Immune cell trafficking and function in allogeneic responses [electronic resource]

Allogeneic hematopoietic cell transplantation (HCT) is an excellent model for the controlled study of adoptively transferred cells in the induction and progression of numerous immune responses in vivo, with the added benefit that these cells can be tracked, identified, and re-isolated. Using this model, we conducted four distinct sets of studies related to the trafficking and function of immune cells in allogeneic responses. In our first project, we focused on the early events of T cell activation leading to the induction of Graft-versus-Host Diesease (GVHD) in a murine model of major histocompatibility complex (MHC)-matched transplantation. Although it is well known GVHD occurs in MHC-matched transplants, little is known about donor T cell activation kinetics in minor-mismatched models. Yet, because these models better approximate clinical transplantation, it is critical to understand how GVHD develops across minor barriers. To investigate temporal and spatial events of GHVD development, side-by-side transplants were conducted into major and minor-mismatched recipients using hematopoietic cells from the same donor mouse strain. In both models, T cells home to nodal sites by day 3, proliferate, and exit to GVHD target tissues by day 6. Additionally, expression of homing and activation markers was equivalent for all markers examined on day 3. However, tissue migration and proliferation were reduced in the minor model. And by day 6, minor-mismatched T cells had increased CD62L retention and reduced P-selectin and CD44 expression. We also found fewer MHC-matched T cells producing interferon-[gamma] (IFN-[gamma]) and tumor necrosis factor-[alpha] (TNF-[alpha]). Our data show that very early events of donor T cell activation are similar in both models, suggesting that delays in GVHD onset across minor barriers arise from temporal differences in the effector phase, rather than the initiation phase, of GVHD. For our second project, we examined the ability of transplanted natural killer (NK) cells to impact GVHD in MHC-matched transplants as allogeneic NK cells are already known to suppress GVHD in transplants across major histocompatibility barriers. Since NK cells can lyse host antigen-presenting cells (APCs) in these transplants due to disparities in MHC class I expression between the recipient and donor, thus reducing the activation of donor T cells, it is thought that this is primary mechanism of NK-mediated reductions in GVHD. However, in MHC-matched transplants, MHC class I expression is shared between donor and host, protecting host APCs from lysis. Previous work from our laboratory has shown that NK cells can also reduce GVHD, in MHC-mismatched transplants, through the inhibition and lysis of autologous donor T cells that have upregulated expression of NKG2D activating ligands. As such, we wondered whether this mechanism may also reduce GVHD in transplants across minor histocompatibility barriers. Our data demonstrate that donor NK cells are able to improve survival and reduce GHVD disease severity in the more clinically relevant model of MHC-matched transplantation. As allorecognition and lysis of MHC-mismatched host APCs is not possible in this model, NK cells must be mediating their benefits through alternate mechanisms. Although we did find that NK cells were capable of reducing the proliferation of syngeneic T cells in an MLR using irradiated MHC-matched splenocytes as stimulators, we were unable to find evidence for the direct inhibition, or lysis, of donor T cells by NK cells in vivo. It seems likely that this was at least partially due to the much lower number of NK cells used in these studies compared to the previous reports. Additionally, as NK cells may impact T cells that are homeostatically proliferating differently then they impact T cells proliferating in response to antigen, it will be critical for future studies to clearly separate these two populations, especially given the reduced frequency of alloreactive T cells in MHC-matched models. Perhaps then we will be able to identify the mechanisms of donor NK mediated reductions in GVHD. In our third project, we studied the in vivo ability of NK cells to clear tumor. NK cells have natural cytotoxicity and cytokine production capabilities, and are known to mount potent responses against both viruses and tumor cells. Preclinical data in combination with results from clinical trials of haploidentical killer immunoglobulin-like receptor (KIR)-ligand mismatched bone marrow transplantation led to interest in the use of adoptive NK immunotherapy for the treatment of malignancy. Recent early-phase clinical trial results have shown that allogeneic NK cells can be safely administered after chemotherapy and/or irradiation but have also demonstrated limited persistence of the infused NK cells. Additionally, in the absence of control arms, it has been difficult to determine whether tumor clearance is due to the preparative regimen, or due to actual immune mediated effects. Here, using several murine models of NK-sensitive hematologic malignancy, we trace the fate of adoptively infused NK cells and show that NK cells traffic to tumor sites but fail to control tumor growth. NK cells rapidly down-regulate effector functions and activating receptor expression upon homeostatic proliferation and upon tumor exposure in syngeneic as well as allogeneic settings. This dysfunction is likely cell-intrinsic as it is accompanied by down-regulation of the canonical transcription factors Eomesodermin and T-bet, and is not impacted by depletion of regulatory T cells from the tumor milieu. These results demonstrate that NK cell capability is intrinsically limited and cannot be relied upon for adoptive anti-tumor immunotherapy. As such, novel approaches may be required in order to circumvent the described dysfunction phenotype. Lastly, we attempted to deplete dendritic cells (DCs) within the host to study impacts on the development of GVHD. To try to delineate whether there is a critical time window during which host DCs cells need to initiate GVHD, we conducted MHC-mismatched transplants into BALB/c mice that were chimeric for the CD11c DTR-GFP transgene in their hematopoietic system, to be able to control the deletion of host DCs at specific time points. Despite initial problems with DT lethality in the original transgenic mice, we were able to achieve successful, transient, depletion of host DCs using these chimeric mice made from the BM of transgenic donors. Although we found that DT treatment of transgenic mice 24 hours to harvesting their spleens for use as stimulators in an MLR greatly reduced allogeneic T cell proliferation, DT treatment of transgenic chimeric recipients prior to irradiation and transplantation with allogeneic T cells did not provide any protection from the development of GVHD, even with up to a 4 day delay in T cell administration and titrating T cell doses down to 1 x 105. In contrast, DT treated mice actually had reduced survival compared to transgenic chimeras given only PBS, suggesting that DC depletion may somehow enhance T cell proliferation. This may be partially due to negative impacts on the activation, expansion, and function of Tregs, which are known to have important cross-talk with DCs. These studies were further complicated by the fact that the chimeric transgenic mice had all previously undergone lethal TBI, as we found that previously irradiated recipients had reduced survival when transplanted with allogeneic T cells, regardless of whether or not they were transgenic of WT or whether they received DT or PBS, compared to mice that had never been irradiated previously. Additionally, since DC depletion is not 100% in these animals, we were unable to precisely determine the contribution of remaining cell types in the outcomes we observed. As such we were unable to conclude anything about the role of host DCs in the development of GVHD from these studies.

Advisor: Negrin Robert S. primary advisor. Thesis advisor
Chawla Ajay advisor. Thesis advisor
Engleman Edgar G. advisor. Thesis advisor
Utz Paul advisor. Thesis advisor