Immune Netw.  2010 Oct;10(5):153-163. 10.4110/in.2010.10.5.153.

CD43 Expression Regulated by IL-12 Signaling Is Associated with Survival of CD8 T Cells

• Lee JB
• Chang J

• KMID: 1456219
• DOI: http://doi.org/10.4110/in.2010.10.5.153

Abstract

BACKGROUND
In addition to TCR and costimulatory signals, cytokine signals are required for the differentiation of activated CD8 T cells into memory T cells and their survival. Previously, we have shown that IL-12 priming during initial antigenic stimulation significantly enhanced the survival of activated CD8 T cells and increased the memory cell population. In the present study, we analyzed the mechanisms by which IL-12 priming contributes to activation and survival of CD8 T cells.
METHODS
We observed dramatically decreased expression of CD43 in activated CD8 T cells by IL-12 priming. We purified CD43(lo) and CD43(hi) cells after IL-12 priming and analyzed the function and survival of each population both in vivo and in vitro.
RESULTS
Compared to CD43(hi) effector cells, CD43(lo) effector CD8 T cells exhibited reduced cytolytic activity and lower granzyme B expression but showed increased survival. CD43(lo) effector CD8 T cells also showed increased in vivo expansion after adoptive transfer and antigen challenge. The enhanced survival of CD43(lo) CD8 T cells was also partly associated with CD62L expression.
CONCLUSION
We suggest that CD43 expression regulated by IL-12 priming plays an important role in differentiation and survival of CD8 T cells.

Keyword

CD8 T cell; IL-12; CD43; Survival

MeSH Terms

Adoptive Transfer
Granzymes
Interleukin-12
Memory
T-Lymphocytes
Granzymes
Interleukin-12

Figure

• 

  Figure 1 CD43 expression is regulated by initial IL-12 priming during primary CD8 T-cell stimulation. (A) OT-I TCR transgenic cells were stimulated with the OVA peptide (OVAp) in the presence or absence of IL-12. On days 1, 3 and 7, cells were harvested, stained with anti-CD8α, CD44, and CD43, and analyzed by flow cytometry. (B) OT-I cells were stimulated with OVA peptide in the presence of IL-12 (5 ng/ml), and on day 7, cells were harvested, stained with anti-CD8α, CD43 and CD127, CD62L or KLRG1, and analyzed by flow cytometry. Histograms show the expression levels of CD127, CD62L or KLRG1 in the gated CD43hi and CD43lo populations and are shown as the mean±SD. The results shown are representative of three independent experiments with similar results. Filled histogram, CD43hi; open histogram, CD43lo.

• 

  Figure 2 CD43lo cells induced by initial IL-12 priming increase their survival rate in vivo, and enhanced survival of CD43lo cells is not due to differential trafficking of transferred cells to various organs. (A) For adoptive transfer experiments, the splenocytes from OT-I TCR transgenic mice were stimulated with the peptide in the presence of IL-12 for 7 days. CD8+CD43hi and CD8+CD43lo cells were MACS-purified and 2×106 cells were i.v. injected into normal C57BL/6 mice. (B) After 3, 10, 17, 34, 45 and 56 days, donor OT-I cells in the blood of recipient mice were identified by CD8α, Kb/OVA-Tet and CD43 staining. (C) On day 7, the frequency of donor OT-I cells in lymphoid and non-lymphoid organs of the recipient mice were measured. The results shown are derived from spleens of 5 mice for each group and are representative of three independent experiments with similar results.

• 

  Figure 3 Attenuation of apoptosis in CD43lo population. Spleen cell suspensions from naïve OT-I mice were cultured for 7 days with 100 nM OVAp in the presence of IL-12. Then, activated OT-I cells were MACS-purified and rested for 3 additional days without any stimulant and with IL-2. At the indicated time points, the cells were harvested and apoptotic cell death was determined by Annexin V and 7-AAD double-staining. The samples were assayed in triplicate and the error bars represent the SD values of the mean.

• 

  Figure 4 Decreased cytolytic functions in CD43lo population. Spleen cell suspensions from naïve OT-I mice were cultured for 7 days with 100 nM OVAp in the presence of IL-12. Then, activated OT-I cells were MACS-sorted into CD8+CD43hi and CD8+CD43lo populations. (A) The levels of granzyme B in each cell population were determined by intracellular staining. Histograms show the expression of granzyme B in the gated CD43hi and CD43lo subsets and are shown as the mean±SD. The results shown are representative of three independent experiments with similar results. Filled histogram, CD43lo; open green histogram, CD43hi. (B) Peptide-pulsed EL4 target cells (1×104/well) were added to serial dilutions of effector cells (prepared as described above) in 96-well round-bottom plates at E:T ratio of 1:1 to 20:1. After 4 h at 37℃, cytotoxicity was quantified by measurement of cytosolic lactate dehydrogenase (LDH) in the culture supernatant (n=3) using a cytotoxicity detection kit. All experimental procedures and assays were performed two or more times, with similar results. (C) IFN-γ production of purified CD43hi and CD43lo cells was assessed by intracellular IFN-γ staining following 5 h stimulation with 1 pM~1µM OVAp. (D) Data for functional avidity of purified CD43hi and CD43lo cells have been normalized to equate the maximum response obtained after stimulation with the OVAp used to establish the line to 100%.

• 

  Figure 5 Recall response of CD43lo population is superior to that of CD43hi population. For recall response, the splenocytes from OT-I TCR transgenic mice were stimulated with the antigenic peptide (OVAp) in the presence of IL-12 for 7 days. Then, CD8+CD43hi and CD8+CD43lo cells were separated by MACS. 1×105 purified CD43hi and CD43lo CD8+ T cells in 200µl of PBS were transferred into naïve C57BL/6 mice via tail vein injection and then 1 day after transfer, the mice were intranasally challenged with 2×107 pfu of recombinant adenovirus expressing OVA (rAd/OVA).

• 

  Figure 6 Enhanced survival of CD43lo cells is partly associated with CD62L expression. (A) OT-I cells were cultured for 7 days with OVAp in the presence of IL-12. After 7 days, purified CD8+CD62Lhi T cells and CD8+CD62Llo T cells were adoptively transferred into C57BL/6 mice. (B) At the indicated time points, donor cell recovery from the peripheral blood of recipient mice was determined by tetramer staining, and (C) on day 32, the frequency of donor OT-I cells in lymphoid and non-lymphoid organs of the recipient mice was measured.

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