Lung-draining Mediastinal Lymph Nodes Research Articles

Cutting Edge: First Lung Infection Permanently Enlarges Lymph Nodes and Enhances New T Cell Responses.

Humans experience frequent respiratory infections. Immunology and vaccinology studies in mice are typically performed in naive specific pathogen-free animals responding to their very first respiratory challenge. We found that the first respiratory infection induces lifelong enlargement of the lung-draining mediastinal lymph nodes (medLNs). Furthermore, infection-experienced medLNs supported better naive T cell surveillance and effector responses to new unrelated infections that exhibited more biased accumulation and memory establishment within the lung. Moreover, we observed that weight loss induced by influenza infection was substantially reduced in mice that had recovered from a previous unrelated respiratory viral challenge. These data show that the lack of infectious history and corresponding medLN hypoplasia in specific pathogen-free mice alter their immune response to lung infections. Preclinical vaccination and immunology studies should consider the previous infectious experience of the model organism.

Runx3 Regulates CD8+ T Cell Local Expansion and CD43 Glycosylation in Mice by H1N1 Influenza A Virus Infection.

We have recently reported that transcription factor Runx3 is required for pulmonary generation of CD8+ cytotoxic T lymphocytes (CTLs) that play a crucial role in the clearance of influenza A virus (IAV). To understand the underlying mechanisms, we determined the effects of Runx3 knockout (KO) on CD8+ T cell local expansion and phenotypes using an inducible general Runx3 KO mouse model. We found that in contrast to the lungs, Runx3 general KO promoted enlargement of lung-draining mediastinal lymph node (mLN) and enhanced CD8+ and CD4+ T cell expansion during H1N1 IAV infection. We further found that Runx3 deficiency greatly inhibited core 2 O-glycosylation of selectin ligand CD43 on activated CD8+ T cells but minimally affected the cell surface expression of CD43, activation markers (CD44 and CD69) and cell adhesion molecules (CD11a and CD54). Runx3 KO had a minor effect on lung effector CD8+ T cell death by IAV infection. Our findings indicate that Runx3 differently regulates CD8+ T cell expansion in mLNs and lungs by H1N1 IAV infection. Runx3 is required for CD43 core 2 O-glycosylation on activated CD8+ T cells, and the involved Runx3 signal pathway may mediate CD8+ T cell phenotype for pulmonary generation of CTLs.

L-selectin-dependent and -independent homing of naïve lymphocytes through the lung draining lymph node support T cell response to pulmonary Mycobacterium tuberculosis infection.

Recruiting large numbers of naïve lymphocytes to lymph nodes is critical for mounting an effective adaptive immune response. While most naïve lymphocytes utilize homing molecule L-selectin to enter lymph nodes, some circulating cells can traffic to the lung-draining mediastinal lymph node (mLN) through lymphatics via the intermediate organ, lung. However, whether this alternative trafficking mechanism operates in infection and contributes to T cell priming are unknown. We report that in pulmonary Mycobacterium tuberculosis-infected mice, homing of circulating lymphocytes to the mLN is significantly less efficient than to non-draining lymph node. CD62L blockade only partially reduced the homing of naïve T lymphocytes, consistent with L-selectin-independent routing of naïve lymphocytes to the site. We further demonstrated that lymphatic vessels in infected mLN expanded significantly and inhibiting lymphangiogenesis with a vascular endothelial growth factor receptor 3 kinase inhibitor reduced the recruitment of intravenously injected naïve lymphocytes to the mLN. Finally, mycobacterium-specific T cells entering via the L-selectin-independent route were readily activated in the mLN. Our study suggests that both L-selectin-dependent and -independent pathways contribute to naïve lymphocyte entry into mLN during M. tuberculosis infection and the latter pathway may represent an important mechanism for orchestrating host defence in the lungs.

Nonclassical monocytes potentiate anti-tumoral CD8+ T cell responses in the lungs.

CD8+ T cells drive anti-cancer immunity in response to antigen-presenting cells such as dendritic cells and subpopulations of monocytes and macrophages. While CD14+ classical monocytes modulate CD8+ T cell responses, the contributions of CD16+ nonclassical monocytes to this process remain unclear. Herein we explored the role of nonclassical monocytes in CD8+ T cell activation by utilizing E2-deficient (E2-/-) mice that lack nonclassical monocytes. During early metastatic seeding, modeled by B16F10-OVA cancer cells injected into E2-/- mice, we noted lower CD8+ effector memory and effector T cell frequencies within the lungs as well as in lung-draining mediastinal lymph nodes in the E2-/- mice. Analysis of the myeloid compartment revealed that these changes were associated with depletion of MHC-IIloLy6Clo nonclassical monocytes within these tissues, with little change in other monocyte or macrophage populations. Additionally, nonclassical monocytes preferentially trafficked to primary tumor sites in the lungs, rather than to the lung-draining lymph nodes, and did not cross-present antigen to CD8+ T cells. Examination of the lung microenvironment in E2-/- mice revealed reduced CCL21 expression in endothelial cells, which is chemokine involved in T cell trafficking. Our results highlight the previously unappreciated importance of nonclassical monocytes in shaping the tumor microenvironment via CCL21 production and CD8+ T cell recruitment.

T cell kinetics reveal expansion of distinct lung T cell subsets in acute versus in resolved influenza virus infection.

Influenza virus infection is restricted to airway-associated tissues and elicits both cellular and humoral responses ultimately resulting in generation of memory cells able to initiate a rapid immune response against re-infections. Resident memory T cells confer protection at the site of infection where lung-resident memory T cells are important for protecting the host against homologous and heterologous influenza virus infections. Mapping kinetics of local and systemic T cell memory formation is needed to better understand the role different T cells have in viral control and protection. After infecting BALB/c mice with influenza virus strain A/Puerto Rico/8/1934 H1N1 the main proportion of activated T cells and B cells expressing the early activation marker CD69 was detected in lungs and lung-draining mediastinal lymph nodes. Increased frequencies of activated cells were also observed in the peripheral lymphoid organs spleen, inguinal lymph nodes and mesenteric lymph nodes. Likewise, antigen-specific T cells were most abundant in lungs and mediastinal lymph nodes but present in all organs studied. CD8+CD103-CD49a+ lung-resident T cells expanded simultaneously with timing of viral clearance whereas CD8+CD103+CD49a+ lung-resident T cells was the most abundant subset after resolution of infection and antigen-specific, lung-resident T cells were detected up to seven months after infection. In conclusion, the results in this detailed kinetic study demonstrate that influenza virus infection elicits adaptive immune responses mainly in respiratory tract-associated tissues and that distinct subsets of lung-resident T cells expand at different time points during infection. These findings contribute to the understanding of the adaptive immune response locally and systemically following influenza virus infection and call for further studies on the roles of the lung-resident T cell subsets.

Pertussis toxin suppresses dendritic cell-mediated delivery of B. pertussis into lung-draining lymph nodes

The adenylate cyclase (ACT) and the pertussis (PT) toxins of Bordetella pertussis exert potent immunomodulatory activities that synergize to suppress host defense in the course of whooping cough pathogenesis. We compared the mouse lung infection capacities of B. pertussis (Bp) mutants (Bp AC− or Bp PT–) producing enzymatically inactive toxoids and confirm that ACT action is required for maximal bacterial proliferation in the first days of infection, whereas PT action is crucial for persistence of B. pertussis in mouse lungs. Despite accelerated and near complete clearance from the lungs by day 14 of infection, the PT− bacteria accumulated within the lymphoid tissue of lung-draining mediastinal lymph nodes (mLNs). In contrast, the wild type or AC− bacteria colonized the lungs but did not enter into mLNs. Lung infection by the PT− mutant triggered an early arrival of migratory conventional dendritic cells with associated bacteria into mLNs, where the PT− bacteria entered the T cell-rich paracortex of mLNs by day 5 and proliferated in clusters within the B-cell zone (cortex) of mLNs by day 14, being eventually phagocytosed by infiltrating neutrophils. Finally, only infection by the PT− bacteria triggered an early production of anti-B. pertussis serum IgG antibodies already within 14 days of infection. These results reveal that action of the pertussis toxin blocks DC-mediated delivery of B. pertussis bacteria into mLNs and prevents bacterial colonization of mLNs, thus hampering early adaptive immune response to B. pertussis infection.

Lung memory B cells promote local IgE production in the respiratory tract

Abstract Allergen-specific IgE mediates allergic asthma. IgE responses including class-switch recombination (CSR) to IgE are tightly regulated. However, the regulation of local CSR to IgE at mucosal sites is poorly understood. This study aims to elucidate cellular sources that promote IgE CSR and mechanisms that regulate IgE production in the respiratory tract. To model allergic airway inflammation, we repeatedly administered intranasal ragweed pollen and OVA to mice. We profiled B cell responses to OVA in the lung, the lung-draining mediastinal lymph nodes (medLN), spleen and bone marrow. We used fluorescent reporter mice to track IgE CSR and identify B cell subsets that promote IgE CSR by flow cytometry and microscopy. We found that intranasal allergen exposure elicited robust local B cell responses with the formation of OVA-specific B cells in the medLN and OVA-specific IgE in the airway. OVA-specific germinal center B cells and memory B cells (MBCs) accumulated in the lungs. Using fluorescent reporter mice, we showed that IgG1+ MBCs are the predominant IgE-switching cells. Importantly, the number of IgE-switching MBCs are the highest in the lungs, and IgG1+ MBCs exhibit an increased frequency of IgE CSR in the lungs compared to lymphoid tissues. These suggest that the lung environment favors IgE production. RNA-seq analyses comparing lung and circulating MBCs revealed elevated expression of Tnfsf4 (OX40L) and Icosl on lung MBCs, suggesting a higher propensity for lung MBCs to engage T cells. Together, these data demonstrate that the allergic lung is a major site of IgE CSR with IgG1+ MBCs promoting local IgE CSR. Our ongoing work focuses on delineating the B cell-intrinsic and extrinsic factors that promote IgE production in the respiratory mucosa. Supported by grants from NIH (1F31 HL156459, 5T32 AI007508, 1R21 AI159456)

Temporal dynamics of persistent germinal centers and memory B cell differentiation following respiratory virus infection.

SummaryFollowing infection or immunization, memory B cells (MBCs) and long-lived plasma cells provide humoral immunity that can last for decades. Most principles of MBC biology have been determined with hapten-protein carrier models or fluorescent protein immunizations. Here, we examine the temporal dynamics of the germinal center (GC) B cell and MBC response following mouse influenza A virus infection. We find that antiviral B cell responses within the lung-draining mediastinal lymph node (mLN) and the spleen are distinct in regard to duration, enrichment for antigen-binding cells, and class switching dynamics. While splenic GCs dissolve after 6 weeks post-infection, mLN hemagglutinin-specific (HA+) GCs can persist for 22 weeks. Persistent GCs continuously differentiate MBCs, with “peak” and “late” GCs contributing equal numbers of HA+ MBCs to the long-lived compartment. Our findings highlight critical aspects of persistent GC responses and MBC differentiation following respiratory virus infection with direct implications for developing effective vaccination strategies.

303 Non-functional T cell responses in non-small cell lung cancer are induced during tumor antigen-specific T cell priming in the mediastinal lymph node

BackgroundIn non-small cell lung cancer (NSCLC), response to checkpoint blockade therapy (CBT) is associated with tumor-infiltrating CD8+ T cells, but not all T cell-infiltrated tumors respond to CBT. The subgroup of T cell-infiltrated but CBT-resistant tumors has been clinically described as containing ”non-functional” T cell responses. Mechanisms governing the generation of non-functional T cell responses remain poorly understood, and treatment options for this subgroup are limited.MethodsWe utilized a transplantable, syngeneic murine NSCLC cell line derived from an autochthonous NSCLC driven by KrasG12D expression and p53 deletion (KP cell line) to model non-functional T cell responses. To study antigen-specific responses, we engineered KP cells to express the model CD8+ T cell antigen SIY for certain experiments. CBT consisted of combined anti-CTLA-4 and anti-PD-L1 therapy.ResultsOrthotopic KP lung tumors failed to respond to CBT, but KP flank tumors were controlled by CBT. Lung and flank tumors contained activated CD8+ T cells, providing a platform to compare functional and non-functional CD8+ T cell responses in NSCLC. Single-cell RNA sequencing revealed that lung tumor-infiltrating CD8+ T cells lacked effector and exhaustion molecules despite clonal expansion. Analysis of antigen-specific CD8+ T cells revealed that this lung cancer-specific T cell dysfunction was established during priming in lung-draining mediastinal lymph nodes (mLN) despite robust T cell proliferation. RNA sequencing and flow cytometry of antigen-specific CD8+ T cells found that T cells primed in the mLN underwent blunted effector differentiation characterized by a lack of effector molecules CD25, Granzyme B, and TIM-3, but retention of TCF-1. This phenotype persisted in lung tumors, consistent with our initial observations of absent effector and exhaustion molecule expression. Many CD8+ T cells from NSCLC patients expressed an analogous gene expression program distinct from T cell exhaustion. TCF-1+ CD8+ T cells in lung tumors did not mediate tumor control and failed to differentiate into effector cells after CBT. To investigate alternative therapeutic strategies of reinvigorating lung tumor-reactive T cells, we focused on IL-2 and IL-12, as expression of their receptors was reduced in mLN-primed T cells. Administering recombinant IL-2 and IL-12 was sufficient to restore effector T cell differentiation, induce lung tumor control, and significantly extend survival of lung tumor-bearing mice.ConclusionsOur results suggest that non-functional CD8+ T cell responses in NSCLC arise from failed effector T cell differentiation during priming. Transient combination therapy with IL-2 and IL-12 overcomes this dysfunctional state to induce protective T cell responses in CBT-resistant tumors.Ethics ApprovalAll mouse experiments were approved by MIT’s Committee on Animal Care (CAC) - DHHS Animal Welfare Assurance # D16-00078

Influenza virus infection selectively triggers the accumulation and persistence of more potent Helios-expressing Foxp3+ regulatory T cells in the lungs.

Foxp3+ regulatory T cells (Tregs) represent a special lineage of CD4+ T cells. Analysis of Treg response during primary and secondary influenza virus infection clearly demonstrates a robust accumulation of Tregs into the infected lungs and the existence of a population of long-lived antigen-specific memory Tregs in the same tissues after resolution of the infection. However, it remains unknown whether these Tregs co-express Helios, a member of the Ikaros transcription factor family. In this study, Foxp3+ Helios+ and Foxp3+ Helios- Tregs in the lungs, mLNs and spleens of influenza virus-infected and uninfected control mice were tracked. The data show that while there is a co-existence of Foxp3+ Helios+ and Foxp3+ Helios- Tregs in the tissues, the accumulated Tregs in the lungs and lung-draining mediastinal lymph nodes (mLNs) of the infected mice are highly enriched for Foxp3+ Helios+ cells. It was further demonstrated that, after the clearance of primary infection, Foxp3+ Helios+ cells have the ability to persist in the tissues over their Helios- counterparts. More importantly, Foxp3+ Helios+ Tregs accumulated in an accelerated kinetics during recall response to reinfection. In vitro analysis of Treg suppressive function revealed that Foxp3+ Helios+ Tregs are more capable of suppressing influenza virus-specific CD8+ T cell activation, cytokine production and proliferation. Together, the data provide new insights into Treg responses during primary and secondary influenza virus infection and suggest that Foxp3+ Helios+ Tregs predominantly drive the Treg responses.

Protective function and durability of mouse lymph node-resident memory CD8+ T cells.

Protective lung tissue-resident memory CD8+T cells (Trm) form after influenza A virus (IAV) infection. We show that IAV infection of mice generates CD69+CD103+and other memory CD8+T cell populations in lung-draining mediastinal lymph nodes (mLNs) from circulating naive or memory CD8+T cells. Repeated antigen exposure, mimicking seasonal IAV infections, generates quaternary memory (4M) CD8+T cells that protect mLN from viral infection better than 1M CD8+T cells. Better protection by 4M CD8+T cells associates with enhanced granzyme A/B expression and stable maintenance of mLN CD69+CD103+4M CD8+T cells, vs the steady decline of CD69+CD103+1M CD8+T cells, paralleling the durability of protective CD69+CD103+4M vs 1M in the lung after IAV infection. Coordinated upregulation in canonical Trm-associated genes occurs in circulating 4M vs 1M populations without the enrichment of canonical downregulated Trm genes. Thus, repeated antigen exposure arms circulating memory CD8+T cells with enhanced capacity to form long-lived populations of Trm that enhance control of viral infections of the mLN.

Vaccination With the Commensal Streptococcus mitis Expressing Pneumococcal Serotype 5 Capsule Elicits IgG/IgA and Th17 Responses Against Streptococcus pneumoniae.

Recent studies have identified a clinical isolate of the commensal Streptococcus mitis that expresses Streptococcus pneumoniae serotype 5 capsule (S. mitis serotype 5) and shows serospecificity toward pneumococcal serotype 5. However, it remains unknown whether S. mitis serotype 5 induces protective immunity against pneumococcal serotype 5. In this study, we evaluated the ability of S. mitis serotype 5 to generate protective immunity in a mouse model of lung infection with pneumococcal serotype 5. Upon challenge infection with S. pneumoniae serotype 5, mice intranasally immunized with S. mitis serotype 5 exhibited reduced pneumococcal loads in the lungs, nasal wash, and bronchoalveolar lavage fluid compared with those receiving PBS (control). The immunized mice displayed significantly higher levels of IgG and IgA antibodies reactive to S. mitis serotype 5, S. pneumoniae serotype 5 or S. pneumoniae serotype 4 than the antibody levels in control mice. In vaccinated mice, the IgG/IgA antibody levels reactive to S. mitis serotype 5 or S. pneumoniae serotype 5 were higher than the levels reactive to S. pneumoniae serotype 4. Furthermore, in-vitro restimulation of the lung-draining mediastinal lymph node cells and splenocytes from immunized mice with killed S. mitis serotype 5, S. pneumoniae serotype 5 or S. pneumoniae serotype 4 showed enhanced Th17, but not Th1 and Th2, responses. Overall, our findings show that mucosal immunization with S. mitis serotype 5 protects against S. pneumoniae serotype 5 infection and induces Th17 and predominant serotype-specific IgG/IgA antibody responses against pneumococcal infection.

Dispensable role of chemokine receptors in migration of mycobacterial antigen-specific CD4+ T cells into mycobacteria-infected lung

Abstract Mycobacterial antigen-specific CD4+ Th1 cells have a pivotal role in protective immunity against mycobacterial infections, including pulmonary tuberculosis. In the course of infection, Th1 cells differentiate in the lung-draining lymph nodes and migrate into the infected lung. Chemokine receptors on T cells are involved in T cell migration into the intestine and skin. However, the role of chemokine receptors in the migration of CD4+ T cells into the lung has not yet been determined. To address this issue, the role of chemokine receptors in T cell migration into the mycobacteria-infected lung was analyzed using mycobacterial Ag85B peptide 25-specific T cell receptor-transgenic (P25) CD4+ T cells. P25 T cells in the Mycobacterium bovis BCG-infected lung and lung-draining mediastinal lymph nodes (MedLN) expressed the chemokine receptors, CCR5, CCR6, CXCR3, and CXCR5, which bind chemokines produced by the BCG-infected lung. To further analyze the role of chemokine receptors in the migration of BCG-primed P25 T cells into the lung and medLN, P25 T cells were adoptively transferred into BCG-infected wild-type mice and their migration into the lung was monitored. Unexpectedly, blocking chemokine receptor function with pertussis toxin, a G-protein inhibitor, failed to suppress migration of T cells into the infected lung. However, the treatment completely blocked migration of the MedLN P25 T cells into the recipient lymph node. These results suggest that the interaction of chemokine receptors on mycobacterial antigen-specific Th1 cells with chemokines is dispensable for their migration into the mycobacteria-infected lung.

Site-Specific DC Surface Signatures Influence CD4+ T Cell Co-stimulation and Lung-Homing.

Dendritic cells (DCs) that drain the gut and skin are known to favor the establishment of T cell populations that home to the original site of DC-antigen (Ag) encounter by providing soluble “imprinting” signals to T cells in the lymph node (LN). To study the induction of lung T cell-trafficking, we used a protein-adjuvant murine intranasal and intramuscular immunization model to compare in vivo-activated Ag+ DCs in the lung and muscle-draining LNs. Higher frequencies of Ag+ CD11b+ DCs were observed in lung-draining mediastinal LNs (MedLN) compared to muscle-draining inguinal LNs (ILN). Ag+ CD11b+ MedLN DCs were qualitatively superior at priming CD4+ T cells, which then expressed CD49a and CXCR3, and preferentially trafficked into the lung parenchyma. CD11b+ DCs from the MedLN expressed higher levels of surface podoplanin, Trem4, GL7, and the known co-stimulatory molecules CD80, CD86, and CD24. Blockade of specific MedLN DC molecules or the use of sorted DC and T cell co-cultures demonstrated that DC surface phenotype influences the ability to prime T cells that then home to the lung. Thus, the density of dLN Ag+ DCs, and DC surface molecule signatures are factors that can influence the output and differentiation of lung-homing CD4+ T cells.

CD8+ T Cells require IL-27R for optimal generation of an effector response during influenza infection

Abstract Previous reports have demonstrated the many effects of IL-27 on CD4 T cell activation and differentiation, in addition to a role for IL-27 in cancer therapies mediated by cytolytic CD8 T cells. However, as IL-27 is produced by many cell types during infection, and the IL-27 receptor (IL-27R) is widely expressed on NK cells, as well as on activated CD4 and CD8 T cells, the direct effects of IL-27 signaling on CD8 T cell function are less well understood. We used a murine model of influenza infection in combination with co-transfer of congenic wild type (WT) and IL-27R−/− OT-I CD8 T cells to investigate whether IL-27 signaling had a direct effect on virus-specific CD8 T cell function in vivo. We observed a dramatic reduction in the frequency and number of IL-27R−/− OT-I compared to their WT OT-I counterparts within the same host following influenza infection, indicating a CD8 T cell-intrinsic role for IL-27 in the generation of an acute anti-viral response. This defect was observed in the spleen, lung interstitium, airways and the lung-draining mediastinal lymph node, indicating a systemic defect in development of the CD8 T cell effector response in the absence of IL-27R signaling. Both memory precursor and short lived effector cells were reduced in the absence of IL-27R signaling indicating potential defects in both effector and memory generation. We are currently exploring the mechanism of defective effector CD8 T cell generation in the absence of cell-intrinsic IL-27R signaling. Overall, these findings show a cell-intrinsic role for IL-27 signaling in the generation of a robust effector CD8 T cell response following influenza infection.

Lung-draining lymph-node-resident memory CD8 T cells mediate local protective immunity

Abstract Recent studies indicate tissue-resident memory CD8 T cells (Trms) form at both the sites of initial infection and in lymphoid tissues upon resolution of systemic or localized viral infections. We examined if lymph node Trms were also generated in lung-draining mediastinal lymph nodes (mLN) during intranasal infections. Both transgenic P14 cells and endogenous influenza-specific CD69+/CD103+ Trms were detected in lung-draining mLN but absent in non-draining LNs after intranasal infection with recombinant influenza or vaccinia viruses. Similar to lung Trms, which wane over time, these primary memory (1M) mLN Trms had limited persistence with continual contraction throughout the memory phase. As influenza is a seasonal virus, and we previously observed that repeated antigen exposure extended the durability of lung Trms, we investigated the effects of multiple antigen exposures on the mLN Trm population. Similar to their lung Trm-counterparts, the lung-draining LN Trms derived from repeatedly stimulated (4M) memory CD8 T cells exhibited extended survival, down-regulation of Eomesodermin and the shared IL-2/15Rβ CD122 compared to 1M LN Trms. During parabiosis, both 1M and 4M mLN Trms largely maintained residence within their respective hosts. Importantly, hosts containing 4M P14 mLN Trms provided superior early protection against LCMV challenge in the mLN compared to 1M hosts, demonstrating the functional capacity for mLN Trms to contribute to protective anti-viral immune responses. Overall, these data indicate that lung-draining lymph node Trms co-express CD69 and CD103, largely maintain LN-tissue residency and can contribute to subsequent viral infections.

Dispensable role of chemokine receptors in migration of mycobacterial antigen-specific CD4+ T cells into Mycobacterium-infected lung

Mycobacterial antigen-specific CD4+ Th1 cells have pivotal role in protective immunity against mycobacterial infections including pulmonary tuberculosis. In the course of the infection, Th1 cells differentiate in the lung-draining lymph nodes and migrate into the infected lung. Chemokine receptors on T cells are involved in T cell migration into the intestine and skin. However, role of chemokine receptors in the migration of CD4+ T cells into the lung is not yet established. To address the issue, the role of chemokine receptors in T cell migration into the mycobacteria-infected lung was analyzed using mycobacterial Ag85B peptide 25-specific T cell receptor-transgenic (P25) CD4+ T cells. The P25 T cells in the Mycobacterium bovis BCG-infected lung and lung-draining mediastinal lymph node expressed chemokine receptors CCR5, CCR6, CXCR3 and CXCR5 which bind chemokines expressed by the BCG-infected lung. To further analyze the role of the chemokine receptors in the migration of the BCG-primed P25 T cells into the lung or mediastinal lymph node, the P25 T cells were adoptively transferred into the BCG-infected wild type mice, and their migration into the lung was monitored. Unexpectedly, blocking of chemokine receptor function with pertussis toxin, a G-protein inhibitor, failed to suppress migration of the T cells into the infected lung although the treatment completely blocked migration of the mediastinal lymph node P25 T cells into the recipient lymph node. The results suggest that interaction of chemokine receptors on mycobacterial antigen-specific Th1 cells with chemokines is dispensable in their migration into the mycobacteria-infected lung.

IκB kinase β inhibitor, BMS-345541, suppresses the dendritic cell response in a murine model of allergic airway inflammation

Abstract Pharmacological IκB kinase (IKK) inhibition could be an effective therapeutic strategy for the prevention and treatment of allergic asthma. Recently, we have demonstrated that a selective inhibitor of IKKβ prevented allergic asthma in a mouse model through inhibition of CD4+ effector T cell responses in the lung-draining mediastinal lymph nodes (MLNs). However, the precise mechanism responsible for this effect was not been fully elucidated. The migration of dendritic cells (DCs) from the lungs to the regional lymph nodes is necessary for the development of allergic airway inflammation (AAI). The study attempts to shed light on the involvement of DCs in the anti-asthmatic action of BMS-345541 (BMS), i.e. a selective inhibitor of IKKβ. We studied the effect of a 7-day treatment with BMS on the development of AAI and the absolute count of CD11chighCD11b+CD103− (CD11b+) and CD11chighCD11b−CD103+(CD103+) DCs in the MLNs and lungs of OVA-sensitized/challenged mice. The absolute count of CD11b+ and CD103+ DCs was increased in the MLNs of untreated OVA-immunized mice. Moreover, the absolute number of CD11b+, but not CD103+, DCs was increased in the lungs of these mice. BMS reduced, but did not fully prevent, the OVA-induced increase in the count of migratory CD11b+ DCs in the MLNs. In turn, administration of this agent prevented the OVA-induced increase in absolute counts of CD103+ DCs in the MLNs and CD11b+ DCs in the lungs. Treatment with BMS protected the lungs from OVA-induced AAI. These results strongly suggest that one of the mechanisms, and probably a key one, underlying the anti-asthmatic action of IKKβ inhibitors is the suppression of the formation of inflammatory DCs at the inflammatory site and their accumulation in the draining lymph nodes.

Intranasal delivery of adjuvant-free peptide nanofibers elicits resident CD8+ T cell responses

Influenza vaccines that can be administered intranasally or by other needle-free delivery routes have potential advantages over injected formulations in terms of patient compliance, cost, and ease of global distribution. Supramolecular peptide nanofibers have been investigated previously as platforms for vaccines and immunotherapies and have been shown to raise immune responses in the absence of exogenous adjuvants and without measurable inflammation. However, at present it has not been tested whether the immunogenicity of these materials extends to the intranasal route. Here we investigated the extent to which self-assembled peptide nanofibers bearing an influenza peptide epitope elicit antigen-specific CD8+ T cell responses when delivered intranasally, and we compared these responses with those elicited by subcutaneous immunization. Peptides containing an epitope from influenza acid polymerase (PA) and the Q11 self-assembly domain formed nanofibers that were avidly taken up by dendritic cells in lung-draining mediastinal lymph nodes after intranasal immunization. Intranasally delivered nanofibers generated greater antigen-specific CD8+ T cell responses in the lung-draining lymph nodes than subcutaneous immunizations while retaining the non-inflammatory character of the materials observed in other delivery sites. The CD8+ T cells elicited systemically were functional as assessed by their ability to produce IFN-γ ex vivo, lyse epitope-pulsed target cells in vivo, and diminish viral loads in infected mice. Compared to subcutaneously delivered nanofibers, intranasally delivered peptide nanofibers significantly increased the number of persisting antigen-specific tissue resident memory CD8+ T cells in the lung, allowing for a more rapid response to infection at 6 weeks post-vaccination. These results indicate that intranasally delivered self-assembled peptide nanofibers are immunogenic when delivering CD8+ epitopes without adjuvant or CD4+ epitopes, are non-inflammatory, and promote more lung-resident memory CD8+ T cells compared to subcutaneous immunization.

Increased T-helper 17.1 cells in sarcoidosis mediastinal lymph nodes.

The lung-draining mediastinal lymph nodes (MLNs) are currently widely used to diagnose sarcoidosis. We previously reported that T-helper (Th) 17.1 cells are responsible for the exaggerated interferon-γ production in sarcoidosis lungs. In this study, we aimed to investigate 1) whether Th17.1 cells are also increased in the MLNs of sarcoidosis patients and 2) whether frequencies of the Th17.1 cells at diagnosis may correlate with disease progression.MLN cells from treatment-naive pulmonary sarcoidosis patients (n=17) and healthy controls (n=22) and peripheral blood mononuclear cells (n=34) and bronchoalveolar lavage fluid (BALF) (n=36) from sarcoidosis patients were examined for CD4+ T-cell subset proportions using flow cytometry.Higher proportions of Th17.1 cells were detected in sarcoidosis MLNs than in control MLNs. Higher Th17.1 cell proportions were found in sarcoidosis BALF compared with MLNs and peripheral blood. Furthermore, BALF Th17.1 cell proportions were significantly higher in patients developing chronic disease than in patients undergoing resolution within 2 years of clinical follow-up.These data suggest that Th17.1 cell proportions in pulmonary sarcoidosis can be evaluated as a diagnostic and/or prognostic marker in clinical practice and could serve as a new therapeutic target.