A clathrin/dynamin- and mannose-6-phosphate receptor–independent pathway for granzyme B–induced cell death

The 280-kD cation-independent mannose-6-phosphate receptor (MPR) has been shown to play a role in endocytic uptake of granzyme B, since target cells overexpressing MPR have an increased sensitivity to granzyme B–mediated apoptosis. On this basis, it has been proposed that cells lacking MPR are poor targets for cytotoxic lymphocytes that mediate allograft rejection or tumor immune surveillance. In the present study, we report that the uptake of granzyme B into target cells is independent of MPR. We used HeLa cells overexpressing a dominant-negative mutated (K44A) form of dynamin and mouse fibroblasts overexpressing or lacking MPR to show that the MPR/clathrin/dynamin pathway is not required for granzyme B uptake. Consistent with this observation, cells lacking the MPR/clathrin pathway remained sensitive to granzyme B. Exposure of K44A-dynamin–overexpressing and wild-type HeLa cells to granzyme B with sublytic perforin resulted in similar apoptosis in the two cell populations, both in short and long term assays. Granzyme B uptake into MPR-overexpressing L cells was more rapid than into MPR-null L cells, but the receptor-deficient cells took up granzyme B through fluid phase micropinocytosis and remained sensitive to it. Contrary to previous findings, we also demonstrated that mouse tumor allografts that lack MPR expression were rejected as rapidly as tumors that overexpress MPR. Entry of granzyme B into target cells and its intracellular trafficking to induce target cell death in the presence of perforin are therefore not critically dependent on MPR or clathrin/dynamin-dependent endocytosis

Humans Lack iGb3 Due to the Absence of Functional iGb3-Synthase: Implications for NKT Cell Development and Transplantation

The glycosphingolipid isoglobotrihexosylceramide, or isogloboside 3 (iGb3), is believed to be critical for natural killer T (NKT) cell development and self-recognition in mice and humans. Furthermore, iGb3 may represent an important obstacle in xenotransplantation, in which this lipid represents the only other form of the major xenoepitope Galα(1,3)Gal. The role of iGb3 in NKT cell development is controversial, particularly with one study that suggested that NKT cell development is normal in mice that were rendered deficient for the enzyme iGb3 synthase (iGb3S). We demonstrate that spliced iGb3S mRNA was not detected after extensive analysis of human tissues, and furthermore, the iGb3S gene contains several mutations that render this product nonfunctional. We directly tested the potential functional activity of human iGb3S by expressing chimeric molecules containing the catalytic domain of human iGb3S. These hybrid molecules were unable to synthesize iGb3, due to at least one amino acid substitution. We also demonstrate that purified normal human anti-Gal immunoglobulin G can bind iGb3 lipid and mediate complement lysis of transfected human cells expressing iGb3. Collectively, our data suggest that iGb3S is not expressed in humans, and even if it were expressed, this enzyme would be inactive. Consequently, iGb3 is unlikely to represent a primary natural ligand for NKT cells in humans. Furthermore, the absence of iGb3 in humans implies that it is another source of foreign Galα(1,3)Gal xenoantigen, with obvious significance in the field of xenotransplantation

Recognition of a carbohydrate xenoepitope by human NKRP1A (CD161)

Background: Many immunologically important interactions are mediated by leukocyte recognition of carbohydrates via cell surface receptors. Uncharacterized receptors on human natural killer (NK) cells interact with ligands containing the terminal Gal alpha(1,3)Gal xenoepitope. The aim of this work was to isolate and characterize carbohydrate binding proteins from NK cells that bind alpha Gal or other potential xenoepitopes, such as N-acetyllactosamine (NAcLac), created by the deletion of alpha 1,3galactosyltransferase (GT) in animals. Methods and results: Initial analysis suggested the human C-type lectin NKRP1A bound to a pool of glycoconjugates, the majority of which contained the terminal Gal alpha(1,3)Gal epitope. This was confirmed by high level binding of cells expressing NKRP1A to mouse laminin, which contains a large number of N-linked oligosaccharides with the Gal alpha(1,3)Gal structure. The consequence of removing the terminal alpha Gal was then investigated. Elevated NAcLac levels were observed on thymocytes from GT(-/-) mice. Exposing NAcLac on laminin, by alpha-galactosidase treatment, resulted in a significant increase in NKRP1A binding. Conclusions: NKRPIA binds to the alpha Gal epitope. Moreover, exposing NAcLac by removal of alpha Gal resulted in an increase in binding. This may be relevant in the later phases of xenotransplant rejection if GT(-/-) pigs, like GT(-/-) mice, display increased NAcLac expression

Depletion of p21-activated kinase 1 up-regulates the immune system of APC∆14/+ mice and inhibits intestinal tumorigenesis

Abstract Background P21-activated kinase 1 (PAK1) stimulates growth and metastasis of colorectal cancer (CRC) through activation of multiple signalling pathways. Up-regulation of CRC stem cell markers by PAK1 also contributes to the resistance of CRC to 5-fluorouracil. The aim of this study was to investigate the effect of PAK1 depletion and inhibition on the immune system and on intestinal tumour formation in APC∆14/+ mice. Methods The PAK1 KO APC∆14/+ mice were generated by cross-breeding of PAK1 KO mice with APC∆14/+ mice. Splenic lymphocytes were analysed by flow cytometry, and immunohistochemical staining. The numbers of intestinal tumours were counted. Blood cells were also counted. Results Compared to APC+/+ mice, the numbers of both T- and B- lymphocytes were reduced in the spleen of APC∆14/+ mice. Depletion of PAK1 in APC∆14/+ mice increased the numbers of splenic T- and B- lymphocytes and decreased the numbers of intestinal tumours. Treatment of APC∆14/+ mice with PF-3758309, a PAK inhibitor reduced the numbers of intestinal tumours and increased the numbers of blood lymphocytes. Conclusion Depletion of active PAK1 up-regulates the immune system of APC∆14/+ mice and suppresses intestinal tumour development. These observations suggest an important role for PAK1 in the immune response to tumours