The mechanistic connection between SAP deficiency and excessive lymphoproliferation, particularly in the context of infectious challenge, is a fundamental unanswered question of this severe disease. Here we demonstrate that despite being dispensable for primary stimulation, SAP is critical for ensuring the elimination of activated human T cells following TCR restimulation. Our results also provide what is, to our knowledge, the first insight into the immunological role of NTB-A in T cell regulation.
We propose a model by which SAP plays a key role in setting the threshold for RICD, particularly with respect to CD8+
T cells (Figure ). TCR re-engagement triggers SAP recruitment to immunoreceptor tyrosine-based switch motifs found on the cytoplasmic tail of NTB-A, which can dislodge SHP-1 from the receptor based on its binding properties (33
). We also found that NTB-A is closely complexed with the TCR upon restimulation on activated T cells, an event not previously observed for this SLAM family receptor. SAP association with NTB-A nullified SHP-1 inhibition, and TCR signaling proceeded to turn on key target genes, such as FASL and BIM, that trigger programmed cell death. This leads to a robust homeostatic apoptosis of T cells in restimulation conditions, which would be expected to prevail during a persistent viral infection. SAP association with other SLAM family members may occur upon restimulation, although our RNAi screen data suggest that only NTB-A plays a major role in influencing RICD. Moreover, our data suggest that SAP-dependent recruitment of FYN to NTB-A is not required for RICD, so it remains to be determined whether other kinases act to amplify the apoptosis-promoting signal conveyed by SAP–NTB-A association. For example, SAP can interact with LCK, which has been shown to phosphorylate SLAM and CD84 (27
). Our data provide a coherent molecular model for the severe pathogenic antigen-induced lymphoproliferation observed in children with this genetic defect.
Model for the involvement of SAP/NTB-A signaling in augmenting TCR signal strength for apoptosis.
Enhanced clonotypic expansion of SAP–/–
T cells, concomitant with reduced apoptosis following primary stimulation, was recently described in OT-I TCR transgenic mice and connected to poor upregulation of the p53 homolog p73 in OVA-stimulated naive CD8+
T cells (21
). In that study, death was assessed within 48 hours after a single antigen challenge and by definition did not measure RICD of activated effector cells upon rechallenge with antigen. Moreover, the relative contribution of p73 to T cell apoptosis may be restricted to death immediately following primary stimulation, as p73-deficient T cells show no defect in restimulation-induced apoptosis (35
). However, although we also observed less apoptosis following primary stimulation of XLP T cells, this trend did not correlate with decreased p73 induction relative to controls (Supplemental Figure 18). Moreover, we failed to detect any significant induction of p73 in restimulated human T cells, or any change in apoptosis sensitivity after p73 siRNA transfection (data not shown). Rather, our results suggest that SAP acts as an amplifier of proximal signals triggered by TCR re-engagement that is important for RICD of both CD4+
T cells, ensuring optimal induction of downstream target genes including the pro-apoptotic executioner molecules FASL and BIM.
Regulation of lymphocyte homeostasis by apoptosis adds to the expanding list of immunomodulatory functions attributed to SAP and SLAM family receptors such as NTB-A. However, apoptosis dysregulation in XLP is distinct from the apoptosis defect in autoimmune lymphoproliferative syndrome (ALPS) in several interesting respects. SAP has no bearing on the inherent susceptibility of an activated T cell to extrinsic or intrinsic apoptosis signals; direct crosslinking of Fas or removal of IL-2 effectively kills XLP patient T cells. Hence, cytokine withdrawal–mediated T cell contraction theoretically proceeds normally in XLP patients once antigen is cleared and may be accelerated according to our data (Figure B). In addition to well-established defects in humoral immunity, this explains why XLP patients do not develop chronic lymphadenopathy, splenomegaly, or autoimmune manifestations characteristic of ALPS (37
). However, the paradoxic propensity for B cell lymphomagenesis found in both XLP and ALPS may derive from defective FAS-mediated killing of B cells by activated T cells bearing FASL.
In cloning SAP as the affected gene in XLP, Sayos et al. also noted that SAP could block the recruitment of SHP-2 to SLAM (13
). SAP may serve a similar purpose in augmenting TCR-induced signals by displacing SHP-1 from NTB-A. We observed that NTB-A could colocalize with clustered TCR-CD3 complexes following restimulation, consistent with a function in modulating RICD. This is biochemically analogous to NTB-A signaling in NK cells, which contributes to the cytotoxic potential of NK cells against EBV-infected targets along with 2B4 (28
). SAP also acts as an adaptor protein to recruit SH3 domain–containing kinases such as FYN, LCK, and NCK1 to SLAM family receptors, which can enhance the cascade of protein phosphorylation that accompanies TCR signaling (38
). Our data imply that SAP can potentiate RICD independently of FYN, but more work is required to dissect the specific biochemistry connecting SAP–NTB-A interactions to downstream TCR signal transduction. Whether homotypic NTB-A interactions occur in cis
on activated T cells also remains to be determined in our system. Furthermore, our RNAi screen of the SLAM family suggests that a balance of SLAM receptor expression may control RICD sensitivity, perhaps through differential association with SAP, SHP-1, and/or other binding partners. Although anti-CD3 Ab–induced death of activated T cells has been observed at the single cell level, which would ostensibly preclude a role for NTB-A as we described, physiological antigen stimulation of T cells is invariably a cell-cell transaction, making it possible that SLAM receptor interactions with antigen-presenting cells or adjacent T cells directly influence signaling for RICD. Nevertheless, the global suppression of many TCR-induced target genes in the absence of SAP and NTB-A implies that these molecules directly affect proximal TCR signaling events.
Our observation that the requirement for SAP can be overridden by artificially increasing the TCR signal strength supports the threshold model of RICD. It is generally accepted that a quantitatively and qualitatively strong restimulation signal is required to surpass the threshold necessary for triggering apoptosis. We propose that the SAP–NTB-A interaction plays an integral role in reaching the required threshold. In the absence of SAP, TCR signaling effectively becomes weaker, resembling a partial agonist signal that involves negative feedback loops like those controlled by SHP-1 in response to weakly binding ligands (41
Despite extensive work in vitro, the full importance of RICD in vivo has remained unknown (24
). XLP may now prove illustrative of the significance of this self-regulatory mechanism. Defective RICD likely contributes to several clinical features of the disease, notably T cell–driven LPD and HLH. Without SAP, unbridled effector T cell expansion secondary to viral infection or other antigenic stimuli, especially in antigen-rich environments such as the lungs, proceeds without counterbalancing propriocidal death. Indeed, we noted lung nodules with T cell infiltrates in XLP Pt2, Pt3, and Pt8, anecdotally linked to VZV vaccination and detection of HHV6 in lung tissue or blood, respectively. In turn, this overly expanded T cell compartment can drive activation and proliferation of other hematopoietic cell subsets. Hence, the apoptotic defect that we have discovered provides a unifying mechanism for the diverse pathological features of XLP, including pulmonary/CNS vasculitis, pulmonary nodules, and EBV-associated FIM.
We believe this scenario is particularly relevant to the unique susceptibility of XLP patients to EBV infection for several reasons. First, EBV normally induces a vigorous cellular immune response, greatly expanding the pool of EBV-specific effector T cells. Second, recent evidence from mice indicates that T cell/B cell interactions are inherently weaker in the absence of SAP (42
). This implies that any encounter between an EBV-specific XLP T cell and an EBV-infected B cell is short lived, constituting an attenuated restimulation signal, likely below the threshold required for RICD. Third, SAP-deficient EBV-specific T cells fail to kill EBV-infected B cells due to impaired 2B4 and NTB-A–mediated cytotoxicity, with the consequence that the source of abundant EBV antigen is never effectively cleared. The defect in FASL upregulation we uncovered in SAP-deficient T cells may contribute to impaired cytotoxic potential in this setting. These elements culminate in the “perfect storm” of EBV-associated FIM/HLH, stemming from unchecked proliferation of EBV-specific T cells (especially CD8+
T cells) that fail to undergo RICD in response to repeated antigenic stimulation.
Without curative allogeneic stem cell transplantation, the prognosis for XLP patients remains poor, as only 30% of XLP patients survive past age 10, and the mortality rate for EBV-associated FIM/HLH is approximately 96% (8
). Our findings can now refocus efforts toward novel therapeutic approaches for XLP and related disorders based on this clear and specific apoptosis defect. Removal of the potential source of EBV-associated antigens via pre-emptive B cell depletion, or administration of SHP-1 inhibitors to generally boost TCR signal strength, could prevent or retard HLH progression by constraining numbers of responding T cells. Collectively, our demonstration of impaired RICD in human SAP–deficient T cells provides mechanistic insight on XLP-associated LPD, underscores the physiological importance of this autoregulatory cell death mechanism in a unique disease setting, and reiterates the fact that XLP manifestations are not dependent on the presence of EBV infection. These observations may stimulate new approaches to the clinical management of XLP and other disorders of lymphocyte apoptosis and homeostasis.