Pharmacodynamic assessment of T-cell-based cancer immunotherapies often focus on detecting rare circulating T-cell populations. The therapy-induced immune cells in blood-derived clinical samples are often present in very low frequencies and with the currently available T-cell analytical assays, amplification of the cells of interest prior to analysis is often required. Current approaches aiming to enrich antigen-specific T cells from human Peripheral Blood Mononuclear Cells (PBMCs) depend on in vitro culturing in presence of their cognate peptides and cytokines. In the present work, we improved a standard, publicly available protocol for T-cell immune analyses based on the in vitro expansion of T cells. We used PBMCs from healthy subjects and well-described viral antigens as a model system for optimizing the experimental procedures and conditions. Using the standard protocol, we first demonstrated significant enrichment of antigen-specific T cells, even when their starting frequency ex vivo was low. Importantly, this amplification occurred with high specificity, with no or neglectable enrichment of irrelevant T-cell clones being observed in the cultures. Testing of modified culturing timelines suggested that the protocol can be adjusted accordingly to allow for greater cell yield with strong preservation of the functionality of antigen-specific T cells. Overall, our work has led to the refinement of a standard protocol for in vitro stimulation of antigen-specific T cells and highlighted its reliability and reproducibility. We envision that the optimized protocol could be applied for longitudinal monitoring of rare blood-circulating T cells in scenarios with limited sample material.

A recent article in Proceedings of the National Academy of Sciences investigated γδ T cell antigen specificity in mice and humans, in which the authors show that γδ T cell antigen specificity is not constrained to one epitope. Rather, γδ T cells recognize a broad range of diverse antigens containing similar chemical structures or properties. In this News and Views, the importance of γδ T cell antigen polyspecificity during immune responses is highlighted.

Vaccination is widely used to control foot-and-mouth disease (FMD), but maternal antibodies may interfere with the response to vaccination in calves. This study, conducted on a regularly vaccinated Malaysian dairy farm, aimed to optimise the vaccination regime by measuring the in vitro neutralising virus antibody responses of 51 calves before and after vaccination with a one or two dose vaccination regime starting at 2-7 months old. The presence of maternal antibodies was associated with poor post-vaccination antibody responses after a single dose of vaccine in calves less than 6 months old. However, a second dose of vaccine given three weeks later, improved the antibody responses in all ages of calves. This confirms the view that in regularly vaccinated farms, some combination of delay and revaccination is needed to achieve effective immunization of calves. Sera from cows and pre-vaccinated calves neutralised homologous serotype A vaccine virus more strongly than a heterologous serotype A field virus, but this pattern was reversed in some calves after vaccination. The strength of heterologous responses in calves 49 days after first vaccination correlated to the amount of transferred maternal antibody, suggesting that pre-existing antibodies could have modulated the specificity of these active antibody responses. If confirmed, such an effect by pre-existing antibodies could have wider implications for broadening the coverage of FMD vaccine responses.

Amyotrophic lateral sclerosis (ALS) is a fatal disease associated with motor neuron degeneration, accumulation of aggregated misfolded proteins and neuroinflammation in motor regions of the central nervous system (CNS). Clinical trials using regulatory T cells (Tregs) are ongoing because of Tregs\' immunomodulatory function, ability to traffic to the CNS, high numbers correlating with slower disease in ALS and disease-modifying activity in ALS mouse models. In the current study, a chimeric antigen receptor (CAR) was developed and characterized in human Tregs to enhance their immunomodulatory activity when in contact with an ALS protein aggregate.
A CAR (DG05-28-3z) consisting of a human superoxide dismutase 1 (hSOD1)-binding single-chain variable fragment, CD28 hinge, transmembrane and co-stimulatory domain and CD3ζ signaling domain was created and expressed in human Tregs. Human Tregs were isolated by either magnetic enrichment for CD4+CD25hi cells (Enr-Tregs) or cell sorting for CD4+CD25hiCD127lo cells (FP-Tregs), transduced and expanded for 17 days.
The CAR bound preferentially to the ALS mutant G93A-hSOD1 protein relative to the wild-type hSOD1 protein. The CAR Tregs produced IL-10 when cultured with aggregated G93A-hSOD1 proteins or spinal cord explants from G93A-hSOD1 transgenic mice. Co-culturing DG05-28-3z CAR Tregs with human monocytes/macrophages inhibited production of tumor necrosis factor alpha and reactive oxygen species. Expanded FP-Tregs resulted in more robust Tregs compared with Enr-Tregs. FP-Tregs produced similar IL-10 and less interferon gamma, had lower Treg-specific demethylated region methylation and expressed higher FoxP3 and CD39.
Taken together, this study demonstrates that gene-modified Tregs can be developed to target an aggregated ALS-relevant protein to elicit CAR-mediated Treg effector functions and provides an approach for generating Treg therapies for ALS with the goal of enhanced disease site-specific immunomodulation.

Breast cancer is the most prevalent malignant neoplasm worldwide, necessitating the development of novel therapeutic strategies owing to the limitations posed by conventional treatment modalities. Immunotherapy is an innovative approach that has demonstrated significant efficacy in modulating a patient\'s innate immune system to combat tumor cells. In the era of precision medicine, adoptive immunotherapy for breast cancer has garnered widespread attention as an emerging treatment strategy, primarily encompassing cellular therapies such as tumor-infiltrating lymphocyte therapy, chimeric antigen receptor T/natural killer/M cell therapy, T cell receptor gene-engineered T cell therapy, lymphokine-activated killer cell therapy, cytokine-induced killer cell therapy, natural killer cell therapy, and γδ T cell therapy, among others. This treatment paradigm is based on the principles of immune memory and antigen specificity, involving the collection, processing, and expansion of the patient\'s immune cells, followed by their reintroduction into the patient\'s body to activate the immune system and prevent tumor recurrence and metastasis. Currently, multiple clinical trials are assessing the feasibility, effectiveness, and safety of adoptive immunotherapy in breast cancer. However, this therapeutic approach faces challenges associated with tumor heterogeneity, immune evasion, and treatment safety. This review comprehensively summarizes the latest advancements in adoptive immunotherapy for breast cancer and discusses future research directions and prospects, offering valuable guidance and insights into breast cancer immunotherapy.

Following viral infection, the human immune system generates CD8+ T cell responses to virus antigens that differ in specificity, abundance, and phenotype. A characterization of virus-specific T cell responses allows one to assess infection history and to understand its contribution to protective immunity. Here, we perform in-depth profiling of CD8+ T cells binding to CMV-, EBV-, influenza-, and SARS-CoV-2-derived antigens in peripheral blood samples from 114 healthy donors and 55 cancer patients using high-dimensional mass cytometry and single-cell RNA sequencing. We analyze over 500 antigen-specific T cell responses across six different HLA alleles and observed unique phenotypes of T cells specific for antigens from different virus categories. Using machine learning, we extract phenotypic signatures of antigen-specific T cells, predict virus specificity for bulk CD8+ T cells, and validate these predictions, suggesting that machine learning can be used to accurately predict antigen specificity from T cell phenotypes.

Sensitization to self-peptides induces various immunological responses, from autoimmunity to tumor immunity, depending on the peptide sequence; however, the underlying mechanisms remain unclear, and thus, curative therapeutic options considering immunity balance are limited. Herein, two overlapping dominant peptides of myelin proteolipid protein, PLP136-150 and PLP139-151, which induce different forms of experimental autoimmune encephalomyelitis (EAE), monophasic and relapsing EAE, respectively, were investigated. Mice with monophasic EAE exhibited highly resistant to EAE re-induction with any encephalitogenic peptides, whereas mice with relapsing EAE were susceptible, and progressed, to EAE re-induction. This resistance to relapse and re-induction in monophasic EAE mice was associated with the maintenance of potent CD69+CD103+CD4+CD25high regulatory T-cells (Tregs) enriched with antigen specificity, which expanded preferentially in the central nervous system with sustained suppressive activity. This tissue-preferential sustainability of potent antigen-specific Tregs was correlated with the antigenicity of PLP136-150, depending on its flanking residues. That is, the flanking residues of PLP136-150 enable to form pivotally arranged strong hydrogen bonds that secured its binding stability to MHC-class II. These potent Tregs acting tissue-preferentially were induced only by sensitization of PLP136-150, not by its tolerance induction, independent of EAE development. These findings suggest that, for optimal therapy, \"benign autoimmunity\" can be critically achieved through inverse vaccination with self-peptides by manipulating their flanking residues.

The number of regulatory T cells (Tregs) and how they behave in the pathogenesis of atopic dermatitis (AD) are still controversial. We identified and quantified Tregs, mite-specific Tregs, and mite-specific effector T cells (Teffs) in patients with AD and healthy controls (HCs). We collected peripheral blood and analyzed the cells using flow cytometry after stimulation with mite antigens. Mite-specific Tregs and mite-specific Teffs were recognized by the expression of CD137 and CD154, respectively. Patients with AD had more Tregs than HCs; however, when focusing on a single antigen, the ratio of mite-specific Tregs/Teffs was lower in patients with AD than in HCs. Furthermore, the mite-specific Teffs in patients with AD were more likely to produce proinflammatory cytokines interleukin (IL)-4 and IL-13. This Teff-dominant imbalance is thought to be the cause of development of atopic status in patients with AD without immune tolerance.

The emerging virus SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2 virus), agent of COVID-19, appeared in December 2019 in Wuhan, China, and became a serious threat to global health and public safety. Many COVID-19 vaccines have been approved and licensed around the world. Most of the developed vaccines include S protein and induce an antibody-based immune response. Additionally, T-cell response to the SARS-CoV-2 antigens could be beneficial for combating the infection. The type of immune response is greatly dependent not only on the antigen, but also on adjuvants used in vaccine formulation. Here, we compared the effect of four different adjuvants (AddaS03, Alhydrogel/MPLA, Alhydrogel/ODN2395, Quil A) on the immunogenicity of a mixture of recombinant RBD and N SARS-CoV-2 proteins. We have analyzed the antibody and T-cell response specific to RBD and N proteins and assessed the impact of adjuvants on virus neutralization. Our results clearly indicated that Alhydrogel/MPLA and Alhydrogel/ODN2395 adjuvants elicited the higher titers of specific and cross-reactive antibodies to S protein variants from various SARS-CoV-2 and SARS-CoV-1 strains. Moreover, Alhydrogel/ODN2395 stimulated high cellular response to both antigens, as assessed by IFN-γ production. Importantly, sera collected from mice immunized with RBD/N cocktail in combination with these adjuvants exhibited neutralizing activity against the authentic SARS-CoV-2 virus as well as particles pseudotyped with S protein from various virus variants. The results from our study demonstrate the immunogenic potential of RBD and N antigens and point out the importance of adjuvants selection in vaccine formulation in order to enhance the immunological response. IMPORTANCE Although several COVID-19 vaccines have been approved worldwide, continuous emergence of new SARS-CoV-2 variants calls for new efficient vaccines against them, providing long-lasting immunity. As the immune response after vaccination is dependent not only on antigen used, but also on other vaccine components, e.g., adjuvants, the purpose of this work was to study the effect of different adjuvants on the immunogenicity of RBD/N SARS-CoV-2 cocktail proteins. In this work, it has been shown that immunization with both antigens plus the different adjuvants studied elicited higher Th1 and Th2 responses against RBD and N, which contributed to higher neutralization of the virus. The obtained results can be used for design of new vaccines, not only against SARS-CoV-2, but also against other important viral pathogens.

In pancreatic ductal adenocarcinoma (PDAC) patients, we show that response to radiation therapy (RT) is characterized by increased IL-2Rβ and IL-2Rγ along with decreased IL-2Rα expression. The bispecific PD1-IL2v is a PD-1-targeted IL-2 variant (IL-2v) immunocytokine with engineered IL-2 cis targeted to PD-1 and abolished IL-2Rα binding, which enhances tumor-antigen-specific T cell activation while reducing regulatory T cell (Treg) suppression. Using PD1-IL2v in orthotopic PDAC KPC-driven tumor models, we show marked improvement in local and metastatic survival, along with a profound increase in tumor-infiltrating CD8+ T cell subsets with a transcriptionally and metabolically active phenotype and preferential activation of antigen-specific CD8+ T cells. In combination with single-dose RT, PD1-IL2v treatment results in a robust, durable expansion of polyfunctional CD8+ T cells, T cell stemness, tumor-specific memory immune response, natural killer (NK) cell activation, and decreased Tregs. These data show that PD1-IL2v leads to profound local and distant response in PDAC.