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. 2014 Feb 15;192(4):1651-1660.
doi: 10.4049/jimmunol.1301743. Epub 2014 Jan 15.

Pathogen-related differences in the abundance of presented antigen are reflected in CD4+ T cell dynamic behavior and effector function in the lung

Parizad Torabi-Parizi #  1   2 , Nienke Vrisekoop #  1 , Wolfgang Kastenmuller  1 , Michael Y Gerner  1 , Jackson G Egen  1 , Ronald N Germain  1
Affiliations

Pathogen-related differences in the abundance of presented antigen are reflected in CD4+ T cell dynamic behavior and effector function in the lung

Parizad Torabi-Parizi et al. J Immunol. .

Abstract

Exposure to pathogens in the periphery elicits effector T cell differentiation in local lymph nodes followed by migration of activated T cells to and within the infected site. However, the relationships among pathogen abundance, Ag display on MHC molecules, effector T cell dynamics, and functional responses at the infected sites are incompletely characterized. In this study, we compared CD4(+) T cell effector dynamics and responses during pulmonary mycobacterial infection versus acute influenza infection. Two-photon imaging together with in situ as well as ex vivo analysis of cytokine production revealed that the proportion of migration-arrested, cytokine-producing effector T cells was dramatically higher in the influenza-infected lungs due to substantial differences in Ag abundance in the two infectious states. Despite the marked inflammatory conditions associated with influenza infection, histocytometric analysis showed that cytokine production was focal, with a restriction to areas of significant Ag burden. Optimal effector function is thus constrained by the availability of TCR ligands, pointing to the value of increasing Ag stimulation rather than effector numbers in harnessing CD4(+) T cells for therapeutic purposes in such conditions.

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Figures

FIGURE 1
FIGURE 1
Establishment of a robust in vivo system designed to study CD4+ T cell effector responses in lung tissue. A, Day 7 in vitro-generated effector T cells (P25 or OTIIs) were incubated with an equal number of splenocytes pulsed with the cognate antigen at the indicated concentrations for 5 hours. Percentage of IFNγ-producing T cells is represented. Bar graph to the right indicates the percentage of IFNγ-positive P25 and OTII T cells when stimulated in vitro with PMA+ionomycin. B, Naïve mice received ~1 million day 6 in vitro-generated effector T cells (P25s and OTIIs). After 18 hours, the mice received equimolar amounts of both OVA323 and Ag85b. Two hours later, the mice were sacrificed and the percentage IFNγ-producing transferred cells in the spleen was determined. Data are representative of at least 2 independent experiments, done in triplicates. Symbols and bars indicate mean with indicated standard error of the mean. C, Schematic representation of the experimental setup. 2PM=two-photon microscopy; FC=Flow cytometry; IHC=immunohistochemistry. D, Percent weight loss over time. Symbols indicate mean of at least 5 animals, and error bars the standard error of the mean.
FIGURE 2
FIGURE 2
Effector CD4+ T cells display distinct effector behavior in lungs infected with different agents. A and C, Representative FACS plots showing the percentage of all transferred cells producing IFNγ, as measured by intracellular cytokine stain, when taken from BCG- (A) or Influenza-infected mice (C). P/I=PMA+ionomycin. IN=intranasally. IV=intravenously. Numbers indicate percentages. Gating strategy for lung single cell suspensions involved gating on live cells with a generous forward and side scatter gate, then gating on CD45+ cells, followed by gating on the transferred T cell population of interest. Finally, CD4 by IFNγ was plotted. Negative gate set based on isotype control for each experimental condition. B and D, Quantification of the percentage of IFNγ+ P25 T cells or OTII T cells, when taken directly ex vivo from the lungs, after 4 hours of in vitro PMA+ionomycin stimulation, or 2 hours after in vivo administration of cognate antigen. Responses of the control effector population not recognizing antigen are shown in the same graph depicted by different color bars. Data are represented as mean and standard error of the mean. Statistics performed using the unpaired Student's t test. E, Data represented as percentage of maximal IFNγ production potential, obtained by comparing percentage IFNγ+ ex vivo to percentage IFNγ+ after PMA+ionomycin stimulation. ns=non-significant. Data are representative of at least 4 independent experiments with 3-5 mice per group. Data represented as mean with standard error of the mean. F, Mice infected with BCG 11 weeks prior were also infected with PR8OVA323. 7 days post PR8OVA323 infection (and thus 12 weeks post BCG infection), effector T cells were transferred into the animals. Eighteen hours after transfer, transferred T cells were isolated and the percentage of IFNγ-producing cells (gated on P25s or OTIIs) was determined. Data are representative of 2 independent experiments with 3 mice per group. Data represented as mean with standard error of the mean.
FIGURE 3
FIGURE 3
Differences in CD4+ T cell effector function are mirrored by differences in dynamic behavior. A-C, Lung BCG granuloma image acquired by two-photon microscopy and analysis thereof. D-F, Image of inflammatory patch in an influenza-infected lung acquired by two-photon microscopy and analysis thereof. A and D, Maximum projection rendering of imaging volume. Colors of the words correspond to colors in the image, here and throughout. In panel A, the dotted line delineates the margins of the granuloma in the image. B and E, Mean velocity of OTIIs and P25s; red line indicates mean value. C and F, Total displacement over the duration of the imaging session (red line indicates mean value) and representation of the tracks of OTIIs and P25s over the duration of the movie, all translated to the same starting point. G and H, Frequency distribution of P25 and OTII velocities in BCG (G) and influenza-infected tissue (H); bin width of 2μm. Dashed lines indicate mode of the distributions. ns=non-significant. All data are representative of at least 3 similar experiments, with at least 3 mice per group. Statistics performed using the unpaired Student's t test.
FIGURE 4
FIGURE 4
Antigen abundance dictates T cell effector and dynamic behavior. A and B, Immunofluorescent staining of BCG-infected mouse lung with anti-Ag85b antibody (arrow indicates Ag85b stain) (A) or of influenza-infected mouse lung with anti-PR8 antibody (B). P25 and OTII T cells were labeled with an intracellular dye prior to transfer. In panel A, the dotted line delineates the margins of the granuloma in the image. C, IFNγ staining in an influenza-infected mouse lung. Insets shown are magnifications of the areas in the grey boxes for panels A-C. D, Two-dimensional contour plot depicting data from fluorescent immunohistochemical analysis of influenza-infected mouse lungs, after importing position and mean fluorescent intensity data obtained in Imaris into FlowJo, as described in the Methods section. Data are representative of two independent experiments. E, Percentage of IFNγ-producing OTIIs within the population of cells that reside either beyond or within 100μm of influenza-infected cells. Each dot represents one experiment and the red line represents the mean.
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