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. 2010 Apr 1;184(7):3461-9.
doi: 10.4049/jimmunol.0902593. Epub 2010 Mar 1.

IL-7 is essential for homeostatic control of T cell metabolism in vivo

Sarah R Jacobs  1 , Ryan D Michalek, Jeffrey C Rathmell
Affiliations

IL-7 is essential for homeostatic control of T cell metabolism in vivo

Sarah R Jacobs et al. J Immunol. .

Abstract

It has become apparent that T cells require growth signals to maintain function and viability necessary to maintain proper immune homeostasis. One means by which cell extrinsic signals may mediate these effects is by sustaining sufficient basal cell metabolism to prevent cell atrophy. The role of metabolism and the specific growth factors essential to maintain metabolism of mature T cells in vivo, however, are poorly defined. As IL-7 is a nonredundant cytokine required for T cell development and survival and can regulate T cell metabolism in vitro, we hypothesized it may be essential to sustain metabolism of resting T cells in vivo. Thus, we generated a model for conditional expression of IL-7R in mature T cells. After IL-7R deletion in a generally normal lymphoid environment, T cells had reduced responses to IL-7, including abrogated signaling and maintenance of antiapoptotic Bcl-2 family expression that corresponded to decreased survival in vitro. T cell survival in vivo was also reduced after loss of the IL-7R in a T cell-intrinsic manner. Additionally, IL-7R deletion resulted in delayed growth and proliferation following stimulation. Importantly, in vivo excision of IL-7R led to T cell atrophy that was characterized by delayed mitogenesis and reduced glycolytic flux. These data are the first to identify an in vivo requirement for a specific cell extrinsic signal to sustain lymphocyte metabolism and suggest that control of glycolysis by IL-7R may contribute to the well-described roles of IL-7 in T cell development, homeostatic proliferation, and survival.

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Conflict of interest statement

Disclosures

The authors have no financial conflicts of interest.

Figures

FIGURE 1
FIGURE 1
T cell size and number in the absence of IL-7. A total of 5 × 106 WT T cells were stained with CFSE and adoptively transferred into IL-7−/− or WT control animals for 7 d. A, The number of transferred cells remaining in the spleen was determined by flow cytometric analysis of CFSE-positive cells. B, Cell size of adoptively transferred cells was determined by mean forward light scatter. Each panel is representative of three independent experiments. *p < 0.05.
FIGURE 2
FIGURE 2
An inducible IL-7R knockout system. A, Schematic of the IL-7Rflox transgene, rescue of IL-7R−/− with the transgene, and incorporation of Cre:ER to allow for in vivo excision. B, DNA extracted from animal tail snips was subjected to PCR and agarose gel electrophoresis to amplify a portion of the IL-7R transgene. C, IL-7R expression on CD4 and CD8 splenic T cells was determined by flow cytometry. D, Numbers of thymocytes and splenocytes from WT, IL-7R−/−, and IL-7Rflox animals were determined. E, CD4 and CD8 expression was determined flow cytometrically in thymocytes and splenocytes. F, CD4 and CD8 T cells were measured for expression levels of CD25, CD44, CD62L, and CD69 flow cytometrically in splenocytes. G, B220, Mac1, Nk1.1, CD11c, and Gr1 expression were determined flow cytometrically in splenocytes. Each panel is representative of two or three independent experiments. *p < 0.01.
FIGURE 3
FIGURE 3
The IL-7Rflox transgene responds to IL-7. A, T cells from WT and IL-7Rflox animals were cultured in the presence or absence of IL-7 for 18 h prior to lysis and immunoblotting of 40 µg protein with the indicated Abs. B, Glycolytic flux was measured in WT and IL-7Rflox-purified resting T cells. C, T cells purified from WT and IL-7Rflox animals were analyzed for ability to uptake glucose when resting or following culture in the presence or absence of IL-7 for 24 h. D, Purified mature T cells from WT and IL-7Rflox animals were cultured in the presence or absence of IL-7 and analyzed for cell survival at indicated times. Each panel is representative of two or three independent experiments. *p < 0.02.
FIGURE 4
FIGURE 4
The IL-7Rflox is efficiently excised. IL-7Rflox animals both with and without the Cre:ER transgene were treated with tamoxifen, and splenocytes were stained for CD4, CD8 and IL-7R and analyzed by flow cytometry after 3 d (A) or the indicated time (B). Numbers indicate percentage of cells in the shown gate. C, IL-7Rflox animals both with and without the Cre:ER transgene were treated with tamoxifen, and 3 d posttreatment splenocytes were stained for CD4, CD8, CD25, CD44, CD62L, and CD69 and analyzed by flow cytometry. D, IL-7Rflox animals both with and without the Cre:ER transgene were treated with tamoxifen, and 3 d posttreatment splenocytes were stained for B220. Each panel is representative of two independent experiments.
FIGURE 5
FIGURE 5
Loss of IL-7 responses. A and B, T cells cultured in the presence or absence of IL-7 for 18 h were lysed, and 35 or 40 µg, respectively, were immunoblotted with the indicated Abs. Quantified protein levels indicated are normalized to actin. C, PI exclusion was used to determine cell survival at 24 h intervals of cells cultured in the presence or absence of IL-7. Each panel is representative of two or three independent experiments.
FIGURE 6
FIGURE 6
Loss of IL-7R signaling and cell survival. A, The number of purified CD4 and CD8 T cells 3 d after tamoxifen treatment was determined from IL-7Rflox and IL-7Rnull spleens. B, Purified T cells were lysed ex vivo, and 40 µg was immunoblotted as indicated. Quantified protein levels shown are normalized to actin. C, Purified T cells were stained intracellularly for Bcl-2 and analyzed by flow cytometry. D and E, A total of 4.5 million cells from IL-7Rflox or WT animals were adoptively transferred in Thy1.1 congenic mice. Twelve hours following transfer, recipient animals were tamoxifen treated, and percentage of transferred cells remaining was determined by staining of splenocyte (D) or lymph node (E) suspension with Thy1.1 and Th1.2 fluorescently conjugated Abs and flow cytometry. Each panel is representative of two to six independent experiments. *p < 0.01.
FIGURE 7
FIGURE 7
Loss of IL-7R and cell growth. A, Purified T cells expressing or lacking expression of IL-7R were examined for cell size by particle size analyzer. *p < 0.01. B, Percentage of live cells was determined following purification by PI exclusion and flow cytometry. C, IL-7Rflox and IL-7Rnull purified T cells were cultured on control or plates coated with 5 µg/ml anti-CD3 with or without 5 µg/ml anti-CD28 Abs and assayed for cell size by mean forward scatter after 18 h in culture (left panel) (*p < 0.05), by particle size analyzer after 20 h in culture (right panel) (*p < 0.05), or by mean forward scatter after 48 h in culture (D). E, Proliferation was measured after 48 h by CFSE dilution. Each panel is representative of two to six independent experiments.
FIGURE 8
FIGURE 8
Loss of IL-7R and glycolysis. IL-7Rflox– and IL-7Rnull–purified T cells were permeabilized and stained with anti-Glut1 Ab followed by flow cytometry 3 or 7 d post tamoxifen treatment (A) or lysed and 10 µg immunoblotted 3 d post tamoxifen treatment (B). IL-7Rflox– and IL-7Rnull–purified resting T cells were analyzed for ability to uptake glucose 3 d post tamoxifen treatment (C) or exposed to [3H]glucose to measure glycolytic flux (D). Each panel is representative of two to six independent experiments. *p < 0.001.
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