A rapid and high-throughput T cell immunophenotyping assay for cellular therapy bioprocess using the Cellaca® PLX image cytometer.

In cellular therapies chimeric antigen receptor (CAR) T or NK cells undergo phenotypic analysis at multiple stages during discovery and development of novel therapies. Patient samples are routinely analyzed via flow cytometry for population identification and distribution of CD3, CD4, and CD8 positive T cells. As an alternative or orthogonal method, image cytometry systems have been used to perform simple cell-based assays in lieu of flow cytometry. Recently, a new image cytometry system, the Cellaca® PLX (Revvity Health Sciences, Inc., Lawrence, MA), was developed for high-throughput cell counting and viability, immunophenotyping, transfection/transduction efficiency, and cell health assays. This novel instrument allows investigators to quickly assess several critical quality attributes (CQAs) such as cell identity, viability, and other relevant biological functions recommended by the International Organization for Standardization using the ISO Cell Characterization documents focused on cellular therapeutic products. In this work, we demonstrate a rapid and high-throughput image cytometry detection method for cellular immunophenotyping and viability using the Cellaca PLX system for samples throughout the cellular therapy workflow. Freshly isolated peripheral blood mononuclear cells (PBMCs) underwent red blood cell (RBC) lysis and CD3 enrichment. Samples were then subsequently stained with Hoechst/CD3/CD4/CD8 or Hoechst/CD3/CD8/RubyDead Dye surface marker kits and measured on the Cellaca PLX and three different flow cytometers for side-by-side comparison and assay validation. Acquisition and analysis of cell viability and cell populations was shown to be faster and more efficient process compared to flow while achieving highly comparable results between the two technology platforms. This data shows that the Cellaca PLX Image Cytometer may provide a rapid alternative or orthogonal method for PBMC immunophenotyping experiments, as well as potentially streamline the workflow to quickly move precious patient samples downstream within the development processes.

Fatty acid amide hydrolase shapes NKT cell responses by influencing the serum transport of lipid antigen in mice.

The potent regulatory properties of NKT cells render this subset of lipid-specific T cells a promising target for immunotherapeutic interventions. The marine sponge glycolipid alpha-galactosylceramide (alphaGalCer) is the proto-typic NKT cell agonist, which elicits this function when bound to CD1d. However, our understanding of the in vivo properties of NKT cell agonists and the host factors that control their bioactivity remains very limited. In this report, we isolated the enzyme fatty acid amide hydrolase (FAAH) from mouse serum as an alphaGalCer-binding protein that modulates the induction of key effector functions of NKT cells in vivo. FAAH bound alphaGalCer in vivo and in vitro and was required for the efficient targeting of lipid antigens for CD1d presentation. Immunization of Faah-deficient mice with alphaGalCer resulted in a reduced systemic cytokine production, but enhanced expansion of splenic NKT cells. This distinct NKT response conferred a drastically increased adjuvant effect and strongly promoted protective CTL responses. Thus, our findings identify not only the presence of FAAH in normal mouse serum, but also its critical role in the tuning of immune responses to lipid antigens by orchestrating their transport and targeting for NKT cell activation. Our results suggest that the serum transport of lipid antigens directly shapes the quality of NKT cell responses, which could potentially be modulated in support of novel vaccination strategies.

Fine specificity of natural killer T cells against GD3 ganglioside and identification of GM3 as an inhibitory natural killer T-cell ligand.

GD3, a ganglioside expressed on melanoma, is the only tumour-associated glycolipid described to date that can induce a CD1d-restricted natural killer T (NKT)-cell response. We analysed the fine specificity of GD3-reactive NKT cells and discovered that immunization with GD3 induced two populations of GD3-reactive NKT cells. One population was CD4+ CD8- and was specific for GD3; the other population was CD4- CD8- and cross-reacted with GM3 in a CD1d-restricted manner, but did not cross-react with GM2, GD2, or lactosylceramide. This indicated that the T-cell receptors reacting with GD3 recognize glucose-galactose linked to at least one N-acetyl-neuraminic acid but will not accommodate a terminal N-acetylgalactosamine. Immunization with GM2, GM3, GD2, or lactosylceramide did not induce an NKT-cell response. Coimmunization of GM3-loaded antigen-presenting cells (APCs) with GD3-loaded APCs suppressed the NKT-cell response to GD3 in a CD1d-restricted manner. This suppressive effect was specific for GM3 and was a local effect lasting 2-4 days. In vitro, GM3-loaded APCs also suppressed the interleukin-4 response, but not the interferon-gamma response, of NKT cells to alpha-galactosylceramide. However, there was no effect on the T helper type 2 responses of conventional T cells. We found that this suppression was not mediated by soluble factors. We hypothesize that GM3 induces changes to the APC that lead to suppression of T helper type 2-like NKT-cell responses.

The Niemann-Pick type C2 protein loads isoglobotrihexosylceramide onto CD1d molecules and contributes to the thymic selection of NKT cells.

The Niemann-Pick type C2 (NPC2) protein is a small, soluble, lysosomal protein important for cholesterol and sphingolipid transport in the lysosome. The immunological phenotype of NPC2-deficient mice was limited to an impaired thymic selection of Valpha14 natural killer T cells (NKT cells) and a subsequent reduction of NKT cells in the periphery. The remaining NKT cells failed to produce measurable quantities of interferon-gamma in vivo and in vitro after activation with alpha-galactosylceramide. In addition, thymocytes and splenocytes from NPC2-deficient mice were poor presenters of endogenous and exogenous lipids to CD1d-restricted Valpha14 hybridoma cells. Importantly, we determined that similar to saposins, recombinant NPC2 was able to unload lipids from and load lipids into CD1d. This transfer activity was associated with a dimeric form of NPC2, suggesting a unique mechanism of glycosphingolipid transfer by NPC2. Similar to saposin B, NPC2 dimers were able to load isoglobotrihexosylceramide (iGb3), the natural selecting ligand of NKT cells in the thymus, into CD1d. These observations strongly suggested that the phenotype observed in NPC2-deficient animals was directly linked to the efficiency of the loading of iGb3 into CD1d molecules expressed by thymocytes. This conclusion was supported by the rescue of endogenous and exogenous iGb3 presentation by recombinant NPC2. Thus, the loading of endogenous and exogenous lipids and glycolipids onto CD1d is dependent on various small, soluble lipid transfer proteins present in the lysosome.

Cutting edge: impaired glycosphingolipid trafficking and NKT cell development in mice lacking Niemann-Pick type C1 protein.

Niemann-Pick type C1 (NPC1) is a late endosomal/lysosomal transmembrane protein involved in the cellular transport of glycosphingolipids and cholesterol that is mutated in a majority of patients with Niemann-Pick C neurodegenerative disease. We found that NPC1-deficient mice lacked Valpha14-Jalpha18 NKT cells, a major population of CD1d-restricted T cells that is conserved in humans. NPC1-deficient mice also exhibited marked defects in the presentation of Sphingomonas cell wall Ags to NKT cells and in bacterial clearance in vivo. A synthetic fluorescent alpha-glycosylceramide analog of the Sphingomonas Ag trafficked to the lysosome of wild-type cells but accumulated in the late endosome of NPC1-deficient cells. These findings reveal a blockade of lipid trafficking between endosome and lysosome as a consequence of NPC1 deficiency and suggest a common mechanism for the defects in lipid presentation and development of Valpha14-Jalpha18 NKT cells.

Lysosomal glycosphingolipid recognition by NKT cells.

NKT cells represent a distinct lineage of T cells that coexpress a conserved alphabeta T cell receptor (TCR) and natural killer (NK) receptors. Although the TCR of NKT cells is characteristically autoreactive to CD1d, a lipid-presenting molecule, endogenous ligands for these cells have not been identified. We show that a lysosomal glycosphingolipid of previously unknown function, isoglobotrihexosylceramide (iGb3), is recognized both by mouse and human NKT cells. Impaired generation of lysosomal iGb3 in mice lacking beta-hexosaminidase b results in severe NKT cell deficiency, suggesting that this lipid also mediates development of NKT cells in the mouse. We suggest that expression of iGb3 in peripheral tissues may be involved in controlling NKT cell responses to infections and malignancy and in autoimmunity.

Editing of CD1d-bound lipid antigens by endosomal lipid transfer proteins.

It is now established that CD1 molecules present lipid antigens to T cells, although it is not clear how the exchange of lipids between membrane compartments and the CD1 binding groove is assisted. We report that mice deficient in prosaposin, the precursor to a family of endosomal lipid transfer proteins (LTP), exhibit specific defects in CD1d-mediated antigen presentation and lack Valpha14 NKT cells. In vitro, saposins extracted monomeric lipids from membranes and from CD1, thereby promoting the loading as well as the editing of lipids on CD1. Transient complexes between CD1, lipid, and LTP suggested a "tug-of-war" model in which lipid exchange between CD1 and LTP is on the basis of their respective affinities for lipids. LTPs constitute a previously unknown link between lipid metabolism and immunity and are likely to exert a profound influence on the repertoire of self, tumor, and microbial lipid antigens.

Mechanism of p21-activated kinase 6-mediated inhibition of androgen receptor signaling.

PAK6 was first identified as an androgen receptor (AR)-interacting protein able to inhibit AR-mediated transcriptional responses. PAK6 is a serine/threonine kinase belonging to the p21-activated kinase (PAK) family implicated in actin reorganization and cell motility, gene transcription, apoptosis, and cell transformation. We investigated the biochemical basis for inhibition of AR signaling by PAK6. We compared the kinase activity of PAK6 with two other well characterized members of the PAK family, PAK1 and PAK4. Like PAK4, PAK6 possesses a constitutive basal kinase activity that, unlike PAK1, is not modulated by the binding of active Rac or Cdc42 GTPases. In order to test the involvement of PAK6 kinase activity in suppression of AR-mediated transcription, we generated kinase-dead (K436A) and kinase-active (S531N) mutants of PAK6. We show that PAK6 kinase activity is required for effective PAK6-induced repression of AR signaling. Suppression does not depend upon GTPase binding to PAK6 and is not mimicked by the closely related PAK1 and PAK4 isoforms. Kinase-dependent inhibition by PAK6 extended to the enhanced AR-mediated transcription seen in the presence of coactivating molecules and to the action of AR coinhibitors. Active PAK6 inhibited nuclear translocation of the stimulated AR, suggesting a possible mechanism for inhibition of AR responsiveness. Finally, we observe that autophosphorylated, active PAK6 protein is differently expressed among prostate cancer cell lines. Modulation of PAK6 activity may be responsible for regulation of AR signaling in various forms of prostate cancer.

IAP suppression of apoptosis involves distinct mechanisms: the TAK1/JNK1 signaling cascade and caspase inhibition.

The antiapoptotic properties of the inhibitor of apoptosis (IAP) family of proteins have been linked to caspase inhibition. We have previously described an alternative mechanism of XIAP inhibition of apoptosis that depends on the selective activation of JNK1. Here we report that two other members of the IAP family, NAIP and ML-IAP, both activate JNK1. Expression of catalytically inactive JNK1 blocks NAIP and ML-IAP protection against ICE- and TNF-alpha-induced apoptosis, indicating that JNK1 activation is necessary for the antiapoptotic effect of these proteins. The MAP3 kinase, TAK1, appears to be an essential component of this antiapoptotic pathway since IAP-mediated activation of JNK1, as well as protection against TNF-alpha- and ICE-induced apoptosis, is inhibited when catalytically inactive TAK1 is expressed. In addition, XIAP, NAIP, and JNK1 bind to TAK1. Importantly, expression of catalytically inactive TAK1 did not affect XIAP inhibition of caspase activity. These data suggest that XIAP's antiapoptotic activity is achieved by two separate mechanisms: one requiring TAK1-dependent JNK1 activation and the second involving caspase inhibition.