Distinct aging profiles of CD8⁺ T cells in blood versus gastrointestinal mucosal compartments

<div><p>A hallmark of human immunosenescence is the accumulation of late-differentiated memory CD8⁺ T cells with features of replicative senescence, such as inability to proliferate, absence of CD28 expression, shortened telomeres, loss of telomerase activity, enhanced activation, and increased secretion of inflammatory cytokines. Importantly, oligoclonal expansions of these cells are associated with increased morbidity and mortality risk in elderly humans. Currently, most information on the adaptive immune system is derived from studies using peripheral blood, which contains approximately only 2% of total body lymphocytes. However, most lymphocytes reside in tissues. It is not clear how representative blood changes are of the total immune status. This is especially relevant with regard to the human gastrointestinal tract (GALT), a major reservoir of total body lymphocytes (approximately 60%) and an anatomical region of high antigenic exposure. To assess how peripheral blood T cells relate to those in other locations, we compare CD8⁺ T cells from peripheral blood and the GALT, specifically rectosigmoid colon, in young/middle age, healthy donors, focusing on phenotypic and functional alterations previously linked to senescence in peripheral blood. Overall, our results indicate that gut CD8⁺ T cells show profiles suggestive of greater differentiation and activation than those in peripheral blood. Specifically, compared to blood from the same individual, the gut contains significantly greater proportions of CD8⁺ T cells that are CD45RA^- (memory), CD28^-, CD45RA^-CD28⁺ (early memory), CD45RA^-CD28^- (late memory), CD25^-, HLA-DR⁺CD38⁺ (activated) and Ki-67⁺ (proliferating); <i>ex vivo</i> CD3⁺ telomerase activity levels are greater in the gut as well. However, gut CD8⁺ T cells may not necessarily be more senescent, since they expressed significantly lower levels of CD57 and PD-1 on CD45RO⁺ memory cells, and had <i>in vitro</i> proliferative dynamics similar to that of blood cells. Compartment-specific age-effects in this cohort were evident as well. Blood cells showed a significant increase with age in proportion of HLA-DR⁺38⁺, Ki-67⁺ and CD25⁺ CD8⁺ T cells; and an increase in total CD3⁺ <i>ex-vivo</i> telomerase activity that approached significance. By contrast, the only age-effect seen in the gut was a significant increase in CD45RA^- (memory) and concurrent decrease in CD45RA⁺CD28⁺ (naïve) CD8⁺ T cells. Overall, these results indicate dynamics of peripheral blood immune senescence may not hold true in the gut mucosa, underscoring the importance for further study of this immunologically important tissue in evaluating the human immune system, especially in the context of chronic disease and aging.</p></div

Significant age-effect differences in blood vs gut T lymphocyte populations.

<p>Age-effect in blood and gut T lymphocyte parameters, and intra-individual age-effect difference between compartments, was tested using generalized linear models using SAS v9.3. For (<b>A-E</b>), blood and gut T cell WB aliquots were phenotyped by multi-color flow cytometry and analyzed (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182498#sec002" target="_blank">methods</a>). Differential age-effect data for proportion of <b>(A)</b> CD8⁺ on CD3⁺ (n = 39), <b>(B)</b> HLA-DR⁺CD38⁺ on CD8⁺ (n = 39), <b>(C)</b> CD25⁺ on CD8⁺ (n = 34), <b>(D)</b> Ki-67⁺ on CD8⁺ (n = 39), and <b>(E)</b> CD45RA^- on CD8⁺ (n = 39) is shown. For <b>(F)</b>, relative telomerase activity of 0.32x10⁶ blood and gut purified CD3⁺ T cells from each donor was determined via the PCR-based TRAP protocol, normalized to a standardized cell number of a telomerase-positive control cell line (Jurkat), and differential age-effect was determined (n = 20). P-values < 0.05 were considered significant. **, p< 0.05, ***, p<0.005.</p

Comparison of senescence-related markers in blood and gut.

<p>Comparison of senescence-related markers in blood and gut.</p

Proliferative capacity of stimulated CD8 T cells in 5 day culture.

<p>Proliferation of CD3⁺/CD8⁺ T cells was determined using a recently developed double—label CFSE/BRDU system on 5-day cultures of blood and gut derived mononuclear cells stimulated with CD3/CD28/CD2 antibodies. All cultures include 1.0x10⁶ blood or gut mononuclear cells and 0.5x10⁶ irradiated autologous PBMC feeders. <b>A.</b> Representative bivariate plots from blood- and gut-derived cultures (stimulated culture and unstimulated control). CFSE-FITC (x-axis) versus BrdU-APC (Y-axis) gating used to enumerate replicating (CFSEloBrdU⁺) verus non-replicating (CFSEhiBrdU^-) CD8⁺ population. <b>B.</b> Day 5 CD3⁺ T cell number fold change (n = 13) <b>C.</b> Percentage of CD8⁺ T cells that are proliferating on Day 5 (n = 13). <b>D.</b> Mean of proliferating CD8⁺ T cells (n = 13). <b>E.</b> Percentage of highly proliferative (≥ 4 divisions) CD8⁺ T cells (n = 13). Intra-individual differences between blood and gut were assessed using the Wilcoxon Signed Rank test for paired data. P-values < 0.05 were considered significant. **, p<0.05, ***, p<0.005.</p

Clinical trial design.

<p>A. CONSORT flowchart of subject enrolment. B. After an initial screening visit, blood and gut mucosal biopsies were obtained 28 and 14 days before initiation of vaccination. Vaccinations (vCP205 or placebo) were administered Days 0, 7, 14, 21. Blood samples then were collected on Days 10, 17, 24, 180, and 360. Gut mucosal biopsies were collected at Days 10, 24, 180, 360.</p

Differential Blood and Mucosal Immune Responses against an HIV-1 Vaccine Administered via Inguinal or Deltoid Injection

<div><p></p><p>Mucosal immunity is central to sexual transmission and overall pathogenesis of HIV-1 infection, but the ability of vaccines to induce immune responses in mucosal tissue compartments is poorly defined. Because macaque vaccine studies suggest that inguinal (versus limb) vaccination may better target sexually-exposed mucosa, we performed a randomized, double-blinded, placebo-controlled Phase I trial in HIV-1-uninfected volunteers, using the recombinant Canarypox (CP) vaccine vCP205 delivered by different routes. 12 persons received vaccine and 6 received placebo, divided evenly between deltoid-intramuscular (deltoid-IM) or inguinal-subcutaneous (inguinal-SC) injection routes. The most significant safety events were injection site reactions (Grade 3) in one inguinal vaccinee. CP-specific antibodies were detected in the blood of all 12 vaccinees by Day 24, while HIV-1-specific antibodies were observed in the blood and gut mucosa of 1/9 and 4/9 evaluated vaccinees respectively, with gut antibodies appearing earlier in inguinal vaccinees (24–180 versus 180–365 days). HIV-1-specific CD8⁺ T lymphocytes (CTLs) were observed in 7/12 vaccinees, and blood and gut targeting were distinct. Within blood, both deltoid and inguinal responders had detectable CTL responses by 17–24 days; inguinal responders had early responses (within 10 days) while deltoid responders had later responses (24–180 days) in gut mucosa. Our results demonstrate relative safety of inguinal vaccination and qualitative or quantitative compartmentalization of immune responses between blood and gut mucosa, and highlight the importance of not only evaluating early blood responses to HIV-1 vaccines but also mucosal responses over time.</p><p>Trial Registration</p><p>ClinicalTrials.gov <a href="http://www.clinicaltrials.gov/ct2/show/NCT00076817?term=NCT00076817&rank=1" target="_blank">NCT00076817</a></p></div

Participant demographics.

<p>Participant demographics.</p

Mucosal and blood antibody responses against Canarypox.

<p>Anti-Canarypox antibodies were measured by ELISA in blood (IgG only, Panel A) and gut mucosal secretions (IgG and IgA, Panels B and C) on Day 24. Responses against placebo or vCP205 vaccine are plotted by vaccination route, as abstract units (normalized against placebo and background-subtracted). Medians and 95% confidence intervals are indicated for each group.</p

Total observed CTL responses against HIV-1 in blood and gut mucosal compartments.

<p>The background-subtracted CTL responses of all participants against HIV-1 vaccine peptide pools were summed for persons who received placebo (circles) and vCP205 vaccinations (squares) and plotted for blood (left) and gut mucosa (right). <i>p</i>-values indicate significant differences between groups by Students t-test.</p

HIV-1-specific CTL responses in blood and gut mucosal compartments divided according to vaccination route.

<p>The results in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088621#pone-0088621-g003" target="_blank">Figure 3</a> are plotted separate depending on vaccination route for recipients of saline (circles) and vCP205 vaccine (squares). The upper and lower panels show blood and gut mucosal responses respectively, and the left and right panels give results for persons who received deltoid and inguinal vaccinations respectively. <i>p</i>-values indicate significant differences between groups by Students t-test.</p