Intestinal cDC1s provide cues required for CD4+ T cell-mediated resistance to Cryptosporidium.

Cryptosporidium is an enteric pathogen and a prominent cause of diarrheal disease worldwide. Control of Cryptosporidium requires CD4+ T cells, but how protective CD4+ T cell responses are generated is poorly understood. Here, Cryptosporidium parasites that express MHCII-restricted model antigens were generated to understand the basis for CD4+ T cell priming and effector function. These studies revealed that parasite-specific CD4+ T cells are primed in the draining mesenteric lymph node but differentiate into Th1 cells in the gut to provide local parasite control. Although type 1 conventional dendritic cells (cDC1s) were dispensable for CD4+ T cell priming, they were required for CD4+ T cell gut homing and were a source of IL-12 at the site of infection that promoted local production of IFN-γ. Thus, cDC1s have distinct roles in shaping CD4+ T cell responses to an enteric infection: first, to promote gut homing from the mesLN, and second, to drive effector responses in the intestine.

Lymphatic vessel transit seeds cytotoxic resident memory T cells in skin draining lymph nodes.

Lymphatic transport shapes the homeostatic immune repertoire of lymph nodes (LNs). LN-resident memory T cells (TRMs) play an important role in site-specific immune memory, yet how LN TRMs form de novo after viral infection remains unclear. Here, we tracked the anatomical distribution of antiviral CD8+ T cells as they seeded skin and LN TRMs using a model of vaccinia virus-induced skin infection. LN TRMs localized to the draining LNs (dLNs) of infected skin, and their formation depended on the lymphatic egress of effector CD8+ T cells from the skin, already poised for residence. Effector CD8+ T cell transit through skin was required to populate LN TRMs in dLNs, a process reinforced by antigen encounter in skin. Furthermore, LN TRMs were protective against viral rechallenge in the absence of circulating memory T cells. These data suggest that a subset of tissue-infiltrating CD8+ T cells egress from tissues during viral clearance and establish a layer of regional protection in the dLN basin.

Atypical memory B cells increase in the peripheral blood of patients with breast cancer regardless of lymph node involvement.

BACKGROUND: Breast cancer is the most common cancer in females. The immune system has a crucial role in the fight against cancer. B and T cells, the two main components of the adaptive immunity, are critical players that specifically target tumor cells. However, B cells, in contrast to T cells, and their role in cancer inhibition or progression is less investigated. Accordingly, in this study, we assessed and compared the frequency of naïve and different subsets of memory B cells in the peripheral blood of patients with breast cancer and healthy women. RESULTS: We found no significant differences in the frequencies of peripheral CD19+ B cells between the patients and controls. However, there was a significant decrease in the frequency of CD19+IgM+ B cells in patients compared to the control group (P=0.030). Moreover, the patients exhibited higher percentages of atypical memory B cells (CD19+CD27‒IgM‒, P=0.006) and a non-significant increasing trend in switched memory B cells (CD19+CD27+IgM‒, P=0.074). Further analysis revealed a higher frequency of atypical memory B cells (aMBCs) in the peripheral blood of patients without lymph node involvement as well as those with a tumor size greater than 2cm or with estrogen receptor (ER) negative/progesterone receptor (PR) negative tumors, compared with controls (P=0.030, P=0.040, P=0.031 and P=0.054, respectively). CONCLUSION: Atypical memory B cells (CD19+CD27‒IgM‒) showed a significant increase in the peripheral blood of patients with breast cancer compared to the control group. This increase seems to be associated with tumor characteristics. Nevertheless, additional research is necessary to determine the precise role of these cells during breast cancer progression.

Dimethyl fumarate modulates the regulatory T cell response in the mesenteric lymph nodes of mice with experimental autoimmune encephalomyelitis.

Dimethyl fumarate (DMF, Tecfidera) is an oral drug utilized to treat relapsing-remitting multiple sclerosis (MS). DMF treatment reduces disease activity in MS. Gastrointestinal discomfort is a common adverse effect of the treatment with DMF. This study aimed to investigate the effect of DMF administration in the gut draining lymph nodes cells of C57BL6/J female mice with experimental autoimmune encephalomyelitis (EAE), an animal model of MS. We have demonstrated that the treatment with DMF (7.5 mg/kg) significantly reduces the severity of EAE. This reduction of the severity is accompanied by the increase of both proinflammatory and anti-inflammatory mechanisms at the beginning of the treatment. As the treatment progressed, we observed an increasing number of regulatory Foxp3 negative CD4 T cells (Tr1), and anti-inflammatory cytokines such as IL-27, as well as the reduction of PGE2 level in the mesenteric lymph nodes of mice with EAE. We provide evidence that DMF induces a gradual anti-inflammatory response in the gut draining lymph nodes, which might contribute to the reduction of both intestinal discomfort and the inflammatory response of EAE. These findings indicate that the gut is the first microenvironment of action of DMF, which may contribute to its effects of reducing disease severity in MS patients.

The compensatory role of T cells from lymph nodes in mice with splenectomy.

The spleen is a vital organ for the immune system, while splenectomy may be necessary for various reasons. However, there is limited research on the impact of splenectomy on T cell function in peripheral lymph nodes as a compensatory mechanism in preventing infections. This study aimed to investigate the characteristics and function of CD8+ and CD4+ T cells in different peripheral lymph nodes during viral infection using a well-established splenectomy model. The results revealed that splenectomy caused an increase in CD8+GP33+ T cells in the mesenteric lymph nodes (MLN). Moreover, we demonstrated that splenectomy resulted in an increase of effector KLRG1+ T cells in the MLN. Additionally, the number of CD4+ cytotoxic T cells (CD4 CTLs) was also elevated in the peripheral lymph nodes of mice with splenectomy. Surprisingly, aged mice exhibited a stronger compensatory ability than adult mice, as evidenced by an increase in effector CD8+ T cells in all peripheral lymph nodes. These findings provide compelling evidence that T cells in MLN play a crucial role in protecting individuals with splenectomy against viral infections. The study offers new insights into understanding the changes in the immune system of individuals with splenectomy and highlights the potential compensatory mechanisms involved by T cells in peripheral lymph nodes.

A non-lipid nucleic acid delivery vector with dendritic cell tropism and stimulation.

Rationale: Nucleic acid constructs are commonly used for vaccination, immune stimulation, and gene therapy, but their use in cancer still remains limited. One of the reasons is that systemic delivery to tumor-associated antigen-presenting cells (dendritic cells and macrophages) is often inefficient, while off-target nucleic acid-sensing immune pathways can stimulate systemic immune responses. Conversely, certain carbohydrate nanoparticles with small molecule payloads have been shown to target these cells efficiently in the tumor microenvironment. Yet, nucleic acid incorporation into such carbohydrate-based nanoparticles has proven challenging. Methods: We developed a novel approach using cross-linked bis succinyl-cyclodextrin (b-s-CD) nanoparticles to efficiently deliver nucleic acids and small-molecule immune enhancer to phagocytic cells in tumor environments and lymph nodes. Our study involved incorporating these components into the nanoparticles and assessing their efficacy in activating antigen-presenting cells. Results: The multi-modality immune stimulators effectively activated antigen-presenting cells and promoted anti-tumor immunity in vivo. This was evidenced by enhanced delivery to phagocytic cells and subsequent immune response activation in tumor environments and lymph nodes. Conclusion: Here, we describe a new approach to incorporating both nucleic acids and small-molecule immune enhancers into cross-linked bis succinyl-cyclodextrin (b-s-CD) nanoparticles for efficient delivery to phagocytic cells in tumor environments and lymph nodes in vivo. These multi-modality immune stimulators can activate antigen-presenting cells and foster anti-tumor immunity. We argue that this strategy can potentially be used to enhance anti-tumor efficacy.

In Situ Sprayed Exosome-Cross-Linked Gel as Artificial Lymph Nodes for Postoperative Glioblastoma Immunotherapy.

One key challenge in postoperative glioblastoma immunotherapy is to guarantee a potent and durable T-cell response, which is restricted by the immunosuppressive microenvironment within the lymph nodes (LNs). Here, we develop an in situ sprayed exosome-cross-linked gel that acts as an artificial LN structure to directly activate the tumor-infiltrating T cells for prevention of glioma recurrence. Briefly, this gel is generated by a bio-orthogonal reaction between azide-modified chimeric exosomes and alkyne-modified alginate polymers. Particularly, these chimeric exosomes are generated from dendritic cell (DC)-tumor hybrid cells, allowing for direct and robust T-cell activation. The gel structure with chimeric exosomes as cross-linking points avoids the quick clearance by the immune system and thus prolongs the durability of antitumor T-cell immunity. Importantly, this exosome-containing immunotherapeutic gel provides chances for ameliorating functions of antigen-presenting cells (APCs) through accommodating different intracellular-acting adjuvants, such as stimulator of interferon genes (STING) agonists. This further enhances the antitumor T-cell response, resulting in the almost complete elimination of residual lesions after surgery. Our findings provide a promising strategy for postsurgical glioma immunotherapy that warrants further exploration in the clinical arena.

Patient-derived follicular lymphoma spheroids recapitulate lymph node signaling and immune profile uncovering galectin-9 as a novel immunotherapeutic target.

Follicular lymphoma (FL), the most common indolent non-Hodgkin lymphoma, constitutes a paradigm of immune tumor microenvironment (TME) contribution to disease onset, progression, and heterogenous clinical outcome. Here we present the first FL-Patient Derived Lymphoma Spheroid (FL-PDLS), including fundamental immune actors and features of TME in FL lymph nodes (LNs). FL-PDLS is organized in disc-shaped 3D structures composed of proliferating B and T cells, together with macrophages with an intermediate M1/M2 phenotype. FL-PDLS recapitulates the most relevant B-cell transcriptional pathways present in FL-LN (proliferation, epigenetic regulation, mTOR, adaptive immune system, among others). The T cell compartment in the FL-PDLS preserves CD4 subsets (follicular helper, regulatory, and follicular regulatory), also encompassing the spectrum of activation/exhaustion phenotypes in CD4 and CD8 populations. Moreover, this system is suitable for chemo and immunotherapy testing, recapitulating results obtained in the clinic. FL-PDLS allowed uncovering that soluble galectin-9 limits rituximab, rituximab, plus nivolumab/TIM-3 antitumoral activities. Blocking galectin-9 improves rituximab efficacy, highlighting galectin-9 as a novel immunotherapeutic target in FL. In conclusion, FL-PDLS maintains the crosstalk between malignant B cells and the immune LN-TME and constitutes a robust and multiplexed pre-clinical tool to perform drug screening in a patient-derived system, advancing toward personalized therapeutic approaches.

Molecular and immunological characteristics of postoperative relapse in lymph node-positive esophageal squamous cell cancer.

BACKGROUND: The molecular and immunological characteristics of primary tumors and positive lymph nodes in esophageal squamous cell carcinoma (ESCC) are unknown and the relationship with recurrence is unclear, which this study attempted to explore. METHODS: A total of 30 ESCC patients with lymph node positive (IIB-IVA) were enrolled. Among them, primary tumor and lymph node specimens were collected from each patient, and subjected to 551-tumor-targeted DNA sequencing and 289-immuno-oncology RNA panel sequencing to identify the different molecular basis and immunological features, respectively. RESULTS: The primary tumors exhibited a higher mutation burden than lymph nodes (p < 0.001). One-year recurrent ESCC exhibited a higher Mucin16 (MUC16) mutation rate (p = 0.038), as well as univariate and multivariate analysis revealed that MUC16 mutation is independent genetic factor associated with reduced relapse-free survival (univariate, HR: 5.39, 95% CI: 1.67-17.4, p = 0.005; multivariate, HR: 7.36, 95% CI: 1.79-30.23, p = 0.006). Transcriptomic results showed non-relapse group had higher cytolytic activity (CYT) score (p = 0.025), and was enriched in the IFN-α pathway (p = 0.036), while those in the relapsed group were enriched in the TNF-α/NF-κB (p = 0.001) and PI3K/Akt pathway (p = 0.014). CONCLUSION: The difference in molecular characteristics between primary lesions and lymph nodes may be the cause of the inconsistent clinical outcomes. Mutations of MUC16 and poor immune infiltration are associated with rapid relapse of nodes-positive ESCC.

Anti-inflammatory role of APRIL by modulating regulatory B cells in antigen-induced arthritis.

APRIL (A Proliferation-Inducing Ligand), a member of the TNF superfamily, was initially described for its ability to promote proliferation of tumor cells in vitro. Moreover, this cytokine has been related to the pathogenesis of different chronic inflammatory diseases, such as rheumatoid arthritis. This study aimed to evaluate the ability of APRIL in regulating B cell-mediated immune response in the antigen-induced arthritis (AIA) model in mice. AIA was induced in previously immunized APRIL-transgenic (Tg) mice and their littermates by administration of antigen (mBSA) into the knee joints. Different inflammatory cell populations in spleen and draining lymph nodes were analyzed using flow cytometry and the assay was performed in the acute and chronic phases of the disease, while cytokine levels were assessed by ELISA. In the acute AIA, APRIL-Tg mice developed a less severe condition and a smaller inflammatory infiltrate in articular tissues when compared with their littermates. We also observed that the total cellularity of draining lymph nodes was decreased in APRIL-Tg mice. Flow cytometry analysis revealed an increase of CD19+IgM+CD5+ cell population in draining lymph nodes and an increase of CD19+CD21hiCD23hi (B regulatory) cells in APRIL-Tg mice with arthritis as well as an increase of IL-10 and CXCL13 production in vitro.

Inhibition of caspase pathways limits CD4+ T cell loss and restores host anti-retroviral function in HIV-1 infected humanized mice with augmented lymphoid tissue.

The study of HIV infection and pathogenicity in physical reservoirs requires a biologically relevant model. The human immune system (HIS) mouse is an established model of HIV infection, but defects in immune tissue reconstitution remain a challenge for examining pathology in tissues. We utilized exogenous injection of the human recombinant FMS-like tyrosine kinase 3 ligand (rFLT-3 L) into the hematopoietic stem cell (HSC) cord blood HIS mouse model to significantly expand the total area of lymph node (LN) and the number of circulating human T cells. The results enabled visualization and quantification of HIV infectivity, CD4 T cell depletion and other measures of pathogenesis in the secondary lymphoid tissues of the spleen and LN. Treatment with the Caspase-1/4 inhibitor VX-765 limited CD4+ T cell loss in the spleen and reduced viral load in both the spleen and axillary LN. In situ hybridization further demonstrated a decrease in viral RNA in both the spleen and LN. Transcriptomic analysis revealed that in vivo inhibition of caspase-1/4 led to an upregulation in host HIV restriction factors including SAMHD1 and APOBEC3A. These findings highlight the use of rFLT-3 L to augment human immune system characteristics in HIS mice to support investigations of HIV pathogenesis and test host directed therapies, though further refinements are needed to further augment LN architecture and cellular populations. The results further provide in vivo evidence of the potential to target inflammasome pathways as an avenue of host-directed therapy to limit immune dysfunction and virus replication in tissue compartments of HIV+ persons.

Vaccine targeting to mucosal lymphoid tissues promotes humoral immunity in the gastrointestinal tract.

Viruses, bacteria, and parasites frequently cause infections in the gastrointestinal tract, but traditional vaccination strategies typically elicit little or no mucosal antibody responses. Here, we report a strategy to effectively concentrate immunogens and adjuvants in gut-draining lymph nodes (LNs) to induce gut-associated mucosal immunity. We prepared nanoemulsions (NEs) based on biodegradable oils commonly used as vaccine adjuvants, which encapsulated a potent Toll-like receptor agonist and displayed antigen conjugated to their surface. Following intraperitoneal administration, these NEs accumulated in gut-draining mesenteric LNs, priming strong germinal center responses and promoting B cell class switching to immunoglobulin A (IgA). Optimized NEs elicited 10- to 1000-fold higher antigen-specific IgG and IgA titers in the serum and feces, respectively, compared to free antigen mixed with NE, and strong neutralizing antibody titers against severe acute respiratory syndrome coronavirus 2. Thus, robust gut humoral immunity can be elicited by exploiting the unique lymphatic collection pathways of the gut with a lymph-targeting vaccine formulation.

Necrosis plays a role in the concentration of mycobacterial antigens in granulomas from Mycobacterium bovis naturally infected cattle.

The dynamics that develop between cells and molecules in the host against infection by Mycobacterium bovis, leads to the formation of granulomas mainly present in the lungs and regional lymph nodes in cattle. Cell death is one of the main features in granuloma organization, however, it has not been characterized in granulomatous lesions caused by M. bovis. In this study we aimed to identify the profiles of cell death in the granuloma stages and its relationship with the accumulation of bacteria. We identified necrosis, activated caspase-3, LC3B/p62 using immunohistochemistry and digital pathology analysis on 484 granulomatous lesions in mediastinal lymph nodes from 23 naturally infected cattle. Conclusions: greater amounts of mycobacterial antigens were identified in granulomas from calves compared with adult cattle. The highest percentage of necrosis and quantity of mycobacterial antigens were identified in granuloma stages (III/IV) from adults. The LC3B/p62 profile was heterogeneous in granulomas between adults and calves. Our data suggest that necrosis is associated with a higher amount of mycobacterial antigens in the late stages of granuloma and the development of autophagy appears to play an heterogeneous effector response against infection in adults and calves. These results represent one of the first approaches in the identification of cell death in the four stages of granulomas in bovine tuberculosis.

Fluorescence tracking demonstrates T cell recirculation is transiently impaired by radiation therapy to the tumor.

T cells recirculate through tissues and lymphatic organs to scan for their cognate antigen. Radiation therapy provides site-specific cytotoxicity to kill cancer cells but also has the potential to eliminate the tumor-specific T cells in field. To dynamically study the effect of radiation on CD8 T cell recirculation, we used the Kaede mouse model to photoconvert tumor-infiltrating cells and monitor their movement out of the field of radiation. We demonstrate that radiation results in loss of CD8 T cell recirculation from the tumor to the lymph node and to distant sites. Using scRNASeq, we see decreased proliferating CD8 T cells in the tumor following radiation therapy resulting in a proportional enrichment in exhausted phenotypes. By contrast, 5 days following radiation increased recirculation of T cells from the tumor to the tumor draining lymph node corresponds with increased immunosurveillance of the treated tumor. These data demonstrate that tumor radiation therapy transiently impairs systemic T cell recirculation from the treatment site to the draining lymph node and distant untreated tumors. This may inform timing therapies to improve systemic T cell-mediated tumor immunity.

Construction of lymph nodes-targeting tumor vaccines by using the principle of DNA base complementary pairing to enhance anti-tumor cellular immune response.

Tumor vaccines, a crucial immunotherapy, have gained growing interest because of their unique capability to initiate precise anti-tumor immune responses and establish enduring immune memory. Injected tumor vaccines passively diffuse to the adjacent draining lymph nodes, where the residing antigen-presenting cells capture and present tumor antigens to T cells. This process represents the initial phase of the immune response to the tumor vaccines and constitutes a pivotal determinant of their effectiveness. Nevertheless, the granularity paradox, arising from the different requirements between the passive targeting delivery of tumor vaccines to lymph nodes and the uptake by antigen-presenting cells, diminishes the efficacy of lymph node-targeting tumor vaccines. This study addressed this challenge by employing a vaccine formulation with a tunable, controlled particle size. Manganese dioxide (MnO2) nanoparticles were synthesized, loaded with ovalbumin (OVA), and modified with A50 or T20 DNA single strands to obtain MnO2/OVA/A50 and MnO2/OVA/T20, respectively. Administering the vaccines sequentially, upon reaching the lymph nodes, the two vaccines converge and simultaneously aggregate into MnO2/OVA/A50-T20 particles through base pairing. This process enhances both vaccine uptake and antigen delivery. In vitro and in vivo studies demonstrated that, the combined vaccine, comprising MnO2/OVA/A50 and MnO2/OVA/T20, exhibited robust immunization effects and remarkable anti-tumor efficacy in the melanoma animal models. The strategy of controlling tumor vaccine size and consequently improving tumor antigen presentation efficiency and vaccine efficacy via the DNA base-pairing principle, provides novel concepts for the development of efficient tumor vaccines.

Dissociation protocols influence the phenotypes of lymphocyte and myeloid cell populations isolated from the neonatal lymph node.

Frequencies and phenotypes of immune cells differ between neonates and adults in association with age-specific immune responses. Lymph nodes (LN) are critical tissue sites to quantify and define these differences. Advances in flow cytometry have enabled more multifaceted measurements of complex immune responses. Tissue processing can affect the immune cells under investigation that influence key findings. To understand the impact on immune cells in the LN after processing for single-cell suspension, we compared three dissociation protocols: enzymatic digestion, mechanical dissociation with DNase I treatment, and mechanical dissociation with density gradient separation. We analyzed cell yields, viability, phenotypic and maturation markers of immune cells from the lung-draining LN of neonatal and adult mice two days after intranasal respiratory syncytial virus (RSV) infection. While viability was consistent across age groups, the protocols influenced the yield of subsets defined by important phenotypic and activation markers. Moreover, enzymatic digestion did not show higher overall yields of conventional dendritic cells and macrophages from the LN. Together, our findings show that the three dissociation protocols have similar impacts on the number and viability of cells isolated from the neonatal and adult LN. However, enzymatic digestion impacts the mean fluorescence intensity of key lineage and activation markers that may influence experimental findings.

Immunology is for everyone.

This "Focus on Immunology" issue brings Cell's 50th anniversary celebrations straight to your lymph nodes! Special Leading Edge articles highlight the exciting past, present, and future of the increasingly interdisciplinary field of immunology.

A novel FK506-loading mesoporous silica nanoparticle homing to lymph nodes for transplant rejection treatment.

Tacrolimus (FK506) is an effective therapeutic for transplant rejection in clinical practice, primarily inhibiting rejection by suppressing the activation and proliferation of allogeneic T cells in the lymph nodes (LNs). However, conventional administration methods face challenges in directly delivering free FK506 to the LNs. In this study, we introduce a novel LN-targeted delivery system based on mesoporous silica nanoparticles (MSNs-FK506-MECA79). These particles were designed to selectively target high endothelial venules in LNs; this was achieved through surface modification with MECA79 antibodies. Their mean size and zeta potential were 201.18 ± 5.98 nm and - 16.12 ± 0.36 mV, respectively. Our findings showed that MSNs-FK506-MECA79 could accumulate in LNs and increase the local concentration of FK506 from 28.02 ± 7.71 ng/g to 123.81 ± 76.76 ng/g compared with the free FK506 treatment group. Subsequently, the therapeutic efficacy of MSNs-FK506-MECA79 was evaluated in a skin transplantation model. The treatment with MSNs-FK506-MECA79 could lead to a decrease in the infiltration of T cells in the grafts, a reduction in the grade of rejection, and a significant prolongation of survival. Consequently, this study presents a promising strategy for the active LN-targeted delivery of FK506 and improving the immunotherapeutic effects on transplant rejection.