Listeria monocytogenes infection enhances the interaction between rat non-classical MHC-Ib molecule and Ly49 receptors.

Murine NK cell Ly49 receptors, functionally analogous to KIRs in humans recognize MHC class I molecules and play a key role in controlling NK cell function. We have previously shown that the paired activating Ly49s4 and inhibitory Ly49i4 receptors recognize undefined non-classical MHC-Ib ligands from the RT1-CE region in rats. Here, the RT1-CE16 gene of the RT1d haplotype was stably transfected into the mouse RAW macrophage cell line, termed RAW-CE16d cells. Combining RAW-CE16d cells with Ly49 expressing reporter cells demonstrated Ly49i4 and Ly49s4 specificity for CE16d. The Ly49s4/i4:CE16d interaction was confirmed by specific MHC-I blocking monoclonal Abs. Further, we used our in vitro model to study the effect of Listeria monocytogenes (LM) on CE16d after infection. LM infection and IFN-γ stimulation both led to enhanced CE16d expression on the surface of transfected RAW-CE16d cells. Interestingly, the reporter cells displayed increased response to LM-infected RAW-CE16d cells compared with IFN-γ-treated RAW-CE16d cells, suggesting a fundamental difference between these stimuli in supporting enhanced Ly49 recognition of CE16d. Collectively, our data show that Ly49s4 and Ly49i4 recognize the non-classical RT1-CE16d molecule, which in turn is up-regulated during LM infection and thereby may contribute to NK-mediated responses against infected cells.

Listeria monocytogenes infection differentially affects expression of ligands for NK cells and NK cell responses, depending on the cell type infected.

The pivotal role of NK cells in viral infection is extensively studied, whereas the role of NK cells in bacterial infection has been poorly investigated. Here, we have examined how Listeria monocytogenes (LM) affects expression of ligands for NK cell receptors and subsequent NK cell responses, depending on the type of cell infected. LM infected rat cell lines derived from different tissues were coincubated with splenic NK cells, and NK cell proliferation and IFN-γ production were measured. In addition, expression of ligands for the NK cell receptors Ly49 and NK cell receptor protein 1 (NKR-P1), MHC class I and C-type lectin-related molecules, respectively, was assessed. Infected pleural R2 cells, but not epithelium-derived colon carcinoma cell line CC531 cells, induced proliferation of NK cells. Reporter cells expressing the inhibitory NKR-P1G receptor or the activating NKR-P1F receptor were less stimulated under incubation with infected CC531 cells versus uninfected CC531 controls, suggesting that the ligand(s) in question were down-regulated by infection. Conversely, LM infection of R2 cells did not affect reporter cell stimulation compared with uninfected R2 controls. We characterized a rat monocyte cell line, termed RmW cells. In contrast to LM infected R2 cells that up-regulate MHC class I molecules, RmW cells displayed unchanged MHC class I expression following infection. In line with MHC class I expression, more NK cells produced a higher amount of IFN-γ against infected R2 cells compared with RmW cells. Together, L. monocytogenes infection may variously regulate cellular ligands for NK cells, depending on the cell type infected, affecting the outcome of NK cell responses.

Oncolytic peptide LTX-315 induces an immune-mediated abscopal effect in a rat sarcoma model.

LTX 315 is an oncolytic peptide with potent immunological properties. In the present study, we demonstrate that intratumoral treatment with LTX-315 resulted in a complete regression and systemic immune response in a rat fibrosarcoma model. The treatment was T-cell dependent, and also resulted in an abscopal effect as demonstrated by the regression of distal non-treated lesions. Significant infiltration of CD8+ T cells was observed in both treated and non-treated lesions, as shown by immunohistochemical and flow cytometric analysis. LTX-315 rapidly killed the cells in vitro with a lytic mode of action followed by the subsequent release of Danger-Associated Molecular Pattern (DAMP) molecules such as HMGB1, ATP and Cytochrome c. Together, our data demonstrate that LTX-315 represents a new approach to cancer immunotherapy, which has the potential as a novel immunotherapeutic agent.

A novel rat fibrosarcoma cell line from transformed bone marrow-derived mesenchymal stem cells with maintained in vitro and in vivo stemness properties.

Increasing evidence suggests a possible relationship between mesenchymal stem cells (MSCs) and sarcoma. MSCs are hypothesized to be the cells initiating sarcomagenesis, and cancer stem cells (CSCs) sharing features of MSCs have been identified in sarcomas. Here, we report on the characteristics of a bone marrow-derived rat mesenchymal stem cell line that spontaneously transformed in long-term culture. The rat transformed mesenchymal stem cells (rTMSCs) produced soft-tissue fibrosarcomas in immunocompromised mice and immunocompetent rats. In vitro, the rTMSCs displayed increased proliferation capacity compared to the untransformed cell line. The transformed MSCs maintained the mesenchymal phenotype by expression of the stem cell marker CD 90 and the lack of hematopoietic and endothelial markers. Cytogenetic analysis detected trisomy 6 in the rTMSCs. Side population (SP) isolation and tumorsphere cultivation of the transformed cells confirmed the presence of CSCs among the rTMSCs. Importantly, the rTMSCs retained their differentiation capacity towards osteogenic and adipogenic lineages. This transformed MSC-based cell line may be valuable in examining the balance in a mixed cell population between cancer stem cell properties and the ability to differentiate to specific non-transformed cell populations. Moreover, it may also be a useful tool to evaluate the efficacy of novel targeted immunotherapies in vivo.

Irradiation and bone marrow reconstitution affect the functional Ly49 natural killer cell repertoire in rats.

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 Ly49s3(bright) 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.

Partial NK cell tolerance induced by radioresistant host cells in rats transplanted with MHC-mismatched bone marrow.

We have studied the effect of radioresistant host cells in inducing tolerance and adaptation of the MHC recognition repertoire of donor-derived NK cells in stem cell allotransplanted (allo-SCT) rats. Sub-lethally irradiated PVG.1AV1 rats (RT1(av1)) were transplanted with bone marrow from fully MHC-mismatched allotype-marked PVG.7B (RT1(c)) rats; MHC-identical PVG (RT1(c)) controls were transplanted in parallel. In the PVG.7B → PVG.1AV1 allogeneic chimeras, NK cells were donor derived and showed partial tolerance toward host cells. Allogeneic chimeras failed to efficiently reject PVG.1AV1 cells by an NK-mediated mechanism in vivo (allogeneic lymphocyte cytotoxicity), and IL-2-cultured NK cells derived from these chimeras showed diminished cytolytic activity against PVG.1AV1 cells in vitro. There were corresponding changes in the phenotype and function of the highly alloreactive Ly49i2(+) NK cells, which are specifically inhibited by a donor MHC class I ligand, RT1-A1(c). The ligand-negative host MHC haplotype apparently induced expression of a second uncharacterized inhibitory MHC receptor responsible for the partial tolerance toward host-derived cells, along with a modest increase in Ly49i2 receptor levels. The host MHC haplotype did not induce a general hyporesponsiveness in Ly49i2(+) NK cells, which showed normal activation responses in a panel of MHC congenic strains. The data suggest that the MHC constitution of radiation-resistant host cells can have permanent, albeit not fully tolerogenic, effects on the development of a functional NK repertoire following allo-SCT.

A second prophylactic MHC-mismatched bone marrow transplantation protects against rat acute myeloid leukemia (BNML) without lethal graft-versus-host disease.

BACKGROUND: We have employed a rat model for human acute myeloid leukemia, a promyelocytic leukemia in the BN rat strain (BNML), to develop new protocols for immunotherapy in combination with allogeneic bone marrow transplantation (alloBMT). The status of mixed chimerism in allotransplanted rats provided an opportunity for immunotherapy using alloreactive donor cells. In addition to T or natural killer (NK) cells, we introduced a second infusion of bone marrow cells as prophylactic donor lymphocyte infusions (DLI) to test whether an effective graft-versus-leukemia (GVL) response could be obtained without clinical graft-versus-host disease (GVHD). METHODS: BN rats were sublethally irradiated and transplanted with T-cell depleted bone marrow cells from either fully major histocompatibility complex (MHC)-mismatched (PVG) donor rats or MHC-matched (PVG.1N) as controls. Seven days after transplantation, rats were given 500 leukemic cells to mimic minimal residual disease. Additional cellular therapy was given at day +7. The efficiency of DLI was monitored by chimerism analysis in peripheral blood. RESULTS: Rats receiving infusions of NK cells succumbed to leukemia. T-DLI induced complete donor T-cell chimerism and lethal GVHD. A second alloBMT protected against leukemia. This effect was dependent on an MHC incompatibility between the donor and host and also on the presence of alloreactive T cells in the second bone marrow inoculum, resulting in an increased, mixed donor T-cell chimerism. CONCLUSION: A second prophylactic transplantation influenced the degree of T-cell chimerism to balance favorably between GVL and GVHD. If applicable to humans, repeated alloBMT may provide a novel approach to leukemia therapy.