Dendritic cell expression of CD24 contributes to optimal priming of T lymphocytes in lymph nodes

Abstract

CD24 is a GPI anchored cell surface glycoprotein whose function as a co-stimulatory molecule has been implicated. However, the function of CD24 on antigen presenting cells during T cell responses is not well understood. Here we show that in the CD24-deficient host, adoptively transferred CD4⁺ T cells undergo inefficient expansion and have accelerated cell death in lymph nodes, which results in insufficient priming of T cells. Insufficient expansion of T cells in the CD24-deficient host was not due to host anti-CD24 response by NK, T and B lymphocytes. Transgenic expression of CD24 on DC in CD24^-/- mice restored T cell accumulation and survival in draining lymph nodes. Consistent with these findings, MHC II tetramer staining also revealed that an antigen-specific polyclonal T cell response was reduced in lymph nodes of CD24^-/- mice. Taken together, we have revealed a novel role of CD24 on DC in optimal T cell priming in lymph nodes. These data suggest that CD24 blockade should lower unwanted T cell responses such as those in autoimmune diseases.

Keywords: CD24; EAE (experimental autoimmune encephalitis); T cell priming; T lymphocytes; dendritic cells.

Figure 1

Proliferation of 2D2 T cells in CD24-deficient mice. CFSE-labeled 2D2 T cells (5 x 10⁶/mouse) were injected into WT or CD24^-/- mice intravenously (i.v.) followed by subcutaneous (s.c.) injection of MOG 35-55 emulsified in CFA. (A, B). Percentages of 2D2 T cells (CFSE⁺Vβ11⁺) in draining lymph nodes and spleens of WT and CD24^-/- mice at 17 h (A) and 65 h (B) after immunization were analyzed by flow cytometry. Data shown in A-B are representative of eight experiments with similar results. (C) Percent of divided 2D2 T cells in lymph nodes and spleens at 65 h. Representative data from one experiment are shown. (D, E). Percentages and total numbers of 2D2 T cells in draining lymph nodes (C) and spleens (D) of WT and CD24^-/- hosts at 65 h were quantified. Data shown in (C, D) were pooled from three independent experiments performed in sex and age-matched mice. SP, spleen; LN, draining lymph nodes. *p<0.05, student’s t test was used for the comparison.

Figure 2

2D2 T cell survival in lymph nodes of CD24^-/- and WT mice. (A). Flow cytometry analysis of 2D2 T cell death in draining lymph nodes of WT and CD24^-/- mice. Data shown are gated on Vβ11⁺CFSE⁺ cells. 5 x 10⁶ CFSE-labeled 2D2 T cells were injected into WT or CD24^-/- mice i.v. followed by immunization with MOG 35-55/CFA. (B). Percent of Annexin V⁺ 2D2 T cells in the draining lymph nodes of WT and CD24^-/- mice. Five mice per group were used for the experiment. *p<0.05, student’s t test was used for the comparison. Data represents 8 experiments with similar results.

Figure 3

Proliferation and survival of OT2 T cells in CD24^-/- mice. (A, B). 1 x 10⁶ CFSE-labeled OT2 T cells were injected into WT or CD24^-/- mice i.v. Percent of OT2 T cells (CFSE⁺Vβ5.1⁺) in draining lymph nodes and spleens were quantified at 65 h after immunization with OVA 323-339/CFA. Five mice per group were used in this experiment. Data shown are Mean ± SD. *p<0.05, ***P<0.001; student’s t test was used for the comparison. (C, D). Percent of 2D2 T cells that underwent apoptosis in draining lymph nodes and spleens of WT and CD24^-/- mice at 65 h after immunization with OVA 323-339/CFA. Five mice per group were used in this experiment. Data shown are Mean ± SD. *p<0.05, student’s t test. Data shown represent two experiments with similar results.

Figure 4

Host response to CD24 is not responsible for insufficient expansion of T cells in lymph nodes of CD24^-/- mice. (A, B). Depletion of NK cells does not rescue 2D2 T cells in the lymphoid organs of CD24^-/- mice. 300 μg of PK136 antibody (anti-NK1.1) was injected into each WT and CD24^-/- mouse i.p. Two days later each mouse received 3 x 10⁶ of CFSE-labeled 2D2 T cells i.v. followed by immunization with MOG 35-55/CFA. Flow cytometry analysis of lymph node cells was performed at 65 h. Three mice per group were used in this experiment. Data shown are Mean ± SD. **p<0.01, student’s t test. Data are representative of two experiments with similar results. (C, D). 5 x 10⁶ of CFSE-labeled CD24-deficient 2D2 T cells (obtained from 2D2⁺CD24^-/-MOG^-/- mice) were injected into each WT and CD24^-/- mice i.v. followed by immunization with MOG 35-55/CFA. Flow cytometry analysis of lymph node cells was performed at 65 h. Five mice per group were used in this experiment. Data shown are Mean ± SD. **p<0.01, student’s t test. Data are representative of two experiments with similar results.

Figure 5

2D2 T cell proliferation and pathogenicity in RAG-1^-/-CD24^-/- mice. (A, B). 2D2 T cells accumulated in the draining lymph nodes [upper panel of (A)] and spleens [lower panel of (A)] of RAG-1^-/- mice but not in RAG-1^-/-CD24^-/- mice. 5 x 10⁶ CFSE-labeled 2D2 T cells were injected into each recipient mouse i.v. followed by immunization with MOG 35-55/CFA. Flow cytometry analysis of lymph node cells were performed at 65 h. Percent of CFSE⁺Vβ11⁺ cells in each group were plotted in (B). **P<0.01, ***P<0.001 by student's t test. Five mice per group were used in this experiment and data shown represents Mean ± SD. Student’s t test was used for the statistical analysis. (C). RAG-1^-/-CD24^-/- mice receiving 2D2 T cells failed to develop EAE. 2 x 10⁶ of CD4 T cells from 2D2 TCR transgenic mice were injected into each RAG-1^-/- and RAG-1^-/-CD24^-/- mouse i.v. The recipients were then immunized for EAE using MOG35-55/CFA/PT. Data shown represents two experiments with similar results.

Figure 6

Expression of CD24 in lymph node cells. (A). Lymph node cells from WT and CD24^-/- mice were stained for CD45 and CD24. (B). CD24 expression on different subsets of CD45⁺ Cells. Lymph node cells from WT and CD24^-/- mice were stained for CD45, CD24 and one of the subset markers. Data represent five experiments with similar results.

Figure 7

CD24 expression on DC contributes to proliferation and survival of 2D2 T cells. 5 x 10⁶ of CFSE-labeled 2D2 T cells were injected into DC^CD24CD24^-/- or CD24^-/- mice i.v. followed by immunization with MOG 35-55 emulsified in CFA. Representative staining (A) and quantification of % CFSE⁺Vβ11⁺ cells (B) are shown. Five mice per group were used in this experiment and data shown are Mean ± SD. ***P < 0.001. Student’s t test was used for the statistical analysis. (C, D). Apoptosis of 2D2 T cells was quantified at 65 h after immunization with MOG 35-55/CFA. Five mice per group were used for this experiment. Data shown are Mean ± SD. Student’s t test was used for the statistical analysis. Data represent two experiments with similar results. *P < 0.05.

Figure 8

Reduced priming of MOG peptide-specific T cells in lymph nodes from CD24^-/- mice. WT and CD24^-/- mice were immunization with MOG 35-55/CFA. On day-10 after immunization, lymph node CD4⁺ T cells were assessed for proliferation (A) and pMOG-MHCII-tetramer staining (B). Data shown represents two (A) and three (B) experiments with similar results. *P<0.05 by student’s t test.