H-2 effects on cell-cell interactions in the response to single non-H-2 alloantigens. II. H-2 D region control of H-7.1-specific stimulator function in mixed lymphocyte culture and susceptibility to lysis by H-7.1- specific cytotoxic cells

The relative immunogenicity of the H-7.1 alloantigen has been shown in a previous communication to be regulated by a gene in the D region of the mouse major histocompatibility (H-2) complex. The level of relative immunogenicity was inferred from survival times of H-7.1-incompatible skin grafts donated by donors with different H-2 haplotype origins of H-2D region genes. In this communication we report the results of an extension of these previous investigations into the possible role of H-2D region genes in controlling the capacity of H-7.1-incompatible lymphocytes to stimulate H-7.1-speciflc mixed lymphocyte culture proliferation and generation of cytotoxic effector cells. The results reported herein demonstrate that the H-2D genotype of H-7.1-incompatible stimulator cells determines the relative H-7.1-specific capacity of those lymphocytes to stimulate H-7.1-specific proliferation of in vivo primed responder T cells in secondary mixed lymphocyte culture. H-2D(b)-bearing, H-7.l-incompatible stimulators were significantly more effective in stimulating H-7.1-specific proliferation than H-2D(d)-bearing stimulators. As expected, H-2D(b), H-7.1-in-compatible stimulators were also more effective than H-2D(d) a stimulators in generating H-7.1- specific cytotoxic effector cells. Further, the susceptibility of (51)Cr- labeled, H-7.1-incompatible lymphoblast targets to H-7.1-specific lysis was similarly regulated by an H-2D gene. Reciprocal H-2 restriction (F(1) cells are capable of killing only the cells bearing the immunizing cell parental H-2 haplotype) observed by other investigators for cytolysis of non-H-2-incompatible targets was not observed. H-2D a-bearing, H-7.1- incompatible stimulators stimulated generation of cytotoxic effectors capable of detectably lysing H-2D(b) but not H-2D(a)-bearing, H-7.1- incompatible targets. The impact of these observations on the proposed models for H-2 restriction of non-H-2 histocompatibility antigen-specific cytolysis is discussed

Functional similarities between pigeon \u27milk\u27 and mammalian milk : induction of immune gene expression and modification of the microbiota

Pigeon ‘milk’ and mammalian milk have functional similarities in terms of nutritional benefit and delivery of immunoglobulins to the young. Mammalian milk has been clearly shown to aid in the development of the immune system and microbiota of the young, but similar effects have not yet been attributed to pigeon ‘milk’. Therefore, using a chicken model, we investigated the effect of pigeon ‘milk’ on immune gene expression in the Gut Associated Lymphoid Tissue (GALT) and on the composition of the caecal microbiota. Chickens fed pigeon ‘milk’ had a faster rate of growth and a better feed conversion ratio than control chickens. There was significantly enhanced expression of immune-related gene pathways and interferon-stimulated genes in the GALT of pigeon ‘milk’-fed chickens. These pathways include the innate immune response, regulation of cytokine production and regulation of B cell activation and proliferation. The caecal microbiota of pigeon ‘milk’-fed chickens was significantly more diverse than control chickens, and appears to be affected by prebiotics in pigeon ‘milk’, as well as being directly seeded by bacteria present in pigeon ‘milk’. Our results demonstrate that pigeon ‘milk’ has further modes of action which make it functionally similar to mammalian milk. We hypothesise that pigeon ‘lactation’ and mammalian lactation evolved independently but resulted in similarly functional products

Significance of the six peptide-binding pockets of HLA-A2.1 in influenza A matrix peptide-specific cytotoxic T-lymphocyte reactivity.

To evaluate the roles of the six peptide-binding pockets of HLA-A2.1 in FMP-specific CTL recognition, we have constructed an extensive library of HMy2.C1R cell lines expressing mutant HLA-A2.1 molecules with different amino acid substitutions in each of the six pockets. These cell lines were tested for their ability to present synthetic FMP 58-66 to FMP-specific, HLA-A2.1-restricted human CTL lines. Six of 12 mutants with amino acid changes in pocket B significantly affect the FMP-specific CTL recognition, suggesting that pocket B plays a critical role in FMP-specific CTL recognition. Surprisingly, mutations in all other pockets, except for pocket F, also have significant effects on the CTL recognition. These results suggest that even the shallow pockets, which are likely to be less critical for peptide binding than the deep pockets, play a crucial role in FMP-specific CTL recognition

Allele-specific B pocket transplant in class I major histocompatibility complex protein changes requirement for anchor residue at P2 of peptide.

To investigate the role of an anchoring pocket in allele-specific peptide presentation by a major histocompatibility complex class I molecule, we "transplanted" a B pocket from HLA-A*0201 into HLA-B*2705 by site-directed mutagenesis. The resulting protein, designated B27.A2B, binds a different set of endogenous peptides than B*2705 as evidenced by complete loss of allorecognition as well as restored expression in the antigen processing-defective mutant cell line T2. B27.A2B also fails to present an HLA-B27-restricted influenza virus peptide [nucleoprotein (383-391)] to cytotoxic T lymphocytes (CTLs). However, substitution of leucine, the predominant P2 anchor residue in A*0201-restricted peptides, for arginine, the P2 anchor in nucleoprotein-(383-391) and other B*2705-restricted peptides, restores recognition of B27.A2B by the same B*2705-restricted peptide-specific CTLs. These results demonstrate that a dominant polymorphic pocket in a class I molecule, through interaction with the anchor residue of an antigenic peptide, can distinguish among peptides differing by only a single amino acid and thus determine the allelic specificity of peptide presentation

Differences in peptide presentation between B27 subtypes: the importance of the P1 side chain in maintaining high affinity peptide binding to B*2703.

Susceptibility to spondyloarthropathies is strongly associated with the MHC class I molecule HLA-B27, and is hypothesized to result from the presentation of arthritogenic peptides. Subtypes of B27 that differ structurally but are disease-associated ought to be capable of presenting such peptides, while nondisease-associated subtypes would not. We demonstrate that B*2703, the predominant West African B27 subtype that may not predispose to disease, is not recognized by most B*2705-alloreactive CTL, and does not efficiently present a known B*2705-restricted influenza A nucleoprotein (NP) peptide. We show inefficient presentation is due to a reduced binding affinity of B*2703 for the NP peptide. Furthermore, substituting Arg for the naturally occurring Ser at P1 of the NP peptide, restores high affinity binding and efficient presentation by B*2703. Our results suggest that B*2703 will bind and present efficiently only a subset of the peptides that bind to B*2705, in particular those with Arg or Lys at P1. The apparent lack of disease in individuals with B*2703 may be due to an inability to bind and present putative arthritogenic peptides

A cluster of mutations in HLA-A2 alpha 2 helix abolishes peptide recognition by T cells.

In order to investigate the regions of HLA-A2 that control peptide-specific cytotoxic T lymphocyte (CTL) recognition, 37 HLA-A2 genes coding for 50 point mutations that span the alpha 2 helix were synthesized by the technique of saturation mutagenesis. Twenty-nine of these genes, which code for 41 point mutations, were transfected into C1R cells and used as targets in cytotoxicity assays, in the presence of influenza-A matrix peptide 58-68 with specific CTL as effectors. All the transfectants were recognized fully by matrix peptide-specific CTL apart from those with amino acid substitutions at positions 152, 154, 155, 156, or 161, which led to a total loss of recognition and those with mutations at residue 27 or a double mutation at 138 and 150, which were recognized in an intermediate manner. The clustering of the crucial residues that emerges may reflect direct interaction of their side-chains with peptide or the CTL receptor

Evidence of widespread binding of HLA class I molecules to peptides.

We have tested the binding of HLA class I proteins to peptides using a solid-phase binding assay. We tested 102 peptides, mostly derived from the HIV gag and HIV pol sequences. Most peptides did not bind to any class I protein tested. The pattern of binding among the three class I proteins tested, HLA-A2, -B27, and -B8, was approximately 85% concordant. Further, all five of the known HIV-1 gag T cell epitopes detected by human CTL bound at least one class I protein. Binding of class I to the peptides could be detected either by directly iodinated class I proteins, or indirectly using monoclonal antibodies specific for class I. The binding to the plates could be blocked with MA2.1, which binds in the alpha 1 region of A2, but not by W6/32, which binds elsewhere. The data presented here show that binding of class I to peptides is specific, but that many peptides bind to more than a single class I protein

Analysis of mutant HLA-A2 molecules. Differential effects on peptide binding and CTL recognition.

Previous studies have identified several residues lining the groove of the HLA-A2.1 molecule that are critical for Ag presentation. However, it is not clear whether these residues are critical for binding of the peptide epitope per se or for determining the appropriate conformation of bound peptide. To distinguish between these possibilities, mutations at eight of these residues have been tested for their effects on the ability of the molecule to bind and present two known peptide epitopes--one derived from the influenza A matrix protein, the other from HIV pol. With only one exception, the mutations were found to affect the binding of the two peptides similarly. Most of the mutations resulted in intermediate deleterious effects on binding, with the B pocket mutant F9Y having the most dramatic negative effect on binding for both peptides. Two of the mutations significantly enhanced binding of both peptides and a peptide-specific effect on binding was seen with the substitution, Y99H, which enhanced binding of the matrix peptide yet diminished binding of the pol peptide. In contrast to the effects on binding, the effects of the mutations on presentation differed considerably for the two peptides. The most striking difference was seen with two alpha 2 alpha helix mutants that are fully recognized by pol peptide-specific CTL but not recognized by matrix peptide-specific CTL even though levels of binding were comparably diminished for the two peptides. These results suggest that some interactions, although not critical for binding per se, are critical for functional binding and the importance of these interactions differs among peptide epitopes

Different MHC class I alleles compete for presentation of overlapping viral epitopes.

We previously identified an HLA-B8+ donor, NW, whose lymphoblastoid cells failed to present a B8-restricted epitope from the influenza A nucleoprotein following viral infection, although added peptide could still be presented. The failure to present through HLA-B8 following viral infection appears to be specific for the NP epitope. Here, we report that donor NW makes an HLA-B2702-restricted influenza-specific CTL response to an epitope in the nucleoprotein that overlaps the B8-restricted epitope by 8 aa. Two mechanisms for the failure of this cell line to present the B8-restricted epitope following viral infection are investigated. One is that there is an antigen processing polymorphism specific to the NW cell line, so that there is either preferential generation or preferential transport of the B2702 epitope. The other is that B8 and B2702 compete for a common peptide fragment in the ER and this leads to suboptimal loading of HLA-B8

Lifelong CMV infection improves immune defense in old mice by broadening the mobilized TCR repertoire against third-party infection

Lifelong interactions between host and the ubiquitous and persistent cytomegalovirus (CMV) have been proposed to contribute to the age-related decline in immunity. Prior work from us and others found some support for that idea, yet evidence that this led to increased vulnerability to other infections was not obtained. Moreover, evidence has accumulated that CMV infection can be beneficial to immune defense in young/adult mice and humans, dominantly via enhanced innate immunity. Here, we describe an unexpected impact of murine CMV (MCMV) upon the T cell response of old mice to Listeria monocytogenes expressing the model antigen, OVA (Lm-OVA). Single-cell sequencing of the OVA-specific CD8 T cell receptor β (TCRβ) repertoire of old mice demonstrated that old MCMV-infected mice recruited many diverse clonotypes that afforded broad and often more efficient recognition of antigenic peptide variants. This stood in contrast to old control mice, which exhibited strong narrowing and homogenization of the elicited repertoire. High-throughput sequencing of the total naïve CD8 TCRβ repertoire showed that many of these diverse OVA-specific clonotypes were present in the naïve CD8 repertoire of mice in all groups (adult, old control, and old MCMV+) yet were only recruited into the Lm-OVA response in MCMV+ old mice. These results have profound implications for our understanding of T cell immunity over a life span and suggest that our coevolution with CMV may include surprising, potentially positive impacts on adaptive heterologous immunity in late life