Lymph Node Germinal Centers Research Articles

IL-21 enhances influenza vaccine responses in aged macaques with suppressed SIV infection.

Natural aging and HIV infection are associated with chronic low-grade systemic inflammation, immune senescence, and impaired antibody responses to vaccines such as the influenza (flu) vaccine. We investigated the role of IL-21, a CD4+ T follicular helper cell (Tfh) regulator, on flu vaccine antibody response in nonhuman primates (NHPs) in the context of age and controlled SIV mac239 infection. Three doses of the flu vaccine with or without IL-21–IgFc were administered at 3-month intervals in aged SIV+ NHPs following virus suppression with antiretroviral therapy. IL-21–treated animals demonstrated higher day 14–postboost antibody responses, which associated with expanded CD4+ T central memory cells and peripheral Tfh–expressing (pTfh–expressing) T cell immunoreceptor with Ig and ITIM domains (TIGIT), expanded activated memory B cells, and contracted CD11b+ monocytes. Draining lymph node (LN) tissue from IL-21–treated animals revealed direct association between LN follicle Tfh density and frequency of circulating TIGIT+ pTfh cells. We conclude that IL-21 enhances flu vaccine–induced antibody responses in SIV+ aged rhesus macaques (RMs), acting as an adjuvant modulating LN germinal center activity. A strategy to supplement IL-21 in aging could be a valuable addition in the toolbox for improving vaccine responses in an aging HIV+ population.

Nano-Enabled COVID-19 Vaccines: Meeting the Challenges of Durable Antibody Plus Cellular Immunity and Immune Escape.

At the time of preparing this Perspective, large-scale vaccination for COVID-19 is in progress, aiming to bring the pandemic under control through vaccine-induced herd immunity. Not only does this vaccination effort represent an unprecedented scientific and technological breakthrough, moving us from the rapid analysis of viral genomes to design, manufacture, clinical trial testing, and use authorization within the time frame of less than a year, but it also highlights rapid progress in the implementation of nanotechnology to assist vaccine development. These advances enable us to deliver nucleic acid and conformation-stabilized subunit vaccines to regional lymph nodes, with the ability to trigger effective humoral and cellular immunity that prevents viral infection or controls disease severity. In addition to a brief description of the design features of unique cationic lipid and virus-mimicking nanoparticles for accomplishing spike protein delivery and presentation by the cognate immune system, we also discuss the importance of adjuvancy and design features to promote cooperative B- and T-cell interactions in lymph node germinal centers, including the use of epitope-based vaccines. Although current vaccine efforts have demonstrated short-term efficacy and vaccine safety, key issues are now vaccine durability and adaptability against viral variants. We present a forward-looking perspective of how vaccine design can be adapted to improve durability of the immune response and vaccine adaptation to overcome immune escape by viral variants. Finally, we consider the impact of nano-enabled approaches in the development of COVID-19 vaccines for improved vaccine design against other infectious agents, including pathogens that may lead to future pandemics.

TFH Cells Induced by Vaccination and Following SIV Challenge Support Env-Specific Humoral Immunity in the Rectal-Genital Tract and Circulation of Female Rhesus Macaques.

T follicular helper (TFH) cells are pivotal in lymph node (LN) germinal center (GC) B cell affinity maturation. Circulating CXCR5+ CD4+ T (cTFH) cells have supported memory B cell activation and broadly neutralizing antibodies in HIV controllers. We investigated the contribution of LN SIV-specific TFH and cTFH cells to Env-specific humoral immunity in female rhesus macaques following a mucosal Ad5hr-SIV recombinant priming and SIV gp120 intramuscular boosting vaccine regimen and following SIV vaginal challenge. TFH and B cells were characterized by flow cytometry. B cell help was evaluated in TFH-B cell co-cultures and by real-time PCR. Vaccination induced Env-specific TFH and Env-specific memory (ESM) B cells in LNs. LN Env-specific TFH cells post-priming and GC ESM B cells post-boosting correlated with rectal Env-specific IgA titers, and GC B cells at the same timepoints correlated with vaginal Env-specific IgG titers. Vaccination expanded cTFH cell responses, including CD25+ Env-specific cTFH cells that correlated negatively with vaginal Env-specific IgG titers but positively with rectal Env-specific IgA titers. Although cTFH cells post-2nd boost positively correlated with viral-loads following SIV challenge, cTFH cells of SIV-infected and protected macaques supported maturation of circulating B cells into plasma cells and IgA release in co-culture. Additionally, cTFH cells of naïve macaques promoted upregulation of genes associated with B cell proliferation, BCR engagement, plasma cell maturation, and antibody production, highlighting the role of cTFH cells in blood B cell maturation. Vaccine-induced LN TFH and GC B cells supported anti-viral mucosal immunity while cTFH cells provided B cell help in the periphery during immunization and after SIV challenge. Induction of TFH responses in blood and secondary lymphoid organs is likely desirable for protective efficacy of HIV vaccines.

Germinal center hypoxia in tumor-draining lymph nodes negatively regulates tumor-induced humoral immune responses in mouse models of breast cancer

ABSTRACT Hypoxia develops in germinal centers (GCs) induced by model antigens; however, it is unknown whether tumor-reactive GCs are also hypoxic. We identified GC hypoxia in lymph nodes (LNs) draining murine mammary tumors and lethally irradiated tumor cells, and found that hypoxia is associated with the levels of antibody-secreting B cells. Hypoxic culture conditions impaired the proliferation of activated B cells, and inhibited class-switching to IgG1 and IgA immunoglobulin isotypes in vitro. To assess the role of the hypoxic response in tumor-reactive GCs in vivo, we deleted von Hippel-Lindau factor (VHL) in class-switched B cells and found decreased GC B cells in tumor-draining LNs, reduced class-switched and tumor-specific antibodies in the circulation, and modified phenotypes of tumor-infiltrating T cells and macrophages. We also detected the hypoxia marker carbonic anhydrase IX in the GCs of LNs from breast cancer patients, providing evidence that GC hypoxia develops in humans. We conclude that GC hypoxia develops in TDLNs, and that the hypoxic response negatively regulates tumor-induced humoral immune responses in preclinical models.

3D investigation shows walls and wall-like structures around human germinal centres, probably regulating T- and B-cell entry and exit.

This study deals with 3D laser investigation on the border between the human lymph node T-zone and germinal centre. Only a few T-cells specific for antigen selected B-cells are allowed to enter germinal centres. This selection process is guided by sinus structures, chemokine gradients and inherent motility of the lymphoid cells. We measured gaps and wall-like structures manually, using IMARIS, a 3D image software for analysis and interpretation of microscopy datasets. In this paper, we describe alpha-actin positive and semipermeable walls and wall-like structures that may hinder T-cells and other cell types from entering germinal centres. Some clearly defined holes or gaps probably regulate lymphoid traffic between T- and B-cell areas. In lymphadenitis, the morphology of this border structure is clearly defined. However, in case of malignant lymphoma, the wall-like structure is disrupted. This has been demonstrated exemplarily in case of angioimmunoblastic T-cell lymphoma. We revealed significant differences of lengths of the wall-like structures in angioimmunoblastic T-cell lymphoma in comparison with wall-like structures in reactive tissue slices. The alterations of morphological structures lead to abnormal and less controlled T- and B-cell distributions probably preventing the immune defence against tumour cells and infectious agents by dysregulating immune homeostasis.

A Spatially Resolved Dark- versus Light-Zone Microenvironment Signature Subdivides Germinal Center-Related Aggressive B Cell Lymphomas.

SummaryWe applied digital spatial profiling for 87 immune and stromal genes to lymph node germinal center (GC) dark- and light-zone (DZ/LZ) regions of interest to obtain a differential signature of these two distinct microenvironments. The spatially resolved 53-genes signature, comprising key genes of the DZ mutational machinery and LZ immune and mesenchymal milieu, was applied to the transcriptomes of 543 GC-related diffuse large B cell lymphomas and double-hit (DH) lymphomas. According to the DZ/LZ signature, the GC-related lymphomas were sub-classified into two clusters. The subgroups differed in the distribution of DH cases and survival, with most DH displaying a distinct DZ-like profile. The clustering analysis was also performed using a 25-genes signature composed of genes positively enriched in the non-B, stromal sub-compartments, for the first time achieving DZ/LZ discrimination based on stromal/immune features. The report offers new insight into the GC microenvironment, hinting at a DZ microenvironment of origin in DH lymphomas.

Organoid Polymer Functionality and Mode of Klebsiella pneumoniae Membrane Antigen Presentation Regulates Ex Vivo Germinal Center Epigenetics in Young and Aged B Cells

Antibiotic-resistant bacteria are a major global health threat that continues to rise due to a lack of effective vaccines. Of concern are Klebsiella pneumoniae that fail to induce in vivo germinal center B cell responses, which facilitate antibody production to fight infection. Immunotherapies using antibodies targeting antibiotic-resistant bacteria are emerging as promising alternatives, however, they cannot be efficiently derived ex vivo, necessitating the need for immune technologies to develop therapeutics. Here, PEG-based immune organoids were developed to elucidate the effects of polymer end-point chemistry, integrin ligands, and mode of K. pneumoniae antigen presentation on germinal center-like B cell phenotype and epigenetics, to better define the lymph node microenvironment factors regulating ex vivo germinal center dynamics. Notably, PEG vinyl sulfone or acrylate failed to sustain primary immune cells, but functionalization with maleimide (PEG-4MAL) led to B cell expansion and germinal center-like induction. RNA sequencing analysis of lymph node stromal and germinal center B cells showed niche associated heterogeneity of integrin-related genes. Incorporation of niche-mimicking peptides revealed that collagen-1 promoted germinal center-like dynamics and epigenetics. PEG-4MAL organoids elucidated the impact of K. pneumoniae outer membrane-embedded protein antigen versus soluble antigen presentation on germinal centers and preserved the response across young and aged mice.

Germinal center formation, immunoglobulin production and hindlimb nociceptive sensitization after tibia fracture

Emerging evidence suggests that Complex Regional Pain Syndrome (CRPS) is in part a post-traumatic autoimmune disease mediated by an adaptive immune response after limb injuries. We previously observed in a murine tibial fracture model of CRPS that pain-related behaviors were dependent upon adaptive immune mechanisms including the neuropeptide-dependent production of IgM for 5 months after injury. However, the time course of induction of this immune response and the demonstration of germinal center formation in lymphoid organs has not been evaluated. Using the murine fracture model, we employed behavioral tests of nociceptive sensitization and limb dysfunction, serum passive transfer techniques, western blot analysis of IgM accumulation, fluorescence-activated cell sorting (FACS) of lymphoid tissues and immunohistochemistry to follow the temporal activation of the adaptive immune response over the first 3 weeks after fracture. We observed that: 1) IgM protein levels in the skin of the fractured mice were elevated at 3 weeks post fracture, but not at earlier time points, 2) serum from fracture mice at 3 weeks, but not 1 and 2 weeks post fracture, had pro-nociceptive effects when passively transferred to fractured muMT mice lacking B cells, 3) fracture induced popliteal lymphadenopathy occurred ipsilateral to fracture beginning at 1 week and peaking at 3 weeks post fracture, 4) a germinal center reaction was detected by FACS analysis in the popliteal lymph nodes from injured limbs by 3 weeks post fracture but not in other lymphoid tissues, 5) germinal center formation was characterized by the induction of T follicular helper cells (Tfh) and germinal center B cells in the popliteal lymph nodes of the injured but not contralateral limbs, and 6) fracture mice treated with the Tfh signaling inhibitor FK506 had impaired germinal center reactions, reduced IgM levels, reduced nociceptive sensitization, and no pronociceptive serum effects after administration to fractured muMT mice. Collectively these data demonstrate that tibia fracture induces an adaptive autoimmune response characterized by popliteal lymph node germinal center formation and Tfh cell dependent B cell activation, resulting in nociceptive sensitization within 3 weeks.

Cyclosporine a directly affects human and mouse b cell migration in vitro by disrupting a hIF-1 \u03b1dependent, o2 sensing, molecular switch

BackgroundHypoxia is a potent molecular signal for cellular metabolism, mitochondrial function, and migration. Conditions of low oxygen tension trigger regulatory cascades mediated via the highly conserved HIF-1 α post-translational modification system. In the adaptive immune response, B cells (Bc) are activated and differentiate under hypoxic conditions within lymph node germinal centers, and subsequently migrate to other compartments. During migration, they traverse through changing oxygen levels, ranging from 1-5% in the lymph node to 5-13% in the peripheral blood. Interestingly, the calcineurin inhibitor cyclosporine A is known to stimulate prolyl hydroxylase activity, resulting in HIF-1 α destabilization and may alter Bc responses directly. Over 60% of patients taking calcineurin immunosuppressant medications have hypo-gammaglobulinemia and poor vaccine responses, putting them at high risk of infection with significantly increased morbidity and mortality.ResultsWe demonstrate that O 2 tension is a previously unrecognized Bc regulatory switch, altering CXCR4 and CXCR5 chemokine receptor signaling in activated Bc through HIF-1 α expression, and controlling critical aspects of Bc migration. Our data demonstrate that calcineurin inhibition hinders this O 2 regulatory switch in primary human Bc.ConclusionThis previously unrecognized effect of calcineurin inhibition directly on human Bc has significant and direct clinical implications.

A Spatially Resolved Dark- Versus Light-Zone Microenvironment Signature Subdivides Germinal Center-Related Aggressive B-Cell Lymphomas

We applied digital spatial profiling for 87 immune and stromal genes to lymph node germinal center (GC) dark- and light-zone (DZ/LZ) regions of interest to obtain a differential gene signature of these two distinct microenvironments. The spatially-resolved 53-genes signature, comprising key genes of the DZ mutational machinery, and LZ immune milieu, was applied to the transcriptomes of 543 GC-related Diffuse Large B-cell Lymphomas and double-hit (DH) lymphomas. According to the DZ/LZ signature, the GC-related lymphomas were sub-classified into two clusters. The two subgroups differed in the distribution of DH cases and survival, with most DH displaying a distinct DZ-like profile. The clustering analysis was also performed using a 25-genes signature composed of DZ/LZ genes represented in the non-B, stromal sub-compartments, for the first time achieving a DZ/LZ discrimination based on stromal microenvironment features. The report offers new insight into the GC' microenvironment, hinting to a DZ origin of DH lymphomas.

Role of Ubiquitin E3 Ligase UBR5 in B-Cell Development and Lymphoma

Mantle Cell Lymphoma (MCL) is a non-Hodgkin's lymphoma (NHL) that typically affects older adults. In MCL, there is uncontrolled growth of B cells within the mantle zone of lymph node and spleen germinal centers. The vast majority of MCL cases have a translocation of chromosome 11 and 14 which juxtaposes the cyclin D1 gene, CCND1, with the immunoglobin heavy chain gene promoter (Eµ) and leads to the overexpression of CCND1. This translocation of CCND1 is not enough to cause MCL by itself and additional mutations are needed for MCL to develop. Although MCL only represents ~5% of NHL patients, it has the poorest survival rate among NHL sub-types due to a lack of successful therapeutic treatments. As such, it is important to identify potential targets for treatment. Recently published data shows that in 18% of all MCL patients have a mutation in the gene encoding the E3 ubiquitin ligase, UBR5. UBR5 is part of the ubiquitin proteasome system, a degradation/recycling pathway in which proteins are tagged with a ubiquitin protein and degraded by the proteasome. Of the identified UBR5 mutations, over 60% of the mutations are truncations at the carboxy terminus that cut off the cysteine residue linked to ubiquitin transfer in exon 59 suggesting a catalytic dead mutant protein is produced. Interestingly, UBR5HECT mutations are specific to MCL and are not found in other sub-types of NHL. By studying UBR5, we can determine the role of UBR5 mutations in MCL, elucidate molecular mechanism of UBR5 in B cell development, and identify potential therapeutic targets. In order to study the role of UBR5 in B cell development and MCL, we generated a conditional mouse model targeting exon 58 similar to mutations in MCL patients and crossed the mice with Mb1CRE/WT mice to delete exon 58 specifically in B cells at the pro-pre B cell stage of B cell development. Mb1CRE/WT; Ubr5fl/fl mice shows that mice lacking the carboxy terminus of UBR5 have a block in B cell differentiation at the mature B cell IgM+ IgD+ stage within the spleen. Mb1CRE/WT; Ubr5fl/fl mice showed a marked decrease of mature IgM+ IgD+ B cells in the BM and spleen. Specifically, within the spleen, Mb1CRE/WT; Ubr5fl/fl mice produce abnormal follicular B cells (higher IgM and CD23 expression and lower IgD and CD22 expression) and significant reductions in marginal zone B cells, plasma cells, size of germinal centers, and number of germinal centers. Mass spectrometry comparing mouse B220+ splenocytes in Mb1CRE/WT; Ubr5fl/fl compared to Mb1CRE/WT; Ubr5+/+ mice showed that UBR5 in B cells in Mb1CRE/WT; Ubr5fl/fl mice has over two-fold more expression. Additionally, IgD was the most downregulated protein, B cell specific proteins were downregulated, and proteins involved with mRNA splicing were upregulated within the mass spectrometry. The loss of the UBR5 HECT domain leads to increased UBR5 half-life and upregulation of spliceosome proteins. Coupled together, this suggests that the loss of the carboxy terminus of UBR5 impedes B cell maturation by disrupting IgD expression, potentially by interfering with mRNA splicing. Finally, we have crossed and are currently aging our Mb1CRE/WT; Ubr5fl/fl mice with an EµCCND1 mouse model to determine if Ubr5 mutations lead to tumorigenesis. These studies aim to identify the role of UBR5 in the context of normal B cell development and lymphomagenesis with the goal of identifying therapeutic targets for drug discovery. Disclosures No relevant conflicts of interest to declare.

Quantitative Changes in Serum Proteins Including CXCL13 Are Early Indicators of Response to Anti-IL6 Therapy in Idiopathic Multicentric Castleman Disease

Background Human herpes virus-8 (HHV8) negative/idiopathic multicentric Castleman disease (iMCD) is a rare hematological disorder with one FDA-approved treatment and no early indicators of patient response after treatment is commenced. Clinical onset can be sudden and severe, involving polyclonal lymphoproliferation with characteristic dysmorphic germinal centers, constitutional symptoms, systemic inflammation and life-threatening cytokine storm-driven multi-organ failure. CXCL13, a key regulator of lymph node germinal center development, was recently found to be the most elevated cytokine in iMCD flare, but the clinical significance of this finding is not yet clear. Interleukin-6 (IL-6) is the known driver of pathogenesis in a portion of patients and the target of the only FDA-approved treatment, siltuximab. In the Phase II study of siltuximab (NCT01024036), one-third of patients met response criteria, which were assessed after a minimum of 48 weeks. Early indicators of response to siltuximab are urgently needed to inform clinicians about the likelihood of patient response to therapy, adjust treatments if needed, and identify novel therapeutic targets for siltuximab non-responders. Methods Clinical data and serum samples were collected as part of NCT01024036. We measured serum protein analytes in the 52 subjects who were treated with anti-IL6 therapy, as well as the 26 patients in the control arm, at day 1, day 8, and day 64 of therapy (infusions administered every 21 days). Serum samples from 44 healthy donors were also analyzed. Of the 1,305 analytes measured using SomaLogic SOMAscan, 1,178 passed QC and were included in analyses. Each analyte was log2 transformed and capped at the 2.5th and 97.5th percentiles. Response to anti-IL6 therapy was determined by independent review in NCT01024036. Data processing was performed using the Medidata Rave Omics machine learning platform and R v3.4.4. Linear mixed effects models were used to detect whether kinetic changes in protein expression were associated with anti-IL6 response. Upon running the full model with the selected covariates, the p-values of the interaction between time point and response were used to test for differences between responders and non-responders. A separate model was fitted using each protein, and False Discovery Rates (FDR) were estimated by the Benjamini-Hochberg method with alpha < 0.05. Results Seven days after siltuximab was first administered (day 8), 9 proteins were significantly different between responders and non-responders: IgA, BCMA, NPS-PLA2, ART, IL-18 BPa, CD5L, b2-Microglobulin, CXCL13, and NRP1. All 9 of these proteins were significantly decreased in responders compared to non-responders. At day 64, the number of significantly different proteins increased to 121, including 8 of the 9 proteins from day 8; NPS-PLA2 did not achieve significance at day 64. This result indicates that there may be early indicators of response in serum as early as day 8. Given that CXCL13 was recently discovered as a key cytokine in iMCD, the early and significant decline of CXCL13 in responders versus non-responders was highly notable (Day 8: FDR = 0.02, Day 64: FDR = 0.005). Prior to treatment, CXCL13 was significantly higher in this cohort of iMCD patients than in a group of age-matched healthy donors (p = 8.19e-09). By day 64, CXCL13 levels in siltuximab responders decreased to levels approaching the healthy donor range but remained elevated in non-responders and placebo patients. Conclusions This analysis represents the first use of high-quality serum proteomics data to study early indicators of response to treatment in a rare hyperinflammatory, lymphoproliferative disorder. The decline in CXCL13 levels in responders and continued elevation in non-responders suggests that CXCL13 is downstream of IL-6 in responders and independent of IL-6 signaling in non-responders. CXCL13, along with several other proteins that demonstrated significant decline by day 8 including IgA and beta2-microglobulin, can be routinely measured and could serve as a panel that indicates the likelihood of response soon after commencing therapy, if validated in a separate cohort. These proteins could also provide a more continuous scale of response than traditional outcome measures. Given that iMCD may have a sudden and severe onset, early indicators of response to anti-IL6 therapy are critical for timely treatment administration. Figure Disclosures Katz: Medidata Solutions: Employment. Nabel:Thermo Fisher: Patents & Royalties: royalty income. Diamond:Brigham and Women's Hospital and Harvard Medical School's Department of Medicine, Division of Genetics, Genomes2People Translational Genomics Research Program: Consultancy. Karvir:Medidata Solutions: Employment. Zhang:Medidata Solutions: Employment. Reddy:Janssen Pharmaceuticals: Employment, Equity Ownership. Guilfoyle:Janssen Pharmaceuticals: Employment, Equity Ownership. Tendler:Janssen Pharmaceuticals: Employment, Equity Ownership. van Rhee:Takeda: Consultancy; Sanofi Genzyme: Consultancy; Castleman Disease Collaborative Network: Consultancy; EUSA: Consultancy; Adicet Bio: Consultancy; Kite Pharma: Consultancy; Karyopharm Therapeutics: Consultancy. Beineke:Medidata Solutions: Employment. Oromendia:Medidata Solutions: Employment, Equity Ownership. Fajgenbaum:Janssen Pharmaceuticals: Research Funding.

Assessments of cancer-free lymph nodes for the prediction of disease progression

Abstract Background Histological changes in the cancer-free lymph nodes (LNs) can add to the risk prediction of developing distant metastasis for LN-positive breast cancer patients. As Whole Slide Images (WSI) demonstrated suitability for detection of LN metastasis with high accuracy based on Convolutional Neural Networks (CNN), we extended the assessments to cancer-free LNs to determine whether early signs of disease progression could be identified. Methods Clinico-pathological data, including level of LNs in the axilla, were available for 143 LN-positive breast cancer patients. All resected LNs were digitised (×40 magnification), totalling to 1,054 WSI. CNN models were based on standard ResNet architecture, and trained using 224x224 sized WSI patches, to classify germinal centres (GC), follicles, sinus histiocytosis, and adipose tissue as defined by 2 independent pathologists. To increase the robustness of the CNN, training data was augmented using image rotation and colour perturbation. A weighted cross entropy was used as a training loss function to account for the imbalance in the four types of patches. Confusion matrix and one-vs-all AUC scores were computed on WSIs patches extracted from held-out test patients to assess the discriminative power of the trained CNN models. Multivariate and cross-validated L2-regularised proportional hazard regression analyses were used for outcome prediction. Results An increased number and smaller sized GCs in cancer-free LNs, the prevalence of metastatic LNs in the low axilla, the location of GCs in metastatic LNs, and lymphocytic infiltration at the tumour invasive front were all indicative of developing distant metastasis in LN-positive breast patients. Metastatic LNs were found exclusively in the low axilla in 77% of patients. Amongst the 913 H&E WSI of cancer-free LNs, 21% presented with GC. Patch level AUC scores of detecting each type of tissue region ranged between 0.82 to 0.94 on held-out patients quantifying the effectiveness of CNN to be used as topological feature detector. Conclusions Addition planned WSI analyses of large breast cancer cohorts will expand on these data, capturing histological features in LNs, with the aim of identifying high-risk and low-risk disease, and aiding in pathological prognostication. Legal entity responsible for the study The authors. Funding Breast Cancer Research Trust, Breast Cancer Now, CRUK. Disclosure All authors have declared no conflicts of interest.

Abstract # 3252 Healthy aging – A role for autonomic function?

Emerging evidence suggests that Complex Regional Pain Syndrome (CRPS) is in part a post-traumatic autoimmune disease mediated by an adaptive immune response after limb injuries. We previously observed in a murine tibial fracture model of CRPS that pain-related behaviors were dependent upon adaptive immune mechanisms including the neuropeptide-dependent production of IgM for 5 months after injury. However, the time course of induction of this immune response and the demonstration of germinal center formation in lymphoid organs has not been evaluated. Using the murine fracture model, we employed behavioral tests of nociceptive sensitization and limb dysfunction, serum passive transfer techniques, western blot analysis of IgM accumulation, fluorescence-activated cell sorting (FACS) of lymphoid tissues and immunohistochemistry to follow the temporal activation of the adaptive immune response over the first 3 weeks after fracture. We observed that: 1) IgM protein levels in the skin of the fractured mice were elevated at 3 weeks post fracture, but not at earlier time points, 2) serum from fracture mice at 3 weeks, but not 1 and 2 weeks post fracture, had pro-nociceptive effects when passively transferred to fractured muMT mice lacking B cells, 3) fracture induced popliteal lymphadenopathy occurred ipsilateral to fracture beginning at 1 week and peaking at 3 weeks post fracture, 4) a germinal center reaction was detected by FACS analysis in the popliteal lymph nodes from injured limbs by 3 weeks post fracture but not in other lymphoid tissues, 5) germinal center formation was characterized by the induction of T follicular helper cells (Tfh) and germinal center B cells in the popliteal lymph nodes of the injured but not contralateral limbs, and 6) fracture mice treated with the Tfh signaling inhibitor FK506 had impaired germinal center reactions, reduced IgM levels, reduced nociceptive sensitization, and no pronociceptive serum effects after administration to fractured muMT mice. Collectively these data demonstrate that tibia fracture induces an adaptive autoimmune response characterized by popliteal lymph node germinal center formation and Tfh cell dependent B cell activation, resulting in nociceptive sensitization within 3 weeks.

Abstract # 4278 Intra-hippocampal depletion of microglia as a tool to examine the role of microglia in the ontogeny of learning and learning deficits produced by immune challenge

Emerging evidence suggests that Complex Regional Pain Syndrome (CRPS) is in part a post-traumatic autoimmune disease mediated by an adaptive immune response after limb injuries. We previously observed in a murine tibial fracture model of CRPS that pain-related behaviors were dependent upon adaptive immune mechanisms including the neuropeptide-dependent production of IgM for 5 months after injury. However, the time course of induction of this immune response and the demonstration of germinal center formation in lymphoid organs has not been evaluated. Using the murine fracture model, we employed behavioral tests of nociceptive sensitization and limb dysfunction, serum passive transfer techniques, western blot analysis of IgM accumulation, fluorescence-activated cell sorting (FACS) of lymphoid tissues and immunohistochemistry to follow the temporal activation of the adaptive immune response over the first 3 weeks after fracture. We observed that: 1) IgM protein levels in the skin of the fractured mice were elevated at 3 weeks post fracture, but not at earlier time points, 2) serum from fracture mice at 3 weeks, but not 1 and 2 weeks post fracture, had pro-nociceptive effects when passively transferred to fractured muMT mice lacking B cells, 3) fracture induced popliteal lymphadenopathy occurred ipsilateral to fracture beginning at 1 week and peaking at 3 weeks post fracture, 4) a germinal center reaction was detected by FACS analysis in the popliteal lymph nodes from injured limbs by 3 weeks post fracture but not in other lymphoid tissues, 5) germinal center formation was characterized by the induction of T follicular helper cells (Tfh) and germinal center B cells in the popliteal lymph nodes of the injured but not contralateral limbs, and 6) fracture mice treated with the Tfh signaling inhibitor FK506 had impaired germinal center reactions, reduced IgM levels, reduced nociceptive sensitization, and no pronociceptive serum effects after administration to fractured muMT mice. Collectively these data demonstrate that tibia fracture induces an adaptive autoimmune response characterized by popliteal lymph node germinal center formation and Tfh cell dependent B cell activation, resulting in nociceptive sensitization within 3 weeks.

Abstract # 4268 Alterations of the serum N-glycan profile in female patients with Major Depressive Disorder

Emerging evidence suggests that Complex Regional Pain Syndrome (CRPS) is in part a post-traumatic autoimmune disease mediated by an adaptive immune response after limb injuries. We previously observed in a murine tibial fracture model of CRPS that pain-related behaviors were dependent upon adaptive immune mechanisms including the neuropeptide-dependent production of IgM for 5 months after injury. However, the time course of induction of this immune response and the demonstration of germinal center formation in lymphoid organs has not been evaluated. Using the murine fracture model, we employed behavioral tests of nociceptive sensitization and limb dysfunction, serum passive transfer techniques, western blot analysis of IgM accumulation, fluorescence-activated cell sorting (FACS) of lymphoid tissues and immunohistochemistry to follow the temporal activation of the adaptive immune response over the first 3 weeks after fracture. We observed that: 1) IgM protein levels in the skin of the fractured mice were elevated at 3 weeks post fracture, but not at earlier time points, 2) serum from fracture mice at 3 weeks, but not 1 and 2 weeks post fracture, had pro-nociceptive effects when passively transferred to fractured muMT mice lacking B cells, 3) fracture induced popliteal lymphadenopathy occurred ipsilateral to fracture beginning at 1 week and peaking at 3 weeks post fracture, 4) a germinal center reaction was detected by FACS analysis in the popliteal lymph nodes from injured limbs by 3 weeks post fracture but not in other lymphoid tissues, 5) germinal center formation was characterized by the induction of T follicular helper cells (Tfh) and germinal center B cells in the popliteal lymph nodes of the injured but not contralateral limbs, and 6) fracture mice treated with the Tfh signaling inhibitor FK506 had impaired germinal center reactions, reduced IgM levels, reduced nociceptive sensitization, and no pronociceptive serum effects after administration to fractured muMT mice. Collectively these data demonstrate that tibia fracture induces an adaptive autoimmune response characterized by popliteal lymph node germinal center formation and Tfh cell dependent B cell activation, resulting in nociceptive sensitization within 3 weeks.

Abstract # 3228 Primary cultures of the rat dorsal root ganglia: Influence of lipopolysaccharide and cisplatin on properties of neuronal and glial elements

Emerging evidence suggests that Complex Regional Pain Syndrome (CRPS) is in part a post-traumatic autoimmune disease mediated by an adaptive immune response after limb injuries. We previously observed in a murine tibial fracture model of CRPS that pain-related behaviors were dependent upon adaptive immune mechanisms including the neuropeptide-dependent production of IgM for 5 months after injury. However, the time course of induction of this immune response and the demonstration of germinal center formation in lymphoid organs has not been evaluated. Using the murine fracture model, we employed behavioral tests of nociceptive sensitization and limb dysfunction, serum passive transfer techniques, western blot analysis of IgM accumulation, fluorescence-activated cell sorting (FACS) of lymphoid tissues and immunohistochemistry to follow the temporal activation of the adaptive immune response over the first 3 weeks after fracture. We observed that: 1) IgM protein levels in the skin of the fractured mice were elevated at 3 weeks post fracture, but not at earlier time points, 2) serum from fracture mice at 3 weeks, but not 1 and 2 weeks post fracture, had pro-nociceptive effects when passively transferred to fractured muMT mice lacking B cells, 3) fracture induced popliteal lymphadenopathy occurred ipsilateral to fracture beginning at 1 week and peaking at 3 weeks post fracture, 4) a germinal center reaction was detected by FACS analysis in the popliteal lymph nodes from injured limbs by 3 weeks post fracture but not in other lymphoid tissues, 5) germinal center formation was characterized by the induction of T follicular helper cells (Tfh) and germinal center B cells in the popliteal lymph nodes of the injured but not contralateral limbs, and 6) fracture mice treated with the Tfh signaling inhibitor FK506 had impaired germinal center reactions, reduced IgM levels, reduced nociceptive sensitization, and no pronociceptive serum effects after administration to fractured muMT mice. Collectively these data demonstrate that tibia fracture induces an adaptive autoimmune response characterized by popliteal lymph node germinal center formation and Tfh cell dependent B cell activation, resulting in nociceptive sensitization within 3 weeks.

Perturbation of Thymocyte Development Underlies the PRRS Pandemic: A Testable Hypothesis.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes immune dysregulation during the Critical Window of Immunological Development. We hypothesize that thymocyte development is altered by infected thymic antigen presenting cells (TAPCs) in the fetal/neonatal thymus that interact with double-positive thymocytes causing an acute deficiency of T cells that produces “holes” in the T cell repertoire allowing for poor recognition of PRRSV and other neonatal pathogens. The deficiency may be the result of random elimination of PRRSV-specific T cells or the generation of T cells that accept PRRSV epitopes as self-antigens. Loss of helper T cells for virus neutralizing (VN) epitopes can result in the failure of selection for B cells in lymph node germinal centers capable of producing high affinity VN antibodies. Generation of cytotoxic and regulatory T cells may also be impaired. Similar to infections with LDV, LCMV, MCMV, HIV-1 and trypanosomes, the host responds to the deficiency of pathogen-specific T cells and perhaps regulatory T cells, by “last ditch” polyclonal B cell activation. In colostrum-deprived PRRSV-infected isolator piglets, this results in hypergammaglobulinemia, which we believe to be a “red herring” that detracts attention from the thymic atrophy story, but leads to our second independent hypothesis. Since hypergammaglobulinemia has not been reported in PRRSV-infected conventionally-reared piglets, we hypothesize that this is due to the down-regulatory effect of passive maternal IgG and cytokines in porcine colostrum, especially TGFβ which stimulates development of regulatory T cells (Tregs).

Cell-specific inhibition of SMAD2/3 restores lymph node cellularity and germinal center function in aged mice responding to acute chikungunya virus (CHIKV) infection

Abstract Targeted delivery of small-molecule inhibitors increases drug efficacy by focusing its action upon specific cell-types without global disruption of signaling pathways. TGFβ is a pleotropic cytokine involved in many cellular processes, including specific regulatory functions during anti-viral immune responses. Previously, we have shown that in aged mice, over-production of TGFβ correlates with decreased immune function. TGFβ neutralization during acute chikungunya virus (CHIKV) infection decreases acute- and chronic- disease severity and improves neutralizing antibody titers. Herein, we selectively interrupted TGFβ signaling during acute CHIKV infection via the delivery of lymph node (LN)-targeting nanoparticles coupled to a small-molecule inhibitor of SMAD2/3 phosphorylation. We show that LN-targeted inhibition of SMAD phosphorylation during the initiation of a viral immune response can restore – to the levels measured in adult mice - the cellular content of the draining lymph node. We further demonstrate that inhibition of TGFβ signaling decreases LN fibrosis and improves the germinal center reaction in aged mice.

Presentation of HIV-1 envelope glycoprotein trimers on diverse nanoparticle platforms.

Purpose of reviewWe will discuss recent advances in the development of nanoparticle vaccines presenting HIV-1 envelope trimer vaccines and the immunological mechanisms by which they act.Recent findingsThe multivalent presentation of Env trimers on nanoparticles is a promising strategy to increase Env immunogenicity. Recent studies have shed light on how Env nanoparticles increase lymph node trafficking and germinal center formation by using the lectin-mediated complement pathway and enhancing the interaction with naïve B cells. Meanwhile, research on different nanoparticle platforms has resulted in improved designs, such as liposomes with improved stability, and the emergence of novel platforms such as protein nanoparticles that self-assemble in vitro. Immmunogenicity studies with these nanoparticles delineate the advantages and expose the limitations of the different nanoparticle platforms.SummaryIt is becoming increasingly clear that HIV-1 vaccine research might benefit greatly from using nanoparticles presenting Env trimers, particularly during the priming stage of immunization. Among the different nanoparticles that are being pursued, in vitro-assembling nanoparticles allow for greater control of Env quality making them a promising nanoparticle platform.