Non-Hematopoietic Lymphoid Stromal Cells Prime Alloreactive CD4⁺ T Cells in Acute Graft-Versus-Host Disease

Allogeneic T cell priming is considered as an essential event determining the outcome of allogeneic hematopoietic stem cell transplantation (allo-HCT), ideally triggering anti-leukemic responses (GvL effect) or, at worst, causing life-threatening acute graft-versus-host disease (aGvHD). During acute GvHD initiation, alloreactive T cells are activated by host antigen presenting cells (APCs), rapidly expand and subsequently exert tissue damage. Recently, it was discovered that absence of host hematopoietic APCs does not prevent acute GvHD, suggesting a crucial role of non-hematopoietic APCs for priming alloreactive T cells (Toubai et al., Blood 2012, Li et al., J Immunol. 2012). Furthermore, it was even suggested that in the absence of professional APCs allogeneic CD4⁺ T cells can be activated in the lamina propria by MHC class II expressing myofibroblasts (Koyama et al., Nat Med 2012). As exact location and identity of host non-hematopoietic APCs triggering alloreactive T cell responses are essential to dissect the priming of GvHD-inducing vs. GvL-mediating allogeneic T cells, we investigated the role of lymph node stromal cells (LNSCs) in the initiation phase of aGvHD and their potential role as non-hematopoietic APCs.

Employing allo-HCT mouse models in combination with flow cytometry and advanced microscopy techniques, we explored early alloreactive T cells activation first in a myeloablatively conditioned MHC major mismatch allo-HCT setting (FVB→B6). Under these conditions, CD4⁺ and CD8⁺ T cells activation and proliferation occurred exclusively in secondary lymphoid organs (SLOs) and not in the intestinal lamina propria early after allo-HCT within the first three days after allo-HCT. To study non-hematopoietic antigen presentation early after allo-HCT, we generated bone marrow B6.MHCII^Δ/Δ→B6.WT chimeras that lacked MHC II expression in the host hematopoietic compartment. Subsequent allo-HCT of these chimeras revealed activation and expansion of allogenic donor CD4⁺ T-cells exclusively in SLOs and not the lamina propria in the first three days after transplantation.

Next, we generated recipient mice that selectively lacked MHCII expression either in CD11c expressing cells or under the control of the Vav1-promoter in all hematopoietic cells. In both type of recipients, allogenic donor CD4⁺ T-cells were activated within 3 days after allo-HCT in SLOs and no other tissues.

After irradiation of B6.WT mice we observed LNSCs upregulate co-stimulatory receptors early after irradiation (24 and 72 hours), in vitro and in vivo suggesting that they may act as active non-hematopoietic APCs.

Next, we performed mixed lymphocyte reactions (MLRs) of irradiated APCs derived from Tg^Vav1-Cre+MHCII^Δ/Δmice with alloreactive CD4⁺ T cells from FVB mice. Here, we observed in the total absence of hematopoietic MHCII antigen presentation reduced but still pronounced activation of alloreactive CD4⁺ T cells. Therefore, in these transgenic allo-HCT models, hematopoietic cells in the SLOs were dispensable for alloreactive CD4⁺ T cell activation in the initiation phase of aGvHD.

To ascertain that SLOs are the exclusive priming sites of alloreactive CD4⁺ T cells, we transplanted mesenteric lymph nodes (mLNs) from a B6.CD11c.DTR mice, depleted of CD11c⁺ cells into B6.MHCII^Δ/Δmice. After successful engraftment of donor mLNs in these mice, allo-HCT revealed these as unique priming sites in MHCII deficient hosts for alloreactive CD4⁺ T cells, which differentiated into CD44^hiCD62L^low effector T cells that were detectable in the transplanted mLNs.

Conclusively, these results indicate that specialized non-hematopoietic lymph node stromal cells prime alloreactive CD4⁺ T cell within the SLOs early after allo-HCT. Pinpointing the molecular pathways in these non-hematopoietic APCs within SLOs that trigger alloreactive T cell responses may prove fruitful for selective therapeutic intervention after allo-HCT.