NKT cells and SAP

Source: Chung B, Aoukaty A, Dutz J, Terhorst C, Tan R. Signaling lymphocytic

activation molecule-associated protein controls NKT cell functions. J Immunol.

2005 Mar 15;174(6):3153-7. PubMed PMID: 15749842.

 

X-linked lymphoproliferative disease (XLP) is a primary immunodeficiency disorder, which is caused by mutation in the gene Src homology 2 (SH2) domain containing gene 1A (SH2D1A). This gene encodes signaling lymphocyte activating molecule (SALM)-associated protein (SAP). There are many evidences that indicate that SAP plays a role in regulation of lymphocytes.

• The clinical course of XLP consists of uncontrolled proliferation of B and T cells.
  
• Studies have observed that SAP knock out (SAPKO) mice generate abnormally large virus specific CD8+ and CD4+ T cell following infection with LCMV.
  
• It has also been observed that SAP binds to intracellular domains of SLAM and B4, both of these are immuno-regulatory molecules, which are primarily expressed on T cells and NK cells, respectively.
  
• SAPKO mice are unable to generate memory B cell response.
  

Despite all these evidences about role of SAP in controlling lymphocyte proliferation, a role for SAP in NKT cells development and function, a type of regulatory cells, is not known. It has been previously reported that CD1d restricted NKT cells control EBV-specific lymphocyte proliferation [Ho et al., 2004 (CD4-CD8alphaalpha subset of CD1d-restricted NKT cells controls T cell expansion)]. This can be due to a possible link between NKT cell function and large T cell expansions seen in XLP patients following EBV infection.

In a beautiful paper published in 2005 from the labs of Dr. Rusung Tan, the investigators sought to determine the role of NKT cells in the immune dysregulation of SAPKO mice.

Their findings are briefly described below:

1. SAPKO mice completely lack CD1d restricted NKT cells.
   
2. When SAPKO mice were injected with potent NKT agonist alpha-GalCer, the mice did not develop or activate NKT cells that produce cytokines (IFN-gamma and IL-4).
   
3. When SAPKO mice were co-injected with OVA and alpha-GalCer, the mice did not show OVA specific CTL responses.
   
4. Patients with XLP did not have a population of CD1d restricted NKT cells in their peripheral blood.
   

All these findings indicated that SAP is critical for normal CD1d restricted NKT cell development and function.

 

Performance of experiments:

• SAPKO mice and wild type mice were taken and SAP gene expression was confirmed by PCR and immunoprecipitaion. Lymphocytes were stained with Abs to CD3 and NK1.1, these two molecules are the markers present on murine NKT cells, and flow-cytometric analyses were performed. The investigators observed that frequency of CD3+ NK1.1+ NKT cells from liver, thymus, spleen and lymph nodes was significantly low in SAPKO mice in comparison to wild type. An earlier study (Hammond et al., 2004) has shown that NK1.1 expression may not be the ideal marker for CD1d restricted NKT cells as many of the CD1d restricted cells fail to express NK1.1. Thus, Dr. Tan and his team used CD1d tetramer and Ab to TCRVbeta8 to stain lymphocytes from spleen, liver, thymus and lymph nodes from SAPKO and wild type mice. The data obtained confirmed that SAPKO mice lack CD1d-restrcited NKT cells.
  
• Up on activation NKT cells rapidly produce cytokines. The investigators thus hypothesized that since SAPKO mice lack CD1D-restricted NKT cells, they would not produce these cytokines upon activation. Thus, SAPKO and wild type mice were intra-peritoneally injected with alpha-GalCer and liver cells were isolates after 2 hrs. These cells were stained with CD1d tetramer to identify CD1d-restricted NKT cells and intracellular cytokine staining was performed using anti-IFN-gamma-allophycocyanin or anti-IL4-allophycocyanin. The authors observed no CD1d restricted NKT cells and no IFN-gamma or IL-4 production in SAPKO mice. In contrast, wild type mice produced significant amount of IFN-gamma and IL-4. These findings indicate that SAPKO mice are unable to mount a rapid NKT-induced cytokine response.
  
• A previously conducted study showed that NKT cells not only regulate LCMV-induce cytokine production, but they also controlled the magnitude of the cell-mediated immune response to an acute viral infection. Thus, NKT cells are important for generation and regulation of antigen specific T cells. Thus, to determine whether NKT cells are important for activation or proliferation of antigen specific T cells, the authors immunized SAPKO and wild type mice with vehicle (a control for alpha-GalCer) and OVA, CFA or alpha-GalCer. At day 14, PBMCs were isolated and stained with Kb-OVA254-267 tetramers and CD8 Ab to measure OVA-CTL expansion. Wild type mice showed significant CTL-expansion in comparison to SAPKO mice immunized with OVA and alpha-GalCer. These data indicate that the generation of Ag-specific CTL response is promoted by CD1d restricted NKT cells and SAPKO mice are unable to generate CTL in response to immunized Ag and NKT cell agonist.
  
• The scientists also took peripheral blood from two XLP patients, who had mutations in the second exon of SAP, and stained the PBMCs with CD1d tetramer and anti-CD3 Ab. They followed the same experiments in eight healthy individuals. The healthy controls showed significant percentage of CD1d-restricted NKT cells in comparison to XLP patients, indicating that CD1d-restricted NKT cells are absent in XLP patients.
  

 

Conclusions:

NKT cells act as a bridge between innate immune response and adaptive immune response by rapidly activating NK cells and helping in maturation of DCs. Thus, the authors hypothesized that in the absence of NKT cells, the immune response to virus is sub-optimal. Thus, viral replication occurs unchecked leading to formation of a chronic viral stage. It has been observed that boys with XLP disease show continuous viremia with EBV during the course of diseases. Such uncontrolled virus infection may lead to chronic stimulation of virus specific CTLs that are not efficient to clear virus-infected cells.