Tuning antiviral CD8 T-cell response via proline-altered peptide ligand vaccination.

Viral escape from CD8+ cytotoxic T lymphocyte responses correlates with disease progression and represents a significant challenge for vaccination. Here, we demonstrate that CD8+ T cell recognition of the naturally occurring MHC-I-restricted LCMV-associated immune escape variant Y4F is restored following vaccination with a proline-altered peptide ligand (APL). The APL increases MHC/peptide (pMHC) complex stability, rigidifies the peptide and facilitates T cell receptor (TCR) recognition through reduced entropy costs. Structural analyses of pMHC complexes before and after TCR binding, combined with biophysical analyses, revealed that although the TCR binds similarly to all complexes, the p3P modification alters the conformations of a very limited amount of specific MHC and peptide residues, facilitating efficient TCR recognition. This approach can be easily introduced in peptides restricted to other MHC alleles, and can be combined with currently available and future vaccination protocols in order to prevent viral immune escape.

Analyses of RAG1 and RAG2 genes suggest different evolutionary rates in the Cetacea lineage.

V(D)J recombination is a process of somatic recombination catalyzed by proteins encoded by RAG1 and RAG2 genes, both restricted to the genome of jawed vertebrates. Their proteins constitute the enzymatic core of V(D)J recombination machinery and are crucial for jawed vertebrate adaptive immunity. Mammals possess great ecological diversity, and their complex evolutionary history associated with radiation to different environments presented many distinct pathogenic challenges from these different habitats. Cetaceans comprise a mammalian order of fully aquatic mammals that have arisen from a complete terrestrial ancestor and, accordingly, was confronted with challenges from changing environmental pathogens while they transitioned from land to sea. In this study we undertook molecular evolutionary analyses of RAG1 and RAG2 genes, exploring the possible role of natural selection acting on these genes focusing on the cetacean lineage. We performed phylogenetic reconstructions on IQ-TREE, together with selection analyses in the codeml program of the PAML package, and in the FITMODEL program for codon evolution and switching on both the RAG1 and RAG2 genes. Our findings demonstrate that RAG1 and RAG2 remained fairly conserved among tetrapods, with purifying selection acting on both genes, with evidence for a few punctuated shifts in nucleotide substitution rates of both genes along tetrapod evolution. We demonstrate differential evolution in the closely linked genes RAG1 and RAG2 specifically in cetaceans.

Characterization of an N-Terminal Non-Core Domain of RAG1 Gene Disrupted Syrian Hamster Model Generated by CRISPR Cas9.

The accumulating evidence demonstrates that Syrian hamsters have advantages as models for various diseases. To develop a Syrian hamster (Mesocricetus auratus) model of human immunodeficiency caused by RAG1 gene mutations, we employed the CRISPR/Cas9 system and introduced an 86-nucleotide frameshift deletion in the hamster RAG1 gene encoding part of the N-terminal non-core domain of RAG1. Histological and immunohistochemical analyses demonstrated that these hamsters (referred herein as RAG1-86nt hamsters) had atrophic spleen and thymus, and developed significantly less white pulp and were almost completely devoid of splenic lymphoid follicles. The RAG1-nt86 hamsters had barely detectable CD3⁺ and CD4⁺ T cells. The expression of B and T lymphocyte-specific genes (CD3γ and CD4 for T cell-specific) and (CD22 and FCMR for B cell-specific) was dramatically reduced, whereas the expression of macrophage-specific (CD68) and natural killer (NK) cell-specific (CD94 and KLRG1) marker genes was increased in the spleen of RAG1-nt86 hamsters compared to wildtype hamsters. Interestingly, despite the impaired development of B and T lymphocytes, the RAG1-86nt hamsters still developed neutralizing antibodies against human adenovirus type C6 (HAdV-C6) upon intranasal infection and were capable of clearing the infectious viruses, albeit with slower kinetics. Therefore, the RAG1-86nt hamster reported herein (similar to the hypomorphic RAG1 mutations in humans that cause Omenn syndrome), may provide a useful model for studying the pathogenesis of the specific RAG1-mutation-induced human immunodeficiency, the host immune response to adenovirus infection and other pathogens as well as for evaluation of cell and gene therapies for treatment of this subset of RAG1 mutation patients.

Ectopic expression of Fas Ligand on cardiomyocytes renders cardiac allografts resistant to CD4(+) T-cell mediated rejection.

Fas Ligand limits inflammatory injury and permits allograft survival by inducing apoptosis of Fas-bearing lymphocytes. Previous studies have shown that the CD4(+) T-cell is both sufficient and required for murine cardiac allograft rejection. Here, utilizing a transgenic mouse that over-expresses Fas Ligand specifically on cardiomyocytes as heart donors, we sought to determine if Fas Ligand on graft parenchymal cells could resist CD4(+) T-cell mediated rejection. When transplanted into fully immunocompetent BALB/c recipients Fas Ligand transgenic hearts were acutely rejected. However, when transplanted into CD4(+) T-cell reconstituted BALB/c-rag(-/-) recipients, Fas Ligand hearts demonstrated long-term survival. These results indicate that Fas Ligand over-expression on cardiomyocytes can indeed resist CD4(+) T-cell mediated cardiac rejection and suggests contact dependence between Fas Ligand expressing graft parenchymal cells and the effector CD4(+) T-cells.

Pathogenic role of anti-M3 muscarinic acetylcholine receptor immune response in Sjögren's syndrome.

M3 muscarinic acetylcholine receptor (M3R) is expressed in exocrine glands (e.g., salivary glands [SGs] and lachrymal glands), and plays a crucial role in exocrine secretion. M3R reactive T cells have been detected in circulating mononuclear cells of 40% of patients with Sjögren's syndrome (SS), and the major T cell epitopes of M3R in those patients with HLA-DR B1×0901 are located in the second loop of M3R. Moreover, autoantibodies (autoAbs) against M3R are also present in sera of around 50% of patients with SS, and several B cell epitopes, such as N-region, 1st, 2nd, and 3rd loop of M3R, have been identified. Functional analysis using human SG cell lines showed that autoAbs against the 2nd loop of M3R suppressed intracellular Ca(2+) influx, suggesting inhibition of saliva secretion. To clarify whether the M3R reactive immune response induces autoimmune sialadenitis (AIS), M3R(-/-) mice were immunized with M3R synthetic peptides and their splenocytes transferred into Rag1(-/-) mice. The recipients developed severe sialadenitis, and cell transfer studies indicated that T cells are key factors in the pathogenesis of AIS. These results indicate that the M3R immune reaction plays a key pathogenic role in AIS, suggesting that M3R molecule acts as an autoantigen in the pathogenesis of SS.

NK cells prevalence, subsets and function in viral hepatitis C.

Innate immunity appears to play an important role in the pathogenesis of viral hepatitis C. Among various cell subsets of this immunity natural killer (NK) cells raised particular interest. These cells are abundant in liver, possess significant cytotoxic potential and show links with adaptive immunity. They play important role, particularly in the acute phase of viral infections, including hepatitis C. They exhibit various types of receptors, either inhibitory or activating, that are able to react with distinct ligands on infected cells. Homozygosity of some receptors, namely KIR2DL3 reacting with recipient HLA-C1 antigens is a herald of good prognosis in hepatitis C virus (HCV) infection. In the early stage of the latter, both the prevalence and the cytotoxicity of NK cells are increased. Their inhibitory receptors are down regulated whereas activating ones are up regulated. Interferon-γ secreted by NK56(+bright) NK cells has a direct cytotoxic effect on infected hepatocytes. In contrast, in the chronic phase of HCV liver disease both, the prevalence and function of NK cells are impaired. Nevertheless, their cytotoxicity contributes to liver injury. Cells show change in the polarization profile from NK1 to NK2, manifested by secretion of immunosuppressive cytokines. Some HCV peptides are inhibitory for NK cells leading to the reduction of their antiviral activity. The unwanted effects of HCV peptides can be at least partly reversed by the antiviral therapy.

Bcl-2-interacting mediator of cell death influences autoantigen-driven deletion and TCR revision.

Peripheral CD4(+)Vß5(+) T cells are tolerized to an endogenous mouse mammary tumor virus superantigen either by deletion or TCR revision. Through TCR revision, RAG reexpression mediates extrathymic TCRß rearrangement and results in a population of postrevision CD4(+)Vß5(-) T cells expressing revised TCRß chains. We have hypothesized that cell death pathways regulate the selection of cells undergoing TCR revision to ensure the safety and utility of the postrevision population. In this study, we investigate the role of Bcl-2-interacting mediator of cell death (Bim)-mediated cell death in autoantigen-driven deletion and TCR revision. Bim deficiency and Bcl-2 overexpression in Vß5 transgenic (Tg) mice both impair peripheral deletion. Vß5 Tg Bim-deficient and Bcl-2 Tg mice exhibit an elevated frequency of CD4(+) T cells expressing both the transgene-encoded Vß5 chain and a revised TCRß chain. We now show that these dual-TCR-expressing cells are TCR revision intermediates and that the population of RAG-expressing, revising CD4(+) T cells is increased in Bim-deficient Vß5 Tg mice. These findings support a role for Bim and Bcl-2 in regulating the balance of survival versus apoptosis in peripheral T cells undergoing RAG-dependent TCR rearrangements during TCR revision, thereby ensuring the utility of the postrevision repertoire.

The MAPK/ERK and PI3K pathways additively coordinate the transcription of recombination-activating genes in B lineage cells.

Rag-1 and Rag-2 are essential for the construction of the BCR repertoire. Regulation of Rag gene expression is tightly linked with BCR expression and signaling during B cell development. Earlier studies have shown a major role of the PI(3)K/Akt pathway in regulating the transcription of Rag genes. In this study, by using the 38c13 murine B cell lymphoma we show that transcription of Rag genes is also regulated by the MEK/ERK pathways, and that both pathways additively coordinate in this regulation. The additive effect is observed for both ligand-dependent (upon BCR ligation) and ligand independent (tonic) signals. However, whereas the PI(3)K/Akt regulation of Rag transcription is mediated by Foxo1, we show in this study that the MEK/ERK pathway coordinates with the regulation of Rag by controlling the phosphorylation and turnover of E47 and its consequential binding to the Rag enhancer regions. Our results suggest that the PI(3)K and MEK/ERK pathways additively coordinate in the regulation of Rag transcription in an independent manner.

B cell receptor signaling: picky about PI3Ks.

The B cell receptor (BCR) and the pre-BCR control cell fate at many stages of B cell development, survival, and antigen response. Most of these processes require the activation of phosphatidylinositol 3-kinase (PI3K). Previous work has pointed to p110delta as the key catalytic isoform of PI3K for many B cell responses. A study of mice with different combinations of PI3K mutations confirms the central role of p110delta in agonist-mediated signaling, while identifying an unexpected function for the p110alpha isoform in tonic signaling by the pre-BCR and mature BCR.

Safety of probiotic Escherichia coli strain Nissle 1917 depends on intestinal microbiota and adaptive immunity of the host.

Probiotics are viable microorganisms that are increasingly used for treatment of a variety of diseases. Occasionally, however, probiotics may have adverse clinical effects, including septicemia. Here we examined the role of the intestinal microbiota and the adaptive immune system in preventing translocation of probiotics (e.g., Escherichia coli Nissle). We challenged C57BL/6J mice raised under germfree conditions (GF-raised C57BL/6J mice) and Rag1(-/-) mice raised under germfree conditions (GF-raised Rag1(-/-) mice) and under specific-pathogen-free conditions (SPF-raised Rag1(-/-) mice) with probiotic E. coli strain Nissle 1917, strain Nissle 1917 mutants, the commensal strain E. coli mpk, or Bacteroides vulgatus mpk. Additionally, we reconstituted Rag1(-/-) mice with CD4(+) T cells. E. coli translocation and dissemination and the mortality of mice were assessed. In GF-raised Rag1(-/-) mice, but not in SPF-raised Rag1(-/-) mice or GF-raised C57BL/6J mice, oral challenge with E. coli strain Nissle 1917, but not oral challenge with E. coli mpk, resulted in translocation and dissemination. The mortality rate was significantly higher for E. coli strain Nissle 1917-challenged GF-raised Rag1(-/-) mice (100%; P < 0.001) than for E. coli strain Nissle 1917-challenged SPF-raised Rag1(-/-) mice (0%) and GF-raised C57BL/6J mice (0%). Translocation of and mortality due to strain E. coli Nissle 1917 in GF-raised Rag1(-/-) mice were prevented when mice were reconstituted with T cells prior to strain E. coli Nissle 1917 challenge, but not when mice were reconstituted with T cells after E. coli strain Nissle 1917 challenge. Cocolonization experiments revealed that E. coli mpk could not prevent translocation of strain E. coli Nissle 1917. Moreover, we demonstrated that neither lipopolysaccharide structure nor flagella play a role in E. coli strain Nissle 1917 translocation and dissemination. Our results suggest that if both the microbiota and adaptive immunity are defective, translocation across the intestinal epithelium and dissemination of the probiotic E. coli strain Nissle 1917 may occur and have potentially severe adverse effects. Future work should define the possibly related molecular factors that promote probiotic functions, fitness, and facultative pathogenicity.

B-cell development in bovine fetuses proceeds via a pre-B like cell in bone marrow and lymph nodes.

The production of B cells and the primary antibody repertoire in mammalian species other than rodents or man appears to depend on gut-associated lymphoid tissue. Bovine B cells are generated in ileal Peyer's patch from late gestational to juvenile age. However, little is known about where and when the bona fide B lymphopoiesis takes place. We analyzed bovine fetuses for signs of ongoing B lymphopoiesis using a combination of immunohistochemistry, flow cytometry, real-time quantitative PCR and RNA in situ hybridization. In fetal bone marrow and lymph node, we could demonstrate pre-B like cells positive for intracellular Ig mu but negative for membrane IgM. Strong expression of immunoglobulin lambda-like polypeptide 1 and recombination activating genes was also detected in the same tissues. Similar analyses did not reveal pre-B like cells in the corresponding adult tissues. These results suggest that bovine fetal bone marrow and lymph node support B lymphopoiesis via a pre-B cell like stage before and in parallel to the development of the ileal Peyer's patch.

GvHD-associated cytokine polymorphisms do not associate with Omenn syndrome rather than T-B- SCID in patients with defects in RAG genes.

Recombinase activating genes 1/2 (RAG1/2) deficiency, critical to initiate gene rearrangement encoding lymphocyte receptors, causes T-B- severe combined immunodeficiency (SCID) and Omenn syndrome (OS), characterised by erythroderma, hepatosplenomegaly, lymphadenopathy, activated, clonal T cell expansions with restricted TCRVbeta family usage, and opportunistic infection. Many features of OS resemble graft-versus-host disease (GvHD). Frequency of GvHD-associated cytokine gene polymorphisms (CGPs) with OS was investigated to explain phenotypic differences between T-B- SCID and OS. Allele frequencies of IFNgamma T874A, IFNgamma-R1, TNFalphad microsatellites, IL-10 promoter region C592A and A1082G, IL-4 C-590T, IL-6 G-174C, IL-4R Q+576R, IFNgamma-R1 T-56C, TNFalphaRII 196 M/R single-nucleotide polymorphisms and IL-1Ra intron 1 VNTR were examined in 33 OS and 23 SCID patients. No significant differences in allele frequencies were found between the groups, and no trends identified. The mechanisms determining the OS or T-B-NK+ SCID phenotype remain to be determined.

Classical complement activation and acquired immune response pathways are not essential for retinal degeneration in the rd1 mouse.

Misregulation of the innate immune response and other immune-related processes have been suggested to play a critical role in the pathogenesis of a number of different neurodegenerative diseases, including age related macular degeneration. In an animal model for photoreceptor degeneration, several genes of the innate and acquired immune system were found to be differentially regulated in the retina during the degenerative process. In addition to this differential regulation of individual genes, we found that in the rd1 retina a significantly higher number of genes involved in immune-related responses were expressed at any given time during the degenerative period. The peak of immune-related gene expression was at postnatal day 14, coinciding with the peak of photoreceptor apoptosis in the rd1 mouse. We directly tested the potential involvement of acquired and innate immune responses in initiation and progression of photoreceptor degeneration by analyzing double mutant animals. Retinal morphology and photoreceptor apoptosis of rd1 mice on a SCID genetic background (no mature T- and B-cells) or in combination with a RAG1 (no functional B- and T-cells) or a C1qalpha (no functional classical complement activation pathway) knockout was followed during the degenerative process using light microscopy or TUNEL staining, respectively. Although complement factor C1qalpha was highly up-regulated in the rd1 retina concomitantly with the degenerative process, lack of this protein did not protect the rd1 retina. Similarly, retinal degeneration and photoreceptor apoptosis appeared to proceed normally in the rd1 mouse lacking functional B- and T-cells. Our results suggest that both, the classical complement system of innate immunity and a functional acquired immune response are not essential for the degenerative process in the rd1 mouse retina.

T-cell control of IL-12p75 production.

It is currently thought that IL-12, produced by dendritic cells (DC) early after stimulation by bacterial pathogens or lipopolysaccharide (LPS), acts as a pro-inflammatory cytokine bridging the innate and adaptive immune responses. We found, however, that it is only the p40 subunit and not the IL-12p75 heterodimer that is secreted early in copious amounts in response to LPS. Neither naïve T cells, nor a variety of microbial products, were able to induce IL-12p75 production unless the DC were conditioned by the presence of interferon-gamma (IFN-gamma) or by encounter with previously activated T cells. The inability of naïve T cells or of bacterial products to induce IL-12 argues against its early role as the initiator of innate and adaptive immune responses.

Foxp1 is an essential transcriptional regulator of B cell development.

Forkhead transcription factors are key participants in development and immune regulation. Here we demonstrate that absence of the gene encoding the forkhead transcription factor Foxp1 resulted in a profound defect in early B cell development. Foxp1 deficiency was associated with decreased expression of all B lineage genes in B220+ fetal liver cells as well as with a block in the transition from pro-B cell to pre-B cell involving diminished expression of recombination-activating genes 1 and 2. Foxp1 bound to the Erag enhancer and was involved in controlling variable-(diversity)-joining recombination of the gene encoding immunoglobulin heavy chain in a B cell lineage-specific way. Our results identify Foxp1 as an essential participant in the transcriptional regulatory network of B lymphopoiesis.

Novel animal models for Sjögren's syndrome: expression and transfer of salivary gland dysfunction from regulatory T cell-deficient mice.

IL-2 knockout (KO), IL-2Ralpha KO and scurfy mice lack the CD4+CD25+ regulatory T (Treg) cells and develop severe inflammation in multiple organs, although organs affected vary among these strains. We asked if salivary and lacrimal glands, the main organs affected in Sjögren's syndrome, are targeted in these strains. Severe lymphocyte and neutrophil infiltration in the salivary and lacrimal glands and a decrease in salivary secretory function were observed in IL-2 KO and IL-2Ralpha KO mice, but not in scurfy mice. Interestingly, transfer of lymph node cells from scurfy mice to RAG-1 KO recipients rapidly and effectively induced inflammation and loss of function in the salivary glands. Furthermore, we observed that daily LPS feeding in scurfy mice also induced inflammation in the salivary glands. Our study demonstrates several novel models for Sjögren's syndrome, including an adoptive transfer model that shows that scurfy mice have dormant salivary gland-specific autoreactive lymphocytes that can be activated by certain environmental factors, such as those present in RAG-1 KO mice.

Reconstructing immune phylogeny: new perspectives.

Numerous studies of the mammalian immune system have begun to uncover profound interrelationships, as well as fundamental differences, between the adaptive and innate systems of immune recognition. Coincident with these investigations, the increasing experimental accessibility of non-mammalian jawed vertebrates, jawless vertebrates, protochordates and invertebrates has provided intriguing new information regarding the likely patterns of emergence of immune-related molecules during metazoan phylogeny, as well as the evolution of alternative mechanisms for receptor diversification. Such findings blur traditional distinctions between adaptive and innate immunity and emphasize that, throughout evolution, the immune system has used a remarkably extensive variety of solutions to meet fundamentally similar requirements for host protection.

Ontogeny and regulation of IL-7-expressing thymic epithelial cells.

Epithelial cells in the thymus produce IL-7, an essential cytokine that promotes the survival, differentiation, and proliferation of thymocytes. We identified IL-7-expressing thymic epithelial cells (TECs) throughout ontogeny and in the adult mouse thymus by in situ hybridization analysis. IL-7 expression is initiated in the thymic fated domain of the early primordium by embryonic day 11.5 and is expressed in a Foxn1-independent pathway. Marked changes occur in the localization and regulation of IL-7-expressing TECs during development. IL-7-expressing TECs are present throughout the early thymic rudiment. In contrast, a major population of IL-7-expressing TECs is localized to the medulla in the adult thymus. Using mouse strains in which thymocyte development is arrested at various stages, we show that fetal and postnatal thymi differ in the frequency and localization of IL-7-expressing TECs. Whereas IL-7 expression is initiated independently of hemopoietic-derived signals during thymic organogenesis, thymocyte-derived signals play an essential role in regulating IL-7 expression in the adult TEC compartment. Moreover, different thymocyte subsets regulate the expression of IL-7 and keratin 5 in adult cortical epithelium, suggesting that despite phenotypic similarities, the cortical TEC compartments of wild-type and RAG-1(-/-) mice are developmentally and functionally distinct.

T cell-derived tumour necrosis factor is essential, but not sufficient, for protection against Mycobacterium tuberculosis infection.

Tumour necrosis factor (TNF) is critical for sustained protective immunity against Mycobacterium tuberculosis infection. To investigate the relative contributions of macrophage- and T cell-derived TNF towards this immunity T cells from wild-type (WT) or TNF-/- mice were transferred into RAG-/- or TNF-/- mice which were then infected with M. tuberculosis. Infected RAG-/- mice and RAG-/- recipients of TNF deficient T cells developed overwhelming infection, with extensive pulmonary and hepatic necrosis and succumbed with a median of only 16 days infection. By contrast, RAG-/- recipients of WT T cells showed a significant increase in survival with a median of 32 days. Although initial bacterial growth was similar in all groups of RAG-/- mice, the transfer of WT, but not TNF-/-, T cells led to the formation of discrete foci of leucocytes and macrophages and delayed the development of necrotizing pathology. To determine requirements for macrophage-derived TNF, WT or TNF-/- T cells were transferred into TNF-/- mice at the time of M. tuberculosis infection. Transfer of WT T cells significantly prolonged survival and reduced the early tissue necrosis evident in the TNF-/- mice, however, these mice eventually succumbed indicating that T cell-derived TNF alone is insufficient to control the infection. Therefore, both T cell- and macrophage-derived TNF play distinct roles in orchestrating the protective inflammatory response and enhancing survival during M. tuberculosis infection.