Peripheral Lymph Node Lymphocytes Research Articles

Current and future aspects of IBD research and treatment: The 2022 perspective

Inflammatory bowel diseases (IBD) have seen major progress in current concepts and treatment regimes. Based on the theory of an inadequate “overshoot” of the mucosal immune response to the intestinal microbiome, therapies have been developed to interfere with the key mediators of inflammation from cytokines, including TNF and IL12/23, to integrins such as α4ß7 and intracellular cytokine signal transducers such as janus kinases. Recently, sphingosine-1-receptor agonists were marketed to suppress mucosal inflammation by sequestering lymphocytes in peripheral lymph nodes. However, the aim of these regimes targeting immunity to induce a long-term deep remission, including mucosal healing, is missed in most patients. Contrasting these anti-inflammatory mechanisms of action, the pathogenic focus has finally shifted to the mucosal antibacterial barrier in both Crohn´s disease and ulcerative colitis. Translating this novel concept requires a completely different approach but, in the end, may come closer to a cure of these devastating diseases, in which an incomplete immune modulation fails to achieve the key endpoints: halting disease activity and progression. This review aims to give an overview of past, current, and future concepts in IBD, focusing on both pathogenesis and consequent therapy. A cure is in sight only if both reflect the actual key mechanisms of slow bacterial entry into the mucosa and are harmonized and in line.

Atrophy of skin-draining lymph nodes predisposes for impaired immune responses to secondary infection in mice with chronic intestinal nematode infection.

Intestinal nematodes suppress immune responses in the context of allergy, gut inflammation, secondary infection and vaccination. Several mechanisms have been proposed for this suppression including alterations in Th2 cell differentiation and increased Treg cell suppressive function. In this study, we show that chronic nematode infection leads to reduced peripheral responses to vaccination because of a generalized reduction in the available responsive lymphocyte pool. We found that superficial skin-draining lymph nodes (LNs) in mice that are chronically infected with the intestinal nematode Heligmosomides polygyrus, do not reach the same cellularity as worm-free mice upon subsequent BCG infection in the skin. B cells and T cells, all declined in skin-draining LN of H. polygyrus-infected mice, resulting in LNs atrophy and altered lymphocyte composition. Importantly, anti-helminthic treatment improved lymphocyte numbers in skin-draining LN, indicating that time after de-worming is critical to regain full-scale LN cellularity. De-worming, and time for the skin LN to recover cellularity, also mended responses to Bacille Calmette-Guerin (BCG) in the LN draining the footpad injection site. Thus, our findings show that chronic nematode infection leads to a paucity of lymphocytes in peripheral lymph nodes, which acts to reduce the efficacy of immune responses at these sites.

P2.01-055 Lymphocytes' Subtypes Differentiation after Stimulation with Synthetic Antigen-Pulsed Dendritic Cells in Lung Adenocarcinoma Patients

Immunotherapy in lung cancer is currently experiencing huge interest. The greatest efficacy demonstrated the antibodies against immune checkpoints receptors (PD-L1, CTLA-4 or LAG-3). However, an effective anti-tumor response also required tumor antigen presentation to lymphocytes in peripheral lymph nodes. To enhance this effect, vaccination with synthetic tumor antigen was conducted in many clinical trials using antigen presenting cells (APC). Unfortunately, this type of active immunotherapy has not achieved spectacular success (START or MAGRIT studies). The explanation could lie in distorted presentation of tumor antigens in peripheral lymph nodes or in activation of lymphocytes with strong immunosuppressive properties. In our study, we checked which pathways of T helper differentiation are favored by autologous dendritic cells (DCs) after synthetic tumor antigens stimulation. Peripheral blood was acquired from twenty unresectable and treatment-naïve lung adenocarcinoma patients. Using CD14 magnetic beads, two cell populations were isolated. CD14-negative cells were used for CD3-positive cells isolation, which were frozen until further stimulation. CD14-positive cells were cultured in CellDCGrow medium supplemented with IL-4 and GM-CSF. After TNF-a stimulation, mature DCs were incubated with tumor-specific antigens: MUC1, MAGE-A3, EGFR and CMV (positive stimulator) or only with medium (negative control). Flow cytometry and specific monoclonal antibodies were used to analyze immunophenotype of DCs: CD1a, CD11c, CD83, CD80, CD86, B7-H1 (PD-L1), B7-DC (PD-L2). Mature autologous DCs were cultured with fraction of CD3-positive cells and after 48h of mixed cultures, the expression of intracellular factors specific for different T helper cells subpopulation (T-bet – Th1, STAT-6 – Th2, ROR-gT – Th17, FoxP3 – Treg) and expression of extracellular interleukin receptors (IL-12R, IL-4R, IL-23R, TGF-bR, IL-2R) were analyzed. Autologous DCs were well generated and showed phenotype specific for fully-mature DCs. In culture with DCs after MUC1 stimulation, we observed in lymphocytes the highest expression of ROR-gT and IL-23R (p<0.05) compared with lymphocytes from cultures with other synthetic tumor antigens. Expression of IL-4R on lymphocytes was also significantly (0<0.05) higher in the culture stimulated with MUC1. The highest expression of intracellular FoxP3 factor, TGF-bR (p<0.05) and IL-2R (p=0.009) on Th cells were observed in mixed culture with DCs after MAGE-A3 stimulation. Dendritic cells stimulated with synthetic tumor antigens could activate different T helper cell populations. It could depend on the nature of antigens and could influence the effectiveness of stimulation different lymphocyte subtypes in peripheral lymph nodes. Therefore, our study clarifies some failure reasons of large clinical trials using active antigen-specific immunotherapy.

Modulating sphingosine 1‐phosphate signaling with DOP or FTY720 alleviates vascular and immune defects in mouse sepsis

Sepsis is a systemic inflammatory response to pathogens and a leading cause of hospital related mortality worldwide. Sphingosine 1-phosphate (S1P) regulates multiple cellular processes potentially involved in the pathogenesis of sepsis, including antigen presentation, lymphocyte egress, and maintenance of vascular integrity. We thus explored the impact of manipulating S1P signaling in experimental polymicrobial sepsis in mice. Administration of 4-deoxypyridoxine (DOP), an inhibitor of the S1P-degrading enzyme S1P-lyase, or of the sphingosine analog FTY720 that serves as an S1P receptor agonist after phosphorylation ameliorated morbidity, improved recovery from sepsis in surviving mice, and reduced sepsis-elicited hypothermia and body weight loss. Treated mice developed lymphopenia, leading to an accumulation of lymphocytes in peripheral lymph nodes, and reduced bacterial burden in liver, but not in blood. Sepsis-induced upregulation of mRNA expression of cytokines in spleen remained unchanged, but reduction of IL-6, TNF-α, MCP-1, and IL-10 in plasma was evident. DOP and FTY720 treatment significantly reduced levels of Evans blue leakage from blood into liver and lung, decreased hematocrit values, and lowered plasma levels of VEGF-A in septic mice. Collectively, our results indicate that modulation of S1P signaling showed a protective phenotype in experimental sepsis by modulating vascular and immune functions.

HHV-6, Not JC Virus, Causes Demyelination in PML Connections Between HHV-6, HIV-1 and JC Virus in Multiple Sclerosis, Neuro-AIDS and PML A Commentary and Review Offering Guidelines for Treatment

In recent years, two outstandingly effective drugs for the treatment of multiple sclerosis (MS) have appeared on the market: Tysabri® (Biogen Idec/Elan, 2008) and Gilenya® (Novartis, 2010). Tysabri (Natalizumab) is a humanized monoclonal antibody that binds to the cellular adhesion molecule alpha-4 integrin, which is used by lymphocytes to cross vascular walls and penetrate the blood-brain barrier. Tysabri thus reduces the flux of infected or autoimmune lymphocytes into the CNS. Gilenya (Fingolimod) is an immunomodulatory drug that acts on the sphingosine-1-phosphate receptor to sequester lymphocytes in peripheral lymph nodes, thus also indirectly reducing the flux of infected or autoimmune lymphocytes into the CNS. Both drugs have great clinical promise for treating MS, but both have revealed a critical shortcoming: their use increases the risk of Progressive Multifocal Leucoencephalopathy (PML), a demyelinating disease worse than MS and quickly fatal if not controlled. PML arises in individuals whose immune function is compromised, such as patients undergoing chemotherapy for various kinds of cancer. During the early years of the AIDS epidemic (approximately from 1986-1999), the incidence of PML rose in parallel with increasing numbers of immunocompromised AIDS patients, then fell after effective combined aggressive retrovirus treatment (cART) for HIV-1/AIDS became widely available (after 2000-2001). Unfortunately, the incidence of PML is now rising again in conjunction with the increased use of Tysabri and Gilenya in treating MS. It is therefore important to identify effective treatment modalities for PML.

Prevention, but not cure, of autoimmune diabetes in a NOD.scid transfer model by FTY720 despite effective modulation of blood T cells

FTY720 modulates lymphocyte trafficking through blood (peripheral blood lymphocyte, PBL) and peripheral lymph nodes (PLN). Treatment with FTY720 causes retention of most blood lymphocytes in PLN. Long-term treatment can slow and/or prevent Type 1 diabetes (T1D) in the nonobese diabetic (NOD) mouse model. B and T cells are both affected by FTY720 binding to sphingosine-1-phosphate receptor 1 (S1P1). However, little has been done to elucidate which T-cell subsets are differentially affected by FTY720 under healthy conditions, and how this affects disease pathogenesis in T1D. In healthy C57BL/6J (B6) mice, total CD4+ and CD8+ T-cell subsets were diminished by FTY720, but recently activated and memory subsets were spared and constituted significantly higher percentage of remaining T cells in blood. FTY720 also lowered PBL counts in NOD mice, but less severely than in B6 mice. This is consistent with a different ratio of naïve, activated, and memory cells in NOD mice compared to those in B6 mice, as well as alterations in S1P1 and sphingosine-1-phosphate (S1P) levels in PBLs and blood of NOD mice, respectively. To address the functional consequences of PBL T-cell depletion, we studied the effects of FTY720 on disease progression in a timed adoptive transfer model of T1D. Continuous treatment with FTY720 eliminated T1D, if treatment was started before splenocyte transfer. FTY20 treatment started after disease onset slowed disease progression. The inability to fully suppress memory and effector T-cell circulation may explain why FTY720 is only partially effective in the NOD adoptive transfer model of T1D.

FTY720, a Novel Immunosuppressant, Induces Sequestration of Circulating Mature Lymphocytes by Acceleration of Lymphocyte Homing in Rats. I. FTY720 Selectively Decreases the Number of Circulating Mature Lymphocytes by Acceleration of Lymphocyte Homing

FTY720, given i.v. or orally at 0.03 mg/kg or more, significantly prolonged skin allograft survival in a dose-dependent manner and showed more potent immunosuppressive activity than cyclosporin A (CsA) or tacrolimus (FK506) in MHC-incompatible rat strains of WKAH donors and F344 recipients. However, unlike CsA or FK506, FTY720 up to 1000 nM did not affect IL-2 production in allogeneic MLC. Within 3 to 24 h after a single oral administration of FTY720 at 0.1 to 1 mg/kg, the number of lymphocytes in the rats was markedly decreased in the peripheral blood and thoracic duct lymph and partially in spleen. By contrast, the number of lymphocytes in peripheral lymph nodes (PLN), mesenteric lymph nodes (MLN), and Peyer's patches (PP) was significantly increased at the same time. Intravenous transfusion of calcein-labeled rat lymphocytes into rats revealed that FTY720 significantly accelerated lymphocyte homing to PLN, MLN, and PP, dose dependently. Since FTY720-induced lymphocyte homing was completely blocked by simultaneous treatment of the calcein-labeled lymphocytes with mAbs against CD62L, CD49d, and CD11a before the transfusion, the acceleration of lymphocyte homing by FTY720 appears to be mediated by lymphocyte-homing receptors. These findings indicate that FTY720 sequesters circulating mature lymphocytes into PLN, MLN, and PP by acceleration of lymphocyte homing and thereby decreases the number of lymphocytes in peripheral blood, thoracic duct lymph, and spleen. Based on these observations, sequestration of circulating mature-lymphocytes is presumed to be a main mechanism of the immunosuppressive activity of FTY720.

Generation of Intestinal Mucosal Lymphocytes in SCID Mice Reconstituted with Mature, Thymus-Derived T Cells

Transfer of peripheral lymph node lymphocytes to SCID mice leads to the long term establishment of mucosal T lymphocytes within the epithelium and lamina propria of the small and large intestines. Analysis of engrafted intraepithelial lymphocytes (IEL) showed that they had acquired a surface phenotype that in several respects is typical of IEL. In addition, the functional profile of engrafted IEL derived from lymph node T cells was similar to that of normal IEL; as the donor-derived T cells exhibited a strong cytolytic activity, a poor proliferative response to mitogenic stimuli, and a tendency to home and expand specifically in the intestine upon transfer to secondary SCID recipients. Optimal engraftment of intestinal T cells required bacterial flora, as the number of lymphocytes was greatly reduced in SCID recipients with a reduced flora. These results demonstrate that mature, thymus-derived T cells can migrate to the intestine and become functionally specialized to the intestinal milieu. The acquisition of phenotypic markers characteristic of the intestinal microenvironment by engrafted cells suggests that T cell migration of lymphocytes to the SCID intestine is not aberrant, but it may reflect processes that are ongoing in immunocompetent mice. Furthermore, these data suggest that the homing and/or expansion of typical, thymus-derived T cells in the intestine may be driven by luminal Ags such as those derived from bacterial flora.

Factors controlling T-cell migration across rat cerebral endothelium in vitro

The migration of lymphocytes through primary cultures of rat brain microvascular endothelial cell monolayers was examined in vitro by time-lapse videomicroscopy. Antigen-specific T cell line migration was dependent on the duration of culture (post-antigen stimulation) with exogenous interleukin-2 (IL-2). Peak migration (approximately 50% of T-cells during the 4 h migration assay) occurred after 4 days of culture with IL-2 but did not coincide with maximal expression of LFA-1, VLA-4 or the IL-2 receptor. On unstimulated endothelia antibody blockade of LFA-1 or ICAM-1 inhibited T-cell line migration to 8.0% and 6.8% of control values, respectively, whereas blocking VLA-4 and VCAM-1 had no effect. On IL- β activated endothelium blocking LFA-1 and ICAM-1 was less effective (24.9% and 27.3% of control values, respectively) and blockade of VLA-4 and VCAM-1 brought about a reduction to 63.0% and 68.3% of controls respectively. Inhibition of IL-2-dependent proliferation with an IL-2 receptor blocking antibody also significantly inhibited T-cell migration to 22.2% of controls. Peripheral lymph node (PLN) lymphocytes could also be induced to migrate through untreated cerebral endothelial cell monolayers by cross-linking CD3 which was also time and IL-2-dependent with maximal migration (22.7%) occurring after three days in the presence of exogenous IL-2. Blocking LFA-1 or ICAM-1 resulted in a significant reduction in migration across IL-1 β-activated endothelial cells to 17.4% and 20.9% of control values respectively although blocking the VLA-4/VCAM-1 interaction had no significant effect. Activation of PLN lymphocytes with concanavalin A for up to 5 days did not induce migration but when left in contact with the endothelial monolayer for 24 h migration reached 31.0%. These studies indicate that T-cells require a combination of signals to trigger the migratory phenotype which is necessary to enable them to penetrate the blood–brain barrier.

Effects of the route of infection on immunoglobulin G subclasses and specificity of the reovirus-specific humoral immune response.

Reovirus serotype 1, strain Lang (T1/L), a well characterized enteric virus, elicits humoral and cellular immune responses in mice. Although orally and intradermally induced infections generate comparable reovirus-specific serum antibody titers, little is known about the effects of the route of infection on the systemic immunoglobulin G (IgG) response. To assess whether the route of exposure affects virus-specific humoral immunity, we infected various strains of mice with reovirus T1/L by the oral or intradermal routes. At day 10 following infection, virus-specific serum antibody titers and IgG subclasses were determined by enzyme-linked immunosorbent assay. Serum IgG2a and IgG2b antibodies were detected in all mouse strains independent of the route of infection. Mice of the H-2d haplotype that received an intradermal infection also had high levels of reovirus-specific serum IgG1. This dichotomy of responses was not associated with differences in the types of cytokine produced by draining peripheral lymph nodes. However, peripheral lymph node lymphocytes from C3H mice produced significantly higher levels of gamma interferon than did BALB/c, C57BL/6, and B10.D2 mice. Additionally, peripheral lymph node lymphocytes from all strains of mice produced only low levels of interleukin-5, with no detectable level of interleukin-4 or interleukin-6. Analysis of specific antibody at inductive sites of the immune response showed that orally infected Peyer's patches produced predominantly IgA and intradermally infected peripheral lymph nodes produced predominantly IgG2a. Western blot (immunoblot) analysis showed that virus-specific IgA, IgG1, and IgG2a reacted with reovirus structural proteins. These data suggest that the route of infection affects the isotype and IgG subclasses, but not the antigen specificity, of the local antibody response. In addition, virus-specific IgG1 generated following an intradermally induced infection is linked to the H-2d major histocompatibility complex haplotype.

Stress-induced reduction in the rat mixed lymphocyte reaction is due to macrophages and not to changes in T cell phenotypes

Exposure to aversive events or Stressors modulates various aspects of immune function. We have previously reported that exposure to an acute Stressor, inescapable tail shock (IS), resulted in a shift in T cell subpopulations in rat mesenteric lymph nodes but not in cervical lymph nodes (Fleshner et al. (1992) J. Neuroimmunol. 41, 131–142). The mesenteric CD4 + CD8 + ratio was increased immediately after exposure to IS and was due primarily to an increase in the percent of CD4 + cells. The present experiments were designed to determine the relationship between the IS-associated phenotypic shift and its significance in the function of CD4 + T cells. The function assessed was the in vitro proliferative response to alloantigens coded for by the Major Histocompatibility Complex (MHC). Using the mixed lymphocyte reaction (MLR), we report that exposure to IS resulted in a decrease in the MLR response of cells from both cervical and mesenteric lymph nodes. Depletion of macrophages (nylon wool adherent cells) eliminated the IS-induced reduction and co-culture of macrophages (irradiation-insensitive cells) from shocked rats produced the suppression. One interpretation of these data is that exposure to IS resulted in the activation of macrophages and the release of a suppressive factor which reduced the MLR response of peripheral lymph node lymphocytes.

Evidence that indefinite survival of small bowel allografts achieved by a brief course of cyclosporine or FK506 is not due to systemic hyporesponsiveness.

The immunological status of Lewis (LEW) recipients of indefinitely surviving (greater than 400 days) orthotopic Brown-Norway (BN) small bowel allografts was investigated 1 to 1 1/2 years after cessation of immunosuppressive therapy with either cyclosporine or FK506 and compared with recipients of syngeneic grafts. A normal proliferative response (as measured by a mixed lymphocyte culture) of recipient peripheral lymph node lymphocytes in response to the donor-specific (BN) and the third-party (ACI) antigen, was observed in all experimental groups. Cytolytic T cell generation (as measured by a standard 51Cr-release cytotoxicity assay) in response to the donor-specific (BN) and the third-party (ACI) antigen was observed also in all groups. A FACS analysis of allograft-recipient splenocytes showed no evidence for systemic lymphoid chimerism. BN or ACI skin grafts transplanted onto recipients of allogeneic and syngeneic small bowel grafts were rejected completely in 12-17 days, while the intestinal grafts remained functional. Immunohistologic evaluation of the allografts, using anti-BN class I and anti-Lewis class II monoclonal antibodies showed anti-BN staining on the epithelial and endothelial structures, whereas the mononuclear cells in the lamina propria stained positively with the anti-LEW monoclonal antibody. However, lymphoid depletion and scarring of Peyer's patches and mesenteric lymph nodes as well as focal obliterative mesenteric arteriopathy, indicative of an indolent chronic rejection, were observed. These data demonstrate that recipients of indefinitely surviving small bowel allografts remain immune competent and do not retain the intestinal graft on the basis of specific hyporesponsiveness to the donor antigens.

Mechanism of action of thymosin. I. Thymosin fraction 5 increases lymphokine production by mature murine T cells responding in a mixed lymphocyte reaction.

The effect of thymosin on the murine thymocyte mixed lymphocyte response was studied. Thymosin fraction 5 (TF5) caused a two- to threefold enhancement of the proliferative response and production of IL 2 when murine thymocytes were cultured with alloantigenic stimulator cells. Production of a second lymphokine, CSF, was increased up to sevenfold. The target cell for thymosin was a mature T cell, because the PNA- subpopulation of thymocytes, as well as peripheral lymph node lymphocytes, responded to culture with TF5 and alloantigen by enhanced proliferation and lymphokine production. The active component of TF5 appears to be one or more as yet unidentified peptides, because neither of the well-characterized TF5 component peptides, alpha 1 or beta 4, were active. After incubation with TF5 in primary culture, cells remaining after 10 to 14 days were increased both in number and in secondary response to alloantigen, as measured by lymphokine production. These results suggest that TF5 contains one or more biologically active components which can modulate mature T cell activity and lymphokine production, and which provide the basis for understanding some of the previously reported diverse effects of thymosin.

Lymphocyte locomotion and attachment on two-dimensional surfaces and in three-dimensional matrices.

The adhesion and locomotion of mouse peripheral lymph node lymphocytes on 2-D protein- coated substrata and in 3-D matrices were compared. Lymphocytes did not adhere to, or migrate on, 2-D substrata suck as serum- or fibronectin-coated glass. They did attach to and migrate in hydrated 3-D collagen lattices. When the collagen was dehydrated to form a 2-D surface, lymphocyte attachment to it was reduced. We propose that lymphocytes, which are poorly adhesive, are able to attach to and migrate in 3-D matrices by a nonadhesive mechanism such as the extension and expansion of pseudopodia through gaps in the matrix, which could provide purchase for movement in the absence of discrete intermolecular adhesions. This was supported by studies using serum-coated micropore filters, since lymphocytes attached to and migrated into filters with pore sizes large enough (3 or 8 mum) to allow pseudopod penetration but did not attach to filters made of an identical material (cellulose esters) but of narrow pore size (0.22 or 0.45 mum). Cinematographic studies of lymphocyte locomotion in collagen gels were also consistent with the above hypothesis, since lymphocytes showed a more variable morphology than is typically seen on plane surfaces, with formation of many small pseudopodia expanded to give a marked constriction between the cell and the pseudopod. These extensions often remained fixed with respect to the environment as the lymphocyte moved away from or past them. This suggests that the pseudopodia were inserted into gaps in the gel matrix and acted as anchorage points for locomotion.

Inhibition of Lymphocyte Trapping by Cell-Free Ascitic Fluids Cultivated in Syngeneic Mice

Several cellfree ascitic fluids were tested for their influence on the trapping of lymphocytes in peripheral lymph nodes after subcutaneous antigen administration. Fluid from three ascitic tumors cultivated in syngeneic mice diminished the trapping response significantly. Fluid from one of these ascitic tumors cultivated in allogeneic mice showed no inhibition of trapping. Syngeneic normal mouse serum did not significantly affect the trapping phenomenon. It was concluded that syngeneic ascitic fluids contain a substance capable of inhibiting lymphocyte trapping.