T cells expressing the transcription factor PLZF regulate the development of memory-like CD8+ T cells

Abstract

Several gene-deficiency models promote the development of innate CD8(+) T cells that have diverse T cell antigen receptors (TCRs) but have a memory phenotype and rapidly produce cytokines. We demonstrate here that similar cells developed in mice deficient in the transcription factor KLF2. However, this was not due to intrinsic deficiency in KLF2 but instead was due to interleukin 4 (IL-4) produced by an expanded population of T cells expressing the transcription factor PLZF. The development of innate CD8(+) T cells in mice deficient in the tyrosine kinase Itk and coactivator CBP was also attributable to this IL-4-dependent mechanism. Finally, we show that the same mechanism drove the differentiation of innate CD8(+) T cells in BALB/c mice. Our findings identify a previously unknown mechanism of regulation of CD8(+) T cells via the production of IL-4 by PLZF(+) T cells.

Figure 1

KLF2-deficient T cells induce a memory-like phenotype on bystander CD8⁺ thymocytes. (a) Schematic of the bone marrow chimera set-up used in the following experiments. The majority bone marrow is indicated with parentheses. (b) Analysis of the cell surface phenotype of wildtype (WT) bystander CD8⁺ SP thymocytes. Black line WT CD8⁺s from Cd4-cre-Klf2^fl/fl majority, and filled gray all WT chimera. (c) Eomesodermin (Eomes) and T-bet expression on bystander CD8⁺ thymocytes. T-bet staining control is CD1d-tet⁺ cells from the thymus of the WT chimera. Results are representative of more than 5 experiments. (d) Thymocytes were stimulated with PMA and ionomycin and the percent of CD8⁺ SP producing IFNγ was analyzed.

Figure 2

Bystander CD8⁺s have increased in vivo function. (a) Schematic of mixed bone marrow chimeras with OT-I-Rag1^−/− CD8⁺ T cells as minority bystander population. Thick line indicates majority population. (b) CD44 and CD122 expression of bystander OT-I CD8⁺s from combined spleen and lymph node. (c) 3×10⁵ OT-I T cells from indicated chimera was transferred with an equal number of naïve OT-I T cells. Mice were infected with Act^−/− Listeria monocytogenes (LM)-OVA and ratio of OT-I from experimental (exp) chimera to naïve OT-I was analyzed. Blood (left panel) analyzed at day 4 and spleen (right panel) at day 7. n=3 per group. (d) IFN-γ production by OT-I T cells from indicated chimera or intact OT-I-Rag1^−/− splenocytes following stimulation with IL-2, IL-12 and IL-18, left panel, or OVA peptide, right panel. Graphs represent at least 2 mice from 2 separate experiments. Error bars represent s.d.

Figure 3

KLF2 deficiency leads to an expansion of PLZF-expressing T cells. (a) PLZF mRNA expression on sorted CD4 SP thymocytes, quantified by real time PCR. n=5 (b) PLZF and CD3 expression on thymocytes from intact WT and Cd4-cre-Klf2^fl/fl mice. Results are representative of more than ten mice. (c) PLZF expression on donor thymocytes from mixed chimeras of WT and Cd4-cre/Klf2^fl/fl bone marrow. Percentage of PLZF⁺ cells within the CD45 congenic population is indicated in parentheses.

Figure 4

PLZF⁺ cells are responsible for excess IL-4 production and CD8⁺ bystander effects in KLF2 deficient mice. (a) IL-4 and PLZF expression in thymocytes from intact WT and Cd4-cre-Klf2^fl/fl mice. Thymocytes were isolated and stimulated with PMA and ionomycin for 5 hours then intracellular stained for IL-4 and PLZF. (b) Thymocyte expression of CD4 and CD8⁺ and (c) CD8⁺ SP phenotype from WT, Cd4-cre-Klf2^fl/fl and Cd4-cre-Klf2^fl/fl-Plzf^Lu/Lu mice.

Figure 5

Itk^−/− mice have a cell-extrinsic CD8⁺ phenotype and expanded PLZF⁺ population. (a) CD8⁺ SP phenotype from intact Itk^−/− mice (thin line), mixed chimera with Itk^−/− majority (thick line) and an all WT chimera (shaded). Top row shows the majority population and bottom row shows the WT bystander population. (c) Eomesodermin and T-bet expression on the CD8⁺ SP population and (d) PLZF by CD3 expression on all thymocytes from mixed bone marrow chimeras. Results are representative of at least 4 mice.

Figure 6

The memory-like CD8⁺ phenotype in Itk^−/− mice is dependent on IL-4 and PLZF. Thymocyte analysis from WT, Itk^−/−, Itk^−/−-Il4ra^−/−, and Itk^−/−-PLZF^Lu/Lu mice. (a) CD4 by CD8⁺ expression. (b) Histogram overlays of CD8⁺ SP phenotype. Gray shaded = WT and black lines from left to right represent Itk^−/−, Itk^−/−-Il4ra^−/−, and Itk^−/−-PLZF^Lu/Lu mice. Graphs represent at least 2 mice from 2 separate experiments.

Figure 7

An expanded PLZF⁺ population and an IL-4 and NKT dependent memory-like population in BALB/c mice. (a) PLZF and CD3 expression of thymocytes from B6, BALB/c and Il4ra^−/− on BALB/c background and (d) BALB/c and Cd1d^−/− on BALB/c background. (b, e) CD44 by CD122 and (c, f) Eomesodermin (Eomes) by T-bet expression on the CD8⁺ SP thymocyte populations. Results are representative of a minimum of 3 mice per experimental group.