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.

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.

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.

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.

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.

Suppressive role of B cells in chronic colitis of T cell receptor alpha mutant mice.

The role of antibodies (Abs) in the development of chronic colitis in T cell receptor (TCR)-alpha-/- mice was explored by creating double mutant mice (TCR-alpha-/- x immunoglobulin (Ig)mu-/-), which lack B cells. TCR-alpha-/- x Ig mu-/- mice spontaneously developed colitis at an earlier age, and the colitis was more severe than in TCR-alpha-/- mice. Colitis was induced in recombination-activating gene-1 (RAG-1-/-) mice by the transfer of mesenteric lymph node (MLN) cells from TCR-alpha-/- x Ig mu-/- mice. When purified B cells from TCR-alpha-/- mice were mixed with MLN cells before cell transfer, colitis did not develop in RAG-1-/- mice. Administration of the purified Ig from TCR-alpha-/- mice and a mixture of monoclonal autoAbs reactive with colonic epithelial cells led to attenuation of colitis in TCR-alpha-/- x Ig mu-/- mice. Apoptotic cells were increased in the colon, MLN, and spleen of TCR-alpha-/- x Ig mu-/- mice as compared to Ig mu-/- mice and TCR-alpha-/- mice. Administration of the purified Ig from TCR-alpha-/- mice into TCR-alpha-/- x Ig mu-/- mice led to decrease in the number of apoptotic cells. These findings suggest that although B cells are not required for the initiation of colitis, B cells and Igs (autoAbs) can suppress colitis, presumably by affecting the clearance of apoptotic cells.

Disruption of the Bcl6 gene results in an impaired germinal center formation.

The Bcl6 gene has been identified from the chromosomal translocation breakpoint in B cell lymphomas, and its products are expressed highly in germinal center (GC) B cells. To investigate the function of Bcl6 in lymphocytes, we have generated RAG1-deficient mice reconstituted with bone marrow cells from Bcl6-deficient mice (Bcl6(-/-)RM). Lymphogenesis in primary lymphoid tissues of Bcl6(-/-)RM is normal, and Bcl6(-/-)RM produced control levels of primary IgG1 antibodies specific to T cell-dependent antigens. However, GCs were not found in these mice. This defect was mainly due to the abnormalities of B cells. Therefore, Bcl6 is essential for the differentiation of GC B cells.

Peripheral T cell survival requires continual ligation of the T cell receptor to major histocompatibility complex-encoded molecules.

In the thymus, T cells are selected according to their T cell receptor (TCR) specificity. After positive selection, mature cells are exported from primary lymphoid organs to seed the secondary lymphoid tissue. An important question is whether survival of mature T cells is an intrinsic property or requires continuous survival signals, i.e., engagement of the TCR by major histocompatibility complex (MHC) molecules in the periphery, perhaps in a similar way as occurring during thymic positive selection. To address this issue we used recombination-activating gene (Rag)-deficient H-2b mice expressing a transgenic TCR restricted by I-Ed class II MHC molecules. After engraftment with Rag-/- H-2d fetal thymi, CD4+8- peripheral T cells emerged. These cells were isolated and transferred into immunodeficient hosts of H-2b or H-2d haplotype, some of the latter being common cytokine receptor gamma chain deficient to exclude rejection of H-2b donor cells by host natural killer cells. Our results show that in the absence, but not in the presence, of selecting MHC molecules, peripheral mature T cells are short lived and disappear within 7 wk, indicating that continuous contact of the TCR with selecting MHC molecules is required for survival of T cells.

Spontaneous autoimmune diabetes in monoclonal T cell nonobese diabetic mice.

It has been established that insulin-dependent diabetes mellitus (IDDM) in nonobese diabetic (NOD) mice results from a CD4+ and CD8+ T cell-dependent autoimmune process directed against the pancreatic beta cells. The precise roles that beta cell-reactive CD8+ and CD4+ T cells play in the disease process, however, remain ill defined. Here we have investigated whether naive beta cell-specific CD8+ and CD4+ T cells can spontaneously accumulate in pancreatic islets, differentiate into effector cells, and destroy beta cells in the absence of other T cell specificities. This was done by introducing Kd- or I-Ag7-restricted beta cell-specific T cell receptor (TCR) transgenes that are highly diabetogenic in NOD mice (8.3- and 4.1-TCR, respectively), into recombination-activating gene (RAG)-2-deficient NOD mice, which cannot rearrange endogenous TCR genes and thus bear monoclonal TCR repertoires. We show that while RAG-2(-/-) 4.1-NOD mice, which only bear beta cell-specific CD4+ T cells, develop diabetes as early and as frequently as RAG-2+ 4.1-NOD mice, RAG-2(-/-) 8.3-NOD mice, which only bear beta cell-specific CD8+ T cells, develop diabetes less frequently and significantly later than RAG-2(+) 8.3-NOD mice. The monoclonal CD8+ T cells of RAG-2(-/-) 8.3-NOD mice mature properly, proliferate vigorously in response to antigenic stimulation in vitro, and can differentiate into beta cell-cytotoxic T cells in vivo, but do not efficiently accumulate in islets in the absence of a CD4+ T cell-derived signal, which can be provided by splenic CD4+ T cells from nontransgenic NOD mice. These results demonstrate that naive beta cell- specific CD8+ and CD4+ T cells can trigger diabetes in the absence of other T or B cell specificities, but suggest that efficient recruitment of naive diabetogenic beta cell-reactive CD8+ T cells to islets requires the assistance of beta cell-reactive CD4+ T cells.

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.

V(D)J recombination is regulated similarly in RAG-transfected fibroblasts and pre-B cells.

Here we compare the regulation of V(D)J recombination in the fibroblast cell line L4 and the pre-B cell line 38B9. The former has been rendered recombination-competent by stable transfection of a genomic fragment comprising the recombination activating genes, RAG-1 and RAG-2, along with some of their flanking sequences. We show that V(D)J recombination is similarly regulated in these two cell lines. Activating signals are transmitted through the protein kinase A (PKA) pathway, and inhibitory signals through the protein kinase C (PKC) and the calcium signalling pathways. In both cell lines, recombinational activity reflects steady state levels of mRNA transcribed from the RAG-1 and RAG-2 genes. This suggests that transcription of the RAG genes is a major determinant regulating V(D)J recombination. A comparison of RAG-1 and RAG-2 mRNA levels within each cell line reveals almost identical response patterns indicating that RAG-1 and RAG-2 transcription is coordinately regulated. Together, these results imply that the RAG-containing fragment in L4 fibroblasts carries most, if not all, control regions regulating the transcription of the RAG-1 and RAG-2 genes.

Comparative analysis of invertebrate Tc6 sequences that resemble the vertebrate V(D)J recombination signal sequences (RSS).

Invertebrate cells lack the p53 recombination checkpoint but contain mobile DNA sequences that transpose by a mechanism in part shared with excision of the V(D)J recombination signal sequences (RSS). In this work, inversion, deletion, and duplication of sequences associated with an invertebrate C. elegans Tc6 element is described. The structure of this C. elegans sequence and other dispersed Tc6 elements suggests that covalently closed 'hairpin' structures are not unique to excision of the V(D)J RSS by the RAG proteins, but rather can be generated by transposases at transposon termini leading to characteristic inversion and duplication events. Comparative analysis of recombination events at invertebrate sequences resembling the vertebrate V(D)J RSS may be useful in understanding V(D)J recombination-mediated recombination events in malignant vertebrate cells or genetic diseases such as ataxia telangectasia, in which the p53 recombination checkpoint is defective.

Serum starvation induced secondary V lambda J lambda rearrangement in a human plasma B cell line.

HB4C5 is a human antibody producing plasma B cell line that expresses the recombination activating gene-1 (RAG-1) and RAG-2 constitutively, but undergoes few secondary immunoglobulin gene rearrangements when cultured in fetal bovine serum-containing medium. Here, we found that depletion of serum from the culture media induces secondary VlambdaJlambda rearrangement in this cell line. To investigate the induction mechanism of secondary VlambdaJlambda rearrangement, we assessed the expression levels of RAG-1 and RAG-2 products, Vlambda germline transcription level and the amount of Vlambda signal broken ends (SBE) in HB4C5 cells cultured in serum-supplemented or serum-free medium. Western-blot analysis showed that the expression level for the RAG-1 and RAG-2 proteins was not affected by the serum depletion. Vlambda germline transcript was found to be constitutively expressed in HB4C5 cell line and this transcription level was not affected by the lack of serum. On the other hand, the amount of Vlambda SBE was shown to be increased in HB4C5 cells cultured in serum-free medium, suggesting that this increased formation of Vlambda SBE at least partly contributed to the enhanced occurrence of secondary VlambdaJlambda rearrangement in HB4C5 cells cultured in the serum-free condition. These results indicate that expression of RAG proteins and Vlambda germline transcription is not enough to undergo secondary VlambdaJlambda rearrangement in this cell line.

Omenn syndrome in the context of other B cell-negative severe combined immunodeficiencies.

Severe combined immunodeficiencies represent a heterogeneous group of hereditary defects of the immune system that affect both T and B cells and whose etiology has only recently begun to be understood. A portion of these SCID patients bear a defect in either of the two recombination-activating genes, Rag-1 or Rag-2, while others have mutations in a newly identified gene, Artemis. Omenn syndrome is an unusual severe immunodeficiency with T cells but no B cells, and peculiar features also due to a defect in Rag-1 or Rag-2 genes. All these three forms are characterized by an impairment of the VDJ recombination, the process that insures the somatic diversification of immunoglobulin and T cell receptor-encoding genes. Recent findings have enabled us to better understand the pathophysiology of these three immunodeficiencies, which affect the V(D)J recombination process to a different extent and in different ways.

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.

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.

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.