Cell Subsets In Lymph Nodes Research Articles

Assessment of Lymph Node Stromal Cells as an Underlying Factor in Age-Related Immune Impairment.

Aging negatively impacts immunity, resulting in inefficient responses to vaccinations and infections. Fibroblastic reticular cells (FRCs) are the major stromal cell subset in lymph nodes (LNs) and play an intricate role in the orchestration and control of adaptive immune responses. Although stromal cells have a major impact on immune responses, the impact of aging on LN stromal cells remains unclear. Quantitative analysis of LN stromal cells by flow cytometry revealed that there are no significant differences in the number of stromal cells in young and aged LN at steady state but after influenza infection aged FRCs have delayed expansion as a result of reduced proliferation. Aged LNs also produce reduced levels of homeostatic chemokines, which correlates with reduced homing of naive T cells. Image analysis reveals that young and aged T-cell zone FRCs have similar morphology at steady state and after infection. Furthermore, aged FRCs did not appear to be a contributing factor in the reduced proliferation of young T cells transferred into aged LNs after influenza infection. These results demonstrate that aging alters LN stromal cell response to challenge and these age-related changes may be an underlying contributor to impaired immune responses in the elderly people.

Assessment of lymph node stromal cells as an underlying factor in age related immune impairment to influenza infection

Abstract It is well established that aging negatively impacts immunity, resulting in inefficient responses to vaccinations and infections, like influenza. Fibroblastic reticular cells (FRCs) are the major stromal cell subset in lymph nodes and play an intricate role in the orchestration and control of adaptive immune responses. Although stromal cells have a major impact on immune responses, the impact of aging on LN stromal cells has yet to be determined. We sought to determine how aging impacts FRC number, phenotype and morphology at steady state and after influenza infection. An enzymatic and mechanical digestion was used to make single cell suspensions of lymph node stromal cells for analysis. Flow cytometry was performed to quantify the number and frequency of stromal cells in lymph nodes from young (13 weeks) and aged (19 months) C57BL/6 mice. To analyze stromal cell morphology, lymph nodes from young and aged mice were cryopreserved, and then sectioned for confocal microscopy. Quantitative analysis of lymph node stromal cells by flow cytometry revealed that there are no significant differences in the number stromal cells in young and aged lymph nodes at steady state, but after influenza infection aged FRCs fail to expand in number. Reduced proliferation and activation of aged FRCs, but not increased death leads to the decreased expansion. Microscopic analysis revealed that young and aged LN T cell zone FRCs have similar morphology at steady state and after infection. These results are the first evidence that aging alters lymph node stromal cells ability to respond to challenge. These changes in aged FRCs may be an underlying cause behind the impaired immune response to influenza infection found in the elderly.

Shaping of Peripheral T Cell Responses by Lymphatic Endothelial Cells.

Lymph node stromal cells (LNSCs) have newly been promoted to the rank of new modulators of T cell responses. The different non-hematopoietic cell subsets in lymph node (LN) were considered for years as a simple scaffold, forming routes and proper environment for antigen (Ag)-lymphocyte encountering. Deeper characterization of those cells has recently clearly shown their impact on both dendritic cell and T cell functions. In particular, lymphatic endothelial cells (LECs) control lymphocyte trafficking and homeostasis in LNs and limit adaptive immune responses. Therefore, the new role of LECs in shaping immune responses has drawn the attention of immunologists. Striking is the discovery that LECs, among other LNSCs, ectopically express a large range of peripheral tissue-restricted Ags (PTAs), and further present PTA-derived peptides through major histocompatibility class I molecules to induce self-reactive CD8+ T cell deletional tolerance. In addition, both steady-state and tumor-associated LECs were described to be capable of exogenous Ag cross-presentation. Whether LECs can similarly impact CD4+ T cell responses through major histocompatibility class II restricted Ag presentation is still a matter of debate. Here, we review and discuss our current knowledge on the contribution of Ag-presenting LECs as regulators of peripheral T cell responses in different immunological contexts, including autoimmunity and cancer.

Characterization of Immune System Cell Subsets in Fixed Tissues from Alpine Chamois (Rupicapra rupicapra)

Immune system cell subsets in lymph nodes and spleen from alpine chamois (Rupicapra rupicapra subspecies rupicapra) living in the Italian Alps were characterized immunohistochemically. Seven primary antibodies (against human CD3, CD79αcy, CD68, or ovine CD4, CD8, CD21 and γδ T-cell receptor [TCR] epitopes) were tested on tissues fixed either in formalin or in zinc salts (ZS) and cross-reactivity with chamois immune cell epitopes was shown. ZS fixation allowed wider identification of immune cells, without the need for antigen retrieval. CD4(+) and CD21(+) cells were labelled only in ZS-fixed tissues. Reagents specific for human CD3, CD79 and CD68 antigens successfully detected chamois immune cells, both in ZS-fixed and formalin-fixed tissues. The reactivity and distribution of immune cells in lymph nodes and spleen were similar to those described in other domestic and wild ruminants. Results from this study may allow future investigation of the immune response and pathogenesis of diseases in the chamois.

Selective tumor necrosis factor receptor I blockade is antiinflammatory and reveals immunoregulatory role of tumor necrosis factor receptor II in collagen-induced arthritis.

Tumor necrosis factor (TNF) signals via 2 receptors, TNFR type I (TNFRI) and TNFRII, with distinct cellular distribution and signaling functions. In rheumatoid arthritis (RA), the net effect of TNFR signaling favors inflammatory responses while inhibiting the activity of regulatory T cells. TNFRII signaling has been shown to promote Treg cell function. To assess the relative contributions of TNFRI and TNFRII signaling to inflammatory and regulatory responses in vivo, we compared the effect of TNF blockade, hence TNFRI/II, versus TNFRI alone in collagen-induced arthritis (CIA) as a model of RA. Mice with established arthritis were treated for 10 days with anti-mouse TNFRI domain antibody (dAb; DMS5540), an isotype control dAb (DMS5538), or murine TNFRII genetically fused with mouse IgG1 Fc domain (mTNFRII-Fc) beginning on the day of arthritis onset, and disease progression was monitored. Systemic cytokine concentrations and numbers of T cell subsets in lymph nodes and spleens were measured, and intrinsic Treg cell function was determined by ex vivo suppression assays. Progression of CIA was suppressed similarly by TNFRI (DMS5540) and TNFRI/II (mTNFRII-Fc) blockade. However, blockade of TNFRI/II led to increased effector T cell activity, which was not observed after selective TNFRI blockade, suggesting an immunoregulatory role of TNFRII. In support of this, TNFRI blockade, but not TNFRI/II blockade, expanded and activated Treg cells. Furthermore, a dramatic increase in expression of the Treg cell signature genes FoxP3 and TNFRII was observed in joints undergoing remission, which supports the notion that these molecules have a physiologic role in the resolution of inflammation. We propose that a therapeutic strategy that targets TNFRI while sparing TNFRII has the potential to both inhibit inflammation and promote Treg cell activity, which might be superior to TNF blockade.

AB0050 Irf8 Promotes TH17 Differentiation via Activation of Integrin-Mediated Tgfbeta Signaling in Neuroinflammation

BackgroundThere is accumulating evidence that Th17 cells promote inflammation and tissue injury in many autoimmune diseases. Although recent epidemiological studies identified IRF8 as a susceptibility factor for the autoimmune diseases,...

Characterisation of Dendritic Cell Subsets in Lymph Nodes of Multiple Sclerosis Patients Treated with Transdermally Delivered Myelin Peptides (P02.100)

Characterisation of Dendritic Cell Subsets in Lymph Nodes of Multiple Sclerosis Patients Treated with Transdermally Delivered Myelin Peptides (P02.100)

Immunohistochemical Characterisation of Immune Cell Subsets in Lymph Nodes from Water Buffaloes

Immunohistochemical Characterisation of Immune Cell Subsets in Lymph Nodes from Water Buffaloes

T cell subpopulations in lymph nodes may not be predictive of patient outcome in colorectal cancer

BackgroundThe immune response has been proposed to be an important factor in determining patient outcome in colorectal cancer (CRC). Previous studies have concentrated on characterizing T cell populations in the primary tumour where T cells with regulatory effect (Foxp3+ Tregs) have been identified as both enhancing and diminishing anti-tumour immune responses. No previous studies have characterized the T cell response in the regional lymph nodes in CRC.MethodsImmunohistochemistry was used to analyse CD4, CD8 or Foxp3+ T cell populations in the regional lymph nodes of patients with stage II CRC (n = 31), with (n = 13) or without (n = 18) cancer recurrence after 5 years of follow up, to determine if the priming environment for anti-tumour immunity was associated with clinical outcome.ResultsThe proportions of CD4, CD8 or Foxp3+ cells in the lymph nodes varied widely between and within patients, and there was no association between T cell populations and cancer recurrence or other clinicopathological characteristics.ConclusionsThese data indicate that frequency of these T cell subsets in lymph nodes may not be a useful tool for predicting patient outcome.

Nano-sized drug-loaded micelles deliver payload to lymph node immune cells and prolong allograft survival

By delivering immunomodulatory drugs in vivo directly to lymph nodes draining an injection site, an opportunity exists to increase drug bioavailability to local immune cells. Importantly, particles smaller than 100 nm are efficiently transported through lymphatic vessels to draining lymph nodes. To investigate whether this approach could be used for local delivery of immunomodulatory drugs, amphiphilic poly(ethylene glycol) -bl-poly(propylene sulfide) (PEG -bl-PPS) block copolymers forming 50 nm micelles were used to encapsulate hydrophobic drugs. Micelle drainage was determined using fluorescent micelles and showed effective targeting of multiple immune cell subsets in lymph nodes. For functional studies of our formulations, two approaches were considered. To evaluate the efficacy of anti-inflammatory drug delivery, dendritic cell activation was shown to be prevented when mice were pretreated with micelles loaded with the glucocorticoid mometasone and then challenged with the TLR9 ligand, CpG. To evaluate whether immunosuppressive drug-loaded micelles were effective in prolonging MHC-mismatched allograft survival, BALB/c mice were treated for 14 consecutive days with drug-loaded micelles following transplantation of allogenic C57BL/6 tail skin. Micelles loaded with a mixture of rapamycin and tacrolimus prolonged allograft survival by 2-fold. Our results indicate that the drug-loaded micelle approach effectively targets the draining lymph nodes and exhibits proper immune regulation.

Distinct spatial localization of resident dendritic cell subsets in lymph nodes (100.30)

Abstract Dendritic cells (DCs) are essential for uptake and presentation of antigens (Ags) for T cell activation and can be divided into subsets with varying Ag-presentation capacities. It is not clear whether intrinsic differences or variation in spatial localization and preferential Ag access both contribute to differential Ag-presentation by DC subsets. We thus developed a novel approach of combining multi-parameter (6 or 7 color) confocal microscopy with analytical masking to allow direct in-situ visualization of cellular populations identified by complex phenotypic surface markers. We determined the spatial localization of major resident lymph node (LN) DC subsets and observed preferential positioning of certain populations to distinct LN zones. Some DCs physically associated with lymphatic sinuses (LS) and extended dendrites directly into the LS. While small protein Ags can be directly delivered to resident DCs via LN conduits, particulate Ags are thought to be presented via migratory DCs arriving from peripheral sites. Using 2-photon microscopy, we observed the LS-associated DCs actively sampling the LS for particulate Ags and the formation of T cell clusters in zones proximal to the LS after particulate Ag injection. These results suggest the existence of specialized micro-domains for DC subsets within LN and suggest that LS-associated DC are important for T cell activation to particulate Ags. This research was supported by the Intramural Research Program of the NIH, NIAID.

Dynamics of CD11c+ dendritic cell subsets in lymph nodes draining the site of intestinal nematode infection

Helminth parasites drive dominant Th2 responses through an as yet unidentified pathway. We have previously shown that the rodent gastrointestinal nematode Nippostrongylus brasiliensis secretes products which selectively activate in vitro-derived dendritic cells to induce Th2 responses on in vivo transfer. We now show that, during active infection with this parasite, the draining mesenteric lymph node dendritic cell population is altered significantly. Although there is substantial expansion of DC numbers during infection, the CD86hi-CD8αint-CD11b− subset is markedly diminished, and expression levels of CD40, CD86 and CD103 are reduced. Notably, the reduced frequency of CD8αint DCs is evident only in those mesenteric lymph nodes draining the anterior site of infestation. In infections with the longer lived Heligmosomoides polygyrus, the proportion of CD8αint DCs in the MLNC falls to below 10% of total DC numbers by 35 days post-infection. Further, infection alters TLR responsiveness, as IL-12 production (as measured by ex vivo intracellular staining of CD11c+ DCs) in response to LPS stimulation is reduced, while IL-6, TNF-α and in particular, IL-10 all increase following infection with either nematode parasite. These changes suggest the possibility that helminth parasites modulate gastrointestinal immunity both by inhibiting migration of CD8αint DCs to the draining lymph nodes, and modifying DC responsiveness in a manner which favours a Th2 outcome.

Orchestrating the orchestrators: chemokines in control of T cell traffic

The understanding of how chemokines orchestrate the trafficking and activity of immune cells has increased considerably. So far, over 50 chemokines and 20 chemokine receptors have been identified. Detailed analyses have demonstrated the function of chemokine receptors on T cell subsets, the temporal and spatial expression patterns of chemokines in vivo and the phenotypes of animals genetically deficient in one component or several components of the chemokine-chemokine receptor system. New microscopy modalities for studying the influence of chemokines on the migratory activity of T cells in the lymph node have also brought new insights. Here we review such advances with particular emphasis on control of the migration of T cell subsets in lymph nodes and in peripheral tissues in homeostasis and inflammation.

Dendritic cell subsets in lymph nodes are characterized by the specific draining area and influence the phenotype and fate of primed T cells

Dendritic cells (DC) are important in differential T-cell priming. Little is known about the local priming by DC in the microenvironment of different lymph nodes and about the fate of the imprinted T cells. Therefore, freshly isolated rat DC from mesenteric lymph nodes (mLN) and axillary lymph nodes (axLN) were phenotyped and cultured with blood T cells in the presence of the superantigen Mycoplasma arthritidis mitogen (MAM). The phenotype, proliferation and apoptosis of the primed T cells were analysed. Our data show that a common DC population exists in both mLN and axLN. In addition, region-specific DC with an organotypical marker expression imprinted by the drained area were found. Coculture of T cells with DC from mLN or axLN resulted in a distinct shift in the CD4 and CD8 expression of T cells and their phenotype. Furthermore, when these differentially primed mLN and axLN T cells were injected into recipients, mLN-primed T cells survived longer in other lymphoid organs. The results show that the region-specific DC have a unique phenotype and an impact on the ratio of CD4 : CD8 T cells during an immune response in vivo.

Mycobacterium tuberculosisInfects Dendritic Cells with High Frequency and Impairs Their Function In Vivo

Mycobacterium tuberculosis (Mtb) is thought to reside in macrophages, although infected dendritic cells (DCs) have been observed. Thus, although cellular associations have been made, global characterization of the cells harboring Mtb is lacking. We have performed temporal and quantitative characterization of the cells harboring Mtb following aerosol infection of mice by using GFP-expressing bacteria and flow cytometry. We discovered that Mtb infects phagocytic cells of diverse phenotypes, that the predominant infected cell populations change with time, and that myeloid DCs are the major cell population infected with Mtb in the lungs and lymph nodes. We also found that the bacteria in the lung-draining lymph node are transported there from the lungs by a CCL19/21-dependent mechanism and that the transport of bacteria to the lymph node is a transient phenomenon despite chronic infection. In addition, we found that the lymph node cell subsets that are most efficacious in stimulating Mtb-specific, TCR-transgenic CD4(+) T lymphocytes are not infected with the bacteria and are scarce or absent from the lungs of infected mice. Finally, we found that the lung cell populations that are infected with Mtb at high frequency are relatively ineffective at stimulating Ag-specific CD4(+) T lymphocytes, and we have obtained evidence that live Mtb can inhibit MHC class II Ag presentation without a decrease in the surface expression of MHC class II. These results indicate that Mtb targets DC migration and Ag presentation in vivo to promote persistent infection.

Coreceptor Usage of Primary HIV Type 1 Isolates Obtained from Different Lymph Node Subsets

Biological characteristics of virus quantitatively rescued from different cell types present in lymph nodes of HIV-1-infected individuals in various stages of their disease were determined, not including patients with AIDS defining illness. Viruses were obtained by cocultivation with donor monocyte-derived macrophages and T-lymphocytes and their biological phenotype compared to viruses obtained from the peripheral blood mononuclear cells of the same patient. The biological phenotype was determined on established cell lines (U937-2, CEM, and MT-2) and on the U87.CD4 coreceptor indicator cell lines and variable region 3 (V3) of the envelope was subjected to direct sequencing. All isolates obtained from lymph node subsets used CCR5 as coreceptor. Furthermore, these viruses were also sensitive to inhibition by beta-chemokines as analyzed for viruses of one patient. All 12 V3 regions showed a unique sequence indicating compartmentalization within each patient. The biological phenotype of CCR5-dependent (R5) HIV-1 isolates obtained from PBMC resembles the phenotype of viruses isolated from different lymph node cell subsets.

Twenty-four-hour rhythms of mitogenic responses, lymphocyte subset populations and amino acid content in submaxillary lymph nodes of growing male rats subjected to calorie restriction

Peripubertal male Wistar rats were submitted to a calorie restriction diet enriched in proteins and low in fat and carbohydrates for 4 weeks, and starting on day 35 of life. Mitogenic responses, lymphocyte subset populations, interferon (IFN)- γ release and amino acid content were determined in submaxillary lymph nodes at six time intervals during the 24-h span. The diet employed completely arrested growth in growing rats. After caloric restriction, mean values of Con A response, lymph node T and CD4 + cell number and CD4 +/CD8 + ratio augmented, whereas those of B cell number, IFN-γ release and glutamine and glutamate concentration decreased. Calorie restriction modified 24-h rhythmicity of lymph node mitogenic responses to Con A and LPS, and of T, T–B, CD4 + and CD4 +–CD8 + lymph node cell subsets. It also changed the 24-h pattern of lymph node IFN-γ release and glutamine, aspartate, glutamate and taurine lymph node content. Availability of nutrients presumably affects the mechanisms that modulate the circadian variation of immune responsiveness in growing rats.

Appearance of Extrathymic Early Differentiated CD4 -CD8 - T Cells with T Cell Receptor γ/δ or α/β after Thymus Grafting to Nude Mice: Influence of Thymus on Extrathymic T Cell Differentiation

Fetal thymus grafting into athymic nude mice has been used as an experimental model of T cell development. To understand the early events of T cell development, we have examined the sequence of appearance of T cell subsets in lymph nodes (LN) of BALB/c nu/nu mice after grafting with syngeneic fetal thymus. T cells expressing T cell receptor (TCR) α/β or γ/δ increased in LN from 1 week after grafting, although no host-derived CD3 + T cells were detected in the grafted thymus and no donor thymus-derived T cells were detected in the LN. The early appearing T cells of both TCR α/β and TCR γ/δ showed a CD4 -CD8 - phenotype. V region usage analysis of the early appearing TCR γ/β T cells revealed that they contained cells bearing Vβ3 or Vβ11, which are potentially reactive to self-superantigen MIs-2 a or Dvb11, respectively, and are deleted in the course of T cell development in the thymus of euthymic BALB/c mice. The early appearing T cells showed neither mixed lymphocyte reaction nor cytotoxic T cell activity against allogeneic cells. In contrast, lymphokine-activated killer cells from early appearing T cells, which contained high percentages of TCR γ/δ T cells, exhibited higher cytotoxic activity against P815 mastocytoma than those from euthymic mice or untreated nude mice. All these results suggest that the early appearing T cells are developed extrathymically. We propose that the thymus may induce extrathymic T cell development without direct cell-to-cell interaction. It seems likely that the extrathymically developed T cells, especially TCR γ/δ T cells, induced by the thymus have some role in the defense mechanism in the absence of conventional thymus-derived T cells.

Antigen-induced changes in B cell subsets in lymph nodes: analysis by dual fluorescence flow cytofluorometry.

Changes in the representation and surface phenotype of defined B cell subsets in murine lymph nodes stimulated with keyhole limpet hemocyanin or sheep red blood cells have been analyzed by two-color immunofluorescence fluorocytometric analysis. Shortly after immunization with either antigen there is a dramatic increase in both the frequency and absolute number of IgM+, IgD+ B cells, which is followed by the formation of germinal centers. Germinal center cells, as soon as they appear on day 3 after primary immunization, bind high levels of peanut agglutinin, bear low levels of surface IgM but no detectable surface IgD, and are characterized by lack of staining with MEL-14, a monoclonal antibody which recognizes a lymphocyte surface receptor involved in lymphocyte homing. The level of I-A and H-2K region-encoded surface antigens on early germinal center cells is higher than on PNAlo B cells. During the first 7 days of the germinal centers there is a progressive decrease in the average level of H-2K but not of Ia antigens. A similar decrease was observed for ThB. It is confirmed that the germinal center cell population contains the majority of antigen-binding cells in the stimulated lymph node. These findings indicate that B cells are recruited nonspecifically to antigen-stimulated lymph nodes, and that the antigen-specific cells then selectively participate in the formation of germinal centers where they undergo specific differentiation events.