, but also are also capable of influencing immune responses through the cleavage and activation of inflammatory mediators. Specifically, MMP-3 has been demonstrated to not only cleave ECM structural proteins such as collagens, fibronectin and laminin, but also can activate other MMP, TNF-α precursors and IL-1β. We therefore examined the expression patterns and potential function of MMP-3 in the development of GVHD.

Small intestine tissue samples were collected from either lethally irradiated (9 Gy, split dose) BALB.B or CXB-2 mice receiving only 2 X 10⁶ anti-Thy 1 mAb + C’ treated (T cell- depleted) B6 bone marrow cells (ATBM) for hematopoietic reconstitution, or from BALB.B and MMP3-KO mice (on a BALB.B background) exposed to irradiation (9 Gy, split dose) and transplanted with 1 X 10⁷ BALB.B presensitized B6 T cells along with 2 X 10⁶ATBM. Samples were collected on days 8, 20 and 30 post-irradiation and homogenized using a mechanical dissociator (Miltenyi Biotec). Total RNA was converted into cDNA using oligo(dT) as a primer for reverse transcription. Expression of MMP-3 was determined using real time PCR analysis. IFN-γ production by donor T cells was evaluated using ELISPOT assays where responder presensitized B6 enriched T cells were cocultured with either BALB.B or MMP-3 KO stimulator splenocytes (exposed to 15 Gy irradiation).

Relative gene expression of MMP-3 using GAPDH as an endogenous control was significantly lower (39.9% decrease, p<0.05) at day 8 following irradiation of hosts in BALB.B mice when compared to CXB2 mice. No significant differences in expression levels were observed at later timepoints (20 and 30 days post-irradiation). GVHD experiments comparing MMP-3 knockout (KO) mice on a BALB.B background with BALB.B controls, revealed that a lack of MMP-3 caused a decrease in the median survival time (MST) of recipient mice from day 40 to day 31(p=0.06). ELISPOT assays studying B6 T cell alloresponses against BALB.B or MMP-3 KO splenocytes showed an increase in mean IFNγ+ spots in MMP-3 KO-stimulated wells from 205.7 to 241.7 (p=0.098) when compared to BALB.B controls, Taken together, these data suggest: 1) a protective role for MMP-3 in the early stages of GVHD development in the B6–> BALB.B model; and 2) a potential involvement of both tissue and immune associated MMP-3 in the development of GVHD.

No relevant conflicts of interest to declare.