Retinoic acid induces homing of protective T and B cells to the gut after subcutaneous immunization in mice

Diarrheal diseases represent a major health burden in developing countries. Parenteral immunization typically does not induce efficient protection against enteropathogens because it does not stimulate migration of immune cells to the gut. Retinoic acid (RA) is critical for gut immunity, inducing upregulation of gut-homing receptors on activated T cells. In this study, we have demonstrated that RA can redirect immune responses elicited by s.c. vaccination of mice from skin-draining inguinal LNs (ingLNs) to the gut. When present during priming, RA induced robust upregulation of gut-homing receptors in ingLNs, imprinting gut-homing capacity on T cells. Concurrently, RA triggered the generation of gut-tropic IgA+ plasma cells in ingLNs and raised the levels of antigen-specific IgA in the intestinal lumen and blood. RA applied s.c. in vivo induced autonomous RA production in ingLN DCs, further driving efficient induction of gut-homing molecules on effector cells. Importantly, RA-supplemented s.c. immunization elicited a potent immune response in the small intestine that protected mice from cholera toxin–induced diarrhea and diminished bacterial loads in Peyer patches after oral infection with Salmonella. Thus, the use of RA as a gut-homing navigator represents a powerful tool to induce protective immunity in the intestine after s.c. immunization, offering what we believe to be a novel approach for vaccination against enteropathogens

Characterization of “nodular inflammatory foci” in the neonatal lung 5 days after MCMV-3D infection.

<p>(A–E, G–J) Wildtype or (F) Ncr1^gfp/+ neonatal mice were l.p. infected with 5×10⁴ PFU MCMV-3D, 5 days later mice were sacrificed and lungs explanted. (A, C–E, G–J) Frozen sections were stained with antibodies as indicated and DAPI nuclear staining. (B) Paraffin embedded sections were analyzed after hematoxylin and eosin staining. (C–E) Cellular composition of nodular inflammatory foci. (F) Frozen sections from Ncr1^gfp/+ mice were analyzed by 2-photon microscopy. (A, B) Overview with multiple nodular inflammatory foci (frames in A and B) and solitary mCherry⁺ infected cells (arrowheads in A), (B) one nodular inflammatory focus is magnified. (G) Morphological discrimination between viable infected cells (arrows) and remnants (arrowheads) of infected cells. (H–J) Remnants engulfed by phagocytes are indicated by arrowheads. Scale bars: (A+B) 100 µm, (C–E, G) 50 µm, (F, H–J) 20 µm. AF = autofluorescence of tissue, (A–J) representative of n>6 from >3 independent experiments.</p

MCMV infects the neonatal lung but not the mucosa of the gastrointestinal tract.

<p>Neonatal mice were (A–E) fed or (F–K) l.p. infected with either (A) latex microspheres, (C, E, F, H) mock inoculums, (H, I–K) 5×10⁴ PFU or (B, D, E, G, H) 10⁶ PFU of MCMV-3D and analyzed one day after application. (A) Histological analysis of distal colon after inoculation of latex microspheres. (B) Oral cavity with bony palate was analyzed by epifluorescence microscopy, green and red channels show autofluorescent tissue. (C–E) Intestines were explanted from the proximal esophagus to distal colon in one piece, (F–H) respiratory tract with trachea. (C) Mock inoculum and (D) MCMV-3D inoculum show only autofluorescent tissue but no mCherry⁺ cells in the intestine, inlay in (D) shows autofluorescence in the red channel in high magnification. Luciferase activity from homogenates of (E) flushed intestines or (H) lungs. (F) Mice after mock inoculation show autofluorescent tissue or (G) multiple mCherry⁺ cells, inlay displays single mCherry⁺ cells in high magnification. (I–K) Frozen sections of infected neonatal lungs. (I) Overview with solitary mCherry⁺ infected cells (arrows). (J, K) Solitary mCherry⁺ infected cell with (J) pro-surfactant protein C (proSPC) or (K) CD45 staining. (A–D, F, G, I–K) Representative from >3 experiments with n = 2–3 animals per experiment. (E) Pooled from two independent experiments, n = 4–5, median & range. (H) Pooled from two independent experiments, n = 3–4, median & range. Scale bars: (B–D, F, G) 1000 µm, (A, I) 100 µm and (J, K) 10 µm.</p

Visualization of cross-presentation within nodular inflammatory foci of neonatal mice.

<p>(A) Experimental setup: neonatal CD11c-YFP transgenic mice were infected with 5×10⁴ PFU SIINFEKL-encoding MCMV-3D, 4 days after infection 5×10⁶ naïve OTIxCFP T cells were adoptively i.p. transferred, 1 day after transfer lungs were analyzed by 2-photon microscopy. (B) Representative 2-photon microscopy image from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003828#ppat.1003828.s012" target="_blank">Movie S4</a> of nodular inflammatory foci in surpass mode and z-axis sequential images from one time-point of framed area are depicted, lines in z4 indicate distances from OTI to APC or infected cell, arrow indicates synapse between infected cell (red) and APC (yellow), arrow head indicates synapse between OTI T cell (blue) and APC (yellow). (C) Distances from OTIxCFP T cell center to surface of either mCherry⁺ or APCs at first time-point of Movies are depicted. (D) Contact-duration of OTIxCFP with APCs was estimated from 12–31 min Movies. (E) Percentage of OTIxCFP T cells with APC contacts where APCs are either <i>in</i> or <i>not in</i> contact with mCherry⁺ signal. (F) Neonatal mice were l.p. infected with 5×10⁴ PFU MCMV-3D, 5 days p.i. 5×10⁶ naïve OTIxGFP T cells were adoptively i.p. transferred and one day later mice were sacrificed and lungs explanted, frozen sections were stained with indicated antibodies and DAPI. (C–E) Means & SD. (B–E) Data from n = 4 animals from 2 independent experiments, (F) representative from n = 4 animals from 3 independent experiments. Scale bars (B) 15 µm, (F) 10 µm.</p

Delayed expansion of MCMV-specific T cells contributes to susceptibility of neonates.

<p>(A–C) Neonatal or adult mice were infected with either 5×10⁴ PFU or 10⁶ PFU MCMV-3D, respectively. Cell suspensions were generated from organs and time-points indicated and analyzed for the frequency of M45 tetramer-binding CD8⁺ T cell fraction. (A) Representative FACS plots and (B+C) quantification are depicted, median & IQR. (D) Experimental setup for E+F: neonatal mice were infected with 5×10⁴ PFU MCMV-3D, treated with anti-CD8b antibody or PBS and analyzed at the time-points indicated. (E) Luciferase activity of homogenates from organs depicted; means & SD. (F) Frozen sections of neonatal lung were analyzed with the antibodies indicated and representative images of NIFs are depicted. (G) Experimental setup for H+I: neonatal mice were infected with either 5×10⁴ PFU MCMV-3D or MCMV-3DΔvRAP; 2 days after infection none, 3×10³ or 3×10⁶ naïve OTIxGFP T cells were adoptively i.p. transferred and animals were analyzed 6 days after transfer. (H) Luciferase activity of homogenates from depicted organs, means & SD. (I) Frozen sections of neonatal lung were analyzed with antibodies indicated; representative images of NIFs or NIF residues. (A–C) Data from n = 3–5 animals per time-point from 2 independent experiments, (H+I) data from n = 5–8 animals from 5 independent experiments. (C) Scale bars, 50 µm.</p

Delayed control of lung MCMV infection in neonatal mice.

<p>Neonatal (A–F) or adult (A, C–F) mice were infected as follows: neonates l.p. with 5×10⁴ PFU MCMV-3D, adults intranasal with 10⁶ PFU MCMV-3D. (A–F) Animals were sacrificed at indicated time-points after infection and frozen sections of explanted lungs were performed and analyzed after antibody and DAPI staining. (A) Upper rows show merged images of immune infiltrates associated with MCMV-infected cells (mCherry⁺), lower rows only mCherry signals. (B) Examples and relative distribution of infected cells that are either “contacted” or “not contacted” by neonatal immune cells. (C) Luciferase activity of homogenized lung explants from n = 3–5 animals from 1–2 experiments per time point. (D+E) Number of mCherry+ viable cells or remnants per lung slice, determined by 2 counted slices per animal, normalized to (D) mean of infected cells determined at day 1 p.i or (E) normalized to the maximal value of remnants per group; zero counts were set to <10⁻³ to allow logarithmic illustration, t-test between groups at indicated time-points, n = 2–4 animals per indicated time-point from >3 independent experiments. (F) Number of mCherry⁺ viable cells per lung slice at day 5 p.i., frozen sections of n = 6–7 animals from 2 independent experiments. (B) Median & range, (C–E) each value and a connecting line of medians, (F) mean & SD.</p

APCs in NIFs prime MCMV-specific CD8⁺ T cells.

<p>(A) Experimental setup for B+C: neonatal mice were infected with 5×10⁴ PFU MCMV-2D or MCMV-3D, 3 days p.i. 3×10⁶ naïve eFluor® 670 Proliferation Dye labeled CD8⁺ GFPxOTI T cells were adoptively i.p. transferred, some animals received daily s.c. FTY720 until analysis at day 7 p.i.. (B) Frequency of GFP⁺ cells in leukocytes in different compartments, mean & SD. (C) Representative proliferation profiles of GFP⁺ cells from organs indicated. (D) Experimental setup for (E+F): neonatal mice were infected with 5×10⁴ PFU MCMV-3D and at the same time received polyclonal CD8⁺ T cells from CD45.1⁺ mice. FTY720 was given daily subcutaneously. (E) Representative FACS plot and quantification of CD45.1⁺CD8⁺ T cells from lung stained with M45 tetramers, median & IQR. (F) Frozen sections of neonatal lungs were analyzed with antibodies indicated, representative image of a NIF. (B+C) Data from n = 4–7 animals from 2–3 independent experiments, (E–F) Data from n = 4 animals from 2 independent experiments. Scale bar: 50 µm.</p

Viral dissemination establishes infection in multiple organs in neonatal but not in adult mice.

<p>(A–C) Neonatal or adult mice were infected as follows: neonates l.p. with 5×10⁴ PFU MCMV-3D, adults intranasal with 10⁶ PFU MCMV-3D. (A) Frozen sections of explanted neonatal organs were performed at day 8 after infection and analyzed after indicated antibody and DAPI staining, n>2 animals from >2 independent experiments. (B, C) Animals were sacrificed at indicated time-points after infection and homogenates of single organ preparations were measured for luciferase activity, shown are median & range and connecting line of medians, n = 3 animals per indicated time-points >3 independent experiments, dashed line = detection limit. (D+E) Body weight gain after MCMV-3D infection in (D) neonatal or (E) adult mice with the virus doses as indicated, median+IQR, pooled from two independent experiments each, n = 6–8 per group. Student's t-test between (D) mock and 5×10⁴ PFU group and (E) mock and 10⁶ PFU group.</p

In Vivo Killing Capacity of Cytotoxic T Cells Is Limited and Involves Dynamic Interactions and T Cell Cooperativity.

According to in vitro assays, T cells are thought to kill rapidly and efficiently, but the efficacy and dynamics of cytotoxic T lymphocyte (CTL)-mediated killing of virus-infected cells in vivo remains elusive. We used two-photon microscopy to quantify CTL-mediated killing in mice infected with herpesviruses or poxviruses. On average, one CTL killed 2-16 virus-infected cells per day as determined by real-time imaging and by mathematical modeling. In contrast, upon virus-induced MHC class I downmodulation, CTLs failed to destroy their targets. During killing, CTLs remained migratory and formed motile kinapses rather than static synapses with targets. Viruses encoding the calcium sensor GCaMP6s revealed strong heterogeneity in individual CTL functional capacity. Furthermore, the probability of death of infected cells increased for those contacted by more than two CTLs, indicative of CTL cooperation. Thus, direct visualization of CTLs during killing of virus-infected cells reveals crucial parameters of CD8(+) T cell immunity