T stem cell-like memory cells (TSCM cells) are considered to be essential for the maintenance of immune memory. The TSCM population has been shown to have the key properties of a stem cell population: multipotency, self-renewal and clonal longevity. Here we show that no single population has all these stem cell properties, instead the properties are distributed. We show that the human TSCM population consists of two distinct cell subpopulations which can be distinguished by the level of their CD95 expression (CD95int and CD95hi). Crucially, using long-term in vivo labelling of human volunteers, we establish that these are distinct populations rather than transient states of the same population. These two subpopulations have different functional profiles ex vivo, different transcriptional patterns, and different tissue distributions. They also have significantly different TREC content indicating different division histories and we find that the frequency of CD95hi TSCM increases with age. Most importantly, CD95hi and CD95int TSCM cells also have very different dynamics in vivo with CD95hi cells showing considerably higher proliferation but significantly reduced clonal longevity compared with CD95int TSCM. While both TSCM subpopulations exhibit considerable multipotency, no single population of TSCM cells has both the properties of self-renewal and clonal longevity. Instead, the "stemness" of the TSCM population is generated by the complementary dynamic properties of the two subpopulations: CD95int TSCM which have the property of clonal longevity and CD95hi TSCM which have the properties of expansion and self-renewal. We suggest that together, these two populations function as a stem cell population.

CAR-T cell therapy has revolutionized cancer treatment, with approvals for conditions like acute B-leukemia, large B cell lymphoma (LBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), and multiple myeloma. However, its high costs limit accessibility. Key factors driving these costs include the need for personalized, autologous treatments, transportation to specialized facilities, reliance on viral vectors requiring advanced laboratories, and lengthy cell expansion processes. To address these challenges, alternative strategies aim to simplify and reduce production complexity. Non-viral vectors, such as Sleeping Beauty, piggyBac, and CRISPR, delivered via nanoparticles or electroporation, present promising solutions. These methods could streamline manufacturing, eliminate the need for viral vectors, and reduce associated costs. Furthermore, shortening cell expansion periods and optimizing protocols could significantly accelerate production. An emerging approach involves using genetically edited T cells from healthy donors to create universal CAR-T products capable of treating multiple patients. Finally, decentralized point-of-care (POC) manufacturing of CAR-T cells minimize logistical expenses, eliminating the need for complex infrastructure, and enabling localized production closer to patients. This innovative strategy holds potential for broadening access and reducing costs, representing a step toward democratizing CAR-T therapy. Combined, these advances could make this groundbreaking treatment more feasible for healthcare systems worldwide.

Recently, mRNA/lipid nanoparticle (LNP)-based vaccines have been successfully applied to prevent infectious diseases, and several types of neoantigen-encoding mRNA cancer vaccines are currently under clinical trials. While mRNA vaccines effectively induce adaptive immune responses to antigens, mRNA vaccine-induced immunity is shortly maintained, and the longevity of the immune memory, especially improving the CD8+ T cell memory potential, could be even more important. Previously, microbiome metabolites have shown T cell memory potential-augmenting effects via regulating the immunometabolism. Herein, we develop microbiome metabolite-incorporated LNPs (mmi-LNPs) and evaluate their potential to enhance T cell memory responses following mRNA vaccination. In various ionizable LNP formulations, mmi-LNPs elicited more stem cell-like memory T cells (T-SCMs) and augmented central and effector memory T cell responses, which indicates the general applicability of mmi-LNPs. Notably, butyrate-incorporated mmi-LNP exhibited the strongest effects. In conclusion, we suggest microbiome metabolite-incorporated LNP as a next-generation delivery vehicle for mRNA vaccines.

Chimeric antigen receptor (CAR) T-cell therapy represents a breakthrough in the treatment of B-cell malignancies. CAR-T cells infusion generally follows a chemotherapy regimen whose lymphodepleting properties create a favorable environment for the expansion of engineered T cells. While this process appears straightforward, emerging evidence reveals that complex mechanisms, collectively representing immune dynamics following CAR-T cell infusion, influence CAR-T cells behavior. In advance of infusion, a final-product enriched with less stressed CAR-T cells can improve their expansion and persistence, providing a biological rationale for early apheresis and administration. Following infusion, the emergence of dysfunctional CAR-T subpopulations, like regulatory or NK-like CAR-T cells, can impair efficacy. The recovery of non-CAR transduced T cells adds further complexity, as these cells could either impact outcomes or exacerbate complications, such as infections or prolonged cytopenia. In this review, we summarize the latest advances in understanding the immune dynamics following CAR-T cell infusion for large B-cell lymphomas, with a focus on both CAR-engineered and native T cell populations, and their impact on treatment efficacy and patient outcomes.

Although T-cell immunotherapies have been effective in the treatment of hematological malignancies, solid tumors have proven challenging due to the immunosuppressive microenvironment and lack of viable target antigens. The immune checkpoint ligand CD70, overexpressed in several solid tumors, yet with limited expression in healthy tissue, has emerged as a promising immunotherapeutic target.

This study describes the generation and preclinical characterization of ADP-520, a high-affinity, fratricide-resistant, CD70-targeted T-cell receptor fusion construct (TRuC) T-cell therapy enhanced with constitutively expressed mbIL-15, a membrane-bound fusion protein comprising interleukin-15 (IL-15) linked to full-length IL-15 receptor-alpha. The phenotypic distribution, expansion and persistence of ADP-520 TRuC T cells were measured in vitro under autonomous and antigen-dependent conditions, with the contributions of TCR and IL-15 signaling pathways ascertained using inhibition assays. Chronic antigen stimulation was used to evaluate exhaustion-resistance, while anti-tumor potency was explored both in vitro and in vivo.

ADP-520 was found to have potent and antigen-specific activity against hematological and solid CD70-expressing tumors, without apparent fratricide or killing of bystander T cells despite CD70 expression by activated lymphocytes. Engineered co-expression of mbIL-15 augmented antigen-dependent expansion through pro-survival effects and enrichment of an early memory T-cell phenotype, thus enhancing tumor-autonomous, exogenous cytokine-free persistence and bolstering exhaustion resistance during chronic stimulation. mbIL-15 co-expression also enhanced intratumoral T-cell infiltration in vivo for potent and persistent antitumor efficacy.

These findings characterize ADP-520 as a first-in-class, CD70-targeted, fratricide-resistant autologous TRuC T-cell therapy leveraging native TCR signaling combined with constitutive IL-15 signaling to impart T cells with enhanced persistence, tumor penetration, and antitumor efficacy. This makes ADP-520 a promising cell immunotherapy candidate for clinical development, with the potential to overcome hurdles intrinsic to the treatment of solid tumors.

We conducted a randomized, placebo-controlled trial to assess the safety and biological age (BA) effects of various therapeutic plasma exchange (TPE) regimens in healthy adults over 50. Participants received bi-weekly TPE with or without intravenous immunoglobulin (IVIG), monthly TPE, or placebo. Randomization was based on entry date, and treatments were blinded to maintain objectivity. Primary objectives were to assess long-term TPE safety and changes in biological clocks. Secondary goals included identifying optimal regimens. Exploratory analyses profiled baseline clinical features and longitudinal changes across the epigenome, proteome, metabolome, glycome, immune cytokines, iAge, and immune cell composition. We demonstrate in 42 individuals randomized to various treatment arms or placebo that long-term TPE was found to be safe, with only two adverse events requiring discontinuation and one related to IVIG. TPE significantly improved biological age markers, with 15 epigenetic clocks showing rejuvenation compared to placebo (FDR < 0.05). Biweekly TPE combined with intravenous immunoglobulin (TPE-IVIG) proved most effective, inducing coordinated cellular and molecular responses, reversing age-related immune decline, and modulating proteins linked to chronic inflammation. Integrative analysis identified baseline biomarkers predictive of positive outcomes, suggesting TPE-IVIG is particularly beneficial for individuals with poorer initial health status. This is the first multi-omics study to examine various TPE modalities to slow epigenetic biologic clocks, which demonstrate biological age rejuvenation and the molecular features associated with this rejuvenation. Trial Registration: Registered trial NCT06534450 on clinicaltrials.gov under the purview of the Diagnostic Investigational Review Board.

Th17 lymphocytes are a distinct subpopulation of T cells that are characterized by the production of interleukins IL-17, IL-21, IL-22, and IL-26, and high expression of RORγt. These cells play an important role in inflammation and autoimmune diseases. Recent studies using rodent and human models have also highlighted their promising properties as agents in cellular immunotherapy for cancer. However, much less is known about the properties of canine Th17 lymphocytes, despite the domestic dog being an important model used in comparative medicine. In this study, we developed methods of activation and differentiation of canine CD4+ T lymphocytes towards the Th17 phenotype. Additionally, we targeted the Wnt/β-catenin signaling pathway to modulate the efficiency of Th17 cells differentiation. CD4+ T cells were successfully activated with magnetic EpoxyBeads, and in combination with the appropriate programming medium, they acquired the Th17 phenotype. Furthermore, indomethacin, an inhibitor of the Wnt/β-catenin pathway, significantly increased the efficiency of differentiation, causing elevated production of IL-17 and changed T cell metabolism by promoting oxidative phosphorylation. The protocol elaborated in our study provides an efficient method of canine Th17 lymphocyte differentiation. Our findings also suggested that the modification of the Wnt/β-catenin signaling pathway could be a valuable strategy for optimizing canine Th17 cell differentiation and advancing cell-based immunotherapy.

Long-lived memory CD8+ T cells are essential for the control of persistent viral infections. The mechanisms that preserve memory cells are poorly understood. Fate mapping of the transcriptional repressor GFI1 identified that GFI1 was differentially regulated in virus-specific CD8+ T cells and was selectively expressed in stem cell memory and central memory cells. Deletion of GFI1 led to reduced proliferation and progressive loss of memory T cells, which in turn resulted in failure to maintain antigen-specific CD8+ T cell populations following infection with chronic lymphocytic choriomeningitis virus or murine cytomegalovirus. Ablation of GFI1 resulted in downregulation of the transcription factors EOMES and BCL-2 in memory CD8+ T cells. Ectopic expression of EOMES rescued the expression of BCL-2, but the persistence of memory CD8+ T cells was only partially rescued. These findings highlight the critical role of GFI1 in the long-term maintenance of memory CD8+ T cells in persistent infections by sustaining their proliferative potential.

CD95L is a transmembrane cytokine mainly expressed by activated T and natural killer cells to contract the immune response through cell-cell contact. Conversely, after cleavage by metalloproteases, this ligand releases a soluble CD95L (sCD95L) that stimulates the immune response and its antitumor activity. In posttransplant lymphoproliferative disorders (PTLDs), we hypothesized that the concentration of sCD95L could exert a biological function and affect clinical outcomes by modulating the immune response. Using the K-VIROGREF biobank, we quantified sCD95L in 163 patients with PTLD, 16 transplant controls, and 28 healthy donors. Transplant recipients had higher plasma levels of sCD95L than healthy donors. More interestingly, patients with PTLD and high concentration of sCD95L had better clinical outcomes than patients with lower concentration, particularly those with central nervous system (CNS) involvement, who are known to have poor survival. At the cellular level, only natural killer and natural killer T-like cell fractions were reduced in the blood of patients with CNS-PTLD and high concentration of sCD95L, suggesting that sCD95L may either promote the trafficking of these cells within tumors or modulate their differentiation/survival. In conclusion, we showed in this exploratory analysis that plasma concentration of sCD95L might be a prognostic marker in patients with PTLD, particularly in those with CNS involvement.

Developing vaccines that promote CD8+ T cell memory is a challenge for infectious disease and cancer immunotherapy. TCF-1+ stem cell-like memory CD8+ T (TSCM) cells are important determinants of long-lived memory. Yet, the developmental requirements for TSCM cell formation are unclear. Here, we identify the temporal window for type I interferon receptor (IFNAR) blockade to drive TSCM cell generation following viral infection and mRNA-lipid nanoparticle vaccination. We reveal a reversible developmental trajectory where transcriptionally distinct TSCM cells emerged from a transitional precursor of exhausted T cellular state concomitant with viral clearance. TSCM cell differentiation correlated with T cell retention within the lymph node paracortex due to disrupted CXCR3 chemokine gradient formation. These effects were linked to increased antigen load and a counterintuitive increase in IFNγ, which controlled cell location. Vaccination with the IFNAR blockade promoted TSCM cell differentiation and enhanced protection against chronic infection. These findings propose an approach to vaccine design whereby modulation of inflammation promotes memory formation and function.

Although transcatheter arterial chemoembolization (TACE) is one of the first-line treatments for unresectable HCC (uHCC) patients, its overall efficacy varies significantly. Therefore, the identification of reliable biomarkers capable of effectively distinguishing TACE-responsive populations is clinically critical.

Our research aims to investigate T-lymphocyte subpopulations and associated pathways in peripheral blood that contribute to TACE refractoriness, as well as to develop effective methods for predicting TACE efficacy.

This is an observational study.

A total of 50 patients who underwent standard TACE-based therapy between January 2020 and December 2022 were included in this study. TACE response was evaluated within 1-3 months following two consecutive TACE sessions. Patients with TACE failure were assigned to the Non-Response group, whereas the remaining were categorized into the Response group. Blood samples were collected prior to treatment and subsequently analyzed using flow cytometry and RNA sequencing. Predictors were analyzed using univariate and multivariate analyses within the bivariate logistic regression models. Pathway enrichment analysis was performed using gene set enrichment analysis (GSEA).

A total of 24 of 50 (48%) exhibited TACE failure (Non-Response). Baseline peripheral T-lymphocyte analysis revealed that the Non-Response group had a higher abundance of senescent phenotype (TSenescence, CD27-CD28-) in both CD4/CD8+ T cells (p < 0.0001), but a lower proportion of memory stem cell (TSCM) subpopulation (CD4+ TSCM: p = 0.0411; CD8+ TSCM: p < 0.0001). Furthermore, in CD8+ T cells, they exhibited higher expression of exhaustion marks (PD-1: p = 0.0005; LAG-3: p = 0.0026; TIGIT: p = 0.0014) and significantly lower production of effector molecules (TNF-α: p < 0.0001; IFN-γ: p = 0.0018; GZMB: p < 0.0001). Transcriptomics revealed that the Response group was enriched in pathways associated with energy and drug metabolism. Univariate and multivariate analyses demonstrated that the baseline CD8+ TSCM and CD8+ TSenescence subpopulations were significant predictive factors for TACE efficacy.

Our study demonstrated significant differences in the immune characteristics of peripheral T lymphocytes between the Non-Response and Response groups. The CD8+ TSCM and CD8+ TSenescence subsets are potential predictors of TACE efficacy and long-term survival. These insights into peripheral blood T lymphocytes offer valuable evidence to help clinicians more effectively identify potential TACE-responsive populations, predict survival, and develop personalized treatment regimens for patients with uHCC.

T cell responses are rarely measured in large-scale human vaccine studies due to the sample volumes required, as well as the logistical, technical, and financial challenges associated with available assays. Fluorescent cell barcoding has been proposed in other contexts to allow for more high-throughput flow cytometry-based assays. Here, we aimed to expand on existing barcoding approaches to develop a reagent and sample-sparing assay for in-depth assessment of T cell responses to vaccine antigens. By using various concentrations of two fixable viability dyes in a matrix format, up to 25 samples that were pooled and acquired together could be successfully deconvoluted based on their unique fluorescent signature. This fluorescent cell barcoding approach was then combined with extracellular and intracellular staining to identify functional (i.e., producing at least one cytokine) and polyfunctional (i.e., producing multiple cytokines) T cells in response to vaccine antigen stimulation. As a proof-of-concept, we plated just 200,000 peripheral blood mononuclear cells (PBMC) per condition, and by staining and acquiring only two pooled samples, we were able to detect rare antigen-specific T cell responses in eight donors to four stimulants each. The frequencies of antigen-induced cytokine-positive cells detected in barcoded samples with 200,000 input PBMC were strongly correlated with those detected in non-barcoded samples from the same donors with 1 million input PBMC, demonstrating the validity of this approach. In conclusion, by reducing the number of PBMC needed by five-fold, and the volume of staining reagents needed by 25-fold, this assay has widespread potential applications to human vaccine studies.

The use of deuterated water (also known as 'heavy water') as a tracer to measure human in vivo cell proliferation rates for specific cell subsets has expanded significantly in recent years. Although there have been several published methods papers, investigators developing new applications may be confused by differences in study design and deuterated water dose/duration. Furthermore, this approach may be met with regulatory difficulties and participant concerns about toxicity. This scoping review explores lessons that can be learnt from the current literature on the use of deuterated water in human in vivo studies measuring cell proliferation. We identified 29 such studies involving 535 study participants, both healthy volunteers and those with specific clinical conditions. Wide variations in protocols were noted with doses ranging from 40-100 ml/day of pure deuterated water (or equivalent) and durations from 4-12 weeks. Study design usually reflected the kinetics of the cell of interest. No clinical toxicity signals were noted in any studies although four studies did report transient dizziness, a recognized symptom of changing water density. These published studies provide a strong safety signal for potential participants and regulatory authorities and can act as templates for the development of new research applications.

CD30-directed chimeric antigen receptor T-cell therapy (CART30) has limited efficacy in relapsed or refractory patients with CD30+ lymphoma, with a low proportion of durable responses. We have developed an academic CART30 cell product (HSP-CAR30) by combining strategies to improve performance. HSP-CAR30 targets a proximal epitope within the nonsoluble part of CD30, and the manufacturing process includes a modulation of ex vivo T-cell activation, as well as the addition of interleukin-21 (IL-21) to IL-7 and IL-15 to promote stemness of T cells. We translated HSP-CAR30 to a phase 1 clinical trial of 10 patients with relapsed/refractory classic Hodgkin lymphoma (HL) or CD30+ T-cell non-Hodgkin lymphoma. HSP-CAR30 was mainly composed of memory stem-like (TSCM-like) and central memory (TCM) CAR30+ T cells (87.5% ± 5%). No dose-limiting toxicities were detected. Six patients had grade 1 cytokine release syndrome, and no patient developed neurotoxicity. The overall response rate was 100%, and 5 of 8 patients with HL achieved complete remission (CR). An additional patient with HL achieved CR after a second HSP-CAR30 infusion. Remarkably, 60% of patients have ongoing CR after a mean follow-up of 34 months. CAR30+ T cells at expansion peak had a predominance of TSCM and TCM cells, and CAR30+ T cells remained detectable in 3 of 5 evaluable patients at least 12 months after infusion. Our study shows that selection of the epitope targeting CD30 and ex vivo preservation of less-differentiated memory T cells may enhance the efficacy of CART30 in patients with refractory HL. This trial is registered at www.clinicaltrials.gov (NCT04653649).

Many patients with cancer do not benefit from currently approved immune checkpoint inhibitors (ICIs), suggesting that additional immunomodulation of the immunosuppressive tumor microenvironment (TME) is required. MTL-CCAAT enhancer-binding protein alpha (CEBPA) specifically upregulates the expression of the master myeloid transcription factor, CEBPA, relieving myeloid-driven immunosuppression. Here, we report the safety, tolerability, pharmacokinetics, and efficacy of MTL-CEBPA in combination with pembrolizumab in patients with advanced solid tumors that typically show ICI resistance. Multimodal exploratory analyses of paired patient biopsies demonstrate biological changes associated with the combination treatment of MTL-CEBPA and pembrolizumab, including increased infiltration of T cell and antigen-presenting cells supporting conversion from an immune-desert toward a more immune-inflamed TME. Patients with disease stabilization demonstrate reductions in immunosuppressive myeloid cells post treatment. Collectively, these data support a role for MTL-CEBPA in reducing immunosuppression in the TME. This study was registered at ClinicalTrials.gov (NCT04105335).

Tissue-resident memory (TRM) T cells play an important role in protection against respiratory infection but whether this memory is maintained by long-lived or dividing cells remains controversial. To address the rate of division of lung TRM T cells, deuterium-enriched water was administered orally to young pigs to label dividing lymphocytes. T-cell subsets were separated from blood, lymph nodes, and airways [bronchoalveolar lavage (BAL)], the latter comprising almost exclusively TRM. We show that, as in other species, circulating memory T-cell subsets divide more rapidly than naïve T cells. Rates of labelling of memory subsets were similar in blood and lymph nodes, consistent with the rapid and free exchange. Strikingly, the fraction of label in BAL was similar to those in blood/lymph nodes after 5-21 days of labelling, suggesting replacement with recently divided cells, but this was preceded at Day 2 by a phase when labelling was lower in BAL than blood/lymph node in some memory subsets. Our data exclude long-lived TRM as the source of BAL memory cells leaving three possible hypotheses: blood/airway exchange, in situ proliferation, or proliferation in the lung interstitium followed by migration to BAL. When considered in the context of other information, we favour the latter interpretation. These results indicate the dynamic nature of memory in the lung and have implications for harnessing immune responses against respiratory pathogens.

An estimated 32 million people live with HIV-1 globally. Combined antiretroviral therapy suppresses viral replication but therapy interruption results in viral rebound from a latent reservoir mainly found in memory CD4+ T cells. Treatment is therefore lifelong and not curative. Eradication of this viral reservoir requires hematopoietic stem cell transplantation from hemizygous or homozygous ΔCCR5 donors, which is not broadly applicable. Alternative cure strategies include the pharmacological reactivation of latently infected cells to promote their immune-mediated clearance, or the induction of deep latency. HIV-1 latency is multifactorial and linked to the activation status of the infected CD4+ T cell. Hence to perturb latency, multiple pathways need to be simultaneously targeted without affecting CD4+ T cell function. Hsp90 has been shown to regulate HIV-1 latency, although knowledge on the pathways is limited. Because Hsp90 promotes the proper folding of numerous cellular proteins required for HIV-1 gene expression, we hypothesized that Hsp90 might be a master regulator of latency. We tested this hypothesis using a polyclonal Jurkat cell model of latency and ex-vivo latently infected primary CD4+ T cells. We found that, in the Jurkat model, Hsp90 is required for HIV-1 reactivation mediated by the T-cell receptor, phorbol esters, TNF-α, inhibition of FOXO-1, and agonists of TLR-7 and TLR-8. In primary cells, Hsp90 regulates HIV-1 gene expression induced by stimulation of the T-cell receptor or in the presence of IL-7/IL-15 or a FOXO-1 inhibitor. Chemical inhibition of Hsp90 abrogated activation of the NF-kB, NFAT and AP-1 signal transduction pathways. Within the CD4+ T cell population, CDRA45+ CCR7+ "naïve" and CD45RA- CCR7- "effector memory" cells were most sensitive to Hsp90 inhibition, which did not perturb their phenotype or activation state. Our results indicate that Hsp90 is a master regulator of HIV-1 latency that can potentially be targeted in cure strategies.

Tumor heterogeneity and an immunosuppressive microenvironment pose significant challenges for immunotherapy against solid tumors, particularly glioblastoma multiforme (GBM). Recent studies have highlighted the crucial role of glioma stem cells (GSCs) in tumor recurrence and therapeutic resistance. In this context, we developed a tandem chimeric antigen receptor (CAR)-T cell targeting CD44 and CD133 (PROM1), containing a truncated IL-7 receptor alpha intracellular domain (Δ7R) between the CD28 costimulatory receptor and the CD3ζ signaling chain (Tanζ-T28-Δ7R). Our target identification and validation were carried out using GSCs, samples from GBM patients, and the corresponding sequencing data. The antitumor efficacy of CAR-T cells was evaluated in patient-derived GSCs, intracranial xenograft models, patient-derived xenograft models, and glioblastoma organoids (GBOs). Single-cell RNA sequencing and mass cytometry were used to determine the immune phenotypes of CAR-T cells. We showed that locoregionally administered Tanζ-T28-Δ7R CAR-T cells induced long-term tumor regression with the desired safety outcomes. Patient-derived autologous Tanζ-T28-Δ7R CAR-T cells showed robust antitumor activity against GBOs. Our pre-clinical data has demonstrated the translational potential of Tanζ-T28-Δ7R CAR-T cell against GBM.

Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by the loss of insulin-producing cells in the pancreatic islets. Patients with T1D have autoreactive CD4+ and CD8+ T cells that show specific features, indicating previous exposure to self-antigens. Despite that memory T cells are vital components of the adaptive immune system, providing enduring protection against pathogens; individuals with T1D have a higher proportion of memory T cells compared to healthy individuals with naїve phenotypes. Targeting memory T cells in newly diagnosed T1D patients has shown promising results, providing evidence for the significant role of memory T cells in this disease. There are various types of memory T cells, each with unique characteristics and functions. Recent advancements in understanding the complexity and heterogeneity of T cell subpopulations have shown that T1D cannot be fully understood through simple categorization. This review aims to discuss various types of memory T cells in the immunopathogenesis of T1D, focusing on their phenotypes and frequencies, as well as epigenetic and metabolic alterations. Additionally, it will address novel immunotherapeutic approaches targeting memory T cell subsets in T1D.

Myasthenia Gravis (MG) is a heterogeneous autoimmune disorder characterized by fluctuating muscle weakness caused by autoantibodies targeting neuromuscular junction components. While the role of CD4 + T cells in MG is well established, the contribution of CD8 + T cells remains poorly understood. In this study, we analyze CD8 + T cells in 36 MG patients and 38 age- and gender-matched controls using flow cytometry to evaluate subset distribution, granzyme expression, and cytokine production. MG patients exhibit an altered CD4 + /CD8 + T cell ratio and significant changes in CD8 + T cell subsets, including increased central memory CD8 + T cell (Tcm) proportions and decreased effector memory CD8 + T cell (Tem) proportions. Granzyme B expression in Tcm cells is significantly elevated in MG patients, whereas no significant changes are observed in other subsets or GZMK expression. Cytokine analysis reveals increased IL-21, GM-CSF, and IL-17A production by CD8 + T cells in MG patients. These phenotypic and functional alterations of CD8 + T cells persist during the acute phase of the disease, regardless of immunotherapy usage, and vary between ocular and generalized MG. Subgroup and correlation analyses further identify age-dependent and age-independent dysregulations of CD8 + T cells, indicating complex and subtype-specific roles of CD8 + T cells in the immunopathological processes underlying MG. Our findings provide novel insights into the involvement of CD8 + T cells in MG pathogenesis, laying a foundation for future research and potential therapeutic strategies targeting CD8 + T cells.

One of the most successful treatments in hematologic cancer is chimeric antigen receptor (CAR)-T cell-based immunotherapy. However, CAR-T therapy is not without challenges like the costly manufacturing process required to personalize each treatment for individual patients or graft-versus-host disease. Umbilical cord blood (UCB) has been most commonly used for hematopoietic cell transplant as it offers several advantages, including its rich source of hematopoietic stem cells, lower risk of graft-versus-host disease, and easier matching for recipients due to less stringent HLA requirements compared to bone marrow or peripheral blood stem cells. In this review, we have discussed the advantages and disadvantages of different CAR-T cell manufacturing strategies with the use of allogeneic and autologous peripheral blood cells. We compare them to the UCB approach and discuss ongoing pre-clinical and clinical trials in the field. Finally, we propose a cord blood bank as a readily available source of CAR-T cells.

The recurrence of inflammatory skin diseases represents a significant challenge in clinical practice, primarily mediated by immune memory. In inflammatory skin diseases, immune memory encompasses adaptive immune memory, trained immunity, and inflammatory memory, which are conducted by adaptive immune cells, innate immune cells, and structural cells, respectively. Adaptive immune memory is established through gene rearrangement, leading to antigen-specific immune memory. In contrast, trained immunity and inflammatory memory are formed through epigenetic and metabolic reprogramming, resulting in non-specific immune memory. Different types of immune memory work synergistically to aggravate localized inflammation in recurrent inflammatory skin diseases. However, immune memory in specific cells, such as macrophages, may also play an immunoregulatory role under certain conditions. We reviewed the immune memory mechanisms in different inflammatory skin diseases and discussed future strategies for targeted regulation of the molecular mechanisms underlying immune memory, such as targeted biological agents and epigenetic modifications. Additionally, we explored the potential for precise regulation of immune memory and its application in personalized treatment for recurrent inflammatory skin diseases.

Chimeric antigen receptor T cell (CAR-T) therapy has shown success in treating hematological malignancies, but its effectiveness against solid tumors is hindered by T cell exhaustion. During in vitro expansion, tonic signaling induced by CAR expression contributes to CAR-T cell exhaustion, which can be mitigated by inhibiting calcium signaling. Given that sodium citrate can chelate calcium ions and inhibit calcium signaling, in this study, we investigated whether sodium citrate could reduce exhaustion and enhance CAR-T cell function.

We constructed anti-CD70 CAR-T cells and cultured them in the presence of sodium citrate. The characteristics and functionality of sodium citrate-pretreated CAR-T cells were assessed through in vitro and in vivo experiments. To further validate our observation, we also treated anti-mesothelin (MSLN) CAR-T cells with sodium citrate and detected the phenotypes and anti-tumor function of CAR-T cells.

We found that sodium citrate-pretreated anti-CD70 CAR-T cells exhibited reduced exhaustion, increased memory T cell proportions, and enhanced anti-tumor efficacy both in vitro and in vivo. Notably, sodium citrate treatment improved the in vivo persistence of CAR-T cells and prevented tumor recurrence. These beneficial effects were also observed in anti-MSLN CAR-T cells. Transcriptomic and metabolite analyses revealed that sodium citrate inhibited calcium signaling, mTORC1 activity, and glycolysis pathways, thus modulating T cell exhaustion and differentiation.

Our findings suggest that sodium citrate supplementation during CAR-T cell expansion could be a promising strategy to improve CAR-T therapy for solid tumors by preventing exhaustion and promoting memory T cell formation.

While most people contain Mycobacterium tuberculosis infection, some individuals develop active disease, usually within two years of infection. Why immunity fails after initially controlling infection is unknown. C57BL/6 mice control Mycobacterium tuberculosis for up to a year but ultimately succumb to disease. We hypothesize that the development of CD4 T cell dysfunction permits bacterial recrudescence. We developed a reductionist model to assess antigen-specific T cells during chronic infection and found evidence of CD4 T cell senescence and exhaustion. In C57BL/6 mice, CD4 T cells upregulate coinhibitory receptors and lose effector cytokine production. Single cell RNAseq shows that only a small number of CD4 T cells in the lungs of chronically infected mice are polyfunctional. While the origin and causal relationship between T-cell dysfunction and recrudescence remains uncertain, we propose T cell dysfunction leads to a feed-forward loop that causes increased bacillary numbers, greater T cell dysfunction, and progressive disease.

Chimeric antigen receptor (CAR)-T cell therapy represents a breakthrough for the treatment of hematological malignancies. However, to treat solid tumors and certain hematologic cancers, next-generation CAR-T cells require further genetic modifications to overcome some of the current limitations. Improving manufacturing processes to preserve cell health and function of edited T cells is equally critical. Here, we report that Solupore, a Good Manufacturing Practice-aligned, non-viral physicochemical transfection system, can be used to manufacture multi-edited CAR-T cells using CRISPR-Cas9 ribonucleoproteins while maintaining robust cell functionality. When compared to electroporation, an industry standard, T cells that were triple edited using Solupore had reduced levels of apoptosis and maintained similar proportions of stem cell memory T cells with higher oxidative phosphorylation levels. Following lentiviral transduction with a CD19 CAR, and subsequent cryopreservation, triple-edited CAR-T cells manufactured using Solupore demonstrated improved immunological synapse strength to target CD19+ Raji cells and enhanced cellular cytotoxicity compared with electroporated CAR-T cells. In an in vivo mouse model (NSG), Solupore triple-edited CAR-T cells enhanced tumor growth inhibition by more than 30-fold compared to electroporated cells.

There is a growing demand for chimeric antigen receptor (CAR) -T cells for clinical trials. Consequently, new centers capable of manufacturing advanced therapy medicinal products (ATMPs) are needed. In this study, we established a good manufacturing practice -compliant manufacturing process and phase-appropriate analytics for a novel autologous CD19-targeted CAR T-cell product, 19-FiCART. We evaluated the stability of fresh, healthy donor-derived leukapheresis products (LPs), produced 19-FiCART using a 12-day semi-automated process with CD4/CD8-positive cell enrichment and lentiviral transduction, and evaluated the in vivo efficacy of 19-FiCART in a xenograft mouse lymphoma model. The optimal hold time and temperature to maintain LP stability were up to 73 h at 2-8 °C. The 19-FiCART manufacturing process consistently yielded more than 2 × 109 highly viable CAR+ T cells, which is considered sufficient for a clinical product. The 19-FiCART products also demonstrated potent anti-tumor activity both in vitro and in vivo. This paper provides a detailed description of the manufacturing process and analytics for 19-FiCART and provides insights into the development of a release strategy for novel CAR T-cell products intended for early clinical studies. Additionally, we present data on LP stability, which has broader implications for the development of various immune cell-based ATMPs.

Acute HIV-1 infection (AHI) is a transient period where the virus causes evident damage to the immune system, including an extensive apoptosis of CD4+ T cells associated with a high level of activation and a major cytokine storm to fight the invading virus. HIV infection establishes persistence by integrating the viral genome into host cell DNA in both replicating and non-replicating forms, effectively hiding from immune surveillance within infected lymphocytes as cellular reservoirs. The measurement of total HIV-1 DNA in peripheral blood mononuclear cells (PBMCs) is a reliable reflection of this reservoir. Initiating treatments during AHI with nucleoside reverse transcriptase inhibitors (NRTIs) and/or integrase strand transfer inhibitors (INSTIs) is essential to alter the dynamics of the global reservoir expansion, and to reduce the establishment of long-lived cellular and tissue reservoirs, while preserving and enhancing specific and non-specific immune responses. Furthermore, some of the patients treated at the AHI stage may become post-treatment controllers and should be informative regarding the mechanism of viral control, so patients treated during AHI are undoubtedly the best candidates to test innovative remission strategies toward a functional cure that could play a pivotal role in long-term HIV control. AHI is characterized by high levels of viral replication, with a significant increase in the risk of HIV transmission. Detecting AHI and initiating early treatment following diagnosis provides a window of opportunity to control the epidemic, particularly in high-risk populations.

Cell-based immunotherapy has revolutionized cancer treatment in recent years and is rapidly expanding as one of the major therapeutic options in immuno-oncology. So far ten adoptive T cell therapies (TCTs) have been approved by the health authorities for cancer treatment, and they have shown remarkable anti-tumor efficacy with potent and durable responses. While adoptive T cell therapies have shown success in treating hematological malignancies, they are lagging behind in establishing promising efficacy in treating solid tumors, partially due to our incomplete understanding of the cellular kinetics (CK) and biodistribution (including tumoral penetration) of cell therapy products. Indeed, recent clinical studies have provided ample evidence that CK of TCTs can influence clinical outcomes in both hematological malignancies and solid tumors. In this review, we will discuss the current knowledge on the CK and biodistribution of anti-tumor TCTs. We will first describe the typical CK and biodistribution characteristics of these "living" drugs, and the biological factors that influence these characteristics. We will then review the relationships between CK and pharmacological responses of TCT, and potential strategies in enhancing the persistence and tumoral penetration of TCTs in the clinic. Finally, we will also summarize bioanalytical methods, preclinical in vitro and in vivo tools, and in silico modeling approaches used to assess the CK and biodistribution of TCTs.

Selecting a booster vaccine strategy that generates cellular immune breadth is crucial for effectively recalling cellular reservoirs upon infection with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants. This post hoc analysis from a multicentre, randomized phase 3 study (CTRI/2022/10/046475) compared the cellular immune breadth induced by self-replicating mRNA (samRNA) vaccine GEMCOVAC-OM, encoding Omicron B.1.1.529 Spike protein, with the adenovector vaccine ChAdOx1 nCoV-19, encoding Wuhan variant Spike protein, when administered as a booster. GEMCOVAC-OM elicited significant expansion of memory B-cells (MBCs) specific to Omicron B.1.1.529, compared to ChAdOx1 nCoV-19. GEMCOVAC-OM also induced more B-cells reactive to Omicron XBB.1.5 and BA.2.86 Spike proteins. Additionally, GEMCOVAC-OM triggered higher frequencies of Omicron-Spike-specific T-cells, including stem cell, central, and effector memory subsets. In summary, while ChAdOx1 nCoV-19 showed some cross-reactivity, GEMCOVAC-OM induced a more targeted immune response. GEMCOVAC-OM offers a broader, longer-lasting immunity, making it a promising candidate for future vaccine development and global distribution.

Chimeric antigen receptor T (CAR-T) cells have significantly advanced the treatment of cancers such as leukemia and lymphoma. Traditionally, T cells are collected from patients through leukapheresis, an expensive and potentially invasive process that requires specialized equipment and trained personnel. Although whole blood collections are much more technically straightforward, whole blood starting material has not been widely utilized for clinical CAR-T cell manufacturing, in part due to lack of manufacturing processes designed for use in a good manufacturing practice (GMP) environment. Collecting cellular starting material from whole blood without leukapheresis could reduce manufacturing complexity and cost, thereby improving accessibility to CAR-T cell therapy.

Whole blood samples were collected from eight healthy donors and one pediatric B-cell acute lymphoblastic leukemia (B-ALL) patient. These samples were processed using the Sepax C-Pro (Cytiva) instrument to isolate mononuclear cells (MNCs) via density gradient separation. CAR-T cells were then manufactured from the isolated MNCs using a GMP-compliant 7-day protocol, whereby T cells were activated with anti-CD3 and IL-2, transduced with GMP lentiviral vector encoding a CD19/CD22 bispecific CAR, and expanded in gas permeable cell culture bags. The resulting CAR-T cells were then evaluated for their phenotypic and functional properties using flow cytometry, cytokine release and cytotoxicity assays.

From an average 77.7 mL of whole blood from healthy donors (range = 29-96 mL), we isolated an average of 42.2 × 106 CD3⁺ T cells (range 7.3-63.0) postprocessing. CAR-T cell cultures were initiated from thaw using 1-10 × 106 starting CD3+ T cells, yielding a median T cell number of 105 × 106 cells on day 7 (range 61-188 × 106). We observed 66 ± 11% mean transduction efficiency and produced a mean of 77.4 × 106 transduced CAR-T cells (range 30.8-143.5 × 106). Similar results were obtained when using a blood sample (28mL) obtained from a patient with relapsed B-ALL who had received recent chemotherapy.

Therapeutically relevant doses of CD19/CD22 CAR-T cells can be successfully manufactured from whole blood. On average, 80 mL of whole blood yields enough CAR-T cells to create a single dose for a pediatric patient (50 kg) at a dosage of 1 × 106 CAR-T cells/kg. For larger patients, scaling up is straightforward by collecting a larger blood volume. This method also demonstrates a cost-effective approach to T cell activation and expansion which, alongside a more straightforward collection of whole blood, makes it more widely accessible especially for middle- and low-income countries. By reducing costs and labor, this strategy has the potential to significantly expand global access to CAR-T cell therapy.

Multiple myeloma (MM) remains incurable and patients eventually face the relapse/refractory dilemma. B cell maturation antigen (BCMA)-targeted immunotherapeutic approaches have shown great effectiveness in patients with relapsed/refractory MM, mainly including chimeric antigen receptor T cells (CAR-T), bispecific T cell engagers (TCEs), and antibody-drug conjugates (ADCs). However, their impact on long-term survival remains to be determined. Nonetheless, resistance to these novel therapies is still inevitable, raising a challenge that we have never met in both laboratory research and clinical practice. In this scenario, the investigation aiming to enhance and prolong the anti-MM activity of BCMA-targeted therapies has been expanding rapidly. Despite considerable uncertainty in our understanding of the mechanisms for their resistance, they have mainly been attributed to antigen-dependency, T cell-driven factors, and (immune) tumor microenvironment. In this review, we summarize the current understanding of the mechanisms for resistance to BCMA-targeted immunotherapies and discuss potential strategies for overcoming it.

CD8 T cells play a key role in elimination of intracellular pathogens. Here, we have carried out a detailed phenotypic and functional analysis of swine blood CD8β T cells, after vaccination with a live attenuated PRV vaccine. Based on the expression of six surface molecules (CD11a, CD27, CD45RA, CD95, CCR7 and SLA-DR), up to eight subsets can be identified within the circulating compartment of CD8β T cells of adult pigs; six of which correlate phenotypically with naïve, stem cell memory, central memory, transitional memory, effector memory, and terminal effector subsets described in humans. The remaining two subsets appear to correspond to intermediate stages between naïve and central memory cells, and between transitional memory and effector memory or terminal effector cells, respectively. Although CD45RA has been proposed as a marker to distinguish between porcine naïve and central memory CD8β T cells, we found that a substantial proportion of naïve T cells, which varies among animals, lack this marker and that the combination of CD95 and CD11a allows a more accurate discrimination of these subsets among CD27hi CCR7+ cells. The majority of virus-specific IFN-γ-producing CD8β T cells in the blood of PRV-vaccinated pigs, collected at 10-16-days postvaccination, showed a CD27+ CD11ahi CD45RA- phenotype, consistent with that of central, and/or transitional memory T cells.

Chlamydia trachomatis (CT) is the most common bacterial sexually transmitted infection globally. Understanding natural immunity to CT will inform vaccine design. This study aimed to profile immune cells and associated functional features in CT-infected women and determine immune profiles associated with reduced risk of ascended endometrial CT infection and CT reinfection. PBMCs from CT-exposed women were profiled by mass cytometry, and random forest models identified key features that distinguished outcomes. CT+ participants exhibited higher frequencies of CD4+ Th2, Th17, and Th17 double-negative (Th17 DN) CD4+ T effector memory (TEM) cells than uninfected participants with decreased expression of T cell activation and differentiation markers. Minimal differences were detected between women with or without endometrial CT infection. Participants who remained follow-up negative (FU-) showed higher frequencies of CD4+ T central memory (TCM) Th1, Th17, Th1/17, and Th17 DN but reduced CD4+ TEM Th2 cells than FU+ participants. Expression of markers associated with central memory and Th17 lineage was increased on T cell subsets among FU- participants. These data indicate that peripheral T cells exhibit distinct features associated with resistance to CT reinfection. The highly plastic Th17 lineage appears to contribute to protection. Addressing these immune nuances could promote efficacy of CT vaccines.