Michael D. Stadnisky

Self MHC class I licensed NK cells enhance adaptive CD8 T cell viral immunity

MHC class I (MHC I) is essential to NK- and T-cell effector and surveillance functions. However, it is unknown whether MHC I polymorphism influences adaptive immunity through NK cells. Previously, we found that MHC I D(k), a cognate ligand for the Ly49G2 inhibitory receptor, was essential to NK control of murine (M)CMV infection. Here we assessed the significance of NK inhibitory receptor recognition of MCMV on CD8 T cells in genetically defined MHC I D(k) disparate mice. We observed that D(k)-licensed Ly49G2⁺ NK cells stabilized and then enhanced conventional dendritic cells (cDCs) recovery after infection. Furthermore, licensed NK support of cDC recovery was essential to enhance the tempo, magnitude, and effector activity of virus-specific CD8 T cells. Minimal cDC and CD8 T-cell number differences after low-dose MCMV in D(k) disparate animals further implied that licensed NK recognition of MCMV imparted qualitative cDC changes to enhance CD8 T-cell priming.

MHC class I Dk expression in hematopoietic and nonhematopoietic cells confers natural killer cell resistance to murine cytomegalovirus

NK cell-mediated murine cytomegalovirus (MCMV) resistance (Cmvr) is under H-2k control in MA/My mice, but the underlying gene(s) is unclear. Prior genetic analysis mapped Cmvr to the MHC class I (MHC-I) Dk gene interval. Because NK cell receptors are licensed by and responsive to MHC class I molecules, Dk itself is a candidate gene. A 10-kb genomic Dk fragment was subcloned and microinjected into MCMV-susceptible (Cmvs) (MA/My.L-H2b × C57L)F1 or (B6 × DBA/2)F2 embryos. Transgenic founders, which are competent for Dk expression and germline transgene transmission, were identified and further backcrossed to MA/My.L-H2b or C57L mice. Remarkably, Dk expression delivered NK-mediated resistance in either genetic background. Further, NK cells with cognate inhibitory Ly49G receptors for self-MHC-I Dk were licensed and critical in protection against MCMV infection. In radiation bone marrow chimeras, NK resistance was significantly diminished when MHC-I Dk expression was restricted to only hematopoietic or nonhematopoietic cells. Thus, MHC-I Dk is the H-2k-linked Cmvr locus; these findings suggest a role for NK cell interaction with Dk-bearing hematopoietic and nonhematopoietic cells to shape NK-mediated virus immunity.

MHC class I Dk locus and Ly49G2+ NK cells confer H-2k resistance to murine cytomegalovirus.

Essential NK cell-mediated murine CMV (MCMV) resistance is under histocompatibility-2k (H-2k) control in MA/My mice. We generated a panel of intra-H2k recombinant strains from congenic C57L.M-H2k/b (MCMV resistant) mice for precise genetic mapping of the critical interval. Recombination breakpoint sites were precisely mapped and MCMV resistance/susceptibility traits were determined for each of the new lines to identify the MHC locus. Strains C57L.M-H2k(R7) (MCMV resistant) and C57L.M-H2k(R2) (MCMV susceptible) are especially informative; we found that allelic variation in a 0.3-megabase interval in the class I D locus confers substantial difference in MCMV control phenotypes. When NK cell subsets responding to MCMV were examined, we found that Ly49G2+ NK cells rapidly expand and selectively acquire an enhanced capacity for cytolytic functions only in C57L.M-H2k(R7). We further show that depletion of Ly49G2+ NK cells before infection abrogated MCMV resistance in C57L.M-H2k(R7). We conclude that the MHC class I D locus prompts expansion and activation of Ly49G2+ NK cells that are needed in H-2k MCMV resistance.

Multiparametric Analysis of Host Response to Murine Cytomegalovirus in MHC Class I–Disparate Mice Reveals Primacy of Dk-Licensed Ly49G2+ NK Cells in Viral Control

MHC class I Dk and Ly49G2 (G2) inhibitory receptor–expressing NK cells are essential to murine CMV (MCMV) resistance in MA/My mice. Without Dk, G2+ NK cells in C57L mice fail to protect against MCMV infection. As a cognate ligand of G2, Dk licenses G2+ NK cells for effector activity. These data suggested that Dk-licensed G2+ NK cells might recognize and control MCMV infection. However, a role for licensed NK cells in viral immunity is uncertain. We combined classical genetics with flow cytometry to visualize the host response to MCMV. Immune cells collected from individuals of a diverse cohort of MA/My × C57L offspring segregating Dk were examined before infection and postinfection, including Ly49+ NK subsets, receptor expression features, and other phenotypic traits. To identify critical NK cell features, automated analysis of 110 traits was performed in R using the Pearson correlation, followed with a Bonferroni correction for multiple tests. Hierarchical clustering of trait associations and principal component analyses were used to discern shared immune response and genetic relationships. The results demonstrate that G2 expression on naive blood NK cells was predictive of MCMV resistance. However, rapid G2+ NK cell expansion following viral exposure occurred selectively in Dk offspring; this response was more highly correlated with MCMV control than all other immune cell features. We infer that Dk-licensed G2+ NK cells efficiently detected missing-self MHC cues on viral targets, which elicited cellular expansion and target cell killing. Therefore, MHC polymorphism regulates licensing and detection of viral targets by distinct subsets of NK cells required in innate viral control.

NK gene complex and chromosome 19 loci enhance MHC resistance to murine cytomegalovirus infection

An H-2k MHC locus is critical for murine cytomegalovirus (MCMV) resistance in MA/My mice and virus control is abolished if H-2k is replaced with H-2b MHC genes from MCMV-susceptible C57L mice. Yet, H-2k resistance varies with genetic background; thus, modifiers of virus resistance must exist. To identify non-MHC resistance loci, spleen and liver MCMV levels and genome-wide genotypes were assessed in (C57L × MA/My) and (MA/My × C57L) F2 offspring (representing 550 meioses). Significantly, a non-Mendelian frequency of MHC genotypes was observed for offspring of the latter cross. Quantitative trait loci (QTL) and their interaction potential in MCMV resistance were assessed in R/qtl; QTL on chromosomes 17, 6, and 19 affected MCMV levels in infected animals. A chromosome 6 QTL was linked with the NK gene complex and acted in an additive fashion with an H-2k MHC QTL to mitigate spleen MCMV levels. We provide biological confirmation that this chromosome 6 QTL provided MCMV control independent of H-2k via NK cells. Importantly, both chromosome 6 and 19 QTLs contribute to virus control independent of H-2k. Altogether, MHC and non-MHC MCMV-resistance QTL contribute in early resistance to MCMV infection in this genetic system.

Genomic Modifiers of Natural Killer Cells, Immune Responsiveness and Lymphoid Tissue Remodeling Together Increase Host Resistance to Viral Infection

The MHC class I Dk molecule supplies vital host resistance during murine cytomegalovirus (MCMV) infection. Natural killer (NK) cells expressing the Ly49G2 inhibitory receptor, which specifically binds Dk, are required to control viral spread. The extent of Dk-dependent host resistance, however, differs significantly amongst related strains of mice, C57L and MA/My. As a result, we predicted that relatively small-effect modifier genetic loci might together shape immune cell features, NK cell reactivity, and the host immune response to MCMV. A robust Dk-dependent genetic effect, however, has so far hindered attempts to identify additional host resistance factors. Thus, we applied genomic mapping strategies and multicolor flow cytometric analysis of immune cells in naive and virus-infected hosts to identify genetic modifiers of the host immune response to MCMV. We discovered and validated many quantitative trait loci (QTL); these were mapped to at least 19 positions on 16 chromosomes. Intriguingly, one newly discovered non-MHC locus (Cmv5) controlled splenic NK cell accrual, secondary lymphoid organ structure, and lymphoid follicle development during MCMV infection. We infer that Cmv5 aids host resistance to MCMV infection by expanding NK cells needed to preserve and protect essential tissue structural elements, to enhance lymphoid remodeling and to increase viral clearance in spleen.

Rapid discrimination of MHC class I and killer cell lectin-like receptor allele variants by high-resolution melt analysis

Ly49G and H-2 class I Dk molecules are critical to natural killer cell-mediated viral control. To examine their contributions in greater depth, we established NK gene complex (NKC)/Ly49 congenic strains and a novel genetic model defined by MHC class I Dk disparity in congenic and transgenic mouse strains. Generation and maintenance of Ly49 and H-2 class I select strains require efficient and reproducible genotyping assays for highly polygenic and polymorphic sequences. Thus, we coupled gene- and allele-specific PCR with high-resolution melt (HRM) analysis to discriminate Ly49g and H-2 class I D and K alleles in select strains and in the F2 and backcross hybrid offspring of different genetic crosses. We show that HRM typing for these critical immune response genes is fast, accurate, and dependable. We further demonstrate that H-2 class I D HRM typing is competent to detect and quantify transgene copy numbers in different mice with distinct genetic backgrounds. Our findings substantiate the utility and practicality of HRM genotyping for highly related genes and alleles, even those belonging to clustered multigene families. Based on these findings, we envision that HRM is capable to interrogate and quantify gene- and allele-specific variations due to differential regulation of gene expression.