Age-dependency of terminal ileum tissue resident memory T cell responsiveness profiles to S. Typhi following oral Ty21a immunization in humans.

BACKGROUND: The impact of aging on the immune system is unequivocal and results in an altered immune status termed immunosenescence. In humans, the mechanisms of immunosenescence have been examined almost exclusively in blood. However, most immune cells are present in tissue compartments and exhibit differential cell (e.g., memory T cells -TM) subset distributions. Thus, it is crucial to understand immunosenescence in tissues, especially those that are exposed to pathogens (e.g., intestine). Using a human model of oral live attenuated typhoid vaccine, Ty21a, we investigated the effect of aging on terminal ileum (TI) tissue resident memory T (TRM) cells. TRM provide immediate adaptive effector immune responsiveness at the infection site. However, it is unknown whether aging impacts TRM S. Typhi-responsive cells at the site of infection (e.g., TI). Here, we determined the effect of aging on the induction of TI S. Typhi-responsive TRM subsets elicited by Ty21a immunization. RESULTS: We observed that aging impacts the frequencies of TI-lamina propria mononuclear cells (LPMC) TM and TRM in both Ty21a-vaccinated and control groups. In unvaccinated volunteers, the frequencies of LPMC CD103- CD4+ TRM displayed a positive correlation with age whilst the CD4/CD8 ratio in LPMC displayed a negative correlation with age. We observed that elderly volunteers have weaker S. Typhi-specific mucosal immune responses following Ty21a immunization compared to adults. For example, CD103+ CD4+ TRM showed reduced IL-17A production, while CD103- CD4+ TRM exhibited lower levels of IL-17A and IL-2 in the elderly than in adults following Ty21a immunization. Similar results were observed in LPMC CD8+ TRM and CD103- CD8+ T cell subsets. A comparison of multifunctional (MF) profiles of both CD4+ and CD8+ TRM subsets between elderly and adults also showed significant differences in the quality and quantity of elicited single (S) and MF responses. CONCLUSIONS: Aging influences tissue resident TM S. Typhi-specific responses in the terminal ileum following oral Ty21a-immunization. This study is the first to provide insights in the generation of local vaccine-specific responses in the elderly population and highlights the importance of evaluating tissue immune responses in the context of infection and aging. TRIAL REGISTRATION: This study was approved by the Institutional Review Board and registered on ClinicalTrials.gov (identifier NCT03970304 , Registered 29 May 2019 - Retrospectively registered).

Oral typhoid vaccine Ty21a elicits antigen-specific resident memory CD4+ T cells in the human terminal ileum lamina propria and epithelial compartments.

BACKGROUND: Salmonella enterica serovar Typhi (S. Typhi) is a highly invasive bacterium that infects the human intestinal mucosa and causes ~ 11.9-20.6 million infections and ~ 130,000-223,000 deaths annually worldwide. Oral typhoid vaccine Ty21a confers a moderate level of long-lived protection (5-7 years) in the field. New and improved vaccines against enteric pathogens are needed but their development is hindered by a lack of the immunological correlates of protection especially at the site of infection. Tissue resident memory T (TRM) cells provide immediate adaptive effector immune responsiveness at the infection site. However, the mechanism(s) by which S. Typhi induces TRM in the intestinal mucosa are unknown. Here, we focus on the induction of S. Typhi-specific CD4+TRM subsets by Ty21a in the human terminal ileum lamina propria and epithelial compartments. METHODS: Terminal ileum biopsies were obtained from consenting volunteers undergoing routine colonoscopy who were either immunized orally with 4 doses of Ty21a or not. Isolated lamina propria mononuclear cells (LPMC) and intraepithelial lymphocytes (IEL) CD4+TRM immune responses were determined using either S. Typhi-infected or non-infected autologous EBV-B cell lines as stimulator cells. T-CMI was assessed by the production of 4 cytokines [interferon (IFN)γ, interleukin (IL)-2, IL-17A and tumor necrosis factor (TNF)α] in 36 volunteers (18 vaccinees and 18 controls volunteers). RESULTS: Although the frequencies of LPMC CD103+ CD4+TRM were significant decreased, both CD103+ and CD103- CD4+TRM subsets spontaneously produced significantly higher levels of cytokines (IFNγ and IL-17A) following Ty21a-immunization. Importantly, we observed significant increases in S. Typhi-specific LPMC CD103+ CD4+TRM (IFNγ and IL-17A) and CD103- CD4+TRM (IL-2 and IL-17A) responses following Ty21a-immunization. Further, differences in S. Typhi-specific responses between these two CD4+TRM subsets were observed following multifunctional analysis. In addition, we determined the effect of Ty21a-immunization on IEL and observed significant changes in the frequencies of IEL CD103+ (decrease) and CD103- CD4+TRM (increase) following immunization. Finally, we observed that IEL CD103- CD4+TRM, but not CD103+ CD4+TRM, produced increased cytokines (IFNγ, TNFα and IL-17A) to S. Typhi-specific stimulation following Ty21a-immunization. CONCLUSIONS: Oral Ty21a-immunization elicits distinct compartment specific immune responses in CD4+TRM (CD103+ and CD103-) subsets. This study provides novel insights in the generation of local vaccine-specific responses. Trial registration This study was approved by the Institutional Review Board and registered on ClinicalTrials.gov (identifier NCT03970304, Registered 29 May 2019-Retrospectively registered, http://www.ClinicalTrials.gov/NCT03970304).

Effect of the live oral attenuated typhoid vaccine, Ty21a, on systemic and terminal ileum mucosal CD4+ T memory responses in humans.

Our current understanding of CD4+ T-cell-mediated immunity (CMI) elicited by the oral live attenuated typhoid vaccine Ty21a is primarily derived from studies using peripheral blood. Very limited data are available in humans regarding mucosal immunity (especially CD4+ T) at the site of infection (e.g. terminal ileum; TI). Here using multiparametric flow cytometry, we examined the effect of Ty21a immunization on TI-lamina propria mononuclear cells (LPMC) and peripheral blood CD4+ T memory (TM) subsets in volunteers undergoing routine colonoscopy. Interestingly, we observed significant increases in the frequencies of LPMC CD4+ T cells following Ty21a immunization, restricted to the T effector/memory (TEM)-CD45RA+ (TEMRA) subset. Importantly, Ty21a immunization elicited Salmonella Typhi-responsive LPMC CD4+ T cells in all major TM subsets [interferon (IFN)γ and interleukin (IL)-17A in TEM; IFNγ and macrophage inflammatory protein (MIP)1ß in T central/memory (TCM); and IL-2 in TEMRA]. Subsequently, we analyzed LPMC S. Typhi-responsive CD4+ T cells in depth for multifunctional (MF) effectors. We found that LPMC CD4+ TEM responses were mostly MF, except for those cells exhibiting the characteristics associated with IL-17A responses. Finally, we compared mucosal to systemic responses and observed that LPMC CD4+S. Typhi-specific responses were unique and distinct from their systemic counterparts. This study provides the first demonstration of S. Typhi-specific CD4+ TM responses in the human TI mucosa and provides valuable information about the generation of mucosal immune responses following oral Ty21a immunization.

Early host immune responses in a human organoid-derived gallbladder monolayer to Salmonella Typhi strains from patients with acute and chronic infections: a comparative analysis.

Salmonella enterica serovar Typhi (S. Typhi), a human-restricted pathogen, invades the host through the gut to cause typhoid fever. Recent calculations of the typhoid fever burden estimated that more than 10 million new typhoid fever cases occur in low and middle-income countries, resulting in 65,400-187,700 deaths yearly. Interestingly, if not antibiotic-treated, upon the resolution of acute disease, 1%-5% of patients become asymptomatic chronic carriers. Chronically infected hosts are not only critical reservoirs of infection that transmit the disease to naive individuals but are also predisposed to developing gallbladder carcinoma. Nevertheless, the molecular mechanisms involved in the early interactions between gallbladder epithelial cells and S. Typhi remain largely unknown. Based on our previous studies showing that closely related S. Typhi strains elicit distinct innate immune responses, we hypothesized that host molecular pathways activated by S. Typhi strains derived from acutely and chronically infected patients would differ. To test this hypothesis, we used a novel human organoid-derived polarized gallbladder monolayer model, and S. Typhi strains derived from acutely and chronically infected patients. We found that S. Typhi strains derived from acutely and chronically infected patients differentially regulate host mitogen-activated protein kinase (MAPK) and S6 transcription factors. These variations might be attributed to differential cytokine signaling, predominantly via TNF-α and IL-6 production and appear to be influenced by the duration the isolate was subjected to selective pressures in the gallbladder. These findings represent a significant leap in understanding the complexities behind chronic S. Typhi infections in the gallbladder and may uncover potential intervention targets.

Controlled human infectious models, a path forward in uncovering immunological correlates of protection: Lessons from enteric fevers studies.

Enteric infectious diseases account for more than a billion disease episodes yearly worldwide resulting in approximately 2 million deaths, with children under 5 years old and the elderly being disproportionally affected. Enteric pathogens comprise viruses, parasites, and bacteria; the latter including pathogens such as Salmonella [typhoidal (TS) and non-typhoidal (nTS)], cholera, Shigella and multiple pathotypes of Escherichia coli (E. coli). In addition, multi-drug resistant and extensively drug-resistant (XDR) strains (e.g., S. Typhi H58 strain) of enteric bacteria are emerging; thus, renewed efforts to tackle enteric diseases are required. Many of these entero-pathogens could be controlled by oral or parenteral vaccines; however, development of new, effective vaccines has been hampered by lack of known immunological correlates of protection (CoP) and limited knowledge of the factors contributing to protective responses. To fully comprehend the human response to enteric infections, an invaluable tool that has recently re-emerged is the use of controlled human infection models (CHIMs) in which participants are challenged with virulent wild-type (wt) organisms. CHIMs have the potential to uncover immune mechanisms and identify CoP to enteric pathogens, as well as to evaluate the efficacy of therapeutics and vaccines in humans. CHIMs have been used to provide invaluable insights in the pathogenesis, host-pathogen interaction and evaluation of vaccines. Recently, several Oxford typhoid CHIM studies have been performed to assess the role of multiple cell types (B cells, CD8+ T, Tregs, MAIT, Monocytes and DC) during S. Typhi infection. One of the key messages that emerged from these studies is that baseline antigen-specific responses are important in that they can correlate with clinical outcomes. Additionally, volunteers who develop typhoid disease (TD) exhibit higher levels and more activated cell types (e.g., DC and monocytes) which are nevertheless defective in discrete signaling pathways. Future critical aspects of this research will involve the study of immune responses to enteric infections at the site of entry, i.e., the intestinal mucosa. This review will describe our current knowledge of immunity to enteric fevers caused by S. Typhi and S. Paratyphi A, with emphasis on the contributions of CHIMs to uncover the complex immunological responses to these organisms and provide insights into the determinants of protective immunity.

B and T Cell Immunity in Tissues and Across the Ages.

B and T cells are key components of the adaptive immune system and coordinate multiple facets of immunity including responses to infection, vaccines, allergens, and the environment. In humans, B- and T-cell immunity has been determined using primarily peripheral blood specimens. Conversely, human tissues have scarcely been studied but they host multiple adaptive immune cells capable of mounting immune responses to pathogens and participate in tissue homeostasis. Mucosal tissues, such as the intestines and respiratory track, are constantly bombarded by foreign antigens and contain tissue-resident memory T (TRM) cells that exhibit superior protective capacity to pathogens. Also, tissue-resident memory B (BRM) cells have been identified in mice but whether humans have a similar population remains to be confirmed. Moreover, the immune system evolves throughout the lifespan of humans and undergoes multiple changes in its immunobiology. Recent studies have shown that age-related changes in tissues are not necessarily reflected in peripheral blood specimens, highlighting the importance of tissue localization and subset delineation as essential determinants of functional B and T cells at different life stages. This review describes our current knowledge of the main B- and T-cell subsets in peripheral blood and tissues across age groups.

Elastase-dependent live attenuated swine influenza A viruses are immunogenic and confer protection against swine influenza A virus infection in pigs.

Influenza A viruses cause significant morbidity in swine, resulting in a substantial economic burden. Swine influenza virus (SIV) infection also poses important human public health concerns. Vaccination is the primary method for the prevention of influenza virus infection. Previously, we generated two elastase-dependent mutant SIVs derived from A/Sw/Saskatchewan/18789/02(H1N1): A/Sw/Sk-R345V (R345V) and A/Sw/Sk-R345A (R345A). These two viruses are highly attenuated in pigs, making them good candidates for a live-virus vaccine. In this study, the immunogenicity and the ability of these candidates to protect against SIV infection were evaluated in pigs. We report that intratracheally administrated R345V and R345A induced antigen-specific humoral and cell-mediated immunity characterized by increased production of immunoglobulin G (IgG) and IgA antibodies in the serum and in bronchoalveolar lavage fluid, high hemagglutination inhibition titers in serum, an enhanced level of lymphocyte proliferation, and higher numbers of gamma interferon-secreting cells at the site of infection. Based on the immunogenicity results, the R345V virus was further tested in a protection trial in which pigs were vaccinated twice with R345V and then challenged with homologous A/Sw/Saskatchewan/18789/02, H1N1 antigenic variant A/Sw/Indiana/1726/88 or heterologous subtypic H3N2 A/Sw/Texas/4199-2/9/98. Our data showed that two vaccinations with R345V provided pigs with complete protection from homologous H1N1 SIV infection and partial protection from heterologous subtypic H3N2 SIV infection. This protection was characterized by significantly reduced macroscopic and microscopic lung lesions, lower virus titers from the respiratory tract, and lower levels of proinflammatory cytokines. Thus, elastase-dependent SIV mutants can be used as live-virus vaccines against swine influenza in pigs.

Human Salmonella Typhi exposure generates differential multifunctional cross-reactive T-cell memory responses against Salmonella Paratyphi and invasive nontyphoidal Salmonella.

OBJECTIVE: There are no vaccines for most of the major invasive Salmonella strains causing severe infection in humans. We evaluated the specificity of adaptive T memory cell responses generated after Salmonella Typhi exposure in humans against other major invasive Salmonella strains sharing capacity for dissemination. METHODS: T memory cells from eleven volunteers who underwent controlled oral challenge with wt S. Typhi were characterised by flow cytometry for cross-reactive cellular cytokine/chemokine effector responses or evidence of degranulation upon stimulation with autologous B-lymphoblastoid cells infected with either S. Typhi, Salmonella Paratyphi A (PA), S. Paratyphi B (PB) or an invasive nontyphoidal Salmonella strain of the S. Typhimurium serovar (iNTSTy). RESULTS: Blood T-cell effector memory (TEM) responses after exposure to S. Typhi in humans evolve late, peaking weeks after infection in most volunteers. Induced multifunctional CD4+ Th1 and CD8+ TEM cells elicited after S. Typhi challenge were cross-reactive with PA, PB and iNTSTy. The magnitude of multifunctional CD4+ TEM cell responses to S. Typhi correlated with induction of cross-reactive multifunctional CD8+ TEM cells against PA, PB and iNTSTy. Highly multifunctional subsets and T central memory and T effector memory cells that re-express CD45 (TEMRA) demonstrated less heterologous T-cell cross-reactivity, and multifunctional Th17 elicited after S. Typhi challenge was not cross-reactive against other invasive Salmonella. CONCLUSION: Gaps in cross-reactive immune effector functions in human T-cell memory compartments were highly dependent on invasive Salmonella strain, underscoring the importance of strain-dependent vaccination in the design of T-cell-based vaccines for invasive Salmonella.

Association between S. Typhi-specific memory CD4+ and CD8+ T responses in the terminal ileum mucosa and in peripheral blood elicited by the live oral typhoid vaccine Ty21a in humans.

CD4+ and CD8+ T subsets are essential components of the adaptive immune system which act in concert at the site of infections to effectively protect against pathogens. Very limited data is available in humans regarding the relationship between CD4+ and CD8+ S. Typhi responsive cells in the terminal ileum mucosa (TI) and peripheral blood following Ty21a oral typhoid immunization. Here, we compared TI lamina propria mononuclear cells (LPMC) and peripheral blood CD4+ and CD8+ T memory (TM) subsets responses and their relationship by Spearman's correlation following Ty21a immunization in volunteers undergoing routine colonoscopy. We observed that Ty21a immunization (i) influences the homing and accumulation of both CD4+ and CD8+ T cells in the TI, particularly integrin α4ß7+ CCR9+ CD8+ T cells, (ii) elicits significantly higher frequencies of LPMC S. Typhi-responsive CD8+ T multifunctional (CD107a, IFNγ, IL-17A and/or MIP1ß) cells than their CD4+ T counterparts, and (iii) results in the correlation of LPMC CD4+ Teffector/memory (TEM) S. Typhi responses (CD107a, IFNγ, TNFα, IL-17A and/or MIP1ß) to their LPMC CD8+ TEM counterparts. Moreover, we demonstrated that these positive correlations between CD4+ and CD8+ TEM occur primarily in TI LPMC but not in PBMC, suggesting important differences in responses between the mucosal and systemic compartments following oral Ty21a immunization. This study provides the first demonstration of the correlation of S. Typhi-specific CD4+ and CD8+ TM responses in the human terminal ileum mucosa and provides valuable information regarding the generation of mucosal and systemic immune responses following oral Ty21a-immunization which might impact future vaccine design and development.

Attenuated Oral Typhoid Vaccine Ty21a Elicits Lamina Propria and Intra-Epithelial Lymphocyte Tissue-Resident Effector Memory CD8 T Responses in the Human Terminal Ileum.

Tissue-resident memory T cells (TRM) are newly defined memory T cells (TM) distinct from circulating TM subsets which have the potential to mount rapid protective immune responses at the site of infection. However, very limited information is available regarding the role and contribution of TRM in vaccine-mediated immune responses in humans at the site of infection. Here, we studied the role and contribution of tissue resident memory T cells (TRM) located in the terminal ileum (TI) (favored site of infection for S. Typhi) following oral Ty21a immunization in humans. We examined TI-lamina propria mononuclear cells (LPMC) and intra-epithelial lymphocytes (IEL) CD8+ TRM subsets obtained from healthy volunteers undergoing medically-indicated colonoscopies who were either immunized with Ty21a or unvaccinated. No significant differences in the frequencies of LPMC CD8+ TRM and CD8+CD69+CD103- T cells subsets were observed following Ty21a-immunization. However, LPMC CD8+ TRM exhibited significantly higher levels of cytokines (IFN-γ, IL-17A, and TNF-α) ex-vivo in Ty21a-vaccinated than in unvaccinated volunteers. LPMC CD8+ TRMS. Typhi-specific responses were evaluated using S. Typhi-infected targets and found to produce significantly higher levels of S. Typhi-specific IL-17A. In contrast, LPMC CD8+CD69+CD103- T cells produced significantly increased S. Typhi-specific levels of IFN-γ, IL-2, and IL-17A. Finally, we assessed CD8+ TRM in IEL and observed that the frequency of IEL CD8+ TRM is significantly lower following Ty21a immunization. However, ex-vivo IEL CD8+ TRM elicited by Ty21a immunization spontaneously produced significantly higher levels of cytokines (IFN-γ, IL-17A, IL-2, and TNF-α). This study provides the first demonstration of the effect of oral Ty21a vaccination on CD8+ TRM subsets (spontaneous and S. Typhi-specific) responses in the LPMC and IEL compartment of the human terminal ileum mucosa, contributing novel information to our understanding of the generation of mucosal immune responses following oral Ty21a-immunization.

Innate immune responses induced by classes of CpG oligodeoxynucleotides in ovine lymph node and blood mononuclear cells.

CpG ODN signal through Toll-like receptor 9 (TLR9) and trigger a cascade of events that lead to activation of innate and adaptive immune responses. Our current understanding of the immunobiology of host responses to CpG is based largely on studies on peripheral blood mononuclear cells (PBMC) and splenocytes. Little is known regarding CpG-induced responses in other lymphoid tissues. In the present study, we investigated responses induced by CpG in both PBMC and lymph nodes. Cells were isolated from the superficial cervical lymph node (LNC) and blood and then stimulated with CpG ODN (either A-, or B- or C-class ODN). Cytokine production was assayed by ELISA, and lymphocyte proliferation was determined by (3)H-thymidine incorporation. NK-like cytotoxicity was analyzed by lysis of (51)Cr-labelled target cells. All three classes of CpG induced IFNalpha and IFNgamma in LNC. In contrast, only A and C-class ODN induced IFNalpha and IFNgamma in PBMC. Moreover, the IFN levels in LNC were 20-40-fold higher than in PBMC. Furthermore, all classes of ODN induced higher IL-12 levels in LNC (five- to six-fold) than in PBMC. Both B and C-class ODN induced good proliferative responses in PBMC and LNC, but the A-class ODN did not induce proliferation of PBMC and only induced moderate proliferation of LNC. A-class ODN induced significant NK-like activity in LNC. Thus, all three classes of CpG ODN induced similar responses in LNC, and these responses were consistently higher than in PBMC. These observations indicate that CpG ODN-induced responses differ between blood and lymph nodes, and suggest that the functional classification of CpG ODN based on PBMC responses may not be directly applicable to cells from other immune tissues.

Systemic and Terminal Ileum Mucosal Immunity Elicited by Oral Immunization With the Ty21a Typhoid Vaccine in Humans.

BACKGROUND & AIMS: Systemic cellular immunity elicited by the Ty21a oral typhoid vaccine has been extensively characterized. However, very limited data are available in humans regarding mucosal immunity at the site of infection (terminal ileum [TI]). Here we investigated the host immunity elicited by Ty21a immunization on terminal ileum-lamina propria mononuclear cells (LPMC) and peripheral blood in volunteers undergoing routine colonoscopy. METHODS: We characterized LPMC-T memory (TM) subsets and assessed Salmonella enterica serovar Typhi (S Typhi)-specific responses by multichromatic flow cytometry. RESULTS: No differences were observed in cell yields and phenotypes in LPMC CD8+-TM subsets following Ty21a immunization. However, Ty21a immunization elicited LPMC CD8+ T cells exhibiting significant S Typhi-specific responses (interferon-γ, tumor necrosis factor-α, interleukin-17A, and/or CD107a) in all major TM subsets (T-effector/memory [TEM], T-central/memory, and TEM-CD45RA+), although each TM subset exhibited unique characteristics. We also investigated whether Ty21a immunization elicited S Typhi-specific multifunctional effectors in LPMC CD8+ TEM. We observed that LPMC CD8+ TEM responses were mostly multifunctional, except for those cells exhibiting the characteristics associated with cytotoxic responses. Finally, we compared mucosal with systemic responses and made the important observation that LPMC CD8+S Typhi-specific responses were unique and distinct from their systemic counterparts. CONCLUSIONS: This study provides the first demonstration of S Typhi-specific responses in the human terminal ileum mucosa and provides novel insights into the generation of mucosal immune responses following oral Ty21a immunization.

Mucosal-Associated Invariant T Cells in the Human Gastric Mucosa and Blood: Role in Helicobacter pylori Infection.

Mucosal-associated invariant T (MAIT) cells represent a class of antimicrobial innate-like T cells that have been characterized in human blood, liver, lungs, and intestine. Here, we investigated, for the first time, the presence of MAIT cells in the stomach of children, adults, and the elderly undergoing routine endoscopy and assessed their reactivity to Helicobacter pylori (H. pylori - Hp), a major gastric pathogen. We observed that MAIT cells are present in the lamina propria compartment of the stomach and display a similar memory phenotype to blood MAIT cells. We then demonstrated that gastric and blood MAIT cells are able to recognize H. pylori. We found that CD8(+) and CD4(-)CD8(-) (double negative) MAIT cell subsets respond to H. pylori-infected macrophages stimulation in a MR-1 restrictive manner by producing cytokines (IFN-γ, TNF-α, IL-17A) and exhibiting cytotoxic activity. Interestingly, we observed that blood MAIT cell frequency in Hp(+ve) individuals was significantly lower than in Hp(-ve) individuals. However, gastric MAIT cell frequency was not significantly different between Hp(+ve) and Hp(-ve) individuals, demonstrating a dichotomy between blood and gastric tissues. Further, we observed that the majority of gastric MAIT cells (>80%) expressed tissue-resident markers (CD69(+) CD103(+)), which were only marginally present on PBMC MAIT cells (<3%), suggesting that gastric MAIT cells are readily available to respond quickly to pathogens. These results contribute important new information to the understanding of MAIT cells function on peripheral and mucosal tissues and its possible implications in the host response to H. pylori.

Characterization and functional properties of gastric tissue-resident memory T cells from children, adults, and the elderly.

T cells are the main orchestrators of protective immunity in the stomach; however, limited information on the presence and function of the gastric T subsets is available mainly due to the difficulty in recovering high numbers of viable cells from human gastric biopsies. To overcome this shortcoming we optimized a cell isolation method that yielded high numbers of viable lamina propria mononuclear cells (LPMC) from gastric biopsies. Classic memory T subsets were identified in gastric LPMC and compared to peripheral blood mononuclear cells (PBMC) obtained from children, adults, and the elderly using an optimized 14 color flow cytometry panel. A dominant effector memory T (TEM) phenotype was observed in gastric LPMC CD4(+) and CD8(+) T cells in all age groups. We then evaluated whether these cells represented a population of gastric tissue-resident memory T (TRM) cells by assessing expression of CD103 and CD69. The vast majority of gastric LPMC CD8(+) T cells either co-expressed CD103/CD69 (>70%) or expressed CD103 alone (~20%). Gastric LPMC CD4(+) T cells also either co-expressed CD103/CD69 (>35%) or expressed at least one of these markers. Thus, gastric LPMC CD8(+) and CD4(+) T cells had the characteristics of TRM cells. Gastric CD8(+) and CD4(+) TRM cells produced multiple cytokines (IFN-γ, IL-2, TNF-α, IL-17A, MIP-1ß) and up-regulated CD107a upon stimulation. However, marked differences were observed in their cytokine and multi-cytokine profiles when compared to their PBMC TEM counterparts. Furthermore, gastric CD8(+) TRM and CD4(+) TRM cells demonstrated differences in the frequency, susceptibility to activation, and cytokine/multi-cytokine production profiles among the age groups. Most notably, children's gastric TRM cells responded differently to stimuli than gastric TRM cells from adults or the elderly. In conclusion, we demonstrate the presence of gastric TRM, which exhibit diverse functional characteristics in children, adults, and the elderly.

TLR9 signaling failure renders Peyer's patch regulatory B cells unresponsive to stimulation with CpG oligodeoxynucleotides.

Intestinal Peyer's patch (PP) regulatory CD21+ B cells (B(regs)) suppress TLR9-induced innate immune responses. However, it is not known whether TLR9 activation is regulated in PP B(regs). Here, we investigated the responses of PP B(regs) to stimulation with the TLR9 agonist CpG oligodeoxynucleotides (ODN). We observed that PP CD21+ B(regs) express high levels of TLR9 mRNA, but fail to proliferate when stimulated with CpG ODN. Furthermore, unlike CD21+ B cells from blood, PP CD21+ B(regs) do not secrete IgM or IL-12 following CpG ODN stimulation. We hypothesized that the unresponsiveness of PP B(regs) to CpG stimulation was due to an inability of the TLR9 agonist to activate the TLR9 signaling pathway in these cells. This was confirmed by kinome analysis which demonstrated dynamic patterns of phosphorylation of key TLR adaptor proteins such as IRAK1, TAK1, IKK and NF-kappaB-p65 in CpG-stimulated blood CD21+ B cells, consistent with activation of the TLR9 pathway. In contrast, stimulation of PP CD21+ B(regs) with CpG ODN resulted in phosphorylation patterns of these adaptor proteins suggestive of inactivation of the TLR9 pathway. The absence of apparent TLR9 signaling events immediately following stimulation indicated that signaling is blocked close to the receptor. Our observations suggest a novel mechanism by which the host regulates TLR responses in TLR-expressing cells with regulatory functions.

Co-stimulation with TLR7/8 and TLR9 agonists induce down-regulation of innate immune responses in sheep blood mononuclear and B cells.

Toll-like receptors (TLRs) play an important role in the activation of innate and adaptive immune responses. Stimulation with multiple TLR agonists may result in synergistic, complimentary or inhibitory effects on innate immune responses. In this study, we investigated the effects of co-stimulation of sheep peripheral blood mononuclear cells (PBMC) and B cells with agonists for TLR3, 4, 7/8 and 9. Sheep PBMC stimulated with either CpG (TLR9 agonist) or RNA oligoribonucleotides ([ORNs], TLR7/8 agonist) exhibited significant IL-12 production, but only CpG induced IFNalpha, IgM and proliferative responses. In contrast, poly(I:C) (TLR3 agonist) and LPS (TLR4 agonist) did not induce any of these responses. Interestingly, we observed that co-stimulation of PBMC with CpG+ORN or CpG+imiquimod (another TLR7/8 agonist) resulted in significant reduction in CpG-induced IFNalpha production, B cell proliferation and IgM responses. Pre-incubation of cells with CpG prior to exposure of the cells to imiquimod resulted in similar inhibitory responses indicating that the down-regulatory mechanisms are not associated with competition for cellular uptake or for receptors of the two agonists. Sheep B cells constitutively expressed TLR7, TLR8 and TLR9 mRNA transcripts, suggesting a possible role of TLR cross-talk in the down-regulatory mechanisms. Down-regulation of responses by co-stimulation with closely related TLRs may be a regulatory mechanism by which the host prevents overstimulation of innate immune responses.

Subcutaneous, but not intratracheal administration of the TLR9 agonist, CpG DNA transiently reduces parainfluenza-3 virus shedding in newborn lambs.

Synthetic oligodeoxynucleotides (ODN) containing CpG motifs signal through TLR9 and activate innate immunity resulting in protection against a variety of parasitic, bacterial and viral pathogens in mouse models. However, few studies have demonstrated protection in humans and large animals. In the present investigations, we evaluated protection by CpG ODN in a parainfluenza-3 (PI-3) virus infection in neonatal lambs. Subcutaneous (SC) injection of CpG ODN induced high levels of 2'5'-A synthetase and significantly reduced PI-3 virus shedding in newborn lambs. Furthermore, pre-treatment of newborn lambs with SC CpG ODN 2 days, but not 6 days prior to the virus challenge was protective. In contrast, intratracheal (IT) administration of CpG ODN induced 2'5'-A synthetase but had no significant impact on PI-3 virus shedding in nasal secretions. We conclude that a systemic administration of CpG ODN and the timing of the treatment are critical for the protection of neonatal lambs against a respiratory viral infection.