Abstract
To explore the role of interleukin-2 (IL-2) in T cell proliferation, and to circumvent the IL-2 deficiency autoimmune syndrome of conventional il2 gene deletion, mice were created to allow conditional il2 gene deletion when treated with the estrogen analog, tamoxifen (TAM) as adults. Splenocytes from four different mouse strains, C57Bl/6 wild type (WT), conventional IL-2(−/−), TAM-treated Cre recombinase-negative (Cre−)/IL2fl/fl, and Cre recombinase-positive (Cre+)/IL2fl/fl, were activated with anti-CD3 and anti-CD28, and monitored for CD4+ and CD8+ T cell lymphocyte blastogenesis, aerobic glycolysis, BrdU incorporation into newly synthesized DNA, and CFSE dye dilution to monitor cell division. IL-2 production was monitored by quantitative ELISA and multiple additional cytokines were monitored by quantitative protein-bead arrays. Splenocytes from conventional IL-2(−/−) and TAM-treated Cre+ mice resulted in undetectable IL-2 production by ELISA, so that both strains were IL-2-deficient. As monitored by flow cytometry, activated CD4+ and CD8+ T cells from WT, Cre+, and Cre− mice all underwent blastogenesis, whereas far fewer cells from conventional IL-2(−/−) mice did so. By comparison, only cells from IL-2 sufficient WT and Cre− mice switched to aerobic glycolysis as evidenced by a drop in media pH. Blastogenesis was mirrored by BrdU incorporation and CFSE dye dilution by CD4+ and CD8+ T cells from WT, Cre+, and Cre− mice, which were all equivalent, while proliferation of cells from conventional IL-2(−/−) mice was compromised. Splenocytes from IL-2 deficient conventional IL-2(−/−) mice produced low or undetectable other γc-chain cytokines (IL-4, IL-7, IL-9, IL-13, IL-15, and IL-21), whereas production of these γc-chain cytokines from IL-2-deficient conditional IL-2(−/−) Cre+ mice were comparable with WT and Cre− mice. These results indicate that CD4+ and CD8+ T cell blastogenesis cannot be attributable to IL-2 alone, but a switch to aerobic glycolysis was attributable to IL-2, and proliferation after CD3/CD28 activation is dependent on γc-chain cytokines, and not CD3/28 triggering per se.
Highlights
Splenocytes from IL-2 deficient conventional IL-2(−/−) mice produced low or undetectable other γc-chain cytokines (IL-4, IL-7, IL-9, IL-13, IL-15, and IL-21), whereas production of these γc-chain cytokines from IL-2-deficient conditional IL-2(−/−) Cre+ mice were comparable with wild type (WT) and Cre− mice. These results indicate that CD4+ and CD8+ T cell blastogenesis cannot be attributable to IL-2 alone, but a switch to aerobic glycolysis was attributable to IL-2, and proliferation after CD3/CD28 activation is dependent on γc-chain cytokines, and not CD3/28 triggering per se
With the discovery and purification to homogeneity of the first leukocytotrophic hormone, interleukin-2 (IL-2; Smith et al, 1983), and its receptor (IL-2R; Robb et al, 1981) almost 30 years ago, definitive in vitro reductionist experiments became possible for the first time using purified homogeneous IL-2 and cloned T cells (Baker et al, 1979), together with monoclonal antibodies (MoAbs) reactive with both IL-2 (Smith et al, 1983) and the IL-2 receptor (IL-2R; Leonard et al, 1982)
Since the affinity (K d) of the trimeric IL-2 receptor (IL2R) for IL-2 = 10 pM, with a dose–response range between 1 and 100 pM, these results indicated that TAM treatment had rapidly and effectively created a marked state of IL-2 deficiency in the Cre+ IL-2 conditional IL-2(−/−) mice, whereas TAM treatment of Cre− mice was not detrimental to IL-2 gene expression
Summary
With the discovery and purification to homogeneity of the first leukocytotrophic hormone, interleukin-2 (IL-2; Smith et al, 1983), and its receptor (IL-2R; Robb et al, 1981) almost 30 years ago, definitive in vitro reductionist experiments became possible for the first time using purified homogeneous IL-2 and cloned T cells (Baker et al, 1979), together with monoclonal antibodies (MoAbs) reactive with both IL-2 (Smith et al, 1983) and the IL-2 receptor (IL-2R; Leonard et al, 1982) These experiments established that antigen initiates T cell activation, the T cell proliferative response is mediated by the antigen non-specific IL-2/IL-2R interaction (Cantrell and Smith, 1984; Meuer et al, 1984). Subsequent reports using LCMV infection of mixed bone marrow chimeras of WT and IL-2Rα(−/−) mice, revealed that primary immune responses of the IL-2Rα(−/−) T cells were largely intact, but secondary immune responses to LCMV were markedly deficient and the generation of immunological memory was impaired (Williams et al, 2006)
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