Genetic alterations such as SNPs contribute to differential immune responses between individuals. Here, we report that LYP*W620 causes a combination of reduced proximal TCR-mediated signaling and augmented CD28-associated signaling. At the cellular level, these shifts in signaling affect the differentiation and function of CD4+ T cells ultimately leading to exaggerated Th1 responses. Our findings can explain why carriers of LYP*W620 are relatively resistant to intracellular infections (e.g. tuberculosis), but also are more prone to autoimmunity.
LYP*W620 was originally found a gain-of-function variant. This notion was supported by findings with human T and B cells indicating that LYP*W620 is a more potent inhibitor of both TCR and BCR signaling [8
]. Furthermore, the PTP activity of LYP*W620 is approximately 50% higher than that of LYP*R620 [8
]. Two recent studies have challenged this model by suggesting that LYP*W620 acts as a hypomorph (i.e. a loss-of-function allele). The first one of these studies described the effects of overexpression of the two LYP variants in Jurkat T cells [21
]. Importantly, the authors based their conclusions on data from cells with high (and most probably supra-physiological) levels of LYP. In fact, when cells with modest overexpression of LYP (i.e. closer to physiological levels) were analyzed, LYP*W620 was a stronger inhibitor of TCR signaling. These observations are also in agreement with our previously published observations [8
]. The second study described characterization of Pep-R619W knockin mice (mutation corresponding to the LYP-R620W mutation) and suggested that Pep-R619W is an unstable protein compared to wild-type Pep [12
]. The authors extended these findings to LYP*W620 and concluded that LYP*W620 is a hypomorph. In contrast, we previously found comparable expression of the two LYP variants in CD4+
T cells from healthy human donors homozygous for either LYP*R620 or LYP*W620 [4
]. Furthermore, in the present study we demonstrate similar expression of the two LYP variants in all CD4+
T cell subsets tested, with the exception of naive conventional CD4+
T cells where LYP*W620 expression was slightly lower (approximately 9%) than LYP*R620 expression. The only explanation we can offer for the above-mentioned discrepancies regarding LYP stability is differences in reagents used to determine LYP expression.
Given the data presented in this paper, we now propose that the autoimmune-inducing capabilities of LYP*W620 cannot simply be explained by a gain-of-function model. This is based on several observations. First, our phospho-flow cytometry analysis of TCR/CD28-stimulated primary T cells indicated that WW cells displayed weaker TCR-proximal signaling and augmented CD28-associated signaling. Second, the LYP-R620W mutation led to altered ligand binding properties, e.g. LYP*R620 interacted more efficiently with PI3K. Third, we have previously shown that the sub-cellular localization of LYP*W620 was different from that of LYP*R620, more specifically, in resting cells LYP*W620 partitioned more efficiently into lipid rafts [4
]. Here, it should be added that several findings presented in this paper can also be explained by a gain-of-function mechanism, e.g. reduced TCR-induced ζ-chain phosphorylation due to elevated LYP catalytic activity.
The main substrates for LYP in T cells are LCK, FYN, ZAP-70, the CD3/ζ subunits of the TCR complex, and VAV [5
]. While LCK, FYN, ZAP-70 and CD3/ζ subunits are signaling proteins downstream of the TCR, VAV is involved in CD28-mediated signaling. The fact that LYP*W620 lead to reduced TCR-coupled signaling and augmented CD28-associated signaling suggested that LYP*W620 relative to LYP*R620 preferred the substrates LCK and CD3/ ζ subunits over VAV. Here, it should be added that the evidence involving VAV is partly circumstantial; although we did not see significant alterations in VAV phosphorylation in WW T cells (there was only a tendency towards higher VAV phosphorylation in WW CD4+
T cells), we did make significant findings with other (and more downstream) signaling molecules associated with CD28 (e.g. activation of AKT and IKKα). Lastly, the observed signaling differences between LYP*R620 and LYP*W620 could be explained by differences in catalytic activity and/or sub-cellular localization.
A recent study demonstrated that LYP is overexpressed in chronic lymphocytic leukemia B cells that are homozygous for LYP*R620, and that such overexpression promotes cellular survival through activation of AKT [22
]. Interestingly, the authors propose that the effects of LYP overexpression are two-fold: LYP down-modulates both positive and negative regulatory signaling pathways, and the net result is a shift in the signaling balance, eventually leading to diverse outcomes such as reduced BCR signaling and yet elevated AKT activity. There are currently no data indicating that the same mechanisms are operative in T cells. Although we suggest that augmented AKT activation in WW T cells may be explained by altered ligand binding properties for LYP*W620, additional mechanisms can be involved. One such mechanism is elevated LYP activity since LYP*W620 is a more active PTP than LYP*R620.
In this study, we analyzed T cells derived from peripheral blood donated by healthy individuals. The functional properties of these cells are a result of several processes including development in thymus, antigenic challenges in lymphoid organs, and homeostatic mechanisms in the periphery. For disease-mechanistic purposes it is tempting to compare our findings with those obtained with established animal models. Based on the immunological analyses presented here and published epidemiological studies, the phenotype of individuals homozygous for LYP*W620 is characterized by: i) T cells with elevated AKT activity, ii) a shift in the balance between conventional and regulatory T cells resulting in exaggerated Th1 responses, and iii) higher risk for autoimmune diseases. Interestingly, this phenotype is reminiscent of the phenotype observed for mice with Foxo1 or combined Foxo1/Foxo3 deficiencies in T cells [23
]. These animals display defective Treg cell development in thymus (but normal numbers of Tregs in the periphery), enhanced Th1 responses, and severe autoimmune manifestations. Although our study in human could not include analysis of T cell development in thymus, it is tempting to speculate that the elevated AKT activation observed in peripheral WW T cells is also taking place in developing T cells. Such a scenario would lead to defective Treg cell development and function, but homeostatic mechanisms would ensure normal Treg cell numbers in the periphery.
Epidemiological data have indicated that carriers of LYP*W620 are less likely to contract tuberculosis, and that if they do, the disease will be less severe compared to RR individuals [26
]. Our findings provide a cellular and molecular explanation for these data; carriers of LYP*W620 display exaggerated Th1 responses (e.g. augmented IFNγ production), which offer increased protection against tuberculosis. From an evolutionary perspective, carriers of LYP*W620 probably had a survival advantage since tuberculosis has been a common and serious disease throughout history. Thus, our findings may explain why evolution has kept the C1858T SNP in PTPN22
. This is further supported by the fact that WW donors also produced higher amounts of TNFα, which is also a major pro-inflammatory mediator. Given our data, we also speculate that carriers of LYP*W620 may be better protected from other intracellular infections, although this remains to be shown. In this study we also found that production/secretion of the Th17 cytokine IL-17 was reduced in WW donors. This is an interesting finding and may suggest that Th17 responses are not that important in humans with regard to induction of autoimmunity or control of tuberculosis. Finally, we found that healthy WW donors also produced higher amounts of IL-2. In contrast, we previously reported that WW individuals with type 1 diabetes produced lower amounts of IL-2 [8
]. One possible explanation for this discrepancy may be the ongoing immune processes in type 1 diabetes patients.
In conclusion, we provide evidence that the autoimmune-associated version of LYP, LYP*W620, causes reduced proximal TCR-mediated signaling but augmented CD28-coupled signaling. This imbalance in signaling ultimately affects T cell differentiation, which is skewed favoring Th1 responses.