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Comparative Study
. 2007 Mar 19;204(3):619-31.
doi: 10.1084/jem.20061871. Epub 2007 Feb 26.

Impaired CD8 T cell memory and CD4 T cell primary responses in IL-7R alpha mutant mice

Lisa C Osborne  1 , Salim Dhanji, Jonathan W Snow, John J Priatel, Melissa C Ma, M Jill Miners, Hung-Sia Teh, Mark A Goldsmith, Ninan Abraham
Affiliations
Comparative Study

Impaired CD8 T cell memory and CD4 T cell primary responses in IL-7R alpha mutant mice

Lisa C Osborne et al. J Exp Med. .

Abstract

Loss of interleukin (IL)-7 or the IL-7 receptor alpha (IL-7Ralpha, CD127) results in severe immunodeficiencies in mice and humans. To more precisely identify signals governing IL-7 function in vivo, we have disrupted the IL-7Ralpha Y449XXM motif in mice by knock-in mutagenesis (IL-7Ralpha(449F)). Thymic precursors were reduced in number in IL-7Ralpha(449F) mice, but in marked contrast to IL-7Ralpha(-/-) knockout mice, thymocytes and peripheral T cells developed normally. Strikingly, Listeria infection revealed that CD4 and CD8 T cells had different requirements for IL-7Ralpha signals. CD4 T cells failed to mount a primary response, but despite normal CD8 primary responses, maintenance of CD8 memory was impaired in IL-7Ralpha(449F) mice. Furthermore, we show that Bcl-2 is IL-7Ralpha Y449 independent and insufficient for IL-7-mediated maintenance of CD8 memory.

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Figures

Figure 1.
Figure 1.
Generation of IL-7Rα449F knock-in mouse. (A) A targeting vector carrying a partial cDNA of the IL-7Rα transmembrane and cytoplasmic domains with Y449F site-specific mutation (*) were used for homologous recombination in ES cells. WT, targeted, floxed and targeted (IL-7Rα449F) loci are shown (not to scale). Light gray boxes, exons; numerals below thick black lines, exon numbers; dark gray box, IL-7Rα cDNA. Sequences for neomycin (NEO) and thymidine kinase (TK) are shown. H, HindIII; B, BamHI; St, StuI; Bg, BglII; N, NotI, H*, HindIII site introduced. Probe 1 is a 0.65-kb EcoRI-BamHI fragment upstream of the short arm (Supplemental Materials and methods). Probe 2 is a 202-bp fragment amplified from exon 8, and the Neo probe is a 1.6-kb HindIII-SpeI fragment from the targeting vector. (B) Expression of IL-7Rα is maintained in IL-7Rα449F thymocytes. Thymocyte lysates were prepared from WT, IL-7Rα449F, and IL-7Rα−/− mice, and analyzed by immunoblot. Protein loading is indicated by the antitubulin immunoblot (bottom). (C) Site-specific mutation of IL-7Rα Y449 does not affect expression of IL-7Rα on peripheral CD4 and CD8 T cells as assessed by flow cytometry. Experiment is representative of three mice for WT, IL-7Rα−/−, and IL-7Rα449F in two independent experiments. Isotype antibody, dashed trace; anti–IL-7Rα, solid trace. (D) IL-7Rα Y449F mutation abrogates activation of STAT5, but identifies activation of the p38 MAPK pathway as Y449 independent. Neither Akt nor Erk are activated by IL-7 in T cells, but are efficiently activated by α-CD3 and IL-2 stimulation.
Figure 2.
Figure 2.
Bone marrow progenitor populations and IL-7Rα expression are normal in IL-7Rα449F mice. (A) Bone marrow progenitors develop at similar frequencies (left) and total cell numbers (right) in mice with targeted IL-7Rα449F mutation. Bar graphs are gated on Lin−Sca1+c-kit+ HSCs and their Lin−Sca1loc-kit+ derivatives (post-HSCs). (B) IL-7Rα is expressed normally on IL-7Rα449F bone marrow populations as determined by FACS analysis. Isotype antibody, dashed trace; anti–IL-7Rα, solid trace. (C) The frequency of ETPs is decreased in both IL-7Rα−/− and IL-7Rα449F thymi compared with WT. FACS plots shown are gated on Lin−CD44+CD25− DNI populations. WT, black bars; IL-7Rα−/−, unfilled bars; IL-7Rα449F, hatched bars. All experiments are representative of at least three mice for WT, IL-7Rα−/−, and IL-7Rα449F in two independent experiments.
Figure 3.
Figure 3.
An early thymocyte defect is bypassed in IL-7Rα449F mice. (A) FACS analysis shows that IL-7Rα449F thymocytes develop DN, DP, CD4 SP, and CD8 SP populations in frequencies similar to WT mice (left). Total cellularity in IL-7Rα449F mice is reduced in comparison to WT but significantly higher than in IL-7Rα−/− littermates (right). (B) DN thymocyte development is affected by IL-7Rα449F mutation (left). Mean cell numbers are shown in quadrants. The transition to DP thymocytes is unaffected in IL-7Rα449F mice (right). (C) Positive selection is IL-7Rα independent as shown by frequency of TCR-β+ HSAlo cells in WT, IL-7Rα−/−, and IL-7Rα449F DP thymocytes. (D) γδ T cell development is impaired in the absence of IL-7Rα Y449 signaling. Bar charts show the number of anti-γδ TCR+ lymphocytes in the thymus. (A, B, and D) WT, black bars; IL-7Rα−/−, unfilled bars; IL-7Rα449F, hatched bars. (E) BrdU uptake revealed cell cycling is increased in DNIII and DP stages but decreased in DNIV IL-7Rα449F thymocytes. Isotype antibody, dashed trace; anti-BrdU, solid trace. (F) Competitive repopulation shows IL-7Rα449F– derived cells were able to differentiate normally but are poorly competitive. Bar chart (left) and contour plots (right) are gated on CD90.1+ CD45.2+ (black bar) and CD90.1− CD45.2+ (hatched bar) thymocytes. Numbers in bar charts represent the mean thymocyte recovery from each genotype. All experiments are representative of at least three mice for WT and IL-7Rα449F. IL-7Rα−/− samples were pooled from three to six mice.
Figure 4.
Figure 4.
Disruption of IL-7Rα Y449 signaling only partially perturbs peripheral lymphocyte development. (A) Peripheral CD4 and CD8 T cells develop in IL-7Rα449F mice and confirm involvement of IL-7Rα Y449–independent pathways. (B) Quantification of FACS data shows that peripheral T and B cell development is reduced in IL-7Rα449F compared with WT but significantly higher than in IL-7Rα−/− littermates and supports a role for IL-7Rα Y449–independent signaling requirements. Natural killer cells (CD3−NK1.1+) are unaffected by abrogation of IL-7Rα Y449 signals. Data is expressed as the percentage of WT cellularity, IL-7Rα−/− (white bars), and IL-7Rα449F (hatched bars). (C) IL-7Rα Y449–independent signaling allows for accumulation of mature CD3+TCRβ+ T cells in the periphery. See Fig. S2.
Figure 5.
Figure 5.
Signals from IL-7Rα Y449 are essential for IL-7–driven homeostatic proliferation. Experiments are representative of two to four recipient mice from WT, IL-7Rα449F, and IL-7Rα−/− T cells in two independent experiments. Irradiated hosts, black, filled trace; nonirradiated hosts, white trace.
Figure 6.
Figure 6.
IL-7Rα Y449 is essential for the CD4 primary response to L. monocytogenes. (A) CD4 but not CD8 T cells require IL-7Rα Y449 for differentiation into effector cells. Bar charts represent total cell numbers of CD4+ IFN-γ+ or CD8+ IFN-γ+ T cells from WT (black bars) or IL-7Rα449F (hatched bars) spleens at day 7 after infection. (B) Decreased TCR proliferation in IL-7Rα449F CD4 and CD8 T cells at low concentrations of agonist stimulation. The doses of plate-bound α-CD3 are indicated. Histograms show representative results of one to three replicates. WT, solid trace; IL-7Rα449F, dashed trace. (C) Adoptively transferred WT Thy1.1 (CD90.1+) CD4 and CD8 T cells respond to rLM-SIY infection equally well in WT (CD90.1−) and IL-7Rα449F (CD90.1−) hosts. Bar charts represent the number of Thy1.1 antigen–specific T cells recovered from WT hosts (black bars) and IL-7Rα449F hosts (hatched bars). Data are representative of at least three infected mice of each genotype for each experiment.
Figure 7.
Figure 7.
CD8 memory T cells require signals from IL-7Rα Y449 for long-term maintenance. (A) IL-7Rα449F CD8 T cells are defective in their ability to survive and generate a stable pool of antigen-specific memory cells. (B) Rechallenge with rLM-SIY at day 100 showed that recovery of antigen-specific CD8 memory T cells was significantly impaired in IL-7Rα449F mice. (C) Analysis of SIY-specific T cells at days 60 and 100 after rLM-SIY infection using SIY-loaded MHC–Ig dimer showed that IL-7Rα449F memory CD8 T cells numbers are impaired and decrease over time. Data shown is representative of four mice of each genotype. (D) Adoptive transfer of WT CD90.1+ CD4 and CD8 T cells allows generation of WT CD8 memory, but provision of WT CD4 T cell help is insufficient for IL-7Rα449F CD8 T cell memory. % of primary effectors persisting as memory = no. of d45 CD8+ Dimer+ / no. of d7 CD8+Dimer+ × 100. Data shown is based on four transplanted mice at each time point. (E) Intracellular Bcl-2 was measured in peptide-stimulated CD8+IFN-γ+ T cells of WT and IL-7Rα449F mice at memory stages. No significant difference was detected in frequency of Bcl-2+ cells. Isotype antibody, gray filled trace; unstimulated, dashed trace; SIY stimulated, solid trace. Data shown is representative of three mice of each genotype and two independent experiments. (F) BrdU incorporation is similar in WT and IL-7Rα449F memory CD8 T cells at day 45 after rLM-SIY infection. Histograms are gated on CD8+IFN-γ+ cells. Solid trace, WT; dashed trace, IL-7Rα449F. Data is representative of three WT and IL-7Rα449F mice.
Figure 8.
Figure 8.
IL-7Rα449F mutation abrogates activation of STAT5 but Bcl-2 up-regulation is Y449 independent. (A) Intracellular flow cytometry of WT and IL-7Rα449F lymphocytes stimulated ex vivo showed that activation of the STAT5 pathway is dependent on IL-7Rα Y449 in both the thymus and periphery. Isotype antibody, gray filled trace; untreated, dashed trace; +IL-7, solid trace. (B) Bcl-2 up-regulation is IL-7Rα Y449 independent in all thymic subsets and peripheral T cells except DN. Bar charts shown represent WT −IL-7, unfilled bars; WT +IL-7, black bars; IL-7Rα449F –IL-7, gray bars; IL-7Rα449F +IL-7, hatched bars. All experiments are representative of at least three mice for each genotype and two independent experiments.
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