Immunoproteasome-Deficiency Has No Effects on NK Cell Education, but Confers Lymphocytes into Targets for NK Cells in Infected Wild-Type Mice

Natural killer (NK) cells are part of the innate immune system and contribute to the eradication of virus infected cells and tumors. NK cells express inhibitory and activating receptors and their decision to kill a target cell is based on the balance of signals received through these receptors. MHC class I molecules are recognized by inhibitory receptors, and their presence during NK cell education influences the responsiveness of peripheral NK cells. We here demonstrate that mice with reduced MHC class I cell surface expression, due to deficiency of immunoproteasomes, have responsive NK cells in the periphery, indicating that the lower MHC class I levels do not alter NK cell education. Following adoptive transfer into wild-type (wt) recipients, immunoproteasome-deficient splenocytes are tolerated in naive but rejected in virus-infected recipients, in an NK cell dependent fashion. These results indicate that the relatively low MHC class I levels are sufficient to protect these cells from rejection by wt NK cells, but that this tolerance is broken in infection, inducing an NK cell-dependent rejection of immunoproteasome-deficient cells

Epitope mapping and kinetics of CD4 T cell immunity to pneumonia virus of mice in the C57BL/6 strain

Pneumonia virus of mice (PVM) infection has been widely used as a rodent model to study the closely related human respiratory syncytial virus (hRSV). While T cells are indispensable for viral clearance, they also contribute to immunopathology. To gain more insight into mechanistic details, novel tools are needed that allow to study virus-specific T cells in C57BL/6 mice as the majority of transgenic mice are only available on this background. While PVM-specific CD8 T cell epitopes were recently described, so far no PVM-specific CD4 T cell epitopes have been identified within the C57BL/6 strain. Therefore, we set out to map H2-IAb-restricted epitopes along the PVM proteome. By means of in silico prediction and subsequent functional validation, we were able to identify a MHCII-restricted CD4 T cell epitope, corresponding to amino acids 37–47 in the PVM matrix protein (M37–47). Using this newly identified MHCII-restricted M37–47 epitope and a previously described MHCI-restricted N339–347 epitope, we generated peptide-loaded MHCII and MHCI tetramers and characterized the dynamics of virus-specific CD4 and CD8 T cell responses in vivo. The findings of this study can provide a basis for detailed investigation of T cell-mediated immune responses to PVM in a variety of genetically modified C57BL/6 mice

Pre-existing virus-specific CD8+ T-cells provide protection against pneumovirus-induced disease in mice

Pneumoviruses such as pneumonia virus of mice (PVM), bovine respiratory syncytial virus (bRSV) or human (h)RSV are closely related pneumoviruses that cause severe respiratory disease in their respective hosts. It is well-known that T-cell responses are essential in pneumovirus clearance, but pneumovirus-specific T-cell responses also are important mediators of severe immunopathology. In this study we determined whether memory- or pre-existing, transferred virus-specific CD8 + T-cells provide protection against PVM-induced disease. We show that during infection with a sublethal dose of PVM, both natural killer (NK) cells and CD8 + T-cells expand relatively late. Induction of CD8 + T-cell memory against a single CD8 + T-cell epitope, by dendritic cell (DC)-peptide immunization, leads to partial protection against PVM challenge and prevents Th2 differentiation of PVM-induced CD4 T-cells. In addition, adoptively transferred PVM-specific CD8 + T-cells, covering the entire PVM-specific CD8 + T-cell repertoire, provide partial protection from PVM-induced disease. From these data we infer that antigen-specific memory CD8 + T-cells offer significant protection to PVM-induced disease. Thus, CD8 + T-cells, despite being a major cause of PVM-associated pathology during primary infection, may offer promising targets of a protective pneumovirus vaccine

Terminal NK cell maturation is controlled by concerted actions of T-bet and Zeb2 and is essential for melanoma rejection

Natural killer (NK) cell maturation is a tightly controlled process that endows NK cells with functional competence and the capacity to recognize target cells. Here, we found that the transcription factor (TF) Zeb2 was the most highly induced TF during NK cell maturation. Zeb2 is known to control epithelial to mesenchymal transition, but its role in immune cells is mostly undefined. Targeted deletion of Zeb2 resulted in impaired NK cell maturation, survival, and exit from the bone marrow. NK cell function was preserved, but mice lacking Zeb2 in NK cells were more susceptible to B16 melanoma lung metastases. Reciprocally, ectopic expression of Zeb2 resulted in a higher frequency of mature NK cells in all organs. Moreover, the immature phenotype of Zeb2(-/-) NK cells closely resembled that of Tbx21(-/-) NK cells. This was caused by both a dependence of Zeb2 expression on T-bet and a probable cooperation of these factors in gene regulation. Transgenic expression of Zeb2 in Tbx21(-/-) NK cells partially restored a normal maturation, establishing that timely induction of Zeb2 by T-bet is an essential event during NK cell differentiation. Finally, this novel transcriptional cascade could also operate in human as T-bet and Zeb2 are similarly regulated in mouse and human NK cells

NK cells migrate CCR2-dependent to the BAL during influenza virus infection.

<p>Mixed BM chimeric mice were constructed by injecting a mix of BM from CCR2^−/− (CD45.2) and C57BL/6.SJL (CD45.1) into lethally irradiated CD45.1.2. recipients. After 6–8 weeks, mice were infected with influenza virus or left uninfected. (A) Representative FACS plots showing CD45.1 and CD45.2 staining of NK cells (TCRβ⁻NK1.1⁺) recovered from the indicated organs 5 days p.i. (B-D) Ratio of CD45.2 (CCR2^−/−)/CD45.1 (<i>wt</i>) NK cells calculated by dividing absolute numbers of CD45.2⁺ NK cells by absolute numbers of CD45.1⁺ (<i>wt</i>) NK cells. Ratio of CD45.2/CD45.1 NK cells recovered from the indicated organs shown as average ± S.E.M. at the indicated days (B) and shown as individual mice at day 5 (C), and the ratio in the spleen and BM of individual mice are connected by a line at day 5 (D) after influenza virus infection. Representative results of two independent experiments are shown with 4–6 mice per group. Statistical analysis was performed using a Mann-Whitney U test. *, P<0.05.</p

A subset of NK cells expresses the CCR2 receptor.

<p>(A) Gating strategy (upper panel) for NK cells (TCRβ⁻NK1.1⁺DX5⁺) and representative histograms (lower panel) showing CCR2 expression on NK cells recovered from the indicated organs (blood, lung, BM and spleen) of naïve C57BL/6 (<i>wt</i>) or CCR2^−/− mice. (B,C) Expression of CD27 and CD11b (C) or KLRG1 on electronically gated NK cells (TCRβ⁻NK1.1⁺) or CCR2⁺ NK cells. The depicted FACS plots are representatives of 9 mice that were analyzed in two independent experiments. Statistical analysis was performed using a Mann-Whitney U test (C) and KLRG-1-expression on CCR2⁺ or CCR2⁻ NK cells is not significantly different (Mann-Whitney U test, *, P<0.05).</p

MCP-1 is expressed in the airways of influenza virus infected mice.

<p>(A) C57BL/6 mice were infected with HKx31 and the presence of MCP-1 in the BALF was determined by ELISA at the indicated days post-infection. Data shown are means+S.E.M. from one experiment with 5 mice per time point. Similar results were found in an independent experiment at day 2 and 5 post-infection.</p

Immunoproteasome-deficient mice have responsive peripheral NK cells.

<p>(A–C) Splenocytes of RAG1^−/− and RAG1^−/−β2i/MECL-1^−/−β5i/LMP7^−/− (RAG1^−/−IS^−/−) mice were analyzed by flow cytometry (A) Frequencies of NK cells (DX5⁺NKp46⁺) as percentage of total lymphocytes. (B,C) Expression of CD11b and CD27 on DX5⁺NKp46⁺ NK cells. (B) Representative FACS plots and (C) graph showing percentages of subsets for individual mice for the regions indicated in (B). (D–H) Splenocytes of RAG1^−/− and RAG1^−/−IS^−/− mice were incubated on plates coated with anti-NKG2D and anti-NKp46, in the presence or absence of IL-2, or left unstimulated. Frequencies of IFNγ⁺, LAMP-1⁺ NK cells and CD69 mean fluorescent intensity (MFI) on NK cells were determined by flow cytometry. (D) Representative FACS plots showing IFNγ⁺ and LAMP-1⁺ NK cells. (E–H) Bars showing the percentages of IFNγ⁺LAMP-1⁺ NK cells (E), IFNγ⁺ NK cells (F), LAMP-1⁺ NK cells (G), and CD69 MFIs on NK cells (H). Results are shown as mean ± S.E.M. Data are representative for 2 independent experiments with 6 mice per group. Statistical analysis was performed using Mann-Whitney U test. *, P<0.05.</p