Peter J. Skene

Despite decades of HIV-1 vaccine development, the clonal and cellular determinants of durable vaccine-induced T cell memory remain incompletely understood. Here, we combined antigen- specific T cell receptor (TCR) identification, longitudinal TCR sequencing, and single-cell multi- omics to characterize Env- and Gag-specific memory precursor T cells elicited by the HIV Vaccine Trials Network (HVTN) 505 DNA prime–recombinant adenovirus serotype 5 (rAd5) boost (DNA/rAd5) vaccine regimen. We developed a generalizable high-throughput approach to identify HIV-1 Env- and Gag-specific TCRs and found distinct patterns of clonal expansion, persistence, and contribution to memory between Env- and Gag-specific CD8⁺ T cell responses. The DNA prime and rAd5 boost differentially shaped these repertoires, with rAd5-induced clones contributing proportionally more to the Gag-specific than to the Env-specific memory precursor compartment. Single-cell immune profiling further revealed distinct memory precursor states, with Env-specific responses enriched for GZMB ⁺ PRF1 ⁺ cytotoxic effector-memory (EM) CD8⁺ T cells and Gag-specific responses containing a larger cycling/proliferative population. Together, these findings demonstrate that heterologous DNA/rAd5 vaccination generates antigen-specific CD8⁺ T cell memory with distinct clonal trajectories and cellular programs, providing new insights into how vaccine platform and antigen-specificity shape the durability and functional properties of HIV-1 specific cellular immunity.

Lenalidomide (Revlimid™), bortezomib (Velcade™), and dexamethasone, are used alone or in combination (RVd therapy) as first-line therapies for hematologic malignancies including the plasma cell dyscrasia, Multiple Myeloma (MM). The effects of RVd treatment on tumor cells have been documented, but little is known about their impacts on “healthy” immune cells. New therapeutics, including chimeric antigen receptor T cell (CAR-T) and bispecific T-cell engagers (BiTEs) depend on robust T cell function but these are often harvested for cellular therapies after or in tandem with RVd therapy. Understanding the molecular effects of these drugs on healthy T cells is therefore of particular importance. Since the molecular effects of each drug includes regulation of key transcription factors, we used a tri-modal, single-cell assay (TEA-seq), simultaneously profiling mRNA transcripts, cell surface proteins, and chromatin accessibility in primary human peripheral blood T cells treated for 4, 24, or 72 hours in vitro . Synergies and conflicts potentially arising from combinatorial therapy were identified and validated by flow cytometry. Doublet and triplet drug combinations were used to assess effects on cell-surface marker expression and on T cell activation. Our results suggest that T cell function can be optimized by the administration of these drugs individually and that sequential administration may offer an opportunity to enhance T cell function by affecting localization and altering T cell states. This work provides those interested in immunology, oncology, and cell therapies an opportunity to explore molecular pathways that could be manipulated by these commonly used drugs to modify T cell function. One Sentence Summary: Primary human T cells treated with multiple myeloma RVd induction therapy drugs exhibit transcriptomic, epigenetic, and phenotypic changes that should be considered and potentially leveraged in the era of T cell-dependent treatments.

Peripheral blood mononuclear cells (PBMCs) are commonly used for immune profiling in clinical research, but their isolation and cryopreservation present logistical challenges, particularly in settings with limited access to research facilities. To address this, we implemented a whole blood fixation protocol to preserve surface protein epitopes and enable high‐dimensional flow cytometry without the need for immediate sample processing. A 36‐marker lymphoid and 35‐marker myeloid spectral flow cytometry panels were developed and optimized to characterize diverse immune populations, including T cell subsets, B cells, NK cells, monocytes, dendritic cells, and granulocytes. Panel design focused on antibody clone compatibility with fixed antigens, minimizing non‐specific binding, and reliably detecting chemokine receptors. Performance was evaluated by comparing fresh and fixed whole blood, revealing consistent identification of major immune lineages. Long‐term stability was demonstrated by using cryopreserved fixed blood from a healthy donor over two and half years, with immune profiles remaining stable. These findings support the use of fixed whole blood for high‐dimensional spectral cytometry and its utility in longitudinal clinical immune monitoring.

Antibody-secreting cells (ASCs) provide humoral immunity that can mediate lifelong protection against pathogens. Current classifications cannot delineate the heterogenous functionalities, tissue residencies, and lifespans of human ASC subsets, impeding clinical translation. We applied multi-omic sequencing, spatial proteomics, and functional assays to discover and characterize human bone marrow (BM) ASC subsets. We identified two peripheral subsets (ASCp) also present in blood and three BM-resident subsets (ASCr), comprising a maturation continuum associated with increased mitochondrial networking, diminished antibody secretion, differential transcription factor motif accessibility, and preferential co-localization in homotypic niches. CD19+9+ASCr and CD19-ASCr exhibited poor recovery years after BM transplantation, indicating a strong dependence on supportive niches. Childhood vaccine antigens were recognized by long-lived ASCr subsets in adults, as well as by immature HLA DR+ASCp, implying ASCs can differentiate without recent antigen exposure. Our results provide new insights into ASC identity, maturation, and longevity and a generalizable framework for study and manipulation of human ASCs.

Summary: Infections are the most common cause of non-relapse mortality in multiple myeloma (MM), but the basis of persistent immune dysfunction is obscured by patient heterogeneity and complex treatment regimens, including autologous stem cell transplant (ASCT). We performed longitudinal multi-omic profiling of matched bone marrow and peripheral blood from MM patients across diagnosis, induction, ASCT, and recovery. We found the tumor imposes a compartment-specific immune program where the marrow exhibits metabolic and inflammatory changes that bias hematopoiesis and alter cytotoxic effector programs not mirrored in blood. Adaptive immune reconstitution is impaired up to two years post-ASCT. Half of patients fail to mount IgG responses to high-dose non-adjuvanted influenza vaccine, a defect overcome by the lipid nanoparticle (LNP) adjuvanted COVID mRNA vaccine, which elicited responses in all patients, supporting adjuvanted influenza vaccine strategies in MM. Together these findings define how myeloma and its treatment durably reshape immunity from the marrow outward. Highlights: Multiple Myeloma marrow and blood show opposing metabolic and inflammatory states Induction therapy selects durable myeloma plasma-cell transcriptional states B cell and follicular helper T deficits blunt antigen responses after transplant COVID-19 vaccination builds immune memory with variable responses to flu vaccination eTOC: Multiple myeloma and its treatment leave a lasting imprint on the bone marrow niche. By profiling bone marrow and blood longitudinally at diagnosis, through induction, autologous transplant, and recovery, we show that marrow-local metabolic and inflammatory constraints persist and help explain why influenza vaccination often fails while mRNA vaccination succeeds.

Profiling the vast diversity of the T-cell repertoire at single-cell resolution is challenging due to the scale and cost requirements involved. Current technologies capture TCR or perform single-cell sequencing at scale but struggle to combine both. The following protocol describes the steps taken to perform REFLEX TCR on the 10x Genomics, GEM-X v1 platform, using cryopreserved human PBMCs as a starting point. With it, one can expect to generate multi-omic profiling inclusive of FLEX transcriptome, paired alpha and beta TCR (paired efficiency varies with sample context), and protein level information through ADTs if desired.

Type 2 inflammation at barrier surfaces is an evolutionarily conserved response that promotes immunity to helminth parasites, allergic inflammation and tissue repair^1,2,3,4. Direct sensing of environmental triggers by epithelial cells initiates type 2 inflammation, and signals derived from neurons can modulate immune responses^5,6,7,8. However, how diverse sensory inputs from epithelial, neuronal and immune cells are coordinated and integrated remains unclear. Here we identify that TRPV1⁺ pain-sensing nociceptors co-opt chemosensory epithelial tuft cells to initiate a cascade of tissue responses that drive type 2 inflammation. Chemogenetic silencing or chemical ablation of TRPV1⁺ nociceptors results in a significant reduction in intestinal tuft cells and defective anti-helminth type 2 immunity. By contrast, chemogenetic activation of TRPV1⁺ nociceptors leads to remodelling of CGRP⁺ nerve fibres, significantly increased CGRP expression, enhanced tuft cell accumulation and protective anti-helminth type 2 immunity. Using spatial transcriptomic and single-cell RNA sequencing analyses, we reveal that nociceptor activation promotes rapid epithelial progenitor cell proliferation and differentiation. Mechanistically, intestinal epithelial cell-intrinsic and tuft cell-intrinsic expression of CGRP receptor subunits are required for tuft cell responses and type 2 immunity to helminth infection. Together, these results identify sensory convergence of a neuronal–epithelial tuft cell circuit as a critical upstream determinant of type 2 immunity and tissue adaptation.

Interferons (IFNs) orchestrate diverse immune responses, but distinguishing individual IFN contributions in human transcriptomic data is challenging due to overlapping interferon-stimulated gene (ISG) signatures and limited cell-type-specific datasets. To address this, we generated a single-cell transcriptomic atlas of IFN responses by stimulating primary human T, B, NK, and CD14 monocytes with IFN-I, IFN-II, and IFN-III. This revealed core and cell-type-specific ISG programs across 13 subsets, highlighting distinct functions of IFNs. We developed an algorithm to separate IFN-I and IFN-II activity in transcriptomic data. Applied to multiple myeloma samples, it showed elevated IFN-I and IFN-II responses, with induction therapy reducing only IFN-I. Extending to multiple disease datasets provided a cross-disease overview of IFN-I and IFN-II activities and revealed increased IFN-II activities in T cells during lupus flares. This resource and the accompanying analytical framework enable dissection of IFN-driven transcriptional programs in a cell-type specific manner in human disease.

Single-cell profiling of T cell state with immune repertoire is critical for understanding heterogenous T cell phenotypes and responses to antigen, however, existing technologies struggle to generate this information at sufficient throughput to match biological complexity. We present “REFLEX”, a novel single-cell method enabling highly scalable, cost-efficient, multiomic profiling with paired-chain TCR sequencing. REFLEX utilizes in-situ reverse transcription with integrated sample multiplexing barcodes in a way that merges seamlessly with the commonly used 10x FLEX platform to allow capture of TCR sequences at unprecedented scale and depth. We profile >2 million cells from CMV-peptide-pulsed T cell expansions, capturing TCR sequences and rich multiomic information from 1.4M T cells, identifying many putative novel CMV reactive clonotypes and illustrating the scale and transformative impact on our understanding of T cell mediated adaptive immunity achievable with REFLEX.