Antigen availability determines CD8⁺ T cell-dendritic cell interaction kinetics and memory fate decisions

Abstract

T cells are activated by antigen (Ag)-bearing dendritic cells (DCs) in lymph nodes in three phases. The duration of the initial phase of transient, serial DC-T cell interactions is inversely correlated with Ag dose. The second phase, characterized by stable DC-T cell contacts, is believed to be necessary for full-fledged T cell activation. Here we have shown that this is not the case. CD8⁺ T cells interacting with DCs presenting low-dose, short-lived Ag did not transition to phase 2, whereas higher Ag dose yielded phase 2 transition. Both antigenic constellations promoted T cell proliferation and effector differentiation but yielded different transcriptome signatures at 12 hr and 24 hr. T cells that experienced phase 2 developed long-lived memory, whereas conditions without stable contacts yielded immunological amnesia. Thus, T cells make fate decisions within hours after Ag exposure, resulting in long-term memory or abortive effector responses, correlating with T cell-DCs interaction kinetics.

Figure 1. P14 T cells maintain brief interactions with 1μM C-peptide pulsed DCs

(A) Experimental paradigm for study of P14 Tcra−/− (P14) and control (OT-I) T cell interactions with peptide pulsed DCs. Unless otherwise mentioned, this will be the protocol for experiments throughout the paper. (B) Percentage of antigen specific (P14) T cells at 48h that remain unproliferated after exposure in vivo to DCs pulsed with various concentrations of C-peptide (bars are mean +/−SD). (C-G) P14 and control (OT-I Rag1−/−) T cell interactions with DCs pulsed with 1μM or 100μM C-peptide pulsed DCs (1C or 100C, respectively) were visualized in popLNs by MP-IVM at the indicated time points after T cell injection iv. (C) Duration of P14 T cell-1C-peptide pulsed DC (1C DC) contacts at various timepoints after T cell transfer was assessed in 3D reconstructed videos (bar at median, *** = p<0.0001, ** = p =.0013 by Mann-Whitney). (D) Duration of T cell-DC contacts with 1C DC or 100C DC at various timepoints after T cell transfer was assessed in 3D reconstructed videos. P14 and control T cell interactions with 1C vs. 100C DCs were visualized in popliteal LNs by MP-IVM from 0-10h after T cell injection (bar at median, box surrounds durations of 30-60min, percentage of events above box). (E-F) Cumulative distribution plots of interaction durations for P14 (E) and control (F) T cells interacting with 1C or 100C. (G) The bootstrap corrected means of the interaction durations between P14 T cells (blue) control T cells (red) with DCs. (1C: N=2-4 experiments per timepoint; mean ± 95% CI, ** = p<0.0004 and p = 0.0008). (H) The bootstrap corrected means of the meandering indices (MI) of P14 and control T cells when interacting with 1C or 100C DCs. Cell centroids in 3D were measured by semi-automated cell tracking and the MI was calculated by dividing the displacement for each cell track by the total path length for that cell track. (n=2-4 expt per timept; mean ± 95% C.I., ** = p<4×10⁻⁴, * = p=0.017). Figure 1, see also Movies S1-6.

Figure 2. High and low dose antigen both lead to the majority of transferred T cells participating in effector response

Standard protocol (Figure 1A), with CD45.1 recipients who received 1C or 100C DCs in the right footpad and control DCs in the left footpad (to serve as internal controls). 2h after T cell injection, anti-L-selectin antibody (Ab) and FTY-720 (or vehicle alone), were injected to prevent further T cell entry and exit, respectively. At 4h, 24h and 96h recipients were sacrificed and quantitative flow cytometry with beads was used to enumerate the number of remaining transferred cells. (A) Representative flow cytometry plots. (B) Summary of percentage of transferred cells that have proliferated at indicated timepoints (vehicle treated recipients). C) The number of total undivided, transferred cells at indicated timepoints and conditions (N= 4 expt, mean +/− SD, 3-7 mice per condition, except 100C at 4h with 2).

Figure 3. Higher dose antigen eventually yields larger effector pool after a larger early apoptotic loss

Standard protocol (Figure 1a), with CD45.1 recipients who received 1C or 100C DCs in the right footpad and control peptide pulsed DCs in the left footpad (to serve as internal controls). 2h after T cell injection, anti-L-selectin Ab and FTY-720 (or vehicle alone), were injected to prevent further T cell entry and exit, respectively. (A-B; E-F) At 4h, 48h, 96h or 7d recipients were sacrificed and quantitative flow cytometry, with beads, was used to enumerate the number of remaining transferred cells in the popLN. This is presented as (A) the absolute number of recovered CD45.2+ cells recovered at 4h, 48h and 96h in the LN (vehicle treated; mean ±SEM) and (F) vehicle or FTY treated recipient (mean±SEM). Number of CD.45.2 (transferred and progeny of transferred) cells, as a percentage of total LN cells at each timepoint, either additionally treated with vehicle (B) or (E) FTY-720 (A-B; E-F: N=4 expt, 3-7 mice per condition, except 100C at 4h with 2; B and E: mean±SD). (C-D) Percentage of apoptotic transferred CFSE labeled P14 T cells at 24h after T cell transfer (Annexin V+, 7-AAD+), with 1C DC, 100C DC or DCs pulsed with control peptide, (C) representative flow cytometry and (D) summary of percentage of cells which are apoptotic at 24h, (N=3-6 expt and 3-8 mice per condition, bar at median). (G) The percentage at d7 of transferred CD4- B220- cells in the LN, CD8+ T cell negative selected spleen and bone marrow that represents recovered transferred cells. On left, representative flow cytometry and on right, summary of flow cytometry data. (N=2 expt, 4 mice per condition).

Figure 4. Effector function is equivalent on whether or not T cells engage in stable contacts with DCs

(A-B) Standard protocol (Figure 1a), with CD45.1 recipients who received 1C or 100C DCs in the right footpad and control DCs injected in left footpad to serve as internal controls. At 20h and 48h, popLN were harvested and IFN-γ production was measured by cell-surface capture. (A) Representative flow cytometry plots at 48h. (B) %IFN-γ positive transferred cells from 1C or 100C at 20h and 48h after P14 T cell transfer. IFN-γ positivity is calculated based on IFN-γ secretion from P14 T cells exposed to test peptide-pulsed DCs corrected for the amounts of secretion from P14 T cells exposed to control peptide-pulsed DCs. (%IFN-γ positivity by experiment γ SEM and MFI is presented per mouse, mean γ SD.; N = 4-5 mice 1C at 20 and 48h, N = 3-4 mice 100C at 20 and 48h; N=2 expt at 20h, N=3 expt at 48h). (C-D) Control peptide DC (left), 100C DC(middle) or 1C DC (right), were injected into the footpads of recipient congenic (CD45.1) mice. P14 T cells were injected iv 18h later. After an additional 48h, two target polyclonal B cell populations (one pulsed with 10μM M-peptide, labelled with 2μM CFSE; the other non-peptide pulsed, labelled with 0.1μM CFSE), were mixed at a 1:1 ratio and injected iv. 6h later, the ratio of CFSE^high:CFSE^low B cells was assessed in the popLN. (C) Representative plots of target cells after in vivo lysis (representative of 3-9 mice per group, N=2 (100C) or 4 (all other concentrations) expt). (D) Pooled specific lysis of Ag pulsed target cells (per mouse, mean ± SD. N=3-9 mice per group, N=2 (100C) or 4 (all other concentrations) expt). Percent specific lysis is calculated as (1-(ratio of unprimed/ratio primed))*100), where the ratio is %(CFSE^lo non-peptide pulsed)/(%CFSE^hi peptide-pulsed) among transferred CFSE⁺ target B cells. Figure 4, see also Figure S2.

Figure 5. Memory differentiation is impaired without stable DC-T cell contacts

(a, b) Purified CD11c⁺ DCs were pulsed with 10 μM control-peptide (or no peptide), 1C or 100C and injected into the right footpad of recipient mice (in A, peptide depleted C57BL/6 recipients, in B, OT-I recipients) with LPS. 5 ×10⁶ P14 T cells were injected iv 18h later. At d30+ post-T cell transfer each mouse was infected iv with 10³ p.f.u. LCMV Armstrong, the spleens harvested at d5 p.i. and IFN-γ was stained by ICCS after a 5h ex vivo stimulation with (+) or without (−) 1μM C-peptide at 37°. (A) C57BL/6 recipients were treated at d-10, -7 and -4 with high dose M-peptide to deplete them of Ag specific cells. Upper graph shows a summary of CD8+ IFNγ+ and lower graph shows representative flow cytometry. (N=3 expt, 3-5 mice, per condition; mean ± SEM). (B) Recipients are OT-I Rag1−/−. Upper graph shows a CD8+ IFN-γ+ and lower graph shows representative flow cytometry. Of note, in the OT-I recipients there were occasionally animals with extreme splenomegaly and expansion of lymph nodes (in all conditions) which were excluded from analysis in all settings. (N=3-5 expt, 4-7 mice per condition, mean±SD per mouse).

Figure 6. Comparison of cDNA profiles by DNA microarray at 12 and 24hr

(A-E) Standard protocol (Figure 1a), with two types of recipients who received 1C or 100C or no DCs in the right footpad. The right popliteal LN was harvested at 12 or 24h post T cell transfer, single cell suspension created, and stained with CD4, B220 and CD19 (dump channel). Cells were then sorted on a FACSAria as non-doublets, CMFDA+ and dump channel negative. RNA was extracted using standard phenol-chloroform techniques, and then concentrated and cleaned with the Agencourt RNAdvance tissue kit. Of the total RNA extracted, an aliquot was then amplified using commercially available kits (NuGEN Pico). Following RNA amplification, aliquots of cDNA from each sample were assayed using the Agilent 2100 bioanalyzer to ensure high-quality amplification prior to fragmentation, labelling and hybridization to microarray. cDNA was then hybridized on Affymetrix 430_2 arrays for analysis of gene expression patterns at a core facility. (A) Venn diagrams of differentially expressed genes. Left column, 12h timepoint, right column, 24h timepoint. Upper row represents the number of genes that were upregulated for vs naïve, and the lower row represents the number of genes that were downregulated vs naïve. For each Venn diagram, on the left are genes that are differentially expressed between T cells exposed to 1C vs. adoptively transferred naïve T cells not exposed to DCs and on the right are genes differentially expressed between T cells exposed to 100C vs. adoptively transferred naïve T cells not exposed to DCs. Genes included in these diagrams have fold change statistics > 0.5 Wilcoxon p-value <= 0.001. (B) 2D principal components analysis (PCA) of all 6 conditions (control, 1C and 100C, each at 12 and 24h with each color indicating a separate concentration at a specific timept), with arrows drawn from 12h to 24h data for each antigenic dose. Principle component (PC) 1 accounts for 28.3% and PC 2 accounts for 11.32%. (C) Enrichment of the d8 effector signature (Wherry et al., 2007) for the samples from T cells exposed to 1C and 100C samples at 24h. (D) Heatmap of the 30 most differentially expressed genes for each of three sample types (T cells exposed to no DCs, T cells exposed to 1C and T cells exposed to 100C all at 24h) with some genes of interest noted by arrows on the right margin. (E) Gene expression volcano plot, with –log 10 of the SAM p-value on the y axis and log 2 fold change on the x axis, such that genes with higher expression in 1C are on the left and genes with higher expression in 100C are on the right. Genes plotted were expert selected for their known relevance in T cell effector and memory differentiation. (arrays: N = 3 for control T cells at 24h, N= 10 for 1C at 24h, N=7 for 100C at 24h; N= 3 for control T cells at 12h; N= 6 for 1C at 12h; N= 6 for 100C at 12h). Figure 6, see also Figure S1 and Table S1-S3.