IsoPlexis (United States)

Modern dashboard design has revolutionized how organizations interact with and interpret their data across various platforms and devices. From initial visualization principles to security implementation and maintenance protocols, effective dashboard creation encompasses multiple critical elements that together form the foundation of successful business intelligence solutions. The interconnected aspects of intuitive navigation, responsive design, accessibility standards, and robust security measures create user-friendly interfaces that drive engagement and decision-making efficiency. By systematically implementing best practices in visual hierarchy, personalization features, and quality assurance processes, organizations can develop powerful tools that bridge the gap between complex data and actionable insights.

Current HIV vaccines designed to stimulate CD8+ T cells have failed to induce immunologic control upon infection. The functions of vaccine-induced HIV-specific CD8+ T cells were investigated here in detail. Cytotoxic capacity was significantly lower than in HIV controllers and was not a consequence of low frequency or unaccumulated functional cytotoxic proteins. Low cytotoxic capacity was attributable to impaired degranulation in response to the low antigen levels present on HIV-infected targets. The vaccine-induced T cell receptor (TCR) repertoire was polyclonal and transduction of these TCRs conferred the same reduced functions. These results define a mechanism accounting for poor antiviral activity induced by these vaccines and suggest that an effective CD8+ T cell response may require a vaccination strategy that drives further TCR clonal selection.

ObjectivesTo determine the prevalence and associations of autoantibodies targeting a muscle-specific autoantigen, four-and-a-half-LIM-domain 1 (FHL1), in South Australian patients with histologically-confirmed idiopathic inflammatory myopathies (IIM) and in patients with SSc.Material and methodsSera from patients with IIM (n = 267) from the South Australian Myositis Database (SAMD), SSc (n = 174) from the Australian Scleroderma Cohort Study (ASCS) and healthy controls (HC, n = 100) were analysed for anti-FHL1 autoantibodies by Enzyme-Linked ImmunoSorbent Assay (ELISA).ResultsAutoantibodies to FHL1 were more frequent in patients with IIM (37/267, 13.8%) compared with SSc (12/174, 7%) (P < 0.02) and HC (2/100, 2%) (P < 0.001). The most common IIM subtypes among FHL1+ IIM patients were (32%) and IBM (2/37, 32%). No statistically significant differences in muscular or extra-muscular manifestations of IIM were found when comparing patients who were anti-FHL1+ with their anti-FHL1– counterparts. In 29/37 (78%) anti-FHL1+ patients, no myositis-specific autoantibodies (MSA) were present. In FHL1+ muscle biopsies, there was less frequent infiltration by CD45+ cells (P = 0.04). There was a trend for HLA alleles DRB1*07 and DRB1*15 to be more frequent in anti-FHL1+ compared with anti-FHL1– patients (9/25 vs 19/113, P = 0.09 and 8/25 vs 15/114, P = 0.09, respectively).ConclusionsWe report a substantial prevalence (13.8%) of anti-FHL1 autoantibodies in a large cohort of patients with histologically confirmed IIM; 75% of these cases did not have a detectable myositis-specific autoantibody. Anti-FHL1 autoantibodies were also detected in a subgroup of patients with SSc (7%), indicating that anti-FHL1 autoantibodies may not be myositis-specific. The trend towards an HLA-DR association might indicate a specific immune response to the FHL1 protein.

Active co-delivery of tumor antigens (Ag) and α-galactosylceramide (α-GalCer), a potent agonist for invariant Natural Killer T (iNKT) cells, to cross-priming CD8α+ dendritic cells (DCs) was previously shown to promote strong anti-tumor responses in mice. Here, we designed a nanoparticle-based vaccine able to target human CD141+ (BDCA3+) DCs - the equivalent of murine CD8α+ DCs – and deliver both tumor Ag (Melan A) and α-GalCer. This nanovaccine was inoculated into humanized mice that mimic the human immune system (HIS) and possess functional iNKT cells and CD8+ T cells, called HIS-CD8/NKT mice. We found that multiple immunizations of HIS-CD8/NKT mice with the nanovaccine resulted in the activation and/or expansion of human CD141+ DCs and iNKT cells and ultimately elicited a potent Melan-A-specific CD8+ T cell response, as determined by tetramer staining and ELISpot assay. Single-cell proteomics further detailed the highly polyfunctional CD8+ T cells induced by the nanovaccine and revealed their predictive potential for vaccine potency. This finding demonstrates for the first time the unique ability of human iNKT cells to license cross-priming DCs in vivo and adds a new dimension to the current strategy of cancer vaccine development.

Abstract Introduction: GoCAR is a chimeric antigen receptor (CAR) platform that utilizes a rimiducid-dependent activation switch (inducible MyD88/CD40; iMC) to enhance CAR-T cell proliferation and to stimulate the host immune system. To understand how these unique CAR signaling molecules compare to conventional costimulatory domains (e.g., CD28 and 4-1BB), we employed a detection and bioinformatics platform to assess the functional attributes of CD19-specific CAR-T cells by simultaneous detection of 32 proteins secreted from single CAR-T cells upon various stimulation conditions. Methods: T cells were transduced with retrovirus encoding CD19-specific CARs with conventional costimulatory molecules (CD19.ζ, CD19.28.ζ or CD19.BB.ζ) or with GoCAR, a vector encoding iMC and a CD19-specific CAR (iMC-CD19.ζ). Control or modified T cells were enriched by anti-CD4 or anti-CD8 microbeads and stimulated with CD19 or ΔNGFR-expressing K562 cells with or without 10 nM rimiducid. After 24-hour, CD4 and CD8 T cells were collected and loaded into an IsoCode chip containing ~12,000 microchambers. Each chamber (~1.2 nL) was pre-patterned with a complete copy of a 32-plex antibody array. Cells on-chip were imaged, and single-cell cytokine signals were captured with a microarray scanner. The T cell polyfunctional profile (2+ cytokines per cell) of single T cells was evaluated across 5 functional groups: Effector: Granzyme B, TNFα, IFNγ, MIP1α, Perforin, TNFβ; Stimulatory: GM-CSF, IL-2, IL-5, IL-7, IL-8, IL-9, IL-12, IL-15, IL-21; Chemoattractive: CCL11, IP-10, MIP-1β, RANTES; Regulatory: IL-4, IL-10, IL-13, IL-22, sCD137, sCD40L, TGFβ1; Inflammatory: IL-6, IL-17A, IL-17F, MCP-1, MCP-4, IL-1β. Results: Single-cell multiplex proteomic analysis demonstrated the synergistic effects of CD19-specific and rimiducid-induced stimulation on polyfunctional upregulation in both CD4 and CD8 GoCAR-T cells across donors. In the “off” state (i.e., no rimiducid), CD19-specific GoCAR-T showed a reduced polyfunctional strength index (PSI) compared to CD28 and 4-1BB CAR-T cells, which was predominated by antitumor-associated proteins. In contrast, activation of iMC dramatically increased GoCAR-T PSI, which was characterized by elevated “stimulatory” (GM-CSF, IL-2, IL-5, IL-8, IL-9), “chemotactic” (MIP-1b) and “regulatory” (IL-4, sCD137) molecules compared to conventional CAR constructs. Polyfunctional heatmaps revealed that GoCAR-T cells co-secreted combinatorial proteins in response to CD19-expressing target cells in combination with iMC-based costimulation. Further, both CD4+ and CD8+ GoCAR-T cells acquired the ability to produce IL-2 as part of their polyfunctional profile, which was only observed with iMC costimulation. Conclusions: Single-cell multiplexed proteomic profiling reveals that polyfunctionality can be regulated by small molecule activation of an inducible costimulatory switch, producing distinct “on/off” states. iMC signaling also enhances production of important stimulatory and chemoattractive molecules which may improve CAR-T cell activity and provoke a host anti-tumor immune response. Thus, the GoCAR-T platform may potentially be used to optimize CAR-T activity through rimiducid administration to maximize efficacy while mitigating CAR-T-related toxicities. Citation Format: Aaron Foster, Joanne Shaw, Matthew Collinson-Pautz, Aruna Mahendravada, Christine Gagliardi, Mariam Khalil, Patrick Paczkowski, Sean Mackay, Jing Zhou. Single-cell multiplex proteomics reveals synergistic activity of antigen and MyD88/CD40 stimulatory signals on promoting polyfunctional chimeric antigen receptor T cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 898.