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LKLF: A Transcriptional Regulator of Single-Positive T Cell Quiescence and Survival

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Science  26 Sep 1997:
Vol. 277, Issue 5334, pp. 1986-1990
DOI: 10.1126/science.277.5334.1986

Abstract

Mature single-positive (SP) T lymphocytes enter a “resting” state in which they are proliferatively quiescent and relatively resistant to apoptosis. The molecular mechanisms regulating this quiescent phenotype were unknown. Here it was found that the expression of a Kruppel-like zinc finger transcription factor, lung Kruppel-like factor (LKLF), is developmentally induced during the maturation of SP quiescent T cells and rapidly extinguished after SP T cell activation. LKLF-deficient T cells produced by gene targeting had a spontaneously activated phenotype and died in the spleen and lymph nodes from Fas ligand–induced apoptosis. Thus, LKLF is required to program the quiescent state of SP T cells and to maintain their viability in the peripheral lymphoid organs and blood.

Single-positive (CD4+ or CD8+) mature T cells circulate through the blood and peripheral lymphoid organs in a quiescent or resting state until they encounter their cognate antigen bound to a major histocompatibility molecule (MHC) on the surface of an appropriate antigen-presenting cell (1). Engagement of the T cell antigen receptor (TCR) by the peptide antigen– MHC complex leads to T cell activation, a process that involves the highly orchestrated expression of more than 100 new genes and concomitant cell cycle progression and proliferation (2). Activated T cells acquire a characteristic set of cell surface markers and are more sensitive to apoptosis (3, 4). Many activated T cells die in the peripheral lymphoid organs from activation-induced cell death, an apoptotic pathway that has been postulated to protect the host against autoimmune disease (5). Several transcription factors, including activation protein–1 (AP1), nuclear factor of activated T cells (NFAT), nuclear factor–kappa B, (NF-κB), and the cAMP response element binding protein (CREB), are known to be important positive regulators of activation-specific T cell gene expression (6,7). However, it was not known if specific transcription factors are also required to program or maintain the quiescent state in resting SP T cells (8).

The erythroid Kruppel-like factor (EKLF) is an erythroid-specific zinc finger transcription factor that binds to CACCC sequence motifs in the promoter of the β-globin gene and regulates the terminal stages of erythroid development (9). To identify EKLF-related genes that might play a similar role in T cell development, we screened an embryonic mouse cDNA library under low-stringency hybridization conditions with a probe derived from the zinc finger region of EKLF (10). We identified three additional KLF family members, BKLF, GKLF, and LKLF (11). To determine whether any of these other KLF proteins might participate in T cell development, we hybridized Northern (RNA) blots and mouse tissue sections to probes specific for each cDNA. LKLF was expressed in the lung, the vasculature, the heart, skeletal muscle, kidney, and testis as well as in the lymphoid organs of the mouse (Fig.1A) (12). Within the thymus, LKLF was expressed exclusively in lymphoid cells in the thymic medulla, a region that contains mature SP thymocytes (Fig. 1B). LKLF was also expressed in large amounts in lymphoid cells in the white pulp of the spleen (12). To more precisely characterize LKLF expression in the hematopoietic lineages, we did Northern blot analyses on purified populations of hematopoietic cells (Fig. 1A). LKLF was expressed in both CD4+ and CD8+ SP thymocytes and splenocytes, but it was undetectable in less mature double-positive (DP) CD4+CD8+ thymocytes (Fig.1A). LKLF was also expressed in B220+ immunoglobulin M (IgM)–positive splenic B cells and in bone marrow macrophages (Fig.1A). LKLF mRNA and protein were decreased significantly after the TCR-mediated activation of quiescent splenic T cells (Fig. 1, C and D). Degradation of the LKLF protein preceded disappearance of the mRNA, suggesting that both LKLF mRNA and protein are regulated after T cell activation. This developmentally and activation-regulated pattern of expression suggested that LKLF might play an important role in regulating the function of resting SP T cells in vivo.

Figure 1

Expression pattern of LKLF. (A) Top, Northern blots containing RNA prepared from mouse tissues (left) or purified populations of adult C57BL/6 thymocytes, splenocytes, and macrophages (right) were hybridized to a radiolabeled cDNA probe from the 3′ untranslated region (UTR) of LKLF (20). Bottom, equal loading and the integrity of RNA were confirmed by ethidium bromide staining and visualization of 28S RNA or by hybridization to a β-actin cDNA probe. (B) In situ hybridization. Sections of thymii from adult C57BL/6 mice were hybridized to 35S-labeled cRNA probes prepared from the 3′ UTR of LKLF (21). Positive hybridization is pink, negative hybridization is blue (original magnification, ×50). M, thymic medulla; C, thymic cortex. (C) Top, Northern blots containing RNA prepared from purified adult splenic T cells activated for various times (0 to 72 hours) with anti-CD3 were hybridized to a radiolabeled cDNA probe from the 3′ UTR of LKLF (20). Bottom, equal loading and the integrity of RNA were confirmed by ethidium bromide staining and visualization of 28S RNA. (D) Top, protein immunoblots containing protein extracts prepared from purified adult splenic T cells activated for various times (0 to 6 hours) with anti-CD3 were incubated with a rabbit polyclonal antiserum to LKLF (22). Bottom, equal loading of cellular extracts was confirmed by equivalent amounts of a low molecular size band on the same blot.

We used homologous recombination in murine embryonic stem (ES) cells to produce a targeted mutation of the LKLF gene (Fig.2A). The resulting null mutation deleted the entire LKLF gene. Heterozygous (LKLF+/−) mutant ES cell clones produced with a phosphoglycerate kinase-neomycin resistance (PGK-neor ) targeting vector (Fig. 2A) were retransfected with a PGK-hygromycin resistance (PGK-hygror ) targeting vector (Fig. 2A) to produce three independently derived homozygous (LKLF−/−) ES cell clones. The genotypes of these clones were confirmed by Southern (DNA) blot analysis (Fig. 2B). Each clone contained singleneor and hygror integrations. By Northern blot analysis, each of the homozygous clones lacked detectable LKLF mRNA (Fig. 2C).

Figure 2

Targeted disruption of the LKLF gene. (A) Representation of the LKLF targeting construct (23). Top, restriction endonuclease map of the murineLKLF locus. Exons (E1 to E3) are shown as boxes, the coding region is shown in black, the zinc finger encoding region is shaded, and the 5′ and 3′ UTRs are white. E, Eco RI; S, Sal I; H, Hind III; P, Pac I. Middle, structure of the LKLF targeting constructs containing the herpes simplex virus–thymidine kinase (tk) and the neomycin (neor ) and hygromycin (hygror ) resistance genes under the control of the mouse PGK promoter. Bottom, structure of the targetedLKLF alleles, which contain deletions of the entireLKLF gene. (B) Southern blot analysis of the LKLF wild-type (+/+), heterozygous (+/−), and homozygous (−/−) ES cell clones (23). The sizes of the wild-type (WT) and targeted (T) alleles are shown to the right. (C) Top, Northern blot analysis of LKLF expression in the wild-type (+/+), heterozygous (+/−), and homozygous (−/−) ES cell clones with a full-length radiolabeled LKLF cDNA probe. Bottom, equal loading and integrity of the RNA samples were confirmed by ethidium bromide staining of the 28S RNA.

LKLF+/− ES cell clones were used to produce chimeric mice, which transmitted the targeted allele through the germ line (13). Heterozygous (LKLF +/−) mice were phenotypically normal and were bred to produce LKLF-deficient animals. Mice homozygous for the LKLF mutation died between embryonic days 12.5 and 14.5 as a result of intra-amniotic and intra-embryonic hemorrhages (13). To analyze the role of LKLF in lymphoid development and function, we injected three independently derived LKLF−/− ES cell clones and two LKLF+/− ES cell clones into recombinase activating gene 2–deficient (RAG-2−/−) blastocysts (14) to produceLKLF +/− RAG2 −/−and LKLF −/− RAG2 −/−chimeric mice. Because mature B and T cells cannot develop in the absence of RAG-2, all B and T cells in such chimeric mice are derived from the LKLF−/− ES cells. Thus, the RAG-2−/− chimera system provides a stringent test for the intrinsic role of LKLF in B and T cell development and function in an environment of LKLF-expressing tissues (14).

In control experiments, injection of RAG-2−/−blastocysts with LKLF+/− ES cells fully rescued the defects in B and T cell development in the RAG-2−/−animals (Fig. 3, A and B).LKLF +/− RAG2 −/− mice contained normal populations of double-negative (DN) CD4CD8, DP, and SP thymocytes and peripheral T cells (Fig. 3A), normal populations of B220+IgM+ peripheral B cells (Fig. 3A), and normal concentrations of serum immunoglobulins (12). Consistent with these results, LKLF +/− mice also displayed apparently normal lymphocyte development and function (12). In contrast, chimeric mice produced by injection of RAG-2−/− blastocysts with LKLF−/− ES cells displayed profound peripheral T cell defects. Total splenic and lymph node T cell numbers were reduced by more than 90% (Figs. 3, A and B) (12). The LKLF −/− RAG2 −/− animals uniformly lacked circulating CD4+ and CD8+ T cells, as assessed by flow cytometry of peripheral blood leukocytes (Fig. 3A). In addition, both the CD4+ and CD8+LKLF−/− splenic and lymph node T cells displayed an abnormal cell surface phenotype (CD44hi, CD69hi, and l-selectinlo) (Fig.3C). This constellation of cell surface markers is characteristic of an activated as opposed to a quiescent peripheral SP T cell (3) and suggested that most LKLF−/− peripheral T cells were spontaneously activated. However, these cells were not CD25+ (high-affinity interleukin-2 receptor) and were not proliferating as determined by propidium iodide fluorescence-activated cell sorting (FACS) analysis (12).

Figure 3

Peripheral T cell defects in adultLKLF −/− RAG2 −/−chimeric mice. (A) Thymocytes, splenocytes, and peripheral blood leukocytes were analyzed by two-color flow cytometry with antibodies to CD4 and CD8 or B220 and IgM. The results shown are representative of the analyses of at least five animals in each group (24). (B) T and B cell numbers in the spleens of wild-type (WT), heterozygous (+/−), and homozygous (−/−) chimeric animals. T cell numbers represent the numbers of CD4+ and CD8+ SP splenocytes. B cell numbers represent the numbers of B220+IgM+ splenocytes. The results are shown as the mean ± SEM for at least five animals in each group. (C) Flow cytometric analyses of splenocytes fromLKLF −/− RAG2 −/−chimeric mice. CD4+ SP splenocytes [see circled populations in (A)] were analyzed for expression of CD44, CD69, andl-selectin by three-color flow cytometric analyses (24). The results are representative of analyses performed on at least five animals in each group, and identical results were obtained in chimeras produced with three independently derived LKLF−/− ES cell clones. Analyses of the CD8+SP thymocytes from these animals yielded similar results (12).

Several experiments were done to ensure that the peripheral T cell defects seen in theLKLF −/− RAG2 −/− mice were not due to insufficient chimerism. Southern blot analysis of DNA isolated from multiple organs including heart, kidney, liver, skeletal muscle, and brain showed that all of the animals used in these studies were 60 to 90% chimeric (12). The SP T cell abnormalities described above were seen in all of theLKLF −/− RAG2 −/−chimeric animals, which were produced by the injection of three independently derived LKLF−/− ES cell clones, but in none of the LKLF +/− RAG2 −/−chimeric animals, nor in any of more than 50 RAG-2−/−chimeras produced with ES cells containing heterozygous or homozygous deletions of the Ets-1, Elf-1, or GATA-3 transcription factors (15). The sameLKLF −/− RAG2 −/−animals that lacked circulating T cells displayed normal numbers of mature (B220+IgM+) peripheral B cells in the spleen, lymph nodes, and blood (Fig. 3, A and B) (12). Purified LKLF−/− splenic B cells from these animals proliferated normally in response to treatment with lipopolysaccharide and to IgM cross-linking and produced normal concentrations of serum immunoglobulins (12). Because it is more difficult to reconstitute the B cell than the T cell compartment in Rag-2−/− chimeric animals (16), the finding of normal numbers of functionally intact B cells in theLKLF −/− RAG2 −/−animals showed that the peripheral T cell defects seen in these animals were the result of LKLF deficiency and were not due to insufficient lymphoid compartment chimerism. Although selective defects in the lymphoid cell lineages in the RAG-2−/− chimera system are typically cell intrinsic, the high levels of chimerism of theLKLF −/− RAG2 −/−animals used in these experiments did not allow us to exclude the possibility that LKLF−/− cells of other lineages contributed to the observed T cell phenotype. To examine this possibility further, we analyzedLKLF −/− RAG2 −/− mice ranging in chimerism from 5 to 90%, each of which had fully reconstituted their B cell compartments. All of these animals displayed an identical T cell phenotype, suggesting that the observed T cell defects were cell intrinsic (12).

Several mechanisms might have been responsible for the severe peripheral T cell defects seen in theLKLF −/− RAG2 −/−mice. First, it was possible that LKLF was required for the production or survival or both of SP thymocytes. However, by flow cytometry, there was no evidence of a block in SP thymocyte production in theLKLF −/− RAG2 −/−animals (Fig. 3A). TheLKLF +/− RAG2 −/− andLKLF −/− RAG2 −/−animals contained equivalent total numbers of thymocytes (12). Moreover, thymii from theLKLF −/− RAG2 −/−animals contained relatively increased numbers of SP cells and decreased numbers of DP cells as compared with the controlLKLF +/− RAG2 −/− thymii (Fig. 3A). This reversal of the normal ratio of SP to DP thymocytes may have been primary or secondary. It is possible that the severely reduced numbers of peripheral SP T cells in theLKLF −/− RAG2 −/− mice fed back on the thymus to accelerate the differentiation of DP to SP cells or that there was a defect in the export of SP LKLF−/− thymocytes. It is also possible that there were defects in the survival or expansion of the DP population in these animals, although this seems less likely, because LKLF is not normally expressed in DP cells (Fig. 1A). Like the peripheral LKLF−/− T cells, both the CD4+ and CD8+ LKLF−/− SP thymocytes displayed a spontaneously activated cell surface phenotype (CD44hi, CD69hi, l-selectinlo, CD25) yet were not proliferating as determined by propidium iodide FACS analysis (12). Finally, TdT-mediated dUTP nick end-labeling (TUNEL) assays failed to demonstrate evidence of increased thymocyte apoptosis in the LKLF −/− RAG2 −/− animals (12). Taken together, these experiments demonstrated that LKLF is not required for the production or survival of SP thymocytes, but instead, appears to play a critical role in programming the quiescent phenotype of SP thymocytes in vivo.

Because the LKLF−/− peripheral T cells displayed a spontaneously activated cell surface phenotype, and because there was no evidence of decreased production or survival of LKLF−/− SP thymocytes, we tested the hypothesis that increased apoptosis in the spleen and lymph nodes was responsible for the severely reduced numbers of peripheral T cells seen in theLKLF −/− RAG2 −/− mice. TUNEL assays demonstrated markedly increased numbers of apoptotic CD3+ T cells in the white pulp of the spleen of theLKLF −/− RAG2 −/−mice (Fig. 4A). Consistent with this finding, 38% of freshly isolated LKLF−/− splenic T cells were apoptotic and 55% were dead (as compared with wild-type splenic T cells, 2% of which were apoptotic and 1% of which were dead) as determined by simultaneous staining with CD4, CD8, and 7-amino-actinomycin D (Fig. 4B). In addition, purified LKLF−/− splenic T cells displayed a greater than fivefold increased rate of cell death as compared with wild-type T cells during in vitro culture for 14 hours in medium alone (12). Thus, the severe reduction in peripheral SP T cells seen in theLKLF −/− RAG2 −/−animals correlated with significantly increased rates of apoptosis of these cells both in vitro and in the peripheral lymphoid organs in vivo.

Figure 4

FasL-induced apoptosis of peripheral T cells inLKLF −/− RAG2 −/−mice. (A) CD3 immunohistochemistry and TUNEL assays were done on adjacent tissue sections of spleens from wild-type (WT) andLKLF −/− RAG2 −/−chimeric animals (25). Arrows show CD3+ (brown staining) T cells that are undergoing apoptosis (black staining by TUNEL). Top panels (TUNEL) were photographed at low magnification (×150), bottom panels (CD3 and TUNEL) were photographed at higher magnification (×400). (B) Freshly isolated splenocytes from wild-type andLKLF −/− RAG2 −/−(−/−) chimeric mice were stained with 7-amino-actinomycin D and anti-CD4 and anti-CD8 and analyzed by three-color flow cytometry. Live, apoptotic, and dead cells were quantitated as described (7). (C) Freshly isolated splenocytes from wild-type andLKLF −/− RAG2 −/−chimeric mice were analyzed by three-color flow cytometry for the expression of CD4, CD8, and Fas or FasL (24). The data are displayed as Fas and FasL expression on the SP (CD4+ and CD8+) T cell populations. As a control, purified wild-type splenic T cells were activated for 24 hours with monoclonal antibody to CD3 and analyzed for FasL expression by three-color flow cytometry (α-CD3; unshaded curves).

The finding that LKLF−/− SP T cells were highly susceptible to apoptosis suggested that LKLF might regulate the expression of genes such as Fas or Fas ligand (FasL) that are known to be involved in peripheral T cell apoptosis (4, 5). Resting peripheral T cells normally express the Fas receptor but fail to express FasL. TCR-mediated activation induces the expression of FasL on both CD4+ and CD8+ T cells. Engagement of Fas by FasL is thought to mediate the subsequent elimination of such activated T cell clones by apoptosis. Wild-type and LKLF−/− CD4+ and CD8+ splenic T cells expressed equivalent amounts of Fas (Fig. 4C). In contrast, both CD4+ and CD8+ LKLF−/− splenic T cells displayed significantly increased amounts of cell surface FasL as assayed by FACS. Amounts of FasL on the LKLF−/− SP T cells were equivalent to (on the CD4+ cells) or higher than (on the CD8+ cells) those observed after TCR-mediated activation of wild-type splenic T cells (Fig. 4C). Taken together, these results were consistent with a model in which LKLF−/− T cells undergo spontaneous activation, express FasL, and die from subsequent Fas-mediated apoptosis in the peripheral lymphoid organs.

Our findings suggest that T cell quiescence and survival, like T cell activation, are programmed by specific transcriptional regulatory pathways and identify LKLF as an important regulator of one such pathway. The evolution of this pathway may reflect the need to maintain a large pool of quiescent peripheral T cell clones while preventing spontaneous T cell activation and the concomitant risk of autoimmune disease. The identification of this pathway suggests future studies aimed at understanding both the potential targets of LKLF and its developmental and posttranslational regulation. Preliminary experiments suggest that LKLF degradation is mediated by a protein kinase C–dependent pathway and that degradation is preceded by alterations in the electrophoretic mobility of the protein, suggesting that phosphorylation or ubiquitination might regulate the stability of LKLF in T cells (12). Our finding of spontaneous FasL expression on the LKLF−/− SP T cells suggests that LKLF might negatively regulate the FasL promoter. Understanding how LKLF is regulated and what it is regulating should help to elucidate the molecular basis of T cell quiescence and may also be relevant for understanding and modulating pathophysiological T cell activation that contributes to the pathogenesis of human autoimmune disease.

  • * To whom correspondence should be addressed. E-mail: jleiden{at}medicine.bsd.uchicago.edu

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