The magnitude of response elicited by CTL-inducing vaccines correlates with the density of MHC class I (MHC-I)-peptide complexes formed on the APC membrane. The MHC-I L chain, β2-microglobulin (β2m), governs complex stability. We reasoned that genetically converting β2m into an integral membrane protein should exert a marked stabilizing effect on the resulting MHC-I molecules and enhance vaccine efficacy. In the present study, we show that expression of membranal human β2m (hβ2m) in mouse RMA-S cells elevates MHC-I thermal stability. RMA-S transfectants bind an exogenous peptide at concentrations 104- to 106-fold lower than parental RMA-S, as detected by complex-specific Abs and by T cell activation. Moreover, saturation of the transfectants’ MHC-I by exogenous peptide occurs within 1 min, as compared with ∼1 h required for parental cells. At saturation, however, level of peptide bound by modified cells is only 3- to 5-fold higher. Expression of native hβ2m only results in marginal effect on the binding profile. Soluble β2m has no effect on the accelerated kinetics, but the kinetics of transfectants parallel that of parental cells in the presence of Abs to hβ2m. Ab inhibition and coimmunoprecipitation analyses suggest that both prolonged persistence of peptide-receptive H chain/β2m heterodimers and fast heterodimer formation via lateral diffusion may contribute to stabilization. In vivo, peptide-loaded transfectants are considerably superior to parental cells in suppressing tumor growth. Our findings support the role of an allosteric mechanism in determining ternary MHC-I complex stability and propose membranal β2m as a novel scaffold for CTL induction.

Level and persistence of antigenic peptides presented by APCs on MHC class I (MHC-I) molecules influence the magnitude and quality of the ensuing CTL response. We recently demonstrated the unique immunological properties conferred on APCs by expressing β2-microglobulin (β2m) as an integral membrane protein. In this study, we explored membrane-anchored β2m as a platform for cancer vaccines using as a model MO5, an OVA-expressing mouse B16 melanoma. We expressed in mouse RMA-S cells two H-2Kb binding peptides from MO5, OVA257–264, and TRP-2181–188, each genetically fused with the N terminus of membranal β2m via a short linker. Specific Ab staining and T cell hybridoma activation confirmed that OVA257–264 was properly situated in the MHC-I binding groove. In vivo, transfectants expressing both peptides elicited stronger CTLs and conferred better protection against MO5 than peptide-saturated RMA-S cells. Cells expressing OVA257–264/β2m were significantly superior to OVA257–264-charged cells in their ability to inhibit the growth of pre-established MO5 tumors. Our results highlight the immunotherapeutic potential of membranal β2m as a universal scaffold for optimizing Ag presentation by MHC-I molecules.

Objectivesγδ T cells, a non-conventional innate lymphocyte subset containing cells that can be activated by lipids and phosphoantigens, are abnormally regulated in systemic sclerosis (SSc). To further evaluate the significance of this dysregulation, we compared how exposure to an autoantigenic lipid, cardiolipin (CL), during co-stimulation with an amino-bisphosphonate (zoledronate, zol), affects the activation and cytokine production of SSc and healthy control (HC) γδ T cells.MethodsExpression of CD25 on Vγ9+, Vδ1+, and total CD3+ T cells in cultured peripheral blood mononuclear cells (PBMCs), their binding of CD1d tetramers, and the effect of monoclonal antibody (mAb) blockade of CD1d were monitored by flow cytometry after 4 days of in vitro culture. Intracellular production of IFNγ and IL-4 was assessed after overnight culture.ResultsPercentages of CD25+ among CD3+ and Vδ1+ T cells were elevated significantly in short-term cultured SSc PBMC compared to HC. In SSc but not HC, CL and zol, respectively, suppressed %CD25+ Vγ9+ and Vδ1+ T cells but, when combined, CL + zol significantly activated both subsets in HC and partially reversed inhibition by the individual reagents in SSc. Importantly, Vδ1+ T cells in both SSc and HC were highly reactive with lipid presenting CD1d tetramers, and a CD1d-blocking mAb decreased CL-induced enhancement of %SSc CD25+ Vδ1+ T cells in the presence of zol. %IFNγ+ cells among Vγ9+ T cells of SSc was lower than HC cultured in medium, CL, zol, or CL + zol, whereas %IFNγ+ Vδ1+ T cells was lower only in the presence of CL or CL + zol. %IL-4+ T cells were similar in SSc and HC in all conditions, with the exception of being increased in SSc Vγ9+ T cells in the presence of CL.ConclusionAbnormal functional responses of γδ T cell subsets to stimulation by CL and phosphoantigens in SSc may contribute to fibrosis and immunosuppression, characteristics of this disease.

BackgroundT cells (TC) expressing Vδ1+ TC receptors recognize lipids presented by CD1d (1). Oligoclonal Vδ1+ TC infiltrate skin and internal organs and are activated in the peripheral blood (PB) in systemic sclerosis (SSc) suggesting lipids as pathogenic immunogens (2,3).ObjectivesTo determine effects and molecular pathways of the auto antigenic lipid – cardiolipin- in SSc and healthy controls (HC).MethodsMononuclear cells (MC) were isolated from PB of 12 SSc patients and 8 HC by density centrifugation and cultured in IL2C (RPMI-1640 with 10% fetal bovine serum + 100 international units of interleukin-2), CARDC or OCHC [IL2C+cardiolipin 2.5μg/ml or OCH 50ng/ml (analogue of α-galactosyl ceramide) respectively]. Zoledronate (zol, 2μM), a Vγ9+ γδTC stimulant that induces their CD1d expression, was added to parallel cultures (labeled ILCzol etc.) (4,5). Flow-cytometry (FC) of 4–5 day cultures after staining with monoclonal antibodies (mAb) or CD1d tetramers to detect %CD25+ cells (%CD25+) or tetramer+ cells, was performed.Means were compared by two tailed student t-test; p<0.05 was considered significant.ResultsVδ1+ and Vγ9+TC consisted 11.85±2.0% and 14.7±3.5% vs 10.42±2.4% and 7.08±1.2% of the CD3+ TC in SSc and HC IL-2C respectively (values not significantly different). In HC cultures, %CD25+ was non significantly different in IL2C, OCHC and CARDC for all TC subsets.Furthermore, IL2Czol increased %CD25+ on all TC subsets relative to IL2C, which was further amplified on Vδ1+ TC in CARDCzol (33.16±7.0% to 56.93±6.3% p<0.007). In SSc, in contrast OCHC and CARDC enhanced %CD25+Vδ1+TC whereas CARDC %CD25+Vγ9+TC was suppressed (76.06±8.7% vs 66.9±10.9%, p<0.036) relative to IL2C. In IL2Czol, however, %CD25+Vγ9+TC increased to 73.93±8.12%. In contrast, IL2Czol decreased SSc %CD25+Vδ1+TC (69.58±8.29% to 55.02±10.54%, p<0.028), but this decrease was partially abrogated in CARDCzol. Both SSc and HC IL2C Vδ1+TC were highly reactive with native and OCH-bound CD1d tetramers (25–70%, n=5 and 50–54% n=2 respectively), but binding was suppressed in SSc samples by zol. In addition, blocking mAb against CD1d abolished the increased expression of CD25 in SSc CARDCzol Vδ1+ TC (from +27±3% to -8.9±2.3%, n=5 p<0.0251) but not in HC (n=2).ConclusionsCardiolipin induced suppression of CD25 expression on Vγ9+ TC in SSc, which could be reversed by zol. On the other hand cardiolipin reversed SSc- specific zol induced suppression of CD25 expression on Vδ1+TC in a CD1d dependent-manner. These results suggest that cardiolipin may suppress Vγ9+ TC while augmenting Vδ1+TC activation in SSc, at least in part via CD1d mediated interactions.ReferencesLuoma, A M.; et al.Immunity 39:6,1032, 2014Giacomelli, R et al Arth. and Rheum., 41(2), 327,1998.Yurovsky, VV et al., J of Immunol, 153(2), 881,1994.Marcu Malina, V et al., Frontiers in Immunol 5 AN:414, 2014.Schneiders FL, Cancer Immunol Res.;2(8),732,2014AcknowledgementSupported by a donation to I.B. via the Sheba Research AuthorityDisclosure of InterestNone declared