Dynamic changes during the immune response in T cell-antigen-presenting cell clusters isolated from lymph nodes

Abstract

Activation of antigen-specific T cells by mature dendritic cells in secondary lymphoid organs is a key control point of the adaptive immune response. Here we describe the ex vivo isolation of preformed multicellular clusters between T cells and antigen-presenting cells. Adoptively transferred, antigen-specific T cells segregated into individual clusters where their activation and proliferation was initiated in vivo. Transit of the T cell cohort through the cluster compartment required 32-36 h. The precise timing of the response to agonistic epitopes was remarkably invariant regardless of the T cell lineage, the major histocompatibility complex haplotype, and the antigen dose. Interestingly, initiation of cell division of T cells specific for a subdominant epitope and a weak agonist was delayed by 6 h. The results provide a basis for the analysis of short range, mutual cell-cell interactions within such confined microenvironments.

Figure 1.

Cluster composition. Clusters were prepared from the draining LNs of CFA-immunized animals 4–5 d after immunization. (a) T cells form clusters with CD11c⁺ DCs. (b and c) B cells form tight clusters both with CD11c⁺ DCs (b) and M2⁺ FDC (c). (d) mixed B and T cell cluster with CD11c⁺ DCs. Note that some B cells appear to attach to T cells rather than DCs. Purified clusters were stained with (a) anti–CD3ɛ-FITC/anti–βTCR-FITC and anti–CD11c-biotin/sav-Cy-5, (b) B220-PE and anti–CD11c-biotin/sav-Cy5, (c) B220-PE and anti–FDC-biotin/sav-Cy-5, and (d) anti–CD3ɛ-FITC/anti–βTCR-FITC, anti-B220-PE, and anti–CD11c-biotin/sav-Cy-5. A detailed composition of cluster-associated lymphocytes is provided in Table S1, available at http://www.jem.org/cgi/content/full/jem.20021512/DC1.

Figure 2.

Antigen-specific T cells segregate into individual clusters. Mice were immunized with 50 μg hCRP/CFA (a) or 50 μg OVA/CFA (b) and then received 5 × 10⁶ CFSE-labeled Sep-specific T cells. Clusters were prepared from regional LNs at the indicated times and analyzed by fluorescence microscopy. 100 clusters were scanned randomly and the number of CFSE-stained T cells/cluster was determined. For comparison, the frequencies of nonclustered cells are shown. For each time point LNs of two mice were pooled. One representative out of three experiments is shown for a and one control experiment was performed for b.

Figure 3.

A distinct phenotype of clustered T lymphocytes. Mice were immunized with 50 μg antigen/CFA and 4 d later received 5 × 10⁶ tg, unlabeled T cells intravenously. Clusters were prepared from the regional LNs at the indicated time points after transfer. Cluster-associated and nonassociated cells were then analyzed for CD62L (for expression levels of CD62L, see Fig. S1, available at http://www.jem.org/cgi/content/full/jem.20021512/DC1) (a) and CD69 expression (b). Transferred cells were identified by expression of their tg TCR Vβ chain and the corresponding coreceptor. In clusters, T cells displayed lower levels of CD62L expression regardless of antigen specificity whereas tg CD4⁺ cells preferentially showed up-regulation of CD69. 20,000 events were analyzed.

Figure 4.

T cell proliferation is initiated in clusters. (a) Mice were immunized with 50 μg hCRP/CFA and then received 5 × 10⁶ CFSE-labeled Sep- or Dep-specific T cells intravenously. Cluster-derived and nonassociated cells were analyzed for CFSE dilution at the indicated time points. Note that T cell proliferation was initiated in the cluster-associated fraction and that there was an epitope-dependent shift in the timing of the first T cell division. Representative of three to four independent experiments (see also Fig. S2, available at http://www.jem.org/cgi/content/full/jem.20021512/DC1). (b) Mice were immunized with 50 μg SIINFEKL/CFA (strong agonist) or SIINFEDL/CFA (weak agonist) and then received 5 × 10⁶ CFSE-labeled OVA-specific OT-1 T cells intravenously. The strong and weak agonists differed with respect to the kinetics and extent of proliferation. Black lines depict the cluster-associated fraction, gray filled histograms depict the respective nonassociated cells. The analysis included “blasts” and small lymphocytes. 100,000 events were collected. The arrow marks the first detectable cell division. CFSE⁻ cells are not shown.

Figure 5.

A threshold dose of antigen is critical for T cell proliferation. BL/6 mice were immunized with varying doses of hCRP/CFA and then received 5 × 10⁶ CFSE-labeled Dep- (a) or Sep-specific T cells intravenously (b). Cluster-derived and nonassociated cells were analyzed for the frequency of CFSE-labeled cells among the blast fraction. The T cell response required a minimal dose of 0.1–0.5 μg antigen/mouse. Above this dose the kinetics and the extent of proliferation were insensitive to additional augmentation. The response to 50 μg is shown for the 48-h time point.

Figure 6.

T cell transit through the cluster compartment. Compiled data of the kinetics of the antigen response of Dep and Sep T cells. ○ and ⋄ represent the nonassociated fractions and • and ♦ represent the cluster-derived fractions. Note the phase shift in the response between both compartments. The fraction of CFSE⁺ lymphocytes of at least three independent experiments (only one experiment for the 72-h value) with two to three mice per time point is shown. There was no expansion in the absence of antigen (Fig. S3, available at http://www.jem.org/cgi/content/full/jem.20021512/DC1).

Figure 7.

Different T cell subsets colocalize in individual clusters. Different pre-enriched and prelabeled tg T cell populations were adoptively transferred into animals previously immunized with the respective antigens. Clusters were isolated 30–36 h after transfer. (a) hCRP-specific Sep (blue) and Dep (green) T cells colocalize to the same MHC class II⁺ (red) stromal cell, (b) OVA-specific CD8 OT-I (green) and CD4 OT-II (red) colocalize to the same DC (blue), and (c) T cells with different protein antigen specificities, Sep (red) and OT-I (green), colocalize to the same DC (blue).

Figure 8.

Summary of dynamic changes in T cell–APC clusters during the antigen-specific immune response in different experimental models.