Although IL-7 has a well-established role in supporting naive CD8+
T-cell homeostasis, it has been generally believed that these cells have a uniform intrinsic reliance on, and responsiveness to, available cytokines. Instead, differences in the homeostatic survival and proliferation of T cells have been so far attributed to variations in the strength and/or frequency with which T cells can actively engage cognate spMHC through their unique TCRs.1, 2
Our study here reveals an additional, complementary layer of functional heterogeneity amongst CD8+
T cells in their responsiveness to IL-7, even when removed from TCR stimulation. Relative CD5 expression was found to be a stable, heritable and IL-7-independent marker for T cells differing in their proliferation and survival responses to IL-7 stimulation. Interestingly, we find that although all T cells survive in response to IL-7 stimulation, only CD5hi
cells proliferate. Conversely, CD5lo
cells have a survival advantage in the absence of homeostatic stimulation. Importantly, variations in IL-7 responsiveness between T-cell clones were shown to be relevant even in the context of other homeostatic stimuli in vivo
, with elevated IL-7 levels leading to selective proliferation of CD5hi
T cells independent of TCR specificity. Although the enhanced LIP of CD5hi
T-cell clones has been previously attributed to their greater avidity for spMHC,11, 13
our data suggest that differential IL-7 responsiveness may also potentiate proliferation. Moreover, it suggests that both IL-7R and TCR signaling contribute to the diversity of the T-cell pool, and that IL-7 does not strictly control population size alone.
Although we found CD5 expression to be a useful marker for heterogeneous IL-7 responsiveness, subtle correlated differences in IL-7R expression were found to mechanistically underlie varying IL-7 sensitivities. Although IL-7R expression is known to vary across the T-cell pool, differences have predominantly been attributed to heterogeneous extracellular environments, and considered insufficient for generating response diversity.8, 9, 15, 23, 31
Nonetheless, our quantitative analysis of IL-7R-mediated signaling reveals shared nonlinear signal-response relationships across T-cell clones that can generate striking differences in functional responses via comparably small variations in receptor expression. A threshold level of signaling required for proliferation well above that required for survival also explains why all T cells could survive, but only those with critically high levels of IL-7Rα could proliferate in response to high doses of IL-7. Thus, although differences in the IL-7-dose requirements for proliferation versus survival have been identified,32
our study reveals that proliferation occurs heterogeneously across polyclonal T-cell populations, and is dictated by the level of receptor expression-limited signaling produced by an individual T cell. Accordingly, signaling thresholds defined here at the population level are likely even more striking when examined for individual cells. We find that even among naïve T cells from TCR-tg Rag−/−
mice, CD5 expression marks variation in mean IL-7R levels and different proliferation capacities at elevated IL-7 levels in vivo
(, Supplementary Figure 4c
). There remains considerable variation in IL-7R expression even within these populations, and this may also explain why only a fraction of the population proliferates.
Very recently, Cho et al.33
also reported enhanced responsiveness of CD5hi
T cells to IL-7, and demonstrated that the selective proliferation of CD5hi
T cells also extends to stimulation by common γ-chain cytokines IL-2 and IL-15. These differences in sensitivity and signaling capacity were attributed to higher expression of GM1-containing lipid rafts in CD5hi
cells, proposed to enhance signaling from cytokine receptors primarily via receptor clustering. Our measurements here indicate that the downstream signaling generated per receptor is essentially equivalent in CD5hi
T cells (), and that even the modest quantitative differences in receptor number per cell that were observed by Cho et al.
, as well as ourselves, can explain the differences in their responses to IL-7. Cho et al.33
also find that lipid-raft expression levels and homeostatic cytokine responsiveness rely upon sustained TCR contact with spMHC, although notably residual cytokine responsiveness persists in MHC-I knockout T cells even after resting for several days in MHC-I deficient hosts. We observe heterogeneous IL-7 responsiveness in MHC-I deficient cells that is correlated with CD5 levels (). We also find that these proliferation differences are transiently increased after first resting the cells overnight in cytokine-free media (), presumably resulting from increased IL-7R expression (). We further see decay in CD5 expression once cells are withdrawn from spMHC (), but this decrease occurs over many days, and the relative expression between T-cell clones is maintained. Persistent effects of TCR signaling received before cell isolation might similarly give rise to long-term differences in expression of other signaling network mediators regulating cytokine responsiveness, including IL-7Rα, though defining the mechanisms involved in establishing and maintaining their expression requires further study. Independent of whether long-term ‘covert' TCR signaling underlies varying cytokine responsiveness, our data highlight that even mild quantitative differences in cytokine receptor expression can have a surprisingly critical role in directly giving rise to substantive functional differences in proliferation responses.
Diversity in CD5, CD8α and IL-7Rα expression amongst naïve T cells in vivo
has been largely attributed to extrinsic heterogeneities in the local spMHC and IL-7 environments.9, 15, 23, 31
On the basis of the requirement of naïve T cells to engage spMHC for the maintenance of their CD5 levels in vivo
CD5 expression has been interpreted as a surrogate measure for the strength of spMHC-induced signaling in the periphery.11, 13, 15, 17
However, we find that differences in basal CD5 levels among naive T cells are stably maintained even after withdrawal from spMHC signals. Similarly, although the broad distribution of IL-7R expression among naïve T cells in vivo
has been attributed solely to the heterogeneity in the spatio-temporal presentation of IL-7 between T cells cycling in and out of lymphoid organs,8
our data suggest that intrinsic differences in basal IL-7R expression also contribute to the heterogeneity in IL-7R expression levels in naïve CD8+
T cells. Although we find that variable IL-7R expression is sufficient to explain heterogeneous IL-7 responsiveness of mature T cells, the observed correlation between CD5 and IL-7R levels may reflect underlying co-regulation of these genes that is established during T-cell development, and possibly reinforced by interactions in the periphery. For instance, CD5 expression in T cells is thought to reflect the strength of thymic selection,18
and signaling network changes during selection may also predetermine IL-7R expression levels in mature T cells. Although determination of potential developmental connections was outside the scope of this study, future work should address the role of TCR signaling received during thymic selection versus interactions with spMHC in the periphery in maintaining differences in expression patterns between CD5hi
A study by Park et al.9
has suggested that mutual feedback between TCR and IL-7R signaling pathways underlies correlations between IL-7R, CD5 and CD8α expression among naïve T cells. In what is termed the ‘co-receptor tuning' model, IL-7R signaling induces the transcription of CD8α to increase TCR signaling, which negatively feeds back to reduce IL-7R signaling, which in turn reduces CD8α and increases IL-7Rα expression. Reduced CD8α expression on CD5hi
T cells is proposed to ‘tune down' their excessive TCR signaling. A lack of IL-7-induced Stat5 phosphorylation in freshly isolated CD5hi
male HY TCR-tg CD8+
T cells has been used to support this model. However, our studies did not reveal any signaling defects in freshly isolated naïve OT-1 or polyclonal CD5hi
T cells (Supplementary Figure 2c
and data not shown). This may reflect differences in their thymic development compared with the male HY T cells used in the Park et al.
study, which, unlike OT-1 cells, are selected on agonist ligands. The ‘co-receptor tuning' model is also supported by a positive correlation of IL-7Rα, and inverse correlation of CD8α, with CD5 expression for a panel of freshly isolated TCR-tg cells. However, our data suggest that increased receptor expression may reflect intrinsic differences in the basal level of IL-7Rα expressed rather than signal inhibition reducing negative feedback. These trends in CD5, CD8α and IL-7Rα expression may also be partly explained by decreased effective IL-7 levels in TCR-tg mice bearing T cells with higher CD5 expression ().
Recent studies have shed light on intra- and extra-cellular mechanisms regulating the ability of T cells to compete for, and respond to, TCR and IL-7R signals. Stronger or more frequent interactions with spMHC may provide preferred access to IL-7 localized at the surface of antigen-presenting cells and/or signal for IL-7 production,10, 34
and cytokine stimulation appears in some cases to ‘prime' T cells for more robust responses to antigen stimulation.35, 36, 37
Yet inhibition of IL-7R signaling by TCR signals has also been proposed to balance overall homeostatic signaling between T-cell clones.9
It remains unclear if and how these complex interactions quantitatively mediate or accentuate differences in TCR–spMHC affinities during homeostasis and under perturbations in the cytokine environment. Rather than attempting to mimic the complex in vivo
environment and identify the nature and extent of cross-talk between IL-7 and spMHC-induced signaling, we chose to carefully quantify IL-7-induced responses in the absence of spMHC, and then determine whether differences in IL-7 sensitivity persist upon reintroduction of other complex homeostatic cues in vivo
. Our study demonstrates intrinsic variations in IL-7 responsiveness across T-cell clones that are not fully balanced by other homeostatic signaling in vivo
upon acute changes in IL-7 concentration. Moreover, T-cell proliferation under supra-physiological IL-7 concentrations in vivo
is more robust than achievable by IL-7 stimulation alone, emphasizing the critical role of spMHC signals.
Our data support the novel finding that reduced IL-7 sensitivity and spMHC affinity in CD5lo
T cells is accompanied by an increased capacity to survive in the absence of homeostatic cues. This coupling may be critical for maintaining and restoring a dynamic homeostatic balance between T cells with different abilities to compete for, and respond to, limited homeostatic resources. Although defining mechanisms supporting prolonged basal survival requires studies beyond the scope of this paper, our data point to intrinsic differences in metabolic function between CD5hi
T cells. CD5lo
T cells have reduced size, indicative of a more quiescent state and preferential utilization of mitochondrial respiration.25
Further, F5 cells exhibit enhanced IL-7-independent glucose uptake, and reduced sensitivity to inhibition of PI3K activity essential for glucose uptake and other processes supporting survival.38
Further, the balance of pro- versus anti-apoptotic proteins regulates susceptibility to apoptosis, and PI3K activity and glucose uptake are known to impact the expression, activity and stability of these proteins.39, 40, 41, 42, 43, 44
cells may have lower basal expression of pro-apoptotic factors, such as Bim,45
cells, whereas CD5hi
cells compensate via their enhanced ability to signal for IL-7-dependent increases in anti-apoptotic proteins, such as Bcl2 ().
Interestingly, recent studies suggest elevated PI3K activity in CD5lo
cells could be connected to their decreased IL-7R expression. Kerdiles et al.46
demonstrated that Foxo1, a target of PI3K, is a transcription factor for IL-7R. Knocking out Foxo1, or inhibition of the PI3K phosphatase PTEN lead to decreased IL-7Rα expression in CD8+
T cells. Decreased sensitivity to PI3K inhibition in F5 cells () is consistent with lower PTEN activity, and our preliminary studies support decreased Foxo1 expression in F5 cells relative to OT-1 cells (data not shown). These data could suggest an interesting model whereby PI3K activity regulates basal IL-7Rα levels and survival in the absence of cytokine, which in turn regulates the ability to activate Jak-dependent signaling pathways in the presence of IL-7.
Because of its diverse role in lymphocyte development and function, IL-7 has prospective therapeutic uses in restoring compromised immune systems following chemotherapy or viral infections, and as an adjuvant for vaccines and cancer immunotherapies.47, 48, 49
Two rhIL-7 phase I clinical trials have recently shown an IL-7-dose-dependent increase in CD8+
T-cell numbers,50, 51
and a concomitant increase in the diversity of TCR Vβ usage.51
Our results raise an additional mechanism by which IL-7 affects the diversity of the T-cell repertoire: regulating the diversity of TCR-spMHC avidities, indicated by CD5 expression, via correlated differences in their IL-7R expression and IL-7 responsiveness. Although TCR Vβ diversity increases with IL-7 therapy, our model suggests that the homeostatic diversity of CD5 expression amongst T-cell clones is maximized at physiological IL-7 levels. Our findings suggest that this equilibrium is achieved by maintaining an IL-7 level that does not promote selective proliferation or persistence of CD5hi
cells. This may result from balancing IL-7 production with the overall size of the T-cell population consuming IL-7.7, 8
Notably, we have shown here that IL-7 treatment can result in the preferential expansion of CD5hi
T cells, which are potentially autoreactive and have been proposed to contribute to the development and progression of autoimmune disorders.52, 53
Nevertheless, this effect may prove to be beneficial in the use of IL-7 as an adjuvant in cancer immunotherapies.48, 54
Enhanced IL-7R signaling has also been proposed to contribute to T-cell leukemogenesis.55
Thus, although IL-7 therapies may expand overall T-cell numbers, it may have the unintended consequence of preferentially expanding undesirable T-cell population subsets. Given the increasing interest in IL-7-based therapies, further investigation of the intrinsic differences in the signaling networks across T-cell populations will help us understand the clinical impact of skewing the T-cell repertoire towards a CD5hi