Natural killer (NK) cells are a type of innate immune cell that can identify and eliminate viral infected cells and cancer cells. NK cells express an array of inhibitory and activating receptors such as natural cytotoxicity receptors, the mouse Ly49 or human KIR family, and NKR-P1 family. The integration of signals that NK cells receive through these receptors controls their activation and ability to kill target cells. Both Ly49 and KIR recognize MHC-I molecules on healthy cells Ly49:MHC-I engagement is essential for functional NK cell development. In the absence of these interactions NK cell is consider as ‘uneducated’ or ‘unlicensed’. Ly49 receptor interactions with MHC-I are critical in an effective NK cell response against cancer. However, the role of unlicensed NK cells in NK-mediated control of viruses is poorly understood. Using NKCKD mice, we sought to determine how the loss of Ly49:MHC-I education, and the concomitant loss of inhibition via MHC-I, affected survival against influenza infection. In this study, we show that Ly49-deficient mice exhibit lower viral load and greater protection than WT mice when infected with influenza. However, this protection was lost when Ly49I was transgenically restored to these mice. Similarly, MHC-I-deficient mice, that also lack educated NK cells, were resistant to influenza infection, and lost this protection when NK cells were depleted before challenge. Based on the markedly reduced inflammation in the Ly49-deficient mice compared to the WT, we conclude that the Ly49-deficient NK cells are swifter and more effective in clearing influenza, resulting in less viral burden and consequentially less need for a dangerously aggressive inflammatory response. Furthermore, influenza infection enhanced MHC-I expression on lung epithelial cells, which could be responsible for inhibition of NK cells. Consequently, blockade of inhibitory Ly49C/I receptors protected WT mice from lethal influenza infection. Additionally, Perforin-deficient NKCKD succumbed to the infection demonstrating that NK cell directly eliminate influenza-infected cells. Collectively, these results confirm that influenza is capable of inhibiting NK cells through MHC-I engagement of KIR/Ly49, and suggests that blocking this interaction may provide a viable therapeutic avenue for severe influenza cases. these results challenge our understanding of basic NK cell function and suggest that, rather than subdividing NK cells into ‘licensed’ and ‘unlicensed’ based on their expression of self-specific Ly49 receptors, a more accurate depiction of these NK subsets would be ‘cancer-specialized’ and ‘pathogen-specialized’. While further work is required to fully test this paradigm of cancer- and pathogen-specialized NK cells, I hope that my findings will stimulate a new appreciation for the role of NK cells in virus control, and lead to a better understanding of this critical immune cell.

Nucleosomes, the primary unit of chromatin structure, are positioned either statistically or specifically. The statistical positioning denotes the arbitrary positioning of nucleosomes on DNA agreeing with the nucleosome’s broad coverage of the genome—however, there is evidence that nucleosomes are also positioned specifically at controlled positions. DNA sequences determine the specific nucleosome positions, and the presence or depletion of nucleosomes affects the availability of the DNA region to other proteins. The DNA sequences of H2A and H2A.Z nucleosomes in Drosophila were analysed in search of nucleosome positioning patterns. Dinucleotide patterns with 10 bp periodicity were identified from the DNA sequences of H2A nucleosomes. Compared with the yeast patterns, the Drosophila patterns had the same periodicity but different dinucleotides near the dyad, which was related to the different H3 structure between them. The nucleosome positioning patterns from the H2A.Z nucleosomes implied the specific histone-DNA interaction as a result of the deviations of the patterns where the different amino acids of H2A and H2A.Z interact with the DNA. The Ly49 gene cluster was selected as a model system to study the interplay between nucleosomes and transcription factors. Ly49 proteins, the surface receptors on NK cells, display variegated expression patterns, and the bidirectional promoter Pro-1 is known as a key determinant of the stochastic expression of each Ly49 gene. The systematic analysis of nucleosome positions based on the genome sequences in the Ly49 gene cluster revealed that the repressing Pro-1 reverse promoters are open, while the activating forward Pro-1 promoters were covered by nucleosomes. Furthermore, specific nucleosome positions covered transcription factor binding sites. The covered factor binding sites were further examined by their periodic appearances on the nucleosome-covered sequences, which revealed the accessibility to the sites. The sequence analysis predicted that the regulation by the transcription factor AML-1 would be sensitive to the nucleosome coverage; the prediction was confirmed by cell line experiments. The 10 bp periodic nucleosome positioning patterns interact with histones specifically. The long nucleosome positioning patterns coexist with the short sequence motifs for transcription factor binding sites adding another layer of the control to the transcriptional regulation.

Impact of NKR-P1 polymorphism on Ly49 receptors expression in hybrid mouse strains (C57BL/6 x Balb/c, F10-12) Abstract Natural killer (NK) cells constitute the subpopulation of large granular lymphocytes which mediate spontaneous immune response against infected, transformed or allogeneic cells and thus represent an important component of the innate immunity. NK cells express a wide repertoir of surface receptors which can be either activating or inhibitory and which mediate NK cell recognition and regulation of cytolytic activity. NKR-P1 and Ly49 receptor families belong to the most important murine NK receptors. Both NKR-P1 and Ly49 families are members of C-type lectin-like superfamily of receptors encoded by natural killer gene complex (NKC) on chromosome 6 and include both activating and inhibitory members. The aim of this diploma thesis was to elucidate the impact of Nkr-p1c gene divergence on Ly49 receptors expression and to find out whether the Ly49 and Nkr-p1 gene clusters (which are localized on opposite ends of NKC) are inherited independently or whether the NKC domain is inherited as a complex. The second research interest was to illustrate the influence of the above mentioned divergence on cytotoxic activity of NK cells and tumor growth. In this study, inbred mouse strains C57BL/6 and Balb/c...