Acute graft-versus-host disease (aGvHD) remains a significant hurdle to successful treatment of many hematological disorders. The disease is caused by infiltration of alloactivated donor T cells primarily into the gastrointestinal tract and skin. Although cytotoxic T cells mediate direct cellular damage, T helper (Th) cells differentially secrete inununoregulatory cytokines. aGvHD is thought to be initiated primarily by Thl cells but a consensus is still lacking regarding the role of Th2 and Th17 cells. The aim of this study was to determine the contribution of distinct T-cell subsets to aGvHD in the rat. aGvHD was induced by transplanting irradiated rats with T-cell depleted major histocompatibility complex-mismatched bone marrow, followed 2 weeks later by donor lymphocyte infusion. Near complete donor T-cell chimerism was achieved in the blood and lymphatic tissues, in contrast to mixed chimerism in the skin and gut. Skin and gut donor T cells were predominantly CD4(+), in contrast to T cells in the blood and lymphatic tissues. Genes associated with Th1 cells were upregulated in gut, liver, lung, and skin tissues affected by aGvHD. Increased serum levels of CXCL10 and IL-18 preceded symptoms of aGvHD, accompanied by increased responsiveness to CXCL10 by blood CD4(+) T cells. No changes in the expression of Th2- or Th17-associated genes were observed, indicating that aGvHD in this rat model is mainly Thl driven. The rat model of aGvHD could be instrumental for further investigations of donor T-cell subsets in the skin and gut and for exploring therapeutic options to ameliorate symptoms of aGvHD. Copyright (C) 2017 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc.

Total body irradiation (TBI) is part of the preconditioning regimen for allogeneic bone marrow transplantation (alloBMT) and the procedure is associated with treatment-related toxicity and delayed immune reconstitution. Natural killer (NK) cells develop and acquire functional competence in close interaction with stromal bone marrow cells that are considered relatively radioresistant compared to the hematopoietic compartment. We thus undertook a study to assess the effect of TBI on the reconstitution of class I MHC-specific Ly49 NK cell receptors in a rat model of alloBMT. In rats subjected to TBI alone or followed by MHC-matched BMT, the irradiation conditioning induced a skewing of the Ly49 repertoire. Specifically, the activating Ly49s3bright subset exhibited increased frequency and receptor density which correlated with augmented alloreactivity relative to untreated control rats. Our results highlight the plasticity of NK cells and indicate that ionizing radiation (IR) affects the stromal compartment and as a consequence the maturation and functional properties of bone marrow-derived NK cells. These changes lasted throughout the 6 months observation period, showing that irradiation induces long term effects on the generation of the NK cell receptor repertoire.

In this review, I summarize some of the early research on NK cell biology and function that led to the discovery of a totally new receptor system for polymorphic MHC class I molecules. That NK cells both could recognize and kill tumor cells but also normal hematopoietic cells through expression of MHC class I molecules found a unifying explanation in the "missing self" hypothesis. This initiated a whole new area of leukocyte receptor research. The common underlying mechanism was that NK cells expressed receptors that were inhibited by recognition of unmodified "self" MHC-I molecules. This could explain both the killing of tumor cells with poor expression of MHC-I molecules and hybrid resistance, i.e., that F1 hybrid mice sometimes could reject parental bone marrow cells. However, a contrasting phenomenon termed allogeneic lymphocyte cytotoxicity in rats gave strong evidence that some of these receptors were activated rather than inhibited by recognition of polymorphic MHC-I. This was soon followed by molecular identification of both inhibitory and stimulatory Ly49 receptors in mice and rats and killer cell immunoglobulin-like receptors in humans that could be either inhibited or activated when recognizing their cognate MHC-I ligand. Since most of these receptors now have been molecularly characterized, their ligands and the intracellular pathways leading to activation or inhibition identified, we still lack a more complete understanding of how the repertoire of activating and inhibitory receptors is formed and how interactions between these receptors for MHC-I molecules on a single NK cell are integrated to generate a productive immune response. Although several NK receptor systems have been characterized that recognize MHC-I or MHC-like molecules, I here concentrate on the repertoires of NK receptors encoded by the natural killer cell gene complex and designed to recognize polymorphic MHC-I molecules in rodents, i.e., Ly49 (KLRA) receptors.

In this review, I summarize some of the early research on NK cell biology and function that led to the discovery of a totally new receptor system for polymorphic MHC class I molecules. That NK cells both could recognize and kill tumor cells but also normal hematopoietic cells through expression of MHC class I molecules found a unifying explanation in the “missing self” hypothesis. This initiated a whole new area of leukocyte receptor research. The common underlying mechanism was that NK cells expressed receptors that were inhibited by recognition of unmodified “self” MHC-I molecules. This could explain both the killing of tumor cells with poor expression of MHC-I molecules and hybrid resistance, i.e., that F1 hybrid mice sometimes could reject parental bone marrow cells. However, a contrasting phenomenon termed allogeneic lymphocyte cytotoxicity in rats gave strong evidence that some of these receptors were activated rather than inhibited by recognition of polymorphic MHC-I. This was soon followed by molecular identification of both inhibitory and stimulatory Ly49 receptors in mice and rats and killer cell immunoglobulin-like receptors in humans that could be either inhibited or activated when recognizing their cognate MHC-I ligand. Since most of these receptors now have been molecularly characterized, their ligands and the intracellular pathways leading to activation or inhibition identified, we still lack a more complete understanding of how the repertoire of activating and inhibitory receptors is formed and how interactions between these receptors for MHC-I molecules on a single NK cell are integrated to generate a productive immune response. Although several NK receptor systems have been characterized that recognize MHC-I or MHC-like molecules, I here concentrate on the repertoires of NK receptors encoded by the natural killer cell gene complex and designed to recognize polymorphic MHC-I molecules in rodents, i.e., Ly49 (KLRA) receptors.

Abstract LTX-315 is a de novo designed peptide derived from a naturally occurring host defence peptide. LTX-315 has the potential to induce long-term specific protective immune responses by stimulating immune cells, inducing tumor cell lysis with subsequent release of danger signals (e.g. HMGB1) and tumor associated antigens (TAA`s). A complete tumor regression has been obtained in several syngenic rodent tumor models by intratumoral (i.t.) injection with LTX-315. The effect was T- cell- dependent since the intervention was inefficient in immune-deficient animals. Studies on treated tumor tissue confirmed infiltration of immune cells and a switch in the cytokine profile towards a Th1 response. Successfully treated animals were protected against re-challenge with the tumor cell type treated, but not against other types of tumor cells. Moreover, tumor resistance could be adoptively transferred by spleen cells from LTX-315-treated animals. The resistance was abrogated by depletion of T- lymphocytes. Additional studies also indicate that LTX-315`s potential to locally activate the innate immune system by the immunogenic stressing of cells, in addition to the subsequent release of endogenous adjuvants and natural danger signals, provides a strong rationale for using LTX-315 as an adjuvant for vaccines based on tumor-associated antigens (TAA) and for combination with other types of immune–modulatory therapies. LTX-315 is currently being tested in a Phase I dose escalation clinical study and may represents a novel strategy for personalized in situ vaccination against cancer. Citation Format: Øystein Rekdal, Gunnar Kvalheim, Pål-Dag Line, Bent Rolstad, Ketil Camilio, Gerd Berge, Janne Nestvold, Mengyu Wang, Jihua Shi, Ali Areffard, Baldur Sveinbjørnsson. Complete regression and long-term specific protective immune responses obtained in rodent tumor models after intratumoral treatment with LTX-315 . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 474. doi:10.1158/1538-7445.AM2013-474

Mesenchymal stromal cells (MSC) can be used to treat graft-versus-host disease (GVHD) caused by allogeneic stem cell transplantation (allo-SCT). The effectiveness of this therapy has been variable in clinical trials and in experimental animal models. In this study, we investigated the ability of bone marrow (BM)-derived MSC to alleviate GVHD in an experimental rat model of allo-SCT using two different combinations of major histocompatibility complex (MHC) mismatch with survival as the primary endpoint. Recipient rats received total body irradiation and a transplant of T cell-depleted donor BM cells with either a full [PVG.7B ? BN] or a partial MHC mismatch [PVG.1U ? PVG.R23] restricted to the class II and non-classical class I sub-regions (RT1-B/D-CE/N/M). GVHD was invoked by infusion of graded doses of donor leukocytes 2 weeks after allo-SCT. Weekly doses of MSC were injected starting on the day of donor leukocyte infusion. No significant overall improvement of mortality and morbidity was observed in the two transplantation settings. Stimulation of MSC with exogenous tumor necrosis factor a and interferon (IFN)? prior to infusion could not rescue BM-transplanted rats from lethal acute GVHD. In conclusion, repeated administrations of MSC failed to alleviate GVHD after fully or partially MHC-mismatched allo-SCT in the rat.

Natural killer cells are able to recognize and kill target cells according to differences in MHC class I expression. In rodents, the Ly49 receptors are primarily responsible for this MHC differentiation. We previously described the cloning of a novel C-type lectin–like receptor, KLRH1, encoded in the NK complex adjacent to the Ly49 genes and expressed by subsets of NK and NKT cells. MHC influence on selection of KLRH1+ NK cells in congenic strains suggested that KLRH1 may have an MHC ligand, although we were unable to identify any such ligand. In this study, we have used a sensitive reporter system and Fc fusion protein to demonstrate that KLRH1 binds specifically to the classical MHC class I molecule RT1-A2 of the RT1n haplotype. Cytolytic activity of KLRH1-transfected RNK-16 cells was also inhibited by target cells expressing RT1-A2n. Thus, KLRH1 represents a novel family of MHC allele–specific inhibitory receptors expressed by NK cells.

Allogeneic hematopoietic cell transplantation (alloHCT) extends the lives of thousands of patients who would otherwise succumb to hematopoietic malignancies such as leukemias and lymphomas, aplastic anemia, and disorders of the immune system. In alloHCT, different immune cell types mediate beneficial graft-versus-tumor (GvT) effects, regulate detrimental graft-versus-host disease (GvHD), and are required for protection against infections. Today, the "good" (GvT effector cells and memory cells conferring protection) cannot be easily separated from the "bad" (GvHD-causing cells), and alloHCT remains a hazardous medical modality. The transplantation of hematopoietic stem cells into an immunosuppressed patient creates a delicate environment for the reconstitution of donor blood and immune cells in co-existence with host cells. Immunological reconstitution determines to a large extent the immune status of the allo-transplanted host against infections and the recurrence of cancer, and is critical for long-term protection and survival after clinical alloHCT. Animal models continue to be extremely valuable experimental tools that widen our understanding of, for example, the dynamics of post-transplant hematopoiesis and the complexity of immune reconstitution with multiple ways of interaction between host and donor cells. In this review, we discuss the rat as an experimental model of HCT between allogeneic individuals. We summarize our findings on lymphocyte reconstitution in transplanted rats and illustrate the disease pathology of this particular model. We also introduce the rat skin explant assay, a feasible alternative to in vivo transplantation studies. The skin explant assay can be used to elucidate the biology of graft-versus-host reactions, which are known to have a major impact on immune reconstitution, and to perform genome-wide gene expression studies using controlled combinations of minor and major histocompatibility between the donor and the recipient.

Abstract Background: In order to develop more effective anticancer agents there is a shift from not only treating the bulk of cells in a tumor but also to target the self renewing cancer stem cells (CSCs). Standard treatment with chemotherapy and radiotherapy is unable to eradicate CSCs and therefore other treatment modalites against CSCs are needed. LTX-315 (Oncopore®) is a chemically modified cytolytic peptide which is equally active against drug-sensitive and drug-resistant cancer cells. Earlier animal studies have demonstrated that treatment of syngenic murine A20 B-lymphomas and CT26WT carcinomas with intratumoral (i.t.) injection with LTX-315 resulted in complete tumor regression. The present study was undertaken to investigate whether LTX-315 induced an antitumor response in vivo in a novel malignant rat mesenchymal tumor model with “stemness” properties. Methods: Following long-term culturing of bone marrow-derived rat mesenchymal cells, a transformed mesenchymal cell line (rTMSC) has been been developed. rTMSC has, as the non-malignant rMSC, maintained osteogenic and adipose differentation properties and side population cells from rTMSC are highly clonogenic both in vitro and in vivo. Previously we have shown that s.c. as well as i.v. or i.p. injection of rTMSCs form immature solid sarcomas both in nude rats as well as in PVG rats. In this study the rTMSCs were marked with a dual reporter gene GFP and Luciferase and a CCD camera was used for dynamic living imaging. rTMSCs (5×105 cells) were subcutaneously inoculated into PVG rats and established tumors treated i.t. with LTX-315. Results: Intratumoral treatment with LTX-315 resulted in a complete regression in the majority of the treated animals. Successfully treated mice were protected against s.c. or i.p. re-challenge with rTMSCs. Conclusions: Taken together, our results suggest that LTX-315 treatment induced complete regression and long-term, specific cellular immunity against malignant rat mesenchymal tumor cells with “stemness” properties”. Thus, intratumoral administration of a cytolytic peptide might, in addition to providing local tumor control, confer a novel strategy for therapeutic vaccination against established cancers containing CSCs Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-161. doi:10.1158/1538-7445.AM2011-LB-161

GVHD causes extensive morbidity and mortality in patients who receive alloHCT. Predictive and reliable markers for GVHD are currently lacking but required to improve the safety and accessibility of alloHCT. We present an experimental rat model of myeloablative total body irradiation and fully mismatched major and minor histoincompatible, T cell-depleted BMT, followed by delayed infusion of donor lymphocytes. This treatment, in contrast to marrow transplantation alone, resulted in severe aGVHD and 100% lethality within 2-6 weeks. We investigated the reconstitution kinetics and phenotypes of donor leukocyte subpopulations as well as the histopathology of selected organs that may correlate with GVHD, with the goal to find potential disease-related markers. We observed histological changes mainly confined to the skin, with degenerative changes in the basal layer. LNs and spleen showed deranged architecture with markedly increased accumulation of lymphocytes, whereas the gut, liver, and lungs appeared normal. Of the lymphocyte markers tested, donor-derived CD62L(+) T cells were markedly decreased in animals suffering from GVHD. Furthermore, we observed peripheral depletion of CD4(+)CD25(hi)FoxP3(+) T(reg), which was in contrast to controls. The relative frequency of these lymphocyte subpopulations in blood may therefore serve as accessible cellular markers of aGVHD. We propose that the animal model presented is instructive for the identification of clinically relevant markers of GVHD, which could improve disease diagnosis and management in alloHCT.

Background Mesenchymal stromal cells (MSC) have important immunomodulatory effects that can be exploited in the clinical setting, e.g. in patients suffering from graft-versus-host disease after allogeneic stem cell transplantation. In an experimental animal model, cultures of rat T lymphocytes were stimulated in vitro either with the mitogen Concanavalin A or with irradiated allogeneic cells in mixed lymphocyte reactions, the latter to simulate allo-immunogenic activation of transplanted T cells in vivo. This study investigated the inhibitory effects of rat bone marrow-derived MSC subsequently found to be infected with a common mycoplasma species (Mycoplasma hyorhinis) on T cell activation in vitro and experimental graft-versus-host disease in vivo. Principal Findings We found that M. hyorhinis infection increased the anti-proliferative effect of MSC dramatically, as measured by both radiometric and fluorimetric methods. Inhibition could not be explained solely by the well-known ability of mycoplasmas to degrade tritiated thymidine, but likely was the result of rapid dissemination of M. hyorhinis in the lymphocyte culture. Conclusions This study demonstrates the potent inhibitory effect exerted by M. hyorhinis in standard lymphocyte proliferation assays in vitro. MSC are efficient vectors of mycoplasma infection, emphasizing the importance of monitoring cell cultures for contamination.

Background The major histocompatibility complex (MHC) is the most important genomic region that contributes to the risk of graft versus host disease (GVHD) after haematopoietic stem cell transplantation. Matching of MHC class I and II genes is essential for the success of transplantation. However, the MHC contains additional genes that also contribute to the risk of developing acute GVHD. It is difficult to identify these genes by genetic association studies alone due to linkage disequilibrium in this region. Therefore, we aimed to identify MHC genes and other genes involved in the pathophysiology of GVHD by mRNA expression profiling. Methodology/Principal Findings To reduce the complexity of the task, we used genetically well-defined rat inbred strains and a rat skin explant assay, an in-vitro-model of the graft versus host reaction (GVHR), to analyze the expression of MHC, natural killer complex (NKC), and other genes in cutaneous GVHR. We observed a statistically significant and strong up or down regulation of 11 MHC, 6 NKC, and 168 genes encoded in other genomic regions, i.e. 4.9%, 14.0%, and 2.6% of the tested genes respectively. The regulation of 7 selected MHC and 3 NKC genes was confirmed by quantitative real-time PCR and in independent skin explant assays. In addition, similar regulations of most of the selected genes were observed in GVHD-affected skin lesions of transplanted rats and in human skin explant assays. Conclusions/Significance We identified rat and human MHC and NKC genes that are regulated during GVHR in skin explant assays and could therefore serve as biomarkers for GVHD. Several of the respective human genes, including HLA-DMB, C2, AIF1, SPR1, UBD, and OLR1, are polymorphic. These candidates may therefore contribute to the genetic risk of GVHD in patients.

Background The use of bone grafting in orthopaedic surgery has increased dramatically in recent years. However, the degree to which immune responses are important for the survival of the allograft is not fully understood. In particular it remains unclear whether differences in the major histocompatibility complex (MHC) influence incorporation of bone allografts and their subsequent biologic performance. Questions/purposes Therefore, we asked whether isolated mismatch for MHC antigens of deep frozen bone allografts in the long-term causes (1) immune reactions, and whether these reactions have any effect on (2) morphologic features of the graft, (3) radiographic graft healing, and (4) graft strength. Methods We used an established orthotopic tibial segment transplantation technique that allows determination of mechanical strength, histologic evaluation, and immune responses. Tibial segments that had been deep-frozen at −80°C for 1 year were transplanted into 24 PVG (RT1c) rats from either 12 syngeneic donors or 12 MHC congenic donors PVG.1U (RT1u). We determined immune responses using an indirect Coombs reaction and determined graft healing radiographically and mechanically after 6 months. Results We detected no alloantibody production to graft MHC-I antigens, and found no differences between syngeneic and MHC mismatched grafts in terms of remodeling with host bone, graft healing, and mechanical strength. Conclusions Mismatches for MHC antigens do not seem to play a decisive role in healing of long-term, deep-frozen bone allografts.

Ly49 receptors in rodents, like KIRs in humans, regulate NK cell activity. Although inhibitory Ly49 receptors clearly recognize MHC‐I molecules, ligands for the activating Ly49 receptors are less well defined. Here, we show that the activating Ly49s4 and the inhibitory Ly49i4 receptors recognize nonclassical MHC‐I molecules on the rat macrophage cell line R2 (RT1d). Listeria infection of R2 macrophages led to increased expression of classical and nonclassical MHC‐I molecules. Coincubation of these infected cells with reporter cells expressing Ly49i4 or Ly49s4 increased the reporter cell responses. These responses were blocked by mAb OX18 (anti‐MHC‐I) and AAS1 (anti‐nonclassical MHC‐I). IFN‐γ treatment of normal R2 cells also increased the MHC‐I expression and enhanced the reporter cell responses. These results suggest that activating and inhibitory Ly49 receptors monitor MHC‐I expression on Listeria‐infected cells.

Ly49 receptors in rodents, like killer cell immunoglobulin-like receptors in humans, regulate natural killer (NK) cell activity. Although inhibitory Ly49 receptors clearly recognize classical major histocompatibility complex class I (MHC-I) molecules, the role for the activating Ly49 receptors has been less well understood. Here, we discuss recent data from a rat model for listeriosis. Rats depleted of NK cells, or more specifically the Ly49 receptor-bearing cells, showed increased bacterial loads in their spleen. Athymic nude rats with no functional T cells but increased numbers of Ly49-expressing NK cells were more resistant to infection, indicating a central role of NK cells in early immune defense against Listeria in this species. Listeria infection of macrophages or enteric epithelial cells led to upregulation of MHC-I, including nonclassical (Ib) molecules not regularly recognized by T cells. We have shown that activating Ly49 receptors are more efficiently stimulated when binding to upregulated class Ib antigens on infected cells. From this we postulate that activating Ly49 receptors may have a sentinel function in the early immune response against Listeria in detecting diseased cells ‘flagged’ by increased MHC-Ib expression.

e21046 Background: LTX-315 (Oncopore) is a chemically designed cationic nonapeptide derived from a natural host defensive CAP, bovine lactoferricin (LfcinB) in development for local treatment of tumours. Earlier studies have demonstrated that treatment of A20 lymphomas and CT26WT carcinomas with LTX-315 in immunocompetent mice resulted in tumour regression. Re-challenge with tumor cells in the cured animals showed that a long- term protection was induced against the tumor. Methods: In the current experiments we have investigated if LTX-315 also can have the same effect on a novel established malignant rat mesenchymal tumor cell line with “stemness” properties. Following long term culturing of bone marrow derived rat mesenchymal cells we developed a rat transformed mesenchymal cell line (rTMSC). Previously we have shown that s.c. as well as i.v. and i.p. injection of rTMSC form immature sarcoma tumors both in nude rats as well as in PVG rats. The rTMSC has been stabled marked with a dual reporter gene GFP and Leuciferase and in the current study CCD camera was used for dynamic living imaging. 5×105 rTMSCs were subcutaneously inoculated into 20 PVG rats. After 7 days measurable tumors could be observed and 18 animals was treated with LTX-315. Results: In spite of repeated injection with the peptide 8 animals lost tumor control, while 9 animals within 38 days achieved complete tumor responses as assessed with CCD camera. Re-challenges of the tumor free animals with injection of 5×104 tRMSCs show no tumor growth while all control animals had a fast tumor formation. Conclusions: 50% of the treated animals have developed an impressive tumor protection of this aggressive mesenchymal tumor with “stemness” properties. Most likely more animals can be cured if the peptide injection procedure can be improved and further studies are underway. Altogether our results bear promises for LTX-315 in future cancer therapy. No significant financial relationships to disclose.

NK cells are protective against certain bacterial and viral infections, and their production of IFN-γ is important for the early innate immune defence against L. monocytogenes. We have previously shown that depletion of NK cells in rats leads to increased bacterial burden upon L. monocytogenes infection, and that a subset of NK cells encompassing the majority of Ly49 receptors (Ly49s3+ NK cells) contributed to this effect. In this study, we have further investigated how the Ly49s3+ NK cell subset is affected by L. monocytogenes infection. We observed an increased percentage of Ly49s3+ NK cells in the spleen and a reduction in the bone marrow within the first 48 hrs of L. monocytogenes infection. Concomitantly, we observed increased expression levels of the inflammatory chemokine receptors CCR5 and CXCR3 by Ly49s3+ bone marrow NK cells, as compared to Ly49s3− NK cells, suggesting involvement of Ly49s3+ NK cells in the early phase of infection. However, NK cell production of IFN-γ was independent of Ly49 receptor expression. Furthermore, we observed increased expression levels of MHC class I molecules on both macrophages and NK cells during the first 48 hrs of infection, paralleled by a reduction in the surface expression of Ly49s3 on NK cells. In conclusion, L. monocytogenes infection modulates the tissue distribution of Ly49s3+ NK cells, and induces increased MHC class I expression and hence reduced surface expression of Ly49 receptors on NK cells. These changes indicate that L. monocytogenes infection may have multiple effects on NK cells in vivo, and suggests the involvement of Ly49-expressing NK cells in the immune responses towards L. monocytogenes.