Heterogeneity of lymphoid tissue inducer cell populations present in embryonic and adult mouse lymphoid tissues

Abstract

Lymphoid tissue inducer (LTi) cells have a well established role in secondary lymphoid tissue development. Here, we report on the heterogeneity of LTi cells based on their CD4 and chemokine receptor expression. The CD4(-) LTi-cell population has a similar phenotype to the CD4(+) population, with similar chemokine-receptor-expressing subsets. In both embryonic and adult spleen the CD4(-) LTi-cell population is comparable as a proportion of total splenocytes to its CD4(+) counterpart. In contrast, different proportions of CD4(+) and CD4(-) LTi cells are found in different lymph nodes. Both CD4(+) and CD4(-) LTi cells share the anatomical location and are associated with vascular cell adhesion molecule-1-positive stromal cells in spleen and lymph nodes. The numbers of both CD4(+) and CD4(-) LTi cells in adult spleen are augmented in the presence of B cells. With the exception of CD4, there is a strong correlation coefficient (0.89) for gene expression between the two populations. Polymerase chain reaction analysis of individual CD4(+) and CD4(-) LTi cells shows that a similar proportion in embryonic and adult spleen co-expressed both CXCR5 and CCR7 or CXCR5 alone: 84.6% for adult CD4(+) and 87.6% for adult CD4(-); 95.3% for embryonic CD4(+) and 91.5% for embryonic CD4(-). Consistently fewer CCR7 single-positive cells were found in the CD4(+) and CD4(-) fractions in the embryo.

Figure 1

Phenotype of CD4⁺ and CD4⁻ lymphoid tissue inducer (LTi) cells from adult and E15 spleens. (a–c) CD11c-depleted and Thy-1-enriched population from adult T-cell-deficient mouse spleens (a), mesenteric lymph nodes (b) and inguinal lymph nodes (c). (d) Embryonic (E15) splenocytes from wild-type mice cultured with interleukin-7 (IL-7) for 1 week. The results are representative of at least five separate experiments. For LTβR-Ig staining adult CD11c⁻ Thy-1⁺ cells were cultured with IL-7 for 1 week. To test IL-7Rα surface expression, E15 spleens cultured with IL-7 were transferred into IL-7-free medium for 1 day before staining. (e) Upregulation of OX40L expression on E15 CD4⁺ and CD4⁻ LTi cells after 2 days culture with 100 ng/ml TL1A. The results are representative of at least 10 separate experiments.

Figure 2

Correlation between gene expressions in CD4⁺ and CD4⁻ lymphoid tissue inducer (LTi) cells isolated from wild-type E15 spleens. (a) CD4⁺ LTi (Thy-1⁺ CD4⁺ c-Kit⁺ CD27⁻) and CD4⁻ LTi (Thy-1⁺ CD4⁻c-Kit⁺ CD27⁻) cells from wild-type E15 spleens were MoFlo-sorted and analysed their mRNA expressions by TaqMan 96-gene arrays for a panel of immunity-related genes (see Materials and methods). Over 0·01% expression of individual genes compared with β-actin mRNA signals (β-actin signal = 100%) was plotted. If the mRNA signal of a gene showed < 0·01% of β-actin signals, the gene was excluded from the plot. x and y axes show relative levels of mRNA expression of the two cell types being correlated by β-actin signals (β-actin signal = 100%). Correlation coefficient (CC) between CD4⁺ and CD4⁻ LTi cells is 0·89. (b) Expression of (i) tumour necrosis factor (TNF) ligands and TNF receptors, (ii) common γ-chain cytokine receptors, and (iii) chemokine receptors, Bcl-2 family members, GATA, and CD4 in E15 CD4⁺ and CD4⁻ LTi cells. * mRNA signals of CD4 in CD4⁻ LTi cells were not detectable (below 0·01% of β-actin signals). Cross line inside graph indicates CC = 1.

Figure 3

Single-cell polymerase chain reaction for the expression of CCR7 and CXCR5 in CD4⁺ and CD4⁻ lymphoid tissue inducer (LTi) cells isolated from adult T-cell-deficient but B-cell-sufficient spleens and wild-type E15 spleens. (a) Expression of CXCR5 and CCR7 in 492 adult CD4⁺ LTi, 516 adult CD4⁻ LTi, 488 E15 CD4⁺ LTi, and 485 E15 CD4⁻ LTi cells. The expression of CXCR5 (x axis) was plotted against CCR7 (y axis) after normalization to β2-microglobulin (β2m) signals [C_t value of β2m –C_t value of chemokine receptor (log 2)]. Each dot indicates the gene expression of a single cell. For each population, 525 total single cells were MoFlo-sorted in three separate plates (175 cells/plate). (b) Percentage of CCR7⁺ CXCR5⁻, CCR7⁺ CXCR5⁺, CCR7⁻ CXCR5⁺, and CCR7⁻ CXCR5⁻ cells in four LTi populations from the three separate experiments. Error bar shows the standard deviation.

Figure 4

Both CD4⁺ and CD4⁻ lymphoid tissue inducer (LTi) cells interact with vascular cell adhesion molecule 1-positive (VCAM-1⁺) stroma in the spleen and lymph node. Spleen (a) and mesenteric lymph node (b) from T-cell-deficient mice stained for expression of VCAM-1 (white), Thy-1 (red) and CD4 (green). CD4⁺ LTi cells are indicated in yellow circles, and CD4⁻ LTi cells in red circles. Left-hand panels show low-power image, scale bar represents 100 μm; right-hand panels show high-power image of demarcated area, scale bar represents 20 μm.

Figure 5

Development of embryonic CD4⁺ and CD4⁻ lymphoid tissue inducer (LTi) cells by interleukin-7 (IL-7). E15 spleens were organ cultured with or without IL-7 up to 7 days and stained for α₄ β₇, lineage marker (lin; CD3, CD8, CD11c, CD27, and B220), CD4 and Thy-1. The α₄ β₇⁺ lin⁻ cells were gated. The results are representative of at least three separate experiments.

Figure 6

Relative cell numbers of adult CD8⁺ DCs, CD8⁻ DCs, CD4⁺ and CD4⁻ lymphoid tissue inducer (LTi) cells in B-cell-sufficient mice to RAG^−/− mice. Age- and sex-matched RAG^−/− (CD45.2) and B-cell-sufficient (CD45.1) adult mouse spleens were mixed and prepared for the isolation of the cells. The mixed cells were distinguished by allotypic marker, CD45. CD11c-enriched population was stained for CD45.1, CD11c, and CD8 (CD45.1⁺ CD11c⁺ CD8⁺ = CD8⁺ DCs and CD45.1⁺ CD11c⁺ CD8⁻ = CD8⁻ DCs from B-cell-sufficient mouse, and CD45.1⁻ CD11c⁺ CD8⁺ = CD8⁺ DCs and CD45.1⁻ CD11c⁺ CD8⁻ = CD8⁻ DCs from RAG^−/− mouse). CD11c-depleted and Thy-1-enriched population was stained for CD45.1, Thy-1, CD4, and all lineage (lin) makers (CD45.1⁺ Thy-1⁺ CD4⁺ lin⁻ = CD4⁺ LTi and CD45.1⁺ Thy-1⁺ CD4⁻ lin⁻ = CD4⁻ LTi cells from B-cell-sufficient mouse, and CD45.1⁻ Thy-1⁺ CD4⁺ lin⁻ = CD4⁺ LTi and CD45.1⁻ Thy-1⁺ CD4⁻ lin⁻ = CD4⁻ LTi cells from RAG^−/− mouse). The relative cell number of each population from B-cell-sufficient mouse was corrected to that from RAG^−/− mouse (the cell number of each population from RAG^−/− mouse is 1). The results are the average of five separate experiments and error bar shows the standard deviation.