The molecular signature underlying the thymic migration and maturation of TCRαβ+ CD4+ CD8 thymocytes

Abstract

Background: After positive selection, the newly generated single positive (SP) thymocytes migrate to the thymic medulla, where they undergo negative selection to eliminate autoreactive T cells and functional maturation to acquire immune competence and egress capability.

Methodology/principal findings: To elucidate the genetic program underlying this process, we analyzed changes in gene expression in four subsets of mouse TCRαβ(+)CD4(+)CD8(-) thymocytes (SP1 to SP4) representative of sequential stages in a previously defined differentiation program. A genetic signature of the migration of thymocytes was thus revealed. CCR7 and PlexinD1 are believed to be important for the medullary positioning of SP thymocytes. Intriguingly, their expression remains at low levels in the newly generated thymocytes, suggesting that the cortex-medulla migration may not occur until the SP2 stage. SP2 and SP3 cells gradually up-regulate transcripts involved in T cell functions and the Foxo1-KLF2-S1P(1) axis, but a number of immune function-associated genes are not highly expressed until cells reach the SP4 stage. Consistent with their critical role in thymic emigration, the expression of S1P(1) and CD62L are much enhanced in SP4 cells.

Conclusions: These results support at the molecular level that single positive thymocytes undergo a differentiation program and further demonstrate that SP4 is the stage at which thymocytes acquire the immunocompetence and the capability of emigration from the thymus.

Figure 1. Isolation of CD4⁺ single positive thymocyte subsets and CD4⁺ naïve T cells from lymph nodes.

(A–B) Thymocytes pooled from 15–20 mice were treated with anti-CD8 (3.155) mAb and complement. Viable cells were then separated by density centrifugation and stained with mAbs against mouse CD4, CD8 (53-6.7), CD69, and 6C10 or Qa-2. To exclude the contamination of NKT and Treg cells, PE-conjugated anti-NK1.1 and anti-CD25 antibodies were included in the mixture of staining Abs. CD4⁺CD8^-CD25^-NK1.1^- medullary thymocytes were gated (A) and sorted into the four subsets (B) defined in the text (i.e., SP1–SP4). The numbers in the top-right corners indicate the purity of the sorted cells. (C) Brachial, mesenteric, and inguinal lymph nodes were pooled from 5 to 8 mice and stained with mAbs against mouse CD4, CD8, CD44, CD62L, NK1.1, and CD25. CD4⁺CD8^-CD44^lo/intCD62L^hiCD25^-NK1.1^- naïve cells were sorted. The number indicates the purity of the sorted cells.

Figure 2. General validation of gene expression.

(A) Gene expressions of Qa2 and Ccr4 in the four thymocyte subsets are revealed by the microarray analysis. (B) Quantitative RT-PCR results of Qa2 and Ccr4 transcripts in the four thymocyte subsets. Asterisks indicate significant (P<0.05) changes in each two-subset comparison. The experiment was performed for three times and similar results were obtained.

Figure 3. Expressions of Il2rb, Cd24, and H2-D1/K1 among the four subsets of CD4⁺ SP thymocytes.

(A) Gene expression of Il2rb, Cd24a, and H2-D1/K1 by the microarray analysis. (B) Expression of IL-2Rβ, CD24, and MHC class I by flow cytometry.

Figure 4. Patterns of expression kinetics among the four subsets of CD4 single positive thymocytes based on SOM analysis.

(A–F) Six patterns of expression kinetics are shown.

Figure 5. CD4 SP thymocytes gradually acquire immuno-competence.

(A) Expression profiles of the immune-related genes among the four subsets. The profile was generated by hierarchical clustering using GeneCluster 2.0 package. All data from three array repeats were shown (080530_SPx, 080722_SPx, and 110413_SPx, x represents 1 to 4). Red represents up-regulation of gene expression, and green represents down regulation of gene expression. The entire list of genes is available in Table S2. (B) Similar activation of SP3, SP4 thymocytes and naïve CD4⁺ T cells upon Listeria Monocytogenes (Lm) infection. Purified SP3, SP4 thymocytes and naïve CD4⁺ T cells from C57BL/6 Ly5.1 congenic mice were adoptively transferred into C57BL/6 Ly5.2 mice (9×10⁶ per host mouse). One day later, the host mice were challenged with 2×10⁴ live Lm via tail vein injection. On day 7 of the infection, the cells from the lymph nodes and spleens were harvested and stained with CD45.1, CD4, CD44, and CD62L. The donor cells with the expression of both CD45.1 and CD4 are gated (shown in left paneal). The expression of CD44 and CD62L of donor cells (CD45.1⁺CD4⁺) are shown on the right panel.

Figure 6. Molecular basis of the adhesion and migration of CD4 SP thymocytes during their final maturation process.

(A) Expression profiles of the adhesion/migration-related genes among the four subsets. The profile was generated by hierarchical clustering using GeneCluster 2.0 package. All data from three array repeats were shown (080530_SPx, 080722_SPx, and 110413_SPx, x represents 1 to 4). Red represents up-regulation of gene expression, and green represents down regulation of gene expression. The entire list of genes is available in Table S2. (B–D) Gene expression of chemotactic molecules that have been reported to change after positive selection. Relative mRNA level of Plxnd1 (B), Ccr9 (C), Cxcr4 (D), and Ccr7 (E) in the four subsets of CD4 SP thymocytes as determined by qRT-PCR. Asterisks indicate significant (p<0.05) changes compared between the two subsets. Three experiments were performed on RNAs from three independently isolated cell populations for each thymocyte subset and similar results were seen.

Figure 7. Expression of genes that are involved in the thymocyte migration and in Foxo1-Klf2 axis.

(A, C–D) Relative mRNA amount of S1pr1, Klf2, and Foxo1 in naïve CD4⁺ T cells and the four subsets of CD4 SP thymocytes as determined by real time RT-PCR. (B) Expression of CD62L as determined by flow cytometry in peripheral naïve CD4⁺ T cells, double positive thymocytes, and the four subsets of CD4 single positive thymocytes. Asterisks indicate significant (p<0.05) change compared between the two subsets. The experiment was repeated for two to three times and similar results were seen.

Figure 8. Cells with the SP4 phenotype are the main population in RTEs.

(A-B) Transwell migration of SP thymocytes and naïve CD4⁺ T cells to S1P (A) and CCL19 (B). Three experiments were performed and the mean and standard deviation were shown. Asterisks indicate significant (P<0.05) difference between SP3 and SP4 subsets. (C) Phenotype of RTEs. Twenty-four hours after intrathymic injection of FITC, the cells from the lymph nodes and spleen were harvested and stained with CD4, Qa-2 and CD69. The surface expression of Qa-2 and CD69 in FITC⁺CD4⁺ T cells are shown. The numbers represent the percentage of Qa-2⁺CD69^- cells in FITC⁺CD4⁺ T cells. The experiment was repeated for two more times and similar results were obtained.