Ag-inexperienced memory-like T (AIMT) cells are functionally unique T cells, representing one of the two largest subsets of murine CD8+ T cells. However, differences between laboratory inbred strains, insufficient data from germ-free mice, a complete lack of data from feral mice, and an unclear relationship between AIMT cells formation during aging represent major barriers for better understanding of their biology. We performed a thorough characterization of AIMT cells from mice of different genetic background, age, and hygienic status by flow cytometry and multiomics approaches, including analyses of gene expression, TCR repertoire, and microbial colonization. Our data showed that AIMT cells are steadily present in mice, independent of their genetic background and hygienic status. Despite differences in their gene expression profiles, young and aged AIMT cells originate from identical clones. We identified that CD122 discriminates two major subsets of AIMT cells in a strain-independent manner. Whereas thymic CD122LOW AIMT cells (innate memory) prevail only in young animals with high thymic IL-4 production, peripheral CD122HIGH AIMT cells (virtual memory) dominate in aged mice. Cohousing with feral mice changed the bacterial colonization of laboratory strains but had only minimal effects on the CD8+ T cell compartment, including AIMT cells.

Chemical communication is mediated by sex-biased signals abundantly present in the urine, saliva and tears. Because most studies concentrated on the urinary signals, we aimed to determine the saliva proteome in wild Mus musculus musculus, to extend the knowledge on potential roles of saliva in chemical communication. We performed the gel-free quantitative LC-MS/MS analyses of saliva and identified 633 proteins with 134 (21%) of them being sexually dimorphic. They include proteins that protect and transport volatile organic compounds in their beta barrel including LCN lipocalins, major urinary proteins (MUPs) and odorant binding proteins (OBPs). To our surprise, the saliva proteome contains one MUP that is female biased (MUP8) and the two protein pheromones MUP20 (or ‘Darcin’) and ESP1 in individuals of both sex. Thus, contrary to previous assumptions, our findings reveal that these proteins cannot function as male-unique signals. Our study also demonstrates that many olfactory proteins (e.g. LCNs and OBPs) are not expressed by submandibular glands but are produced elsewhere–in nasal and lacrimal tissues and potentially also in other oro-facial glands. We have also detected abundant proteins that are involved in wound healing, immune and non-immune responses to pathogens, thus corroborating that saliva has important protective roles.

Female house mice produce pheromone-carrying major urinary proteins (MUPs) in a cycling manner, thus reaching the maximum urinary production just before ovulation. This is thought to occur to advertise the time of ovulation via deposited urine marks. This study aimed to characterize the protein content from the house mouse vaginal flushes to detect putative vaginal-advertising molecules for a direct identification of reproductive states. Here we show that the mouse vaginal discharge contains lipocalins including those from the odorant binding (OBP) and major urinary (MUP) protein families. OBPs were highly expressed but only slightly varied throughout the cycle, whilst several MUPs were differentially abundant. MUP20 or ‘darcin’, was thought to be expressed only by males. However, in females it was significantly up-regulated during estrus similarly as the recently duplicated central/group-B MUPs (sMUP17 and highly expressed sMUP9), which in the mouse urine are male biased. MUPs rise between proestrus and estrus, remain steady throughout metestrus, and are co-expressed with antimicrobial proteins. Thus, we suggest that MUPs and potentially also OBPs are important components of female vaginal advertising of the house mouse.

Background: Chemical communication in mammals involves globular lipocalins that protect and transport pheromones during their passage out of the body. Efficient communication via this protein - pheromone complex is essential for triggering multiple responses including aggression, mate choice, copulatory behaviour, and onset and synchronization of oestrus. The roles of lipocalins in communication were studied in many organisms and especially in mice (i.e. Mus musculus domesticus) which excrete Major Urinary Proteins (Mup) in excessive amounts in saliva and urine. Other mammals, however, often lack the genes for Mups or their expression is very low. Therefore, we aimed at characterization of candidate lipocalins in Myodes glareolus which are potentially linked to chemical communication. One of them is Aphrodisin which is a unique lipocalin that was previously described from hamster vaginal discharge and is known to carry pheromones stimulating copulatory behaviour in males. Results: Here we show that Aphrodisin-like proteins exist in other species, belong to a group of Odorant Binding Proteins (Obp), and contrary to the expression of Aphrodisin only in hamster genital tract and parotid glands of females, we have detected these transcripts in both sexes of M. glareolus with the expression confirmed in various tissues including prostate, prepucial and salivary glands, liver and uterus. On the level of mRNA, we have detected three different gene variants. To assess their relevance for chemical communication we investigated the occurrence of particular proteins in saliva, urine and vaginal discharge. On the protein level we confirmed the presence of Obp2 and Obp3 in both saliva and urine. Appropriate bands in the range of 17-20 kDa from vaginal discharge were, however, beyond the MS detection limits. Conclusion: Our results demonstrate that three novel Obps (Obp1, Obp2, and Obp3) are predominant lipocalins in Myodes urine and saliva. On the protein level we have detected further variants and thus we assume that similarly as Major Urinary Proteins in mice, these proteins may be important in chemical communication in this Cricetid rodent.

Mammalian tears are produced by lacrimal glands to protect eyes and may function in chemical communication and immunity. Recent studies on the house mouse chemical signalling revealed that major urinary proteins (MUPs) are not individually unique inMus musculus musculus. This fact stimulated us to look for other sexually dimorphic proteins that may—in combination with MUPs—contribute to a pool of chemical signals in tears. MUPs and other lipocalins including odorant binding proteins (OBPs) have the capacity to selectively transport volatile organic compounds (VOCs) in their eight-stranded beta barrel, thus we have generated the tear proteome of the house mouse to detect a wider pool of proteins that may be involved in chemical signalling. We have detected significant male-biased (7.8%) and female-biased (7%) proteins in tears. Those proteins that showed the most elevated sexual dimorphisms were highly expressed and belong to MUP, OBP, ESP (i.e., exocrine gland-secreted peptides), and SCGB/ABP (i.e., secretoglobin) families. Thus, tears may have the potential to elicit sex-specific signals in combination by different proteins. Some tear lipocalins are not sexually dimorphic—with MUP20/darcin and OBP6 being good examples—and because all proteins may flow with tears through nasolacrimal ducts to nasal and oral cavities we suggest that their roles are wider than originally thought. Also, we have also detected several sexually dimorphic bactericidal proteins, thus further supporting an idea that males and females may have adopted alternative strategies in controlling microbiota thus yielding different VOC profiles.