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).

Does Mucosal B1 Activation Result in the Accumulation of Peak IgM During Chronic Intrarectal SIVmac239 Exposure to Protect Chinese-Origin Rhesus Macaques From Disease Progression?

Human immunodeficiency virus (HIV) infection is characterized by a dynamic process and highly variable progression. Although extensive comparisons have been reported between the minority of non-progressors (NPGs) and the majority of progressors (PGs), the underlying mechanism is still unclear. One reason for this is that the initial onset of infection is very difficult to track, particularly when men who have sex with men (MSM) are predominantly responsible for the transmission of human HIV. To find potential early protection strategies against later progression during chronic mucosal exposure, 10 Chinese-origin rhesus macaques (ChRhs) that underwent repetitive simian immunodeficiency virus (SIV) intrarectal exposure were longitudinally tracked. The results of the periodic detection of peripheral blood mononuclear cells (PBMCs) and colorectal mucosal lamina propria mononuclear cells (LPMCs) with immunoglobulins in rectal fluid were compared between non-progressive and progressive subgroups, which were classified based on their circulating viral loads. As a result, four NPGs and six PGs were observed after disease onset for 2 months. Upon comparing the mucosal and systemic immune responses, the PBMC response did not differ between the two subgroups. Regarding LPMCs, the increased activation of B1a/B1 cells among B cells and a peak in IgM in rectal fluid was observed approximately 10 days after the first exposure, followed by consistently low viremia in the four non-progressive ChRhs. In the six progressive ChRhs, neither B cell activation nor a peak in IgM was observed, while a robust elevation in IgG was observed, followed by consistently high viremia post exposure. Based on the PBMC-LPMC disparity between the subgroups of monkeys, we hypothesize that early B1 activation in LPMCs that result in an IgM peak might attenuate the entry and acquisition of SIV in the mucosa, resulting in very low dissemination into blood. Our models have suggested that the use of early surveillance both systemically and in the mucosa to comprehensively determine virus-host interactions would be informative for mucosal vaccine development.

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).

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.

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.

Routes and mechanisms of post-endosomal cholesterol trafficking: A story that never ends.

Mammalian cells acquire most exogenous cholesterol through receptor-mediated endocytosis of low-density lipoproteins (LDLs). After internalization, LDL cholesteryl esters are hydrolyzed to release free cholesterol, which then translocates to late endosomes (LEs)/lysosomes (LYs) and incorporates into the membranes by co-ordinated actions of Niemann-Pick type C (NPC) 1 and NPC2 proteins. However, how cholesterol exits LEs/LYs and moves to other organelles remain largely unclear. Growing evidence has suggested that nonvesicular transport is critically involved in the post-endosomal cholesterol trafficking. Numerous sterol-transfer proteins (STPs) have been identified to mediate directional cholesterol transfer at membrane contact sites (MCSs) formed between 2 closely apposed organelles. In addition, a recent study reveals that lysosome-peroxisome membrane contact (LPMC) established by a non-STP synaptotagmin VII and a specific phospholipid phosphatidylinositol 4,5-bisphosphate also serves as a novel and important path for LDL-cholesterol trafficking. These findings highlight an essential role of MCSs in intracellular cholesterol transport, and further work is needed to unveil how various routes are regulated and integrated to maintain proper cholesterol distribution and homeostasis in eukaryotic cells.

Chemokine (C-C Motif) Receptor 2 Mediates Dendritic Cell Recruitment to the Human Colon but Is Not Responsible for Differences Observed in Dendritic Cell Subsets, Phenotype, and Function Between the Proximal and Distal Colon.

BACKGROUND & AIMS: Most knowledge about gastrointestinal (GI)-tract dendritic cells (DC) relies on murine studies where CD103+ DC specialize in generating immune tolerance with the functionality of CD11b+/- subsets being unclear. Information about human GI-DC is scarce, especially regarding regional specifications. Here, we characterized human DC properties throughout the human colon. METHODS: Paired proximal (right/ascending) and distal (left/descending) human colonic biopsies from 95 healthy subjects were taken; DC were assessed by flow cytometry and microbiota composition assessed by 16S rRNA gene sequencing. RESULTS: Colonic DC identified were myeloid (mDC, CD11c+CD123-) and further divided based on CD103 and SIRPα (human analog of murine CD11b) expression. CD103-SIRPα+ DC were the major population and with CD103+SIRPα+ DC were CD1c+ILT3+CCR2+ (although CCR2 was not expressed on all CD103+SIRPα+ DC). CD103+SIRPα- DC constituted a minor subset that were CD141+ILT3-CCR2-. Proximal colon samples had higher total DC counts and fewer CD103+SIRPα+ cells. Proximal colon DC were more mature than distal DC with higher stimulatory capacity for CD4+CD45RA+ T-cells. However, DC and DC-invoked T-cell expression of mucosal homing markers (ß7, CCR9) was lower for proximal DC. CCR2 was expressed on circulating CD1c+, but not CD141+ mDC, and mediated DC recruitment by colonic culture supernatants in transwell assays. Proximal colon DC produced higher levels of cytokines. Mucosal microbiota profiling showed a lower microbiota load in the proximal colon, but with no differences in microbiota composition between compartments. CONCLUSIONS: Proximal colonic DC subsets differ from those in distal colon and are more mature. Targeted immunotherapy using DC in T-cell mediated GI tract inflammation may therefore need to reflect this immune compartmentalization.

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.

The influence of infliximab and adalimumab on the expression of apoptosis-related proteins in lamina propria mononuclear cells and enterocytes in Crohn's disease - an immunohistochemical study.

BACKGROUND AND AIMS: The aim of this study was to assess the influence of anti-TNF agents on the expression of apoptosis-related proteins in Crohn's disease (CD) patients. METHODS: The clinical, biochemical and endoscopic activity of CD was assessed with the use of tissue sampling before the initiation of therapy and after induction doses of infliximab and adalimumab. Additionally, the immunohistochemical expression of active caspase 3, TNFR1, Fas, Bcl-2, Bax, CD4 and CD8 proteins was estimated. Patients achieving deep remission were considered as responders. RESULTS: Of the 35 patients qualified for the study, 60% achieved deep remission. In those patients, a significant decrease in the number of CD4 and CD8 positive cells was noted. Also observed was a significant increase in the expression of active caspase 3 in lamina propria mononuclear cells, which correlated with an increase of the pro-apoptotic Bax/Bcl-2 ratio. No change in Fas and TNFR1 expression was observed in those cells. Moreover, there was a significant decrease in active caspase 3 expression in enterocytes, observed independently of the Bax/Bcl-2 ratio. This correlated with a change in TNFR1 expression. No significant changes in the expression of the investigated proteins were noted in non-responders group. CONCLUSIONS: The efficacy of anti-TNF antibodies is, at least partly, dependent on apoptosis modulation. In lamina propria mononuclear cells, the increase of apoptosis is probably the result of the induction of the intrinsic pathway mediated by Bcl-2 family proteins. In enterocytes - the decrease of apoptosis is mediated by the extrinsic pathway, probably via TNFR1.