Salmonellae are gastrointestinal pathogens of man and animals. However, strains that are host-specific avian pathogens are often avirulent in mammals, and those which are nonspecific are commensal in poultry. The objective of this study was to determine whether host specificity was exhibited by bacterial abilities to invade epithelial cells or resist leukocyte killing. In this study, leukocytes isolated from humans and chickens were used to kill Salmonella in vitro. Both Salmonella pullorum, an avian-specific serotype, and Salmonella typhimurium, a broad-host-range serotype, were sensitive to killing by polymorphonuclear leukocytes isolated from both species. Both serotypes replicated in cells of the MQ-NCSU avian-macrophage cell line. In contrast, S. pullorum was noninvasive for cultured epithelial Henle 407, chick kidney, chick ovary, and budgerigar abdominal tumor cells. In the bird challenge, however, S. typhimurium rapidly caused inflammation of the intestinal mucosa, but S. pullorumpreferentially targeted the bursa of Fabricius prior to eliciting intestinal inflammation. Salmonella serotypes which cause typhoid fever in mice have been shown to target the gut-associated lymphoid tissue. Observations from this study show that S. pullorum initiated a route of infection in chicks comparable to the route it takes in cases of enteric fever.

Ly-6 proteins appear to serve cell adhesion and cell signaling function, but the precise role of Ly-6A.2 in CD4+ T lymphocytes is still unclear. Overexpression of Ly-6A.2 in T lymphocytes has allowed us to analyze the influence of elevated Ly-6A.2 expression on T cell function. In this study we report reduced proliferation of CD4+ T cells overexpressing Ly-6A.2 in response to a peptide Ag. Moreover, the Ly-6A.2-overexpressing CD4+ cells generated elevated levels of IL-4, a key factor that propels the differentiation of naive CD4+ T cells into Th2 subset. The hyporesponsiveness of Ly-6A.2 transgenic CD4+ T cells is dependent on the interaction of Ly-6A.2 T cells with the APCs and can be reversed by blocking the interaction between Ly-6A.2 and a recently reported candidate ligand. Overexpression of Ly-6A.2 in CD4+ T cells reduced their Ca2+ responses to TCR stimulation, therefore suggesting effects of Ly-6A.2 signaling on membrane proximal activation events. In contrast to the observed Ag-specific hyporesponsiveness, the Ly-6A.2 transgenic CD4+ T cells produced IL-4 independent of the interactions between Ly-6A.2 and the candidate Ly-6A.2 ligand. Our results suggest that 1) interaction of Ly-6A.2 with a candidate ligand regulates clonal expansion of CD4+ Th cells in response to an Ag (these results also provide further functional evidence for presence of Ly-6A.2 ligand on APC); and 2) Ly-6A.2 expression on CD4+ T cells promotes production of IL-4, a Th2 differentiation factor.

Stage-specific expression of a number of cell-surface and signaling proteins is critical for normal development of T cells in the thymus. Equally important may be the loss of expression/signaling of developmentally regulated proteins for proper transitioning of developing T cells into thymic subsets. Ly-6A.2 exhibits a regulated pattern of expression on T cells maturing in the thymus, and dysregulating its expression results in arrest of developing T cells within the CD3-CD4-CD8- triple negative (TN) stage where the normal expression of Ly-6A.2 is extinguished. To further characterize the mechanisms underlying this block, we examined whether cell signaling and/or cell adhesion properties of the Ly-6A.2 molecule influenced the block in T-cell development. Analysis of bone marrow chimeras generated by injecting CFSE-labeled Ly-6A.2 transgenic bone marrow cells into irradiated syngeneic non-transgenic mice revealed normal trafficking of developing T cells from the cortex into the medulla. Production of LAT but not p56lck was diminished in CD4-CD8- DN cells from Ly-6A.2 dysregulated mice when compared with control littermates. Dysregulated expression of Ly-6A.2 did not suppress endogenous TCR-Vbeta expression. Finally, dysregulated expression of Ly-6A.2 enhanced apoptosis of an immature CD4+CD8+ (DP) subset of developing cells and altered the selected TCR-Vbeta repertoire. Taken together, these observations indicate that the termination of Ly-6A.2 expression and signaling within the CD4-CD8-CD3- subset of developing T cells is an important checkpoint during normal thymic development.

25The immunosuppressive tumour microenvironment constitutes a significant hurdle to the 26 response to immune checkpoint inhibitors. Both soluble factors and specialised immune cells 27 such as regulatory T cells (TReg) are key components of active intratumoural 28 immunosuppression. Previous studies have shown that Inducible Co-Stimulatory receptor 29 (ICOS) is highly expressed in the tumour microenvironment, especially on TReg, suggesting that 30 it represents a relevant target for preferential depletion of these cells. Here, we used immune 31 profiling of samples from tumour bearing mice and cancer patients to characterise the 32 expression of ICOS in different tissues and solid tumours. By immunizing an Icos knockout 33 transgenic mouse line expressing antibodies with human variable domains, we selected a fully 34 human IgG1 antibody called KY1044 that binds ICOS from different species. Using KY1044, we 35 demonstrated that we can exploit the differential expression of ICOS on T cell subtypes to 36 modify the tumour microenvironment and thereby improve the anti-tumour immune 37 response. We showed that KY1044 induces sustained depletion of ICOS high TReg cells in mouse 38 tumours and depletion of ICOS high T cells in the blood of non-human primates, but was also 39 associated with secretion of pro-inflammatory cytokines from ICOS low TEFF cells. Altogether, 40 KY1044 improved the intratumoural TEFF:TReg ratio and increased activation of TEFF cells, 41 resulting in monotherapy efficacy or in synergistic combinatorial efficacy when administered 42 with the immune checkpoint blocker anti-PD-L1. In summary, our data demonstrate that 43 targeting ICOS with KY1044 can favourably alter the intratumoural immune contexture, 44 promoting an anti-tumour response. 45 46 48 targeting immune checkpoints (e.g. CTLA-4, PD-1 and PD-L1). These immune checkpoint 49 inhibitors (ICIs) are associated with strong and durable responses in patients suffering from 50 advanced malignancies, including but not limited to metastatic melanoma, non-small cell lung 51 cancer (NSCLC), head and neck cancer, renal and bladder cancer (1). However, within all these 52 indications, there are still a high proportion of patients who exhibit intrinsic or acquired 53 resistance to ICIs. These patients represent a population with high unmet medical needs who 54 may benefit from novel combinatory approaches with ICIs. 55 Multiple molecular and cellular mechanisms have been associated with the lack of response to 56 immunotherapies (2). Accumulating evidence has shown that a low incidence of cytotoxic T 57 cells and the presence of immunosuppressive cells represent major barriers to establishing a 58 response to ICIs. One such class of immunosuppressive cells are regulatory T cells (TReg), which 59 are characterised by the expression of the transcription factor FOXP3 and function by blocking 60 the activation and cytotoxic potential of effector T-cells (TEff) through multiple mechanisms (3, 61 4). In fact, numbers of intratumoural TReg cells neg...

The TCRs expressed on T lymphocytes recognize foreign peptides bound to MHC molecules. This reactivity is the basis of specific immune response to the foreign Ag. How such specificities are generated in the thymus is still being debated. Signals generated through TCR upon interaction with self MHC-peptide complexes are critical for maturation of the CD4+ helper and CD8+ cytotoxic subsets. We have observed maturation of CD4+ but not CD8+ T cells in Ly-6A.2 transgenic MHC null mice. Since there can be no interactions with MHC molecules in these mice, these CD4+ cells must express the T cell repertoire that exists before positive and negative selection. Interestingly, despite an absence of selection by MHC molecules, the CD4+ cells that mature recognize MHC molecules at a frequency as high as in CD4+ cells in normal mice. These results demonstrate that: 1) the germline sequences encoding TCRs are biased toward reactivity to MHC molecules; and 2) CD4+ cells as opposed to CD8+ cells have distinct lineage commitment signals. These results also suggest that signals originating from Ly-6 can promote or substitute for signals generated from TCR that are required for positive selection. Moreover, this animal model offers a system to study T cell development in the thymus that can provide insights into mechanisms of lineage commitment in developing T cells.