Using the emerging Collaborative Cross to probe the immune system

The Collaborative Cross (CC) is an emerging panel of recombinant inbred mouse strains. Each strain is genetically distinct but all descended from the same eight inbred founders. In 66 strains from incipient lines of the CC (pre-CC), as well as the 8 CC founders and some of their F1 offspring, we examined subsets of lymphocytes and antigen-presenting cells. We found significant variation among the founders, with even greater diversity in the pre-CC. Genome-wide association using inferred haplotypes detected highly significant loci controlling B-to-T cell ratio, CD8 T-cell numbers, CD11c and CD23 expression. Comparison of overall strain effects in the CC founders with strain effects at QTL in the pre-CC revealed sharp contrasts in the genetic architecture of two traits with significant loci: variation in CD23 can be explained largely by additive genetics at one locus, whereas variation in B-to-T ratio has a more complex etiology. For CD23, we found a strong QTL whose confidence interval contained the CD23 structural gene Fcer2a. Our data on the pre-CC demonstrate the utility of the CC for studying immunophenotypes and the value of integrating founder, CC, and F1 data. The extreme immunophenotypes observed could have pleiotropic effects in other CC experiments

CD4+ cytolytic effectors are inefficient in the clearance of Listeria monocytogenes

Cytotoxic T lymphocytes (CTL) recognize and lyse target cells through the interaction of the T-cell receptor complex with the class I or class II major histocompatibility complex (MHC). The production of class I-restricted CTL has been shown to be critical to the elimination of specific pathogens including . However, the function of class II-restricted CTL in the clearance of intracellular pathogens is poorly understood. H-2β-microglobulin-deficient mice (βM−/−) are not able to produce CD8 CTL in response to infection with . We used this model to evaluate the efficacy of class II-restricted CTL, in the absence of a class I-restricted response, during a primary infection with . We demonstrate that, despite their effectiveness in adoptive transfer of protection, -specific CD4 class II-restricted cytotoxic lymphocytes are ineffective in decreasing titres of in the spleen after an established infection. In βM−/− mice, persistence of in the spleen was found preferentially in class II-negative cells. Surprisingly, class I-restricted CTL from C57BL/6 mice were capable of decreasing bacterial titres during an established infection even in the absence of detectable class I on the surface of cells from βM−/− mice. These data strongly suggest that, in the absence of a class I-restricted response, pathogens that elicit a class II-restricted cytotoxic response may escape prompt eradication by the immune system

Widespread transcription of a Qa region gene in adult mice

The mouse MHC class I family includes genes encoded in four regions: H- 2K, H-2D, Qa and Tla. While K/D genes are well characterized, relatively little is known about Qa or Tla genes. We have studied the transcription of a B10.P Qa region gene. DNA sequence comparisons of the transmembrane region, supported by Southern blot analysis of cosmid and genomic DNAs from BALB/c and C57BL/10, demonstrate the lambda 3a gene corresponds to Q4p. In both Northern blots and RNA protection experiments using probes derived from the 3' noncoding region, we found that Q4, like the H-2K and H-2D genes, is widely transcribed in B10.P tissues. These data demonstrate for the first time widespread transcription of a Qa gene

Evidence for extensive polymorphism of class I genes in the rat major histocompatibility complex (RT1).

The major histocompatibility complex of the rat (RT1) has been poorly characterized with respect to the number, linkage, and polymorphism of class I genes. To estimate the number of class I RT1 genes and the relative extent of their polymorphism, we performed Southern blot analysis with liver DNA from rat strains expressing eight RT1 haplotypes. After digestion with EcoR1 and BamHI, the DNA was separated on agarose gels, blotted onto nitrocellulose, and hybridized with mouse H-2 cDNA probes, pH-2III and pH-2IIa. Ten to 20 EcoRI and 13 to 20 BamHI bands hybridized with pH-2III and pH-2IIa; restriction fragment length patterns were observed to be highly polymorphic. The restriction fragments associated with different RT1 haplotypes differed by 17-70%; this range is similar to the differences observed between mouse H-2 haplotypes. The same restriction fragment pattern was observed in DNA from three different rat strains sharing the same RT1 allele, confirming that the patterns were RT1-associated. Further, the RT11 and RT11v1 haplotypes, which differ at a single previously identified RT1-linked locus, were associated with EcoRI restriction pattern differences of 39-50%, confirming the supposition that RT1 class I genes identified by previous serological and T-cell-mediated assays have identified only a minority of the actual number of RT1-linked class I genes. In summary, the results reported in this communication demonstrate that the RT1 complex encompasses a large family of highly polymorphic class I genes similar to the H-2 and HL-A complexes of mouse and man

The Structural Basis for the Increased Immunogenicity of Two HIV-Reverse Transcriptase Peptide Variant/Class I Major Histocompatibility Complexes

Designing altered peptide ligands to generate specific immunological reactivity when bound to class I major histocompatibility complexes is important for both therapeutic and prophylactic reasons. We have previously shown that two altered peptides, derived from human immunodeficiency virus (HIV)-reverse transcriptase (RT) residues 309-317, are more immunogenic in vitro than the wild-type peptide. One peptide variant, I1Y, was able to stimulate RT-specific cytotoxic T cells from the blood of three HIV-infected individuals better than the wild-type RT peptide. Both I1Y and I1F peptide variants increase the cell surface half-life of the peptide-class I complex approximately 3-fold over that of the RT peptide but have different immunological activities. These peptides are candidates for the design of vaccines for HIV due to their increased immunogenicity. To understand the basis for the increased cell surface stability compared with wild-type peptide and to understand the differences in T cell recognition between I1Y and I1F, we determined the x-ray crystal structures of the two class I MHC-peptide complexes. These structures indicate that the increased cell surface half-life is due to pi-pi stacking interactions between Trp-167 of HLA-A2.1 and the aromatic P1 residues of I1F and I1Y. Comparison of the structures and modeling potential T cell receptor (TCR) interactions suggests that T cell interactions and immunogenicity are different between I1Y and I1F for two reasons. First, subtle changes in the steric and polar properties of the I1Y peptide affect TCR engagement. Second, water-mediated hydrogen bond interactions between the P1-Tyr and the P4-Glu peptide residues increase peptide side chain rigidity of residues critical for TCR engagement

Virus-specific, CD8+ major histocompatibility complex class I-restricted cytotoxic T lymphocytes in lymphocytic choriomeningitis virus-infected beta2-microglobulin-deficient mice.

Following infection with lymphocytic choriomeningitis virus (LCMV), normal adult mice generate virus-specific, major histocompatibility complex (MHC) class I-restricted cytotoxic T lymphocytes (CTL) which clear the virus after intraperitoneal infection or cause death following intracranial (i.c.) infection. We have investigated the response of beta2-microglobulin-deficient (beta2m-) mice of the H-2d haplotype (KOD mice) to LCMV infection. Unlike H-2b beta2m- mice, which generate CD4+ MHC class II-restricted CTL in response to LCMV, KOD mice generate high levels of CD8+ MHC class I-restricted, virus-specific CTL. These CTL are specific for the LCMV nucleoprotein epitope (residues 118 to 126) in association with the Ld class I molecule, analogous to the CTL response in wild-type mice. KOD mice are also susceptible to lethal LCM disease, with 75 to 80% of the mice dying 7 to 9 days following i.c. infection with virus. Similar to results with normal mice, lethal LCM disease in KOD mice is prevented by in vivo depletion of CD8+ T cells prior to i.c. infection. In contrast to wild-type mice, however, KOD mice cannot control LCMV and become persistently infected. Overall, these results demonstrate that beta2m is not an absolute requirement for presentation of endogenous antigen on Ld or for induction of virus-specific Ld-restricted CTL in vivo

Respiratory Francisella tularensis Live Vaccine Strain Infection Induces Th17 Cells and Prostaglandin E2, Which Inhibits Generation of Gamma Interferon-Positive T Cells

Two key routes of Francisella tularensis infection are through the skin and airway. We wished to understand how the route of inoculation influenced the primary acute adaptive immune response. We show that an intranasal inoculation of the F. tularensis live vaccine strain (LVS) with a 1,000-fold-smaller dose than an intradermal dose results in similar growth kinetics and peak bacterial burdens. In spite of similar bacterial burdens, we demonstrate a difference in the quality, magnitude, and kinetics of the primary acute T-cell response depending on the route of inoculation. Further, we show that prostaglandin E2 secretion in the lung is responsible for the difference in the gamma interferon (IFN-γ) response. Intradermal inoculation led to a large number of IFN-γ+ T cells 7 days after infection in both the spleen and the lung. In contrast, intranasal inoculation induced a lower number of IFN-γ+ T cells in the spleen and lung but an increased number of Th17 cells in the lung. Intranasal infection also led to a significant increase of prostaglandin E2 (PGE2) in the bronchoalveolar lavage fluid. Inhibition of PGE2 production with indomethacin treatment resulted in increased numbers of IFN-γ+ T cells and decreased bacteremia in the lungs of intranasally inoculated mice. This research illuminates critical differences in acute adaptive immune responses between inhalational and dermal infection with F. tularensis LVS mediated by the innate immune system and PGE2

A pharmacodynamic comparison of prasugrel vs. high-dose clopidogrel in patients with type 2 diabetes mellitus and coronary artery disease: results of the Optimizing anti-Platelet Therapy In diabetes MellitUS (OPTIMUS)-3 Trial

Aims: Patients with diabetes mellitus (DM) have increased platelet reactivity and reduced platelet response to clopidogrel compared with patients without DM. Prasugrel, a more potent antiplatelet agent, is associated with greater reductions in ischaemic events compared with clopidogrel, particularly in patients with DM. The aim of this study was to perform serial pharmacodynamic assessments of prasugrel with high-dose clopidogrel in patients with DM. Methods and results: Optimizing anti-Platelet Therapy In diabetes MellitUS (OPTIMUS)-3 was a prospective, randomized, double-blind, crossover study in patients with type 2 DM and coronary artery disease (CAD). Patients (n= 35) were randomly assigned to either prasugrel 60 mg loading dose (LD)/10 mg maintenance dose (MD) or clopidogrel 600 mg LD/150 mg MD over two 1-week treatment periods separated by a 2-week washout period. Platelet function was assessed by VerifyNow® P2Y12 assay, light transmission aggregometry, and vasodilator-stimulated phosphoprotein phosphorylation at 0, 1, 4, and 24 h and 7 days. Greater platelet inhibition by VerifyNow® P2Y12 was achieved by prasugrel compared with clopidogrel at 4 h post-LD (least squares mean, 89.3 vs. 27.7%, P< 0.0001; primary endpoint). The difference in platelet inhibition between prasugrel and clopidogrel was significant from 1 h through 7 days (P < 0.0001). Similar results were obtained using all other platelet function measures. Prasugrel resulted in fewer poor responders at all time points irrespective of definition used. Conclusion: In patients with type 2 DM and CAD, standard-dose prasugrel is associated with greater platelet inhibition and better response profiles during both the loading and maintenance periods when compared with double-dose clopidogrel

New family of exon-shuffled recombinant genes reveals extensive interdomain interactions in class I histocompatibility antigens and identifies residues involved

The specificities of an extensive panel of anti-H-2Dd monoclonal antibodies, which had been previously characterized using exon-shuffled H-2Dd/H-2Ld molecules and a number of anti-H-2DP antibodies, were examined using H-2Dd/H-2DP recombinants. The use of this new family of recombinant antigens revealed extensive interaction between the membrane-distal (N and Cl) domains of class I molecules. 20 out of 48 mAbs recognize complex epitopes formed by the interaction of these two domains. These antibodies exhibit a number of distinct patterns of crossreactivity with other class I proteins, revealing the presence of multiple epitopes within the region of domain interaction. Comparison of the data presented here with those from previous work allowed the identification of a small number of residues in the Cl domain that participate in the generation of complex epitopes involving both the N and Cl domains. The results are discussed in terms of the structural information available for these two domains