Multiple Lymphoid Organs Research Articles

JAK1 signaling in dendritic cells promotes peripheral tolerance in autoimmunity through PD-L1-mediated regulatory Tcell induction.

Dendritic cells (DCs) induce peripheral Tcell tolerance, but cell-intrinsic signaling cascades governing their stable tolerogenesis remain poorly defined. Janus Kinase 1 (JAK1) transduces cytokine-receptor signaling, and JAK inhibitors (Jakinibs), including JAK1-specific filgotinib, break inflammatory cycles in autoimmunity. Here, we report in heterogeneous DC populations of multiple secondary lymphoid organs that JAK1 promotes peripheral Tcell tolerance during experimental autoimmune encephalomyelitis (EAE). Mice harboring DC-specific JAK1 deletion exhibit elevated peripheral CD4+ Tcell expansion, less regulatory Tcells (Tregs), and worse EAE outcomes, whereas adoptive DC transfer ameliorates EAE pathogenesis by inducing peripheral Tregs, programmed cell death ligand 1 (PD-L1) dependently. This tolerogenic program is substantially reduced upon the transfer of JAK1-deficient DCs. DC-intrinsic IFN-γ-JAK1-STAT1 signaling induces PD-L1, which is required for DCs to convert CD4+ Tcells into Tregs invitro and attenuated upon JAK1 deficiency and filgotinib treatment. Thus, DC-intrinsic JAK1 promotes peripheral tolerance, suggesting potential unwarranted DC-mediated effects of Jakinibs in autoimmune diseases.

Receptor repertoires of murine follicular T helper cells reveal a high clonal overlap in separate lymph nodes in autoimmunity.

Follicular T helper cells (Tfh) are a specialized subset of CD4 effector T cells that are crucial for germinal center (GC) reactions and for selecting B cells to undergo affinity maturation. Despite this central role for humoral immunity, only few data exist about their clonal distribution when multiple lymphoid organs are exposed to the same antigen (Ag) as it is the case in autoimmunity. Here, we used an autoantibody-mediated disease model of the skin and injected one auto-Ag into the two footpads of the same mouse and analyzed the T cell receptor (TCR)β sequences of Tfh located in GCs of both contralateral draining lymph nodes. We found that over 90% of the dominant GC-Tfh clonotypes were shared in both lymph nodes but only transiently. The initially dominant Tfh clonotypes especially declined after establishment of chronic disease while GC reaction and autoimmune disease continued. Our data demonstrates a dynamic behavior of Tfh clonotypes under autoimmune conditions and emphasizes the importance of the time point for distinguishing auto-Ag-specific Tfh clonotypes from potential bystander activated ones.

Single-cell transcriptome analysis of the B-cell repertoire reveals the usage of immunoglobulins in the gray short-tailed opossum (Monodelphis domestica)

B-cells are key to humoral immunity, are found in multiple lymphoid organs, and have the unique ability to mediate the production of antigen-specific antibodies in the presence of pathogens. The marsupial immunoglobulin (Ig) heavy (H) chain locus encodes four constant region isotypes, IgA, IgG, IgM and IgE, but no IgD, and there are two light (L) chain isotypes, lambda (Igλ) and kappa (Igκ). To gain an understanding of the marsupial humoral immune system, B-cell transcriptomes generated by single-cell RNA sequencing from gray short-tailed opossum (Monodelphis domestica) splenocytes, and peripheral blood mononuclear cells were analysed. The cells used were from a single unimmunized animal and the majority of B-cells were transcribing IgM heavy chains. The ratio of Ig light chain use was roughly 2:1, Igλ:Igκ in this individual. This was not predicted due to Igκ being the more complex of the two L chain loci. The variable (V) gene segment pairs used in individual B-cells confirm greater diversity provided by the L chain V. This study is the first to report on using single cell analysis to investigate Ig repertoires in a marsupial and confirms a number of prior hypothesis, as well as revealing some surprises.

HIV-1 tracing method of systemic viremia in vivo using an artificially mutated virus pool.

The appearance of human immunodeficiency virus type 1 (HIV-1) plasma viremia is associated with progression to symptomatic disease and CD4+ T cell depletion. To locate the source of systemic viremia, this study employed a novel method to trace HIV-1 infection in vivo. We created JRCSFξnef, a pool of infectious HIV-1 (strain JR-CSF) with highly mutated nef gene regions by random mutagenesis PCR and infected this mutated virus pool into both Jurkat-CCR5 cells and hematopoietic stem cell-transplanted humanized mice. Infection resulted in systemic plasma viremia in humanized mice and viral RNA sequencing helped us to identify multiple lymphoid organs such as spleen, lymph nodes, and bone marrow but not peripheral blood cells as the source of systemic viremia. Our data suggest that this method could be useful for the tracing of viral trafficking in vivo.

Using gold nanoparticles for enhanced intradermal delivery of poorly soluble auto-antigenic peptides

Ultra-small 1-2 nm gold nanoparticles (NP) were conjugated with a poorly-soluble peptide auto-antigen, associated with type 1 diabetes, to modify the peptide pharmacokinetics, following its intradermal delivery. Peptide distribution was characterized, in vivo, after delivery using either conventional intradermal injection or a hollow microneedle device. The poorly-soluble peptide was effectively presented in distant lymph nodes (LN), spleen and draining LN when conjugated to the nanoparticles, whereas peptide alone was only presented in the draining LN. By contrast, nanoparticle conjugation to a highly-soluble peptide did not enhance in vivo distribution. Transfer of both free peptide and peptide-NPs from the skin to LN was reduced in mice lacking lymphoid homing receptor CCR7, suggesting that both are actively transported by migrating dendritic cells to LN. Collectively, these data demonstrate that intradermally administered ultra-small gold nanoparticles can widen the distribution of poorly-soluble auto-antigenic peptides to multiple lymphoid organs, thus enhancing their use as potential therapeutics.

Induction of Regulatory B Cells by Bacterial DNA in the Gut Microbiota at Early Age is Beneficial in Lupus-prone Mice

Abstract SLE is a complex autoimmune disorder with no known cure. We previously reported that oral vancomycin treatment of female MRL/lpr mice during active disease (9 to 15 weeks of age) could significantly ameliorate disease symptoms. However, when vancomycin treatment was initiated from an earlier age (from 3 to 15 weeks), the beneficial effect was not observed. Strikingly, mice given vancomycin during the pre-disease stage (from 3 to 8 weeks) exhibited exacerbated lupus disease. As vancomycin works by removing parts of the gut microbiota, we hypothesized that the regulatory immunity induced by gut microbiota at early age is essential in hammering lupus disease development in MRL/lpr mice. To test the hypothesis, we analyzed diverse regulatory cell types from the mice receiving vancomycin from 3 to 8 weeks of age. Regulatory B (Breg) cells, in particular, were found to be reduced in both the percentage and absolute number in multiple lymphoid organs. Importantly, adoptive transfer of Breg cells at 6–7 weeks of age to long-term vancomycin-treated mice improved lupus-like symptoms. This clearly indicates that Breg cells, inducible by vancomycin-sensitive gut microbiota, plays an important role in suppressing lupus disease initiation and progression. We next sought to determine Breg inducers in the gut microbiota. The serum level of bacterial DNA was found to be significantly lower in mice treated with vancomycin. More importantly, early administration of bacterial DNA reproduced the beneficial effect seen in the Breg adoptive transfer experiment. Together, these results suggest an important protective mechanism against lupus initiation that involves bacterial DNA in the gut microbiota and the induction of Breg cells.

Systemic and unconventional antigen presentation generates insulin autoantibodies targeting the islet of Langerhans

Abstract The presence of class-switched insulin autoantibodies (IAAs) is a prominent feature of patients with type 1 diabetes (T1D) and the non-obese diabetic (NOD) mice. Although IAAs are commonly used as a biomarker for T1D prognosis, their genesis and whether they play a pathogenic role is largely unknown. In this study, we developed a novel anti-insulin T cell and B cell receptor transgenic mouse model to investigate cognate interactions between insulin-reactive T and B lymphocytes, a process necessary for IAA production. Strikingly, dynamic anti-insulin T-B cell interactions, spontaneous germinal center formation, and active class-switch recombination occurred in multiple lymphoid organs, leading to remarkable IAA production. Naïve anti-insulin T cells differentiated into competent T follicular helper cells that produced critical factors for reversing B cell tolerance, promoting GC selection, and broadening the repertoire of insulin epitope presentation. Furthermore, the IAAs produced in the peripheral sites relocated into the islets of Langerhans, formed deposits on β-cells, and enhanced presentation of diabetogenic antigens by the resident islet macrophages. Overall, these results indicate that insulin presentation is disseminated throughout secondary lymphoid tissues, and is sufficient for generation of autoantibodies that precipitate previously unrecognized pathogenic outcomes in the target organ.

Of Human DC Migrants and Residents

Migration from peripheral tissues to lymph nodes is a key feature of dendritic cells (DCs), but little is known about the migration patterns of human DCs. By analyzing multiple lymphoid organs and tissues from the same donors, Granot etal. propose that the two main subsets of human DCs display different migratory capacity.

Identification of sea bass pIgR shows its interaction with vitellogenin inducing antibody-like activities in HEK 293T cells

Fish vitellogenin (Vg) has been shown to mediate the phagocytosis via interaction with a Fcγ-like phagocytic receptor on macrophages, but identification of such a receptor and its functional characterization remains lacking. In this study, we isolated a cDNA of polymeric immunoglobulin receptor (pIgR) from sea bass, which encoded a single-spanning transmembrane protein of 326 amino acids including a 21-amino acid signal peptide, an extracellular region, a transmembrane region and a 36-amino acid intracellular region included two Ig-like domains (ILDs), and was expressed in multiple lymphoid organs. We then showed that recombinant extracellular domain of sea bass pIgR was capable of binding to Vg as well as IgG and IgM. We also showed that Vg as well as IgG and IgM interacted with pIgR-expressing HEK 293T cells. Importantly, we demonstrated that Vg as well as IgG and IgM were all capable of enhancing phagocytosis by HEK 293T cells and inducing expression of tnf-α and il-1β, via interacting with pIgR. Collectively, these results suggest that fish Vg, analogous to IgG and IgM, can interact with pIgR and result in similar down-stream immune responses, providing an additional evidence that Vg plays an antibody-like role.

Abstract IA11: Emerging insights into stromal cell function inimmune homeostasis and inflammation

Abstract Stromal cells reside in all bodily organs and have long been viewed as chief structural determinants of tissue organization, function, and regeneration. It is becoming increasingly clear that lymphoid organs contain a developmentally related network of highly specialized myofibroblasts known as fibroblastic reticular cells (FRCs). Our understanding of lymph node and spleen FRC function in immune homeostasis and inflammatory disease is incomplete; however, emerging evidence suggests that these cells are of central importance in immunity and tolerance. Likewise, the precise impact of inflammation on stromal cell numbers, activation, gene expression, and function in situ remains unclear. Using genetic and molecular approaches that target specific subsets of stromal cells in vivo we have begun to examine the identity, plasticity, and immunological functions of FRCs in healthy, inflamed, and malignant tissues. We are systematically defining gene expression and molecular elements that control the activation of FRCs across multiple lymphoid and non-lymphoid organs. Additional studies examining the spatial contexture and molecular interactions between fibroblastic reticular cells and immunocytes have identified previously unknown and functionally significant cross-talk between these compartments in health and inflammatory disease settings. New findings from this work will be presented. Citation Format: Shannon Turley. Emerging insights into stromal cell function inimmune homeostasis and inflammation [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr IA11.

Systemic recognition of insulin by autoimmune T and B cells leads to production of antibodies targeting the islet of Langerhans

Abstract The presence of class-switched insulin autoantibodies (IAAs) is a consistent feature of patients with type 1 diabetes and non-obese diabetic mice. The mechanisms underlying their production and pathogenic role are poorly understood. IAA production is unexpected because insulin-reactive B cells are maintained in a tolerant state. Furthermore, the source of T cell help is limited because T cells recognizing insulin are deleted in the thymus, and those that escape thymic selection recognize insulin peptides but not native insulin. However, increased titers of IAAs to conformational insulin were found in a T cell receptor transgenic strain, 8F10, whose T cell reactivity is restricted to insulin peptides but not insulin protein. Furthermore, when an anti-insulin B cell receptor heavy chain transgene was introduced into the 8F10 mice, a marked increase in IAAs specific for native insulin was observed. Dynamic T-B cell interactions and spontaneous germinal center formation were observed in multiple lymphoid organs indicating widespread insulin presentation. But more pointedly we found that the germinal center B cells were activated and in contrast to naïve B cells, presented a broader repertoire of insulin epitopes. Finally, for the first time we show that IAAs localize in the islets of Langerhans, form deposits on β-cells, and enhance considerably the presentation of diabetogenic antigens. Thus, systemic recognition of a native circulating self-protein is sufficient for generation of autoantibodies that precipitate pathogenic outcomes in the target organ.

Is Dolphin Morbillivirus Virulent for White-Beaked Dolphins (Lagenorhynchus albirostris)?

The virulence of morbilliviruses for toothed whales (odontocetes) appears to differ according to host species. In 4 species of odontocetes, morbilliviruses are highly virulent, causing large-scale epizootics with high mortality. In 8 other species of odontocetes, including white-beaked dolphins (Lagenorhynchus albirostris), morbilliviruses have been found as an incidental infection. In these species, the virulence of morbilliviruses is not clear. Therefore, the admission of 2 white-beaked dolphins with morbillivirus infection into a rehabilitation center provided a unique opportunity to investigate the virulence of morbillivirus in this species. By phylogenetic analysis, the morbilliviruses in both animals were identified as a dolphin morbillivirus (DMV) most closely related to that detected in a white-beaked dolphin in Germany in 2007. Both animals were examined clinically and pathologically. Case No. 1 had a chronic neural DMV infection, characterized by polioencephalitis in the cerebrum and morbillivirus antigen expression limited to neurons and glial cells. Surprisingly, no nervous signs were observed in this animal during the 6 months before death. Case No. 2 had a subacute systemic DMV infection, characterized by interstitial pneumonia, leucopenia, lymphoid depletion, and DMV antigen expression in mononuclear cells and syncytia in the lung and in mononuclear cells in multiple lymphoid organs. Cause of death was not attributed to DMV infection in either animal. DMV was not detected in 2 contemporaneously stranded white-beaked dolphins. Stranding rate did not increase in the region. These results suggest that DMV is not highly virulent for white-beaked dolphins.

77 : The immune system cannot generate immunological memory during infection with the Lyme disease agent B. burgdorfer

In vertebrates including humans, mice and dogs, the bacteria Borrelia burgdorferi (Bb) causes a chronic, non-resolving infection known as Lyme disease, which requires antibiotic treatment to clear the bacteria. Re-infections are common in endemic regions. Similarly, mice can be re-infected with the same strain of Bb, implying a lack of functional immune responses. The mechanisms underlying this lack of effective short and long-term immunity to Bb are unknown. Using a mouse model of Bb-infection we show that infection with Bb produces strong T-dependent and T-independent serum antibodies, characterized by the unusual continued presence of IgM. Remarkably, both T-dependent and T-independent antibodies disappear rapidly when infection is controlled by antibiotic treatment and Bb-specific memory B cells could not be recovered. Thus, maintenance of Bb-specific humoral responses requires ongoing infections. Histological and flow cytometric examination of germinal centers, birthplaces of long-term humoral immunity, demonstrate their induction within 2 weeks of a primary infection and the presence of germinal center follicular helper T and B cells. However, the apparent normal induction of germinal centers is followed by their rapid and global collapse in multiple lymphoid organs by day 45. To determine whether the lack of memory formation is due to the nature of the Bb-antigens or is a sign of Bb-infection-mediated immune suppression, we vaccinated mice with influenza virus during an ongoing Bb-infection. Remarkably, in Bb infected mice the early antibody response to this unrelated antigen was skewed towards increased IgM production compared to that in non-infected mice, and influenza specific IgG responses were strongly reduced. Together our data demonstrate that Bb infection suppresses the development of long-lived antibody production and immunological memory formation and indicates that Bb may achieve this by suppressing the function and/or causing the rapid and global collapse of germinal centers. Supported by NIH AI073911 and T32 AI060555.

Iterative Germinal Center Re-Entries of Memory B-Cells with t(14;18) Translocation and Early Steps of Follicular Lymphoma Progression

AbstractAbstract 150The recent demonstration that memory B-cells can re-enter germinal centers (GCs) and participate to new rounds of GC reactions has opened the possibility that multi-hit B-cell lymphomagenesis could be a much more dynamic process than initially anticipated, gradually progressing throughout the successive passages of memory B-cells in GCs during a lifetime of successive immunological challenges. Here, we provide evidence for this scenario in follicular lymphoma (FL), a GC derived B-cell malignancy initiated in the bone marrow by the hallmark t(14;18) BCL2/IGH translocation. To address this issue, we engineered an original sporadic BCL2tracer mouse model mimicking the rare occurrence of t(14;18) translocation in humans through V(D)J recombination errors (1 in a million B-cells) allowing to track the resultant BCL2-expressing clones; and underwent a molecular/immunofluorescent tracking of t(14;18)+ clones vs. normal memory B-cells in paired lymphoid tissue samples (spleen, lymph nodes, bone marrow) from healthy individuals.We first show that contrary to the current dogma, ectopic BCL2 expression is not sufficient to provoke the FL’s characteristic differentiation arrest of activated B-cells as GC B-cells, thereby suggesting that differentiated BCL2+ memory B-cells must return to the GC to acquire additional oncogenic hits and “fix” in situ growth. Strikingly, we further find that in a small fraction of “healthy” humans, such differentiation arrest already operated, and that a clonally expanded population of t(14;18)+ cells with FL-like features have widely disseminated in blood and in multiple lymphoid organs (spleen, lymph nodes, bone marrow), with unprecedentedly reported frequencies (from 1/million to 1/500 cells in some individuals), shaping the systemic disease presentation observed in FL patients. Using molecular/immunofluorescent backtracking of such clones in various paired and remote lymphoid organs, we further demonstrate that t(14;18)+ clones systematically display an extensive history of AID (activation-induced cytidine deaminase)-mediated events compatible with iterative rounds of GC co-opting, in sharp contrast to single memory B-cell clones from the same individuals. We thus show that BCL2-expressing memory B-cells require multiple GC transits to acquire the distinctive FL-like maturation arrest as centrocyte/centroblasts and to progress to advanced FL precursor stages. Altogether, our data argue for a model of lymphomagenesis, in which progression from precursor stages to FL occurs asymptomatically over an extended period of time by subverting the dynamic and plastic attributes of memory B-cells. This understanding of the pre-clinical phases driving FL development in asymptomatic patients should help rationalize prospective approaches designed to identify biomarkers of risk, and innovative therapeutic targets present in early, potentially more curable phases of the disease. Disclosures:No relevant conflicts of interest to declare.

Experimental evidence of cell dissemination playing a role in pathogenesis of IgA nephropathy in multiple lymphoid organs

Since the pathogenesis of immunoglobulin A (IgA) nephropathy (IgAN) remains unclear, the rationale for current IgAN therapies is still obscure. Recent studies have shown that galactose-deficient IgA1 (GdIgA1) plays a critical role in the pathogenesis of IgAN and can be a non-invasive IgAN biomarker, although the origin of the pathogenic cells producing GdIgA1 is unknown. We examined the cell types and localization of pathogenic cells in IgAN-prone mice. We transplanted bone marrow (BM) or spleen cells with or without specific cell types from IgAN-prone mice, which have many features similar to human IgAN, to identify cell types responsible for the IgAN phenotype and to determine their localization. BM transplantation and whole spleen cell transfer from IgAN-prone mice reconstituted IgAN in normal and severe combined immunodeficiency mice. Depletion of CD90(+) spleen cells had no affect on reconstitution, whereas CD19(+) B cells from the spleen were sufficient to reconstitute IgAN in both recipients. These results indicate that CD19(+) B cells, which can regulate nephritogenic IgA production in a T-cell-independent manner, are responsible for the disease and are disseminated in peripheral lymphoid organs.

TLR-Induced Local Metabolism of Vitamin D3 Plays an Important Role in the Diversification of Adaptive Immune Responses

The addition of monophosphoryl lipid A, a minimally toxic derivative of LPS, to nonmucosally administered vaccines induced both systemic and mucosal immune responses to coadministered Ags. This was dependent on an up-regulated expression of 1alpha-hydroxylase (CYP27B1, 1alphaOHase), the enzyme that converts 25-hydroxycholecalciferol, a circulating inactive metabolite of vitamin D(3), into 1,25(OH)2D(3) (calcitriol). In response to locally produced calcitriol, myeloid dendritic cells (DCs) migrated from cutaneous vaccination sites into multiple secondary lymphoid organs, including classical inductive sites of mucosal immunity, where they effectively stimulated B and T cell immune responses. The endogenous production of calcitriol by monophosphoryl lipid A-stimulated DCs appeared to be Toll-IL-1R domain-containing adapter-inducing IFN-beta-dependent, mediated through a type 1 IFN-induced expression of 1alphaOHase. Responsiveness to calcitriol was essential to promote the trafficking of mobilized DCs to nondraining lymphoid organs. Collectively, these studies help to expand our understanding of the physiologically important roles played by locally metabolized vitamin D(3) in the initiation and diversification of adaptive immune responses. The influences of locally produced calcitriol on the migration of activated DCs from sites of vaccination/infection into both draining and nondraining lymphoid organs create a condition whereby Ag-responsive B and T cells residing in multiple lymphoid organs are able to simultaneously engage in the induction of adaptive immune responses to peripherally administered Ags as if they were responding to an infection of peripheral or mucosal tissues they were designed to protect.

Age-related enlargement of lymphoid tissue and altered leukocyte composition in serglycin-deficient mice

Serglycin (SG) is a proteoglycan that is located predominantly in the secretory granules of hematopoietic cells. Previous studies have established a crucial role for SG in promoting the storage of various secretory granule compounds that are of importance in the immune defense system. Here, we show that mice lacking SG spontaneously develop enlargement of multiple lymphoid organs, including the spleen, Peyer's patches (PP), and bronchus-associated lymphoid tissue. In the spleen, the lack of SG resulted in a significant decrease in the proportion of CD4(+) cells as well as an increase of the CD45RC(+) leukocyte population, indicating an expansion of naïve lymphocytes. In the PP, the lack of SG resulted in a general increase in cellularity, without significant alterations in the proportion of individual leukocyte populations. The enlargement of lymphoid tissues was not accompanied by increased serum levels of inflammatory cytokines. The number of mast cells in the peritoneum was not affected by the lack of SG, as judged by surface staining for CD117 (c-kit). However, the intensity of c-kit staining was reduced significantly in SG null animals. Moreover, the number of peritoneal macrophages, defined by morphological criteria and by CD11b staining, was decreased markedly in older, SG-deficient animals. Finally, experiments in which airway inflammation was induced by bacterial LPS revealed a more pronounced inflammatory response in old, SG-deficient as compared with wild-type mice. Taken together, our data show that SG deficiency causes multiple, age-related effects on the lymphoid system.

Beyond our pages

Beyond our pages

Regulation of T Cell Receptor-induced Activation of the Ras-ERK Pathway by Diacylglycerol Kinase ζ

T cell development in the thymus and activation of mature T cells in the periphery depend on signals stimulated by engagement of the T cell antigen receptor (TCR). Among the second messenger cascades initiated by TCR ligation include the phosphatidylinositol pathway where the membrane phospholipid, phosphatidylinositol 4,5-bisphosphate, is hydrolyzed to inositol 1,4,5-trisphosphate and diacylglycerol (DAG). Inositol 1,4,5-trisphosphate signals a rise in intracellular free calcium, leading to translocation of nuclear factor of activated T cells into the nucleus. DAG activates RasGRP and protein kinase C theta. Because both RasGRP and protein kinase C theta are essential for thymocyte and T cell function, it is critical to understand how DAG is regulated. In this report, we demonstrate expression of DAG kinase zeta (DGKzeta, the enzyme that catalyzes the conversion of DAG to phosphatidic acid) in multiple lymphoid organs, with highest expression observed within the T cell compartment. Overexpression studies in Jurkat T cells indicate that DGKzeta interferes with TCR-induced Ras and ERK activation, AP-1 induction, and expression of the activation marker CD69. In contrast, TCR-stimulated calcium influx is not altered. Mutational analysis indicates that the kinase and DAG binding domains, but not the ankyrin repeats of DGKzeta, are required for its inhibitory effects. Collectively these studies demonstrate a potential role of DGKzeta to function as a selective negative regulator of DAG signaling on T cell activation and provide the first structure/function analysis of this enzyme in T cells.