lck-Driven Cre Expression Alters T Cell Development in the Thymus and the Frequencies and Functions of Peripheral T Cell Subsets

Transgenic mice expressing the bacteriophage DNA-recombinase Cre to generate conditional gene-knockout mice are widely used in biomedical studies. Cre efficiently drives recombination of DNA between two 34-bp-long target sequences (loxP sites) transfected on critical positions of a gene of interest (1). The regulation of Cre expression with inducible or cell-specific promoters is an elegant and valuable tool to study the spatial or temporal function of the targeted genes.

Although conditional-knockout systems using Cre technology have helped to more confidently elucidate the function of genes in specific organs or cells (especially for genes that are embryonically lethal), some shortcomings have been reported. Unreliable tissue or cellular localization of Cre expression (2), a variation in recombination efficiency depending on the loxP-flanked target gene, and genomic toxicity of the Cre recombinase that recognizes pseudo-loxP sites in the mouse DNA were reported (3–5). The potential genomic DNA toxicity of Cre suggested that Cre-deleter mouse strains could be different from wild-type (WT) mice (3, 6–9). The mammalian DNA-damaging effect of Cre probably depends on its expression level and the cell type where Cre is expressed (3). One well-studied example are mice that express Cre under the rat insulin promoter (rip) and become insulin resistant in the absence of any loxP-targeted gene deletion (10, 11). Thus, it was proposed that a large body of published work using rip-cre⁺ mice and claiming effects of several genes on glucose intolerance/diabetes, in which rip-cre⁺ control mice were not included, might be incorrect (10).

The protein tyrosine kinase p56 (Lck) is critical for normal T cell development (12–14). Lck is expressed in the earliest thymic immigrants and in all subsequent T cell lineages (15, 16). Transgenic mice, in which the expression of Cre is controlled by the lck promoter, have been widely used to study the involvement of different genes in T cell development and function.

We hypothesized that lck-cre⁺ (as well as other Cre-expressing) mice may differ from nontransgenic controls as a result of Cre toxicity and, thereby, induced homeostatic compensation mechanisms (3). In support of this, a small number of previous studies found that the expression of Cre under the proximal lck promoter might interfere with normal T cell development (9, 17–21).

In this article, we demonstrate that lck-initiated Cre expression results in strikingly altered thymic T cell development and important qualitative and quantitative differences in T cell numbers, frequencies, and functions in secondary lymphoid organs. Moreover, a meta-analysis of studies using lck-cre⁺ mice to delete genes in T cells showed significantly different results depending on the control used. Results similar to those presented in this article (reduced thymic cellularity, increased apoptosis of double positive [DP] cells, elevated levels of γδ T cells, and an activated phenotype of peripheral T cells) were obtained more frequently when lck-cre⁻ controls were used compared with lck-cre⁺ mice as controls, validating the relevance of our observations.

Mice transgenic for the Cre recombinase under control of the proximal lck promoter (22) backcrossed to C57BL/6 mice for more than nine generations were bred with C57BL/6 mice to obtain lck-cre^+/− (lck-cre⁺) or lck-cre^−/− (WT) littermates. The latter were used as controls. The study was performed under approval of the Stockholm North Ethical Committee on Animal Experiments permit number 197/13. Animals were housed under specific pathogen–free conditions.

Single-cell suspensions from spleen, lymph node (LN), and thymus were obtained by mechanical disruption, straining over a 40-μm nylon mesh, and lysis of erythrocytes. Cells were counted and surface stained with their respective Abs: anti-CD3 (clone 17A2), anti-CD4 (GK1.5), anti-CD8 (53-6.7), anti-CD44 (IM7), anti-CD62L (MEL-14), anti-CD127 (A7R34), anti-βTCR (H57-597), anti-γδTCR (eBioGL3; eBioscience), and anti-CD25 (7D4; BD Pharmingen). Regulatory T cells (Tregs) were additionally stained for Foxp3 (FJK-16s; eBioscience), following the manufacturer’s instructions (Foxp3 buffer set; BD Pharmingen).

Apoptosis was determined by Annexin-V binding, according to the supplier’s protocol (BD Pharmingen). For Cre detection, cells were stained for CD4 and CD8, fixed and permeabilized by Perm buffer III (BD Biosciences), and incubated with anti-Cre (D3U7F; Cell Signaling) and anti-rabbit Ab (DAKO).

For determination of IFN-γ–producing cells, spleen cells were incubated with 50 ng/ml PMA and 2 μg/ml ionomycin (Sigma) in the presence of brefeldin A (5 μg/ml) for 4 h, stained with cell population–specific Abs, fixed, permeabilized using leukocyte permeabilization reagent IntraPrep (Immunotech), and stained with anti–IFN-γ (eBioscience).

Data were acquired using a CyAn ADP flow cytometer (Beckman Coulter) and analyzed using FlowJo software (TreeStar).

We performed a free text search for “lck-cre” using Google Scholar and analyzed the first 480 of 1500 hits. One hundred and forty-eight hits were discarded because they were book chapters or reviews, did not include experiments with lck-cre⁺ mice, or did not properly inform on the nature of the controls used. Only studies of mice in which lck-cre was used to knockout loxP-targeted genes were considered, resulting in 332 publications that were included in our analysis.

Statistical analysis was performed using GraphPad Prism version 6. The p values were calculated by a two-tailed, unpaired t test. χ² tests were used to compare the results from published articles using lck-cre⁺ or lck-cre⁻ mice as controls.

The development of T cells in the thymus of lck-cre⁺ and WT littermates was compared. In the thymus, αβ T cells develop from bone marrow precursors that undergo differentiation through a double-negative (DN) stage (lacking the coreceptors CD4 and CD8), followed by a double-positive (DP) stage to become CD4⁺ or CD8⁺ single-positive (SP) T cells that eventually migrate to the periphery (23).

lck-cre⁺ mice showed a reduced frequency of DP cells and elevated frequencies of DN and CD8⁺ cells compared with WT controls (Fig. 1A, 1B). Thymic cellularity was reduced by 65% in lck-cre⁺ mice as a result of reduced cell numbers of DP, CD4⁺, and CD8⁺ SP cells (Fig. 1C, 1D), whereas DN cell numbers were similar in both strains. The DN population can be subdivided by the expression of CD44 and CD25, with an ordered expression of these markers into DN1–DN4 (24). To determine whether Cre expression altered T cell development at a defined stage of DN development, the composition of the DN compartment was analyzed in lck-cre⁺ and WT mice. Similar frequencies of DN1–4 subpopulations were measured in lck-cre⁺ and WT mice (Fig. 1E, 1F).

Two main lineages of T cells develop in the thymus and branch at the DN stage: cells expressing the αβ TCR and those that express the γδ TCR. The frequency of γδ T cells within the DN stage was increased >2-fold in lck-cre⁺ mice, whereas DN cells expressing the β-TCR were reduced (Fig. 1G, 1H). Despite the reduced thymic cellularity, the numbers of γδ T cells in the thymus of lck-cre⁺ mice were elevated compared with WT controls (Fig. 1I), suggesting a preferential development of the γδ T cell subset.

Binding of IL-7 to its receptor (IL-7R) plays a nonredundant role in T cell development by promoting the survival and proliferation of DN progenitors, as well as during the positive selection of CD8⁺ T cells (25–27). The stage-specific function of IL-7 during intrathymic development is accomplished by a tight regulation of IL-7Rα expression. IL-7Rα is first induced during thymopoiesis in the transition between DN1 and DN2. It declines steadily after the DN2 stage, is terminated before transition to the DP stage, and is upregulated again after positive selection of thymocytes (26). We found an increased frequency of IL-7R⁺ thymocytes in lck-cre⁺ mice (Fig. 2A, 2B). However, total IL-7R⁺ cell numbers were reduced in lck-cre⁺ thymocytes compared with WT thymocytes as a result of the reduced thymic cellularity (Fig. 2C). The median fluorescence intensity (MFI) of γδ and CD8⁺ T cells was increased in lck-cre⁺ thymus (Fig. 2D, 2E), whereas levels of IL-7R expression were similar in DN cells, DP cells, or CD4⁺ cells in lck-cre⁺ and WT mice (Fig. 2D, 2E).

To examine whether the thymus hypoplasia in lck-cre⁺ mice was due to increased cell death, thymocytes were assessed for binding to Annexin-V. We found an elevated frequency of Annexin-V⁺ cells in the thymus of lck-cre⁺ mice (Fig. 2F, 2G) that was due to a increased frequency of Annexin-V⁺ DP cells (Fig. 2H).

We then studied at which stage of thymic development Cre recombinase was expressed in lck-cre⁺ mice by immunolabeling. The highest expression of Cre was found in DP cells, an intermediate expression was found in CD4⁺ and CD8⁺ cells, and DN cells showed the lowest levels (Fig. 2I).

We found that the frequencies of CD3⁺ T cells in spleens and LNs from lck-cre⁺ mice were dramatically reduced (Fig. 3A, 3C, 3E). Because spleens and LNs from WT and lck-cre⁺ mice showed similar cellularity (Fig. 3B), lck-cre⁺ CD3⁺ T cell numbers were reduced accordingly (Fig. 3D, 3F). The frequencies and numbers of CD4⁺ cells in the spleen and LNs were reduced in lck-cre⁺ mice compared with WT mice (Fig. 3A, 3G–J). The CD4/CD8 ratio was also reduced in spleens but not in LNs (Fig. 3K, 3L). The percentage and numbers of CD3⁺CD4⁻CD8⁻ cells were increased in spleen and LNs (Fig. 3G–J). Within this population, the frequency of γδ T cells was elevated (Fig. 3M–O), with a resulting 6-fold increase within the T cell population in lck-cre⁺ mice compared with WT mice and in total cell numbers (Fig. 3P–S).

The levels of naive and memory T cells in spleens and LNs from lck-cre⁺ mice were measured. In mice, T cells can be divided into naive, memory, and effector cells based on the expression of CD44 and CD62L. Naive T cells exhibit high levels of CD62L and low expression of CD44, whereas memory T cells are identified by high CD44, and effector cells show high CD44 and low expression of CD62L (28). A reduction in the frequencies and numbers of naive CD44^lowCD62L⁺ CD4⁺ and CD8⁺ T cells was observed in spleens and LNs from lck-cre⁺ mice (Fig. 4A–I). Frequencies of CD4⁺ and CD8⁺ memory cells (CD44^high/CD62L⁺) were elevated in spleens and LNs, whereas frequencies of effector CD44^high/CD62L⁻ cells were elevated only in the CD4⁺ T cell subset (Fig. 4A–C, 4F, 4G). These differences in the frequencies of memory and effector T cells were not consistently reflected in elevated cell numbers (Fig. 4D, 4E, 4H, 4I).

The ability of T cells from lck-cre⁺ mice to secrete IFN-γ after mitogen stimulation was analyzed. A greater proportion of CD4⁺ and CD8⁺ T cells from lck-cre⁺ spleens produced IFN-γ compared with WT controls (Fig. 4J, 4K). As above, the increased frequencies only compensated for the reduced total T cell numbers in lck-cre⁺ mice, resulting in similar total numbers of IFN-γ–producing cells (Fig. 4L). CD3⁺CD4⁻CD8⁻ and CD3⁺CD4⁻CD8⁺ lck-cre⁺ T cells expressed higher IL-7R levels than did controls (Fig. 4M).

We then evaluated whether the lck-cre transgene also altered the frequency of Tregs. Increased frequencies of Foxp3⁺CD4⁺ T cells were measured in spleens and LNs of lck-cre⁺ mice (Fig. 5A, 5B). Moreover, the frequency of CD4⁺Foxp3⁺ cells was elevated in the thymus of lck-cre⁺ mice, indicating a preferential selection of natural Tregs (nTregs) (Fig. 5D, 5E). Although the proportions of Foxp3⁺ cells within the CD4⁺ T cell compartment were increased in lck-cre⁺ mice, the numbers of Tregs in spleens and LNs were similar and reduced in thymi as a result of the diminished total T cell numbers (Fig. 5C, 5F).

Because the lck-cre transgene had critical effects on T cell differentiation in the thymus and on T cell subpopulation frequencies and functions in secondary lymphoid organs, we performed a survey of published papers in which gene^fl/fl lck-cre⁺ mice were used to study different floxed genes. We hypothesized that the choice of controls (alternatively lck-cre⁺ or lck-cre⁻) may have biased the results obtained.

lck-cre⁺ controls, floxed heterozygous (gene^fl/+) or WT alleles (gene^+/+), were used in 128 of 332 studies analyzed (Fig. 6), whereas lck-cre⁻ controls (gene^fl/fl or just a WT strain) were used in the remaining 204 articles (Supplemental Fig. 1). Within the lck-cre⁺–controlled group, 53 publications contained a further lck-cre⁻ control (WT or gene^fl/fl) in at least one of the experiments (Fig. 6). Only 5 of these 53 studies that included both lck-cre⁺ and lck-cre⁻ mice reported a reduction in thymus size and decreased DP or increased DN cell frequencies in lck-cre⁺ mice in comparison with lck-cre⁻ controls (17–19, 21, 29). Moreover, several articles within this group explicitly reported that there were no differences in thymic T cell development between lck-cre⁺ mice and lck-cre⁻ controls (30–37).

We classified the studies as similar to the lck-cre⁺–transgenic mice described in this article if the lck-cre conditional-knockout mice showed at least one of the following features in comparison with the control: reduced thymic cellularity, increased DN and reduced DP populations, reduced total T cell numbers in the periphery, or increased γδ T cells.

A total of 173 of the 332 studies included the necessary information that allowed us to assess at least one of the parameters above. Although 64% (58/91) of studies using lck-cre⁻ controls reported a similar phenotype, only 32% (26/82) of studies using lck-cre⁺ controls were classified as similar (Fig. 6). Thus, information on the biology of loxP genes deleted via lck-driven Cre is significantly associated with the choice of control (χ² test, p < 0.0001).

This study demonstrates that lck-cre⁺ mice have striking alterations in T cell development in the thymus and altered distributions, numbers, and functions of T cell subsets in the periphery.

lck-cre⁺ mice showed reduced DP thymocyte numbers, as well as decreased αβ and increased γδ T cells in thymus, LNs, and spleen. The thymic alterations described correspond with an early expression of Cre via the proximal lck promoter in the transgene. In line with our results on Cre expression, the proximal promoter was shown to be active at the DN stage and maximally in DP and SP T cells (15, 38). Coincidentally, the highest levels of apoptotic cells were detected at the DP stage. A high level of Cre expression driven by lck probably recognizes the pseudo-loxP sites, causing DNA damage, as suggested in other Cre models (4, 8). For example, basophils and mast cells were found to be highly sensitive to Cre expression because these cell populations are completely deleted in mice expressing Cre under cell-specific promoters (39, 40).

Three different lck-cre⁺ mice using the proximal promoter were reported (22, 41, 42). Although mice generated by Takahama et al. (22) were used in this study, decreased thymic cellularity and increased DP apoptosis in mice from the other founders also were reported (9). Homozygous lck-cre^+/+ mice showed a greater reduction in thymic cell numbers compared with lck-cre^+/− heterozygous mice, implying a dose effect as reported in other Cre strains (43). In line with this suggestion, Cre expression under the control of cd4 and cd2 promoters was reduced compared with lck and did not affect the thymic cellularity (9).

γδ T cells were reported to express lower levels of Lck compared with αβ T cells (44). Lck^−/− mice were shown to have a severe defect in the production of T lymphocytes; however, γδ T cells survived in these mice (14, 45). In line with this, γδ T cells were increased in the thymus, spleen, and LNs of lck-cre⁺ mice, probably as a result of their lower lck-driven Cre expression.

IL-7 is produced by thymic epithelial cells and is needed to promote proliferation, survival, and differentiation of DN thymocytes, stimulate expression of antiapoptotic factors, and initiate V-J recombination. IL-7 produced in the thymus is essential for the development of γδ T cells (46) and for specifying the CD8 lineage choice (27). Thymi from lck-cre⁺ mice showed increased expression of IL-7R in DN and CD8⁺ thymocytes compared with WT controls. Levels of DN and CD8⁺ cell frequencies were increased in lck-cre⁺ mice, suggesting that the elevated IL-7R expression is an adaptive response to Cre toxicity and may bias T cell development toward the γδ T cell lineage.

Ag-specific nTregs develop in the thymus upon TCR-mediated recognition of self-peptide (47). We speculate that higher precursor frequencies or a different sensitivity to cell death of lck-cre⁺ precursors might account for the higher nTreg frequency in the thymus of lck-cre⁺ mice.

Peripheral lck-cre⁺ T cells were characterized by high frequencies of effector and memory cells, an increased IFN-γ secretion, and an elevated frequency of CD4⁺Foxp3⁺ T cells. Lower numbers of naive CD3⁺ T cells in spleens and LNs were also observed. It can be speculated that T cells in lck-cre⁺ mice undergo an increased homeostatic proliferation in response to lymphopenia as a result of lower thymic output. Such homeostatic proliferation may account for the increased frequencies of activated/memory T cells. T cells undergoing homeostatic proliferation showed higher levels of CD44 and increased IFN-γ secretion in response to Ag (48). Cre expression driven by the lck proximal promoter has a lower expression in peripheral sites (15), suggesting that dysfunctional thymopoiesis cannot be fully counterweighed and results in T cell alterations in the periphery.

The findings described in this article can affect the interpretation of results from studies that did not use lck-cre⁺ mice as controls. We performed a literature survey to contrast results from different studies using lck-cre⁺ or lck-cre⁻ mice as controls and found evidence that the obtained results were confounded by the type of lck-cre control used. This also suggests that results from studies lacking lck-cre⁺ controls that measured parameters different from ours should be considered with caution. These might include studies of the role of T cell genes in the outcome of infectious, autoimmune, or inflammatory conditions. Because lck-cre⁺ mice differ from WT mice, care should also be taken when interpreting results using only an lck-cre⁺ control.

Intriguingly, some investigators reported no differences between lck-cre⁺ or lck-cre⁻ mice, whereas others did (17, 19, 21, 29–33, 35–37) (Fig. 6). In our literature survey, the phenotype of lck-cre⁺ mice described in this article was found in all three origins of proximal lck-cre⁺ mice (22, 41, 42). However, no phenotype was observed if Cre was expressed under the distal lck-cre promoter, which is less frequently used and active later during T cell development and in mature T cells (49–51). In most cases, proximal lck-cre⁺ mice are either kept on a mixed Sv129×C57BL/6 background or are backcrossed to C57BL/6 mice. Interestingly, it was observed that an increased proportion of the C57BL/6 genotype showed increased thymic hypocellularity and a block in the transition from DN to DP cells (52). Therefore, it might be speculated that the background of mice used contributes to the observed differences in T cell development.

Our results indicate the need for more stringent controls when using lck-cre mice, as well as other Cre recombinase-targeted mice. When possible, researchers should avoid using controls in which the expression of Cre alters the frequencies or functions of the targeted cell type.

We thank Helen Braxenholm, Torunn Söderberg, and Kenth Andersson (Department of Microbiology, Tumor, and Cell Biology, Karolinska Institutet) for excellent contributions to the animal experiments.

The authors have no financial conflicts of interest.