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Innate lymphoid cells (ILCs) that express NK cell receptors (NCRs) and the transcription factor T-bet populate nonlymphoid tissues and are crucial in immune responses against viral infections and malignancies. Recent studies highlighted the heterogeneity of this ILC population and extended their functional spectrum to include important roles in tissue homeostasis and autoimmunity. In this article, we provide detailed profiling of NCR + T-bet + ILC populations in the murine kidney, identifying conventional NK (cNK) cells and type 1 ILCs (ILC1s) as the two major subsets. Induction of renal inflammation in a mouse model of glomerulonephritis did not substantially influence abundance or phenotype of cNK cells or ILC1s in the kidney. For functional analyses in this model, widely used depletion strategies for total NCR + ILCs (anti-NK1.1 Ab application) and cNK cells (anti-asialoGM1 serum application) were unreliable tools, because they were accompanied by significant off-target depletion of kidney NKT cells and CD8 + T cells, respectively. However, neither depletion of cNK cells and ILC1s in NKT cell-deficient mice nor specific genetic deletion of cNK cells in Ncr1 Cre/wt × Eomes fl/fl mice altered the clinical course of experimental glomerulonephritis. In summary, we show in this article that cNK cells and ILC1s are dispensable for initiation and progression of immune-mediated glomerular disease and advise caution in the use of standard Ab depletion methods to study NCR + ILC function in mouse models., (Copyright © 2022 by The American Association of Immunologists, Inc.)

Regulatory T cells (Treg) are key players in maintaining immune homeostasis but have also been shown to regulate immune responses against infectious pathogens. Therefore, Treg are a promising target for modulating immune responses to vaccines to improve their efficacy. Using a viral vector system, we found that Treg act on the developing immune response early postinfection by reducing the extent of dendritic cell costimulatory molecule expression. Due to this change and the lower IL-2 production that results, a substantial fraction of CD8(+) effector T cells lose CD25 expression several days after activation. Surprisingly, such Treg-dependent limitations in IL-2 signaling by Ag-activated CD8(+) T cells prevent effector differentiation without interfering with memory cell formation. In this way, Treg fine-tune the numbers of effector T cells generated while preserving the capacity for a rapid recall response upon pathogen re-exposure. This selective effect of Treg on a subpopulation of CD8(+) T cells indicates that although manipulation of the Treg compartment might not be optimal for prophylactic vaccinations, it can be potentially exploited to optimize vaccine efficacy for therapeutic interventions.

The size of the adaptive immune system is considered to be kept constant by the attrition of pre-existing memory. However, recently it was shown that the CD8 memory compartment can grow in size and the number of pre-existing memory is largely preserved, predicting that pre-existing immunity should be maintained (Vezys et al.; Nature 457: 196-199). Experimental proof for this assumption is still lacking. We address this question in the Listeria monocytogenes (L.m.) infection model and confirm the growth of size of the memory compartment by subsequent vaccination with modified vaccinia virus Ankara. We also find only modest attrition of pre-existing L.m.-specific memory CD8 T cells. However, pre-existing protective immunity toward L.m. is not preserved. Pre-existing L.m.-specific effector-memory cells, in contrast to central memory cells, become altered, and this results in a significant loss of pre-existing protective immunity. Our findings are clinically relevant for vaccines introducing new CD8 memory cells in high numbers, as this might influence pre-existing immunity.

A major problem of current vaccines is storage stability, often requiring strict maintenance of cold chains. In the course of the eradication of smallpox, a freeze-dried vaccinia virus (Dryvax), which proved to be very stable, was used to overcome this limitation. However, Dryvax needs to be reconstituted before usage and is administered using a bifurcated needle, procedures that pose a number of additional health risks. We report in this study that a stable, lyophilized, modified vaccinia virus Ankara (MVA) vaccine can be directly applied to the nostrils of mice without previous reconstitution. This direct mucosal application induced systemic Ab and T cell responses comparable to those achieved by i.m. administration. Importantly, mucosal application of lyophilized MVA induced long-lasting protective immunity against lethal bacterial and viral challenges. These data clearly demonstrate the potency of a simple needle-free vaccination, combining the advantages of mucosal application with the stability and efficiency of lyophilized MVA.

Viral peptides are presented by HLA class I on infected cells to activate CD8(+) T cells. Several immunogenic peptides have been identified indirectly by epitope prediction and screening of T cell responses to poxviral vectors, including modified vaccinia virus Ankara (MVA) currently being tested as recombinant or smallpox vaccines. However, for the development of optimal vaccination and immunomonitoring strategies, it is essential to characterize the actual viral HLA ligand repertoire of infected cells. We used an innovative approach to identify naturally processed MVA HLA ligands by differential HPLC-coupled mass spectrometry. We describe 12 viral peptides presented by HLA-A*0201 and 3 by HLA-B*0702. All HLA-A*0201 ligands participated in the memory response of MVA-immune donors, and several were immunogenic in Dryvax vaccinees. Eight epitopes were novel. Viral HLA ligand presentation and viral protein abundance did not correlate. All ligands were expressed early during the viral life cycle, and a pool of three of these mediated stronger protection against a lethal challenge in mice as compared with late epitopes. This highlights the reliability of the comparative mass spectrometry-based technique to identify relevant viral CD8(+) T cell epitopes for optimizing the monitoring of protective immune responses and the development of effective peptide-based vaccines.