Summary Proteasomes play a fundamental role in the processing of intracellular antigens into peptides that bind to MHC class I molecules for presentation to CD8 T cells. Three IFNγ-inducible catalytic proteasome (immuno)subunits as well as the IFNγ-inducible proteasome activator PA28 dramatically accelerate the generation of a subset of MHC class I-presented antigenic peptides. To determine whether these IFNγ-inducible proteasome components play a compounded role in antigen processing, we generated mice lacking both PA28 and the immunosubunits β5i/LMP7 and β2i/MECL-1. Analyses of MHC class I cell surface levels ex vivo demonstrated that PA28-deficiency reduced the production of MHC class I-binding peptides both in cells with and without immunosubunits, in the last cells on top of an already diminished production of MHC ligands in the absence of immunoproteasomes. In contrast, the immunosubunits but not PA28 appeared to be of critical importance for the induction of CD8 T cell responses to multiple dominant Influenza and Listeria-derived epitopes. Taken together, our data demonstrate that PA28 and the proteasome immunosubunits use fundamentally different mechanisms to enhance the supply of MHC class I-binding peptides. However, only the immunosubunit-imposed effects on proteolytic epitope processing appear to have substantial effects on the fine-specificity of pathogen-specific CD8 T cell responses.

NK cells play an important role in the early defense against invading pathogens. Although it is well-established that infection leads to a substantial, local increase in NK cell numbers, little is known about the mechanisms that trigger their proliferation and migration. We here investigated the dynamics of NK cell responses following intranasal respiratory virus infection. We show that NK cell numbers increased in the airways following influenza virus infection, but find no evidence of proliferation either at the site of infection or in the draining lymph nodes. Instead, we find that the bone marrow (BM) is the primary site of proliferation of both immature and mature NK cells during infection. Using an adoptive transfer model, we demonstrate that peripheral, long-lived and phenotypically mature NK cells migrate back to the BM and proliferate there, both homeostatically and in response to infection. Thus, the BM is not only a site of NK cell development, but also an important site for proliferation of long-lived mature NK cells.

Natural killer (NK) cells are innate lymphocytes that play an important role in control of viral infections. We recently showed that intranasal infection of mice with influenza virus induced the accumulation of NK cells in the airways. NK cells however did not proliferate in the airways or in the draining lymph node, but in the bone marrow mainly. As also monocyte-precursors undergo vigorous proliferation in the bone marrow (BM) during infections and then egress CCR2-dependently, we decided to determine the role of CCR2 in NK cell migration during intranasal influenza virus infection. We show that a unique population of NK cells in the BM expressed CCR2 and that monocyte chemotactic protein-1 (MCP-1), one of the CCR2 ligands, was produced in the airways of influenza virus infected mice. Analysis of BM chimeric mice reconstituted with a mix of wild-type (wt) and CCR2-deficient BM cells showed that upon influenza virus infection, a significantly lower proportion of CCR2-deficient than wt NK cells was recovered from the bronchoalveolar lavage (BAL). Taken together, our data demonstrate that during influenza virus infection a proportion of NK cells migrate in a CCR2-dependent fashion.

Highlights► NK cells and CD8+ T-cells expand relatively late following pneumovirus infection. ► Memory CD8+ T-cells support type 1 skewing of pneumovirus-specific responses. ► Memory CD8+ T-cells prevent pneumovirus-induced immunopathology. ► CD8+ T-cell targeted immunization protects against pneumovirus-induced disease.

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.