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. 2013 Mar 21;38(3):502-13.
doi: 10.1016/j.immuni.2012.11.012. Epub 2013 Jan 24.

Peripheral prepositioning and local CXCL9 chemokine-mediated guidance orchestrate rapid memory CD8+ T cell responses in the lymph node

Wolfgang Kastenmüller  1 , Marlene Brandes, Ze Wang, Jasmin Herz, Jackson G Egen, Ronald N Germain
Affiliations

Peripheral prepositioning and local CXCL9 chemokine-mediated guidance orchestrate rapid memory CD8+ T cell responses in the lymph node

Wolfgang Kastenmüller et al. Immunity. .

Abstract

After an infection, the immune system generates long-lived memory lymphocytes whose increased frequency and altered state of differentiation enhance host defense against reinfection. Recently, the spatial distribution of memory cells was found to contribute to their protective function. Effector memory CD8+ T cells reside in peripheral tissue sites of initial pathogen encounter, in apparent anticipation of reinfection. Here we show that within lymph nodes (LNs), memory CD8+ T cells were concentrated near peripheral entry portals of lymph-borne pathogens, promoting rapid engagement of infected sentinel macrophages. A feed-forward CXCL9-dependent circuit provided additional chemotactic cues that further increase local memory cell density. Memory CD8+ T cells also produced effector responses to local cytokine triggers, but their dynamic behavior differed from that seen after antigen recognition. These data reveal the distinct localization and dynamic behavior of naive versus memory T cells within LNs and how these differences contribute to host defense.

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Figures

Figure 1
Figure 1. Priming of naïve CD8+ T cell predominates in the cortical ridge
(A–E) Maximum projections of 2P images (z-stack 90–120μm) of pLN acquired in situ. (A) Time course after MVA NP-S-GFP infection; second-harmonic generation (SHG ≈ capsule); time is shown as h:min:sec. (B) Image from the SCS region 4h after infection with MVA NP-S-GFP (nuclear) and s.c. injection with anti-CD169. (C) Image from the SCS region 6h after infection with MVA GFP (cytosolic). (D, E) Images from the SCS region at different time-points after MVA OVA infection showing OT-1 T cells and capsule (D) and at 4h post-infection showing HEV (blood tracer) in relationship to OT-1 T cells (E). (F, G) Track length and average speed of OT-1 T cells and polyclonal control CD8+ T cells 3h pi. (H) Number of OT-1 T cells present in the field of imaging in the course of MVA OVA infection. (I) Positioning of OT-1 T cell clusters (white circles) in relation to virus-infected cells, B cells and collagen VI, 4h after infection with MVA OVA GFP. Inserts show GFP signal within OT-1 clusters. The data are representative of three (A–C) or five (D–F) similar experiments, (** = p≤0.01). See also Figure S1 and Movies S1–4.
Figure 2
Figure 2. CM CD8+ T cells are positioned in LN periphery in the steady state
(A) IF images showing pLN harboring naïve and CM OT-1 T cells. (B, C) Relative distances from a virtual LN center of naïve and CM CD8+ T cells displayed as a dot plot (B) or as a frequency distribution (C). (D, E) Average speed and track length comparing naïve and CM OT-1 T cells in the steady state. (F, G) Average speed and track length of memory OT-1 T cells near to the LN capsule in the steady state. (H) Combined tracks of CM CD8+ T cells (near capsule) over 40min. White lines indicate border to B cell follicles. The data are representative of five similar experiments (*** = p≤0.001, ns = non-significant). See also Figure S2 and Movie S5 and S6.
Figure 3
Figure 3. CM CD8+ T cells are rapidly recruited to the site of infection
(A) Maximum projections of 2P images of pLN acquired in situ showing various time points after MVA OVA infection (z-stack of 120μm, starting beneath the capsule/SHG). (B) Number of naïve and CM OT-1 T cells present in the imaging field (from A) at the indicated times after MVA OVA infection. (C) Average speed of naïve and CM OT-1 T cells 3h pi. (D) Positioning of naïve and CM OT-1 T cell clusters, B cells and collagen VI, 4h after infection with MVA OVA. Inserts show magnification of naïve and CM OT-1 T cell clusters from different locations; yellow numbers indicate distance of cluster from LN center (yellow circle). (E) Maximum projections of 2P images from the pLN acquired in situ (z-stack of 120μm, starting beneath the capsule). White arrows indicate T cell – macrophage interaction before killing and disruption of macrophages. The data are representative of five similar experiments (** = p≤0.01). See also Figure S3 and Movie S7 and S8.
Figure 4
Figure 4. CXCR3 is required for CM T cell SCS recruitment and local swarming
(A) Maximum projections of 2P images of pLN acquired in situ showing various time points after MVA WT infection (z-stack of 120μm, starting near the capsule/SHG). Red square indicates magnified area, yellow square insert shows the combined tracks of cells from the blue squares over 40min. (B) pLN of CD11cYFP mouse 4h after infection with MVA WT harboring WT and CXCR3-deficient (Cxcr3−/−) CM OT-1 T cells. (C) Relative distance of WT and Cxcr3−/−memory OT-1 T cells from the LN capsule 4h after infection with MVA WT. (D) Combined tracks displaying the movement of CD11c YFP cells, WT and Cxcr3−/−CM OT-1 T cells after MVA WT infection (140min–180min), clusters of WT OT-1 are encircled (E) pLN 4h after infection with MVA WT showing WT and Cxcr3−/−CM OT-1 T cells, B cells and collagen IV. The data are representative of five similar experiments (*** = p≤0.001). See also Figure S4 and Movie S9 and S10.
Figure 5
Figure 5. CM CD8+ T cells amplify CXCL9 production
(A) Confocal IF image showing CXCL9 staining in the IFA of a pLN from a CD11cYFP mouse 4h pi MVA WT. (B) Absence of WT OT-1 CM T cell recruitment to the SCS in a pLN from a Cxcl9−/− mouse 4h after MVA WT infection. (C) Quantitative ELISA for CXCL9 in LN homogenates 4h after infection of the indicated mouse strains with MVA OVA; insert shows statistical significance (ns= not significant, * = p≤0.05, ** = p≤0.01*** = p≤0.001). (D) IF images of a pLN 4h pi MVA OVA Tomato showing CM OT-1 and IFNγ staining. (E) IF images of pLN 4h pi MVA OVA of CD11cYFP mice. 7 days before infection mice received an s.c. injection with clodronate liposomes to deplete LN-resident myeloid cells. Data are representative of three independent experiments (A, B, D and E) or show pooled data from 2 independent experiments (C). See also Figure S5.
Figure 6
Figure 6. Non-cognate activation of CM CD8+ T cells leads to extensive non-arrested IFNγ production
(A) Intracellular IFNγ staining of CM OT-1 T cells in the pLN 4h after s.c. infection with MVA OVA or PA. (B) Average speed and (C) track length of CM OT-1 T cells 3h after PA infection. (D) Combined tracks displaying the movement of CM OT-1 T cells 3h after PA infection. (E, F) IF images of CM OT-1 T cells in the pLN 4h after PA infection. (G) IF images of CM OT-1 T cells after PA infection in relation to CXCL9. The data are representative of three similar experiments; p-values and ±SEM are shown. See also Movie S11.
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