Memory Cell Subsets Research Articles

Impaired SARS-CoV-2-Specific CD8+ T Cells After Infection or Vaccination but Robust Hybrid T Cell Immunity in Patients with Multiple Myeloma.

Multiple myeloma (MM) patients are at high risk of severe infections including COVID-19 due to an immune dysregulation affecting both innate and adaptive immune responses. However, our understanding of the immune responses to infection and vaccination in MM patients is limited. To gain more detailed insights into infection- and vaccine-elicited T cell immunity in MM, we studied the CD8+ T cell response on the single-epitope level in SARS-CoV-2 convalescent and mRNA-vaccinated MM patients. We compared peptide/MHC class I tetramer-enriched SARS-CoV-2-specific CD8+ T cells and antibody responses in MM patients (convalescent: n = 16, fully vaccinated: n = 5, vaccinated convalescent: n = 5) and healthy controls (HCs) (convalescent: n = 58, fully vaccinated: n = 7) either after infection with SARS-CoV-2 alone, complete mRNA vaccination or SARS-CoV-2 infection and single-shot mRNA vaccination (hybrid immunity). MM patients have lower frequencies and a lower proportion of fully functional virus-specific CD8+ T cells compared to HCs, after both SARS-CoV-2 infection and vaccination. CD8+ T cell memory subset distribution in MM patients is skewed towards reduced frequencies of central memory (TCM) T cells and higher frequencies of effector memory 1 (TEM1) T cells. In contrast, the humoral immune response was comparable in both cohorts after viral clearance. Notably, CD8+ T cell frequencies as well as the humoral immune response were improved by a single dose of mRNA vaccine in convalescent MM patients. MM patients have relative immunological deficiencies in SARS-CoV-2 immunity but benefit from hybrid immunity. These findings underline the relevance of vaccinations in this vulnerable patient group.

OMIP-109: 45-color full spectrum flow cytometry panel for deep immunophenotyping of the major lineages present in human peripheral blood mononuclear cells with emphasis on the T cell memory compartment.

The need for more in-depth exploration of the human immune system has moved the flow cytometry field forward with advances in instrumentation, reagent development and availability, and user-friendly implementation of data analysis methods. We developed a high-quality human 45-color panel, for comprehensive characterization of major cell lineages present in circulation including T cells, γδ T cells, NKT-like cells, B cells, NK cells, monocytes, basophils, dendritic cells, and ILCs. Assay optimization steps are described in detail to ensure that each marker in the panel was optimally resolved. In addition, we highlight the outstanding discernment of cell activation, exhaustion, memory, and differentiation states of CD4+ and CD8+ T cells using this 45-color panel. The panel enabled an in-depth description of very distinct phenotypes associated with the complexity of the T cell memory response. Furthermore, we present how this panel can be effectively used for cell sorting on instruments with a similar optical layout to achieve the same level of resolution. Functional evaluation of sorted specific rare cell subsets demonstrated significantly different patterns of immunological responses to stimulation, supporting functional and phenotypic differences within the T cell memory subsets. In summary, the combination of full spectrum profiling technology and careful assay design and optimization results in a high resolution multiparametric 45-color assay. This panel offers the opportunity to fully characterize immunological profiles present in peripheral blood in the context of infectious diseases, autoimmunity, neurodegeneration, immunotherapy, and biomarker discovery.

Targeting CD276 for T cell-based immunotherapy of breast cancer.

Breast cancer (BC) is the most common malignancy in women. Immunotherapy has revolutionized treatment options in many malignancies, and the introduction of immune checkpoint inhibition yielded beneficial results also in BC. However, many BC patients are ineligible for this T cell-based therapy, others do not respond or only briefly. Thus, there remains a high medical need for new therapies, particularly for triple-negative BC. CD276 (B7-H3) is overexpressed in several tumors on both tumor cells and tumor vessels, constituting a promising target for immunotherapy. We analyzed tumor samples of 25 patients using immunohistochemistry to assess CD276 levels. The potential of CC-3, a novel bispecific CD276xCD3 antibody, for BC treatment was evaluated using various functional in vitro assays. Pronounced expression of CD276 was observed in all analyzed tumor samples including triple negative BC. In analyses with BC cells, CC-3 induced profound T cell activation, proliferation, and T cell memory subset formation. Moreover, treatment with CC-3 induced cytokine secretion and potent tumor cell lysis. Our findings characterize CD276 as promising target and preclinically document the therapeutic potential of CC-3 for BC treatment, providing a strong rationale for evaluation of CC-3 in BC patients in a clinical trial for which the recruitment has recently started.

Identifying genetic variants associated with chromatin looping and genome function

Here we present a comprehensive HiChIP dataset on naïve CD4 T cells (nCD4) from 30 donors and identify QTLs that associate with genotype-dependent and/or allele-specific variation of HiChIP contacts defining loops between active regulatory regions (iQTLs). We observe a substantial overlap between iQTLs and previously defined eQTLs and histone QTLs, and an enrichment for fine-mapped QTLs and GWAS variants. Furthermore, we describe a distinct subset of nCD4 iQTLs, for which the significant variation of chromatin contacts in nCD4 are translated into significant eQTL trends in CD4 T cell memory subsets. Finally, we define connectivity-QTLs as iQTLs that are significantly associated with concordant genotype-dependent changes in chromatin contacts over a broad genomic region (e.g., GWAS SNP in the RNASET2 locus). Our results demonstrate the importance of chromatin contacts as a complementary modality for QTL mapping and their power in identifying previously uncharacterized QTLs linked to cell-specific gene expression and connectivity.

Hepatitis B vaccine responders show higher frequencies of CD8+ effector memory and central memory T cells compared to non-responders.

Hepatitis B (HB) infection is a major global health problem. There is limited knowledge about HB vaccination-induced immune memory responses. We compared the frequency of CD8+ memory T cell subsets between responders (RSs) and non-responders (NRs) to HB vaccination. Blood samples were collected from RSs and NRs. PBMCs were cultured in the presence of Hepatitis B surface antigens (HBsAg) and PHA for 48 h to restimulate CD8+ memory T cells and T cell memory subsets were detected by flow cytometry using memory cell markers. The frequency of TEM, TCM, and TCM hi was significantly higher in responders compared to non-responders (p = 0.024, 0.022, and 0.047, respectively). Additionally, we report a positive correlation between the frequency of TEM cells in RSs with age and anti-HBsAb level (p = 0.03 and rs = 0.5; p = 0.01 and rs = 0.06). Responders display a higher level of CD8+ T cell-mediated immunity. Therefore, we suggest a possible defect in the formation of immunological CD8+ memory T cells in NRs and it may reduce antibody production compared to the RSs, although more experiments are needed.

Long-lived central memory γδ T cells confer protection against murine cytomegalovirus reinfection.

The involvement of γδ TCR-bearing lymphocytes in immunological memory has gained increasing interest due to their functional duality between adaptive and innate immunity. γδ T effector memory (TEM) and central memory (TCM) subsets have been identified, but their respective roles in memory responses are poorly understood. In the present study, we used subsequent mouse cytomegalovirus (MCMV) infections of αβ T cell deficient mice in order to analyze the memory potential of γδ T cells. As for CMV-specific αβ T cells, MCMV induced the accumulation of cytolytic, KLRG1+CX3CR1+ γδ TEM that principally localized in infected organ vasculature. Typifying T cell memory, γδ T cell expansion in organs and blood was higher after secondary viral challenge than after primary infection. Viral control upon MCMV reinfection was prevented when masking γδ T-cell receptor, and was associated with a preferential amplification of private and unfocused TCR δ chain repertoire composed of a combination of clonotypes expanded post-primary infection and, more unexpectedly, of novel expanded clonotypes. Finally, long-term-primed γδ TCM cells, but not γδ TEM cells, protected T cell-deficient hosts against MCMV-induced death upon adoptive transfer, probably through their ability to survive and to generate TEM in the recipient host. This better survival potential of TCM cells was confirmed by a detailed scRNASeq analysis of the two γδ T cell memory subsets which also revealed their similarity to classically adaptive αβ CD8 T cells. Overall, our study uncovered memory properties of long-lived TCM γδ T cells that confer protection in a chronic infection, highlighting the interest of this T cell subset in vaccination approaches.

Human T-cell activation withToxoplasma gondiiantigens loaded in maltodextrin nanoparticles

Toxoplasmosis is the most prevalent parasitic zoonosis worldwide, causing ocular and neurological diseases. No vaccine has been approved for human use. We evaluated the response of peripheral blood mononuclear cells (PBMCs) to a novel construct ofToxoplasma gondiitotal antigen in maltodextrin nanoparticles (NP/TE) in individuals with varying infectious statuses (uninfected, chronic asymptomatic, or ocular toxoplasmosis). We analyzed the concentration of IFN-γ after NP/TE ex vivo stimulation using ELISA and the immunophenotypes of CD4+and CD8+cell populations using flow cytometry. In addition, serotyping of individuals with toxoplasmosis was performed by ELISA using GRA6-derived polypeptides. Low doses of NP/TE stimulation (0.9 μg NP/0.3 μg TE) achieved IFN-γ–specific production in previously exposed human PBMCs without significant differences in the infecting serotype. Increased IFN-γ expression in CD4+effector memory cell subsets was found in patients with ocular toxoplasmosis with NP/TE but not with TE alone. This is the first study to show how T-cell subsets respond to ex vivo stimulation with a vaccine candidate for human toxoplasmosis, providing crucial insights for future clinical trials.

Abstract 6707: IgG based B7-H3xCD3 bispecific antibody for treatment of gynecological tumors

Abstract Ovarian cancer is characterized by late stage disease and a poor prognosis. Endometrial cancer is a heterogeneous disease with different prognoses depending on the molecular subtype. Novel treatment strategies are needed to improve patient survival in both entities. T cell-based immunotherapy has significantly improved the treatment options for many malignancies. B7-H3 (CD276) is an interesting target for immunotherapeutic approaches because it is overexpressed on a wide range of tumor entities and has characteristics of immune checkpoint molecules. Its expression not only on tumor cells but also on the tumor vasculature allows improved infiltration of immune effector cells into the tumor to overcome the major limitation of T cell-based therapy of solid tumors, which is the access of immune cells to the tumor site. A novel bispecific antibody in an IgG-based format called CC-3 with B7-H3xCD3 specificity has already been shown to efficiently induce T cell responses against gastrointestinal malignancies. Here we show that B7-H3 is highly expressed on endometrial and ovarian cancer cell lines. Treatment with CC-3 induced T cell activation and degranulation as well as secretion of IL-2, IFNy and perforin. These results demonstrate CC-3 induced T cell reactivity against ovarian and endometrial cancer cells. In addition, CC-3 induced efficient T cell proliferation and formation of T cell memory subsets. This resulted in potent target cell lysis. Taken together, our results underscore the potential of CC-3, which is currently in GMP manufacturing, to enable clinical evaluation for the treatment of gynecologic malignancies. Citation Format: Sarah Maria Greiner, Jonas Mauermann, Martina Lutz, Ilona Hagelstein, Engler Tobias, André Koch, Hartkopf Andreas, Brucker Sara, Latifa Zekri, Melanie Märklin. IgG based B7-H3xCD3 bispecific antibody for treatment of gynecological tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6707.

A prognostic model for SARS-CoV-2 breakthrough infection: Analyzing a prospective cellular immunity cohort

BackgroundFollowing the COVID-19 pandemic, studies have identified several prevalent characteristics, especially related to lymphocyte subsets. However, limited research is available on the focus of this study, namely, the specific memory cell subsets among individuals who received COVID-19 vaccine boosters and subsequently experienced a SARS-CoV-2 breakthrough infection. MethodsFlow cytometry (FCM) was employed to investigate the early and longitudinal pattern changes of cellular immunity in patients with SARS-CoV-2 breakthrough infections following COVID-19 vaccine boosters. XGBoost (a machine learning algorithm) was employed to analyze cellular immunity prior to SARS-CoV-2 breakthrough, aiming to establish a prognostic model for SARS-CoV-2 breakthrough infections. ResultsFollowing SARS-CoV-2 breakthrough infection, naïve T cells and TEMRA subsets increased while the percentage of TCM and TEM cells decreased. Naïve and non-switched memory B cells increased while switched and double-negative memory B cells decreased. The XGBoost model achieved an area under the curve (AUC) of 0.78, with an accuracy rate of 81.8 %, a sensitivity of 75 %, and specificity of 85.7 %. TNF-α, CD27-CD19+cells, and TEMRA subsets were identified as high predictors. An increase in TNF-α, cTfh, double-negative memory B cells, IL-6, IL-10, and IFN-γ prior to SARS-CoV-2 infection was associated with enduring clinical symptoms; conversely, an increase in CD3+ T cells, CD4+ T cells, and IL-2 was associated with clinical with non-enduring clinical symptoms. ConclusionSARS-CoV-2 breakthrough infection leads to disturbances in cellular immunity. Assessing cellular immunity prior to breakthrough infection serves as a valuable prognostic tool for SARS-CoV-2 infection, which facilitates clinical decision-making.

Enhancing the Efficacy of CAR-T Cell Therapy: A Comprehensive Exploration of Cellular Strategies and Molecular Dynamics.

The emergence of chimeric antigen receptor T cell (CAR-T cell) therapy has revolutionized cancer treatment, particularly for hematologic malignancies. This commentary discusses developments in CAR-T cell therapy, focusing on the molecular mechanisms governing T cell fate and differentiation. Transcriptional and epigenetic factors play a pivotal role in determining the specificity, effectiveness, and durability of CAR-T cell therapy. Understanding these mechanisms is crucial to improve the efficacy and decrease the adverse events associated with CAR-T cell therapies, unlocking the full potential of these approaches. T cell differentiation in CAR-T cell product manufacturing plays an important role in clinical outcomes. A positive correlation exists between the clinical efficacy of CAR-T cell therapy and signatures of memory, whereas a negative correlation has been observed with signatures of effector function or exhaustion. The effectiveness of CAR-T cell products is likely influenced by T-cell frequency and by their ability to proliferate, which is closely linked to early T cell differentiation. The differentiation process involving distinct T memory cell subsets is initiated upon antigen elimination, indicating infection resolution. In chronic infections or cancer, T cells may undergo exhaustion, marked by continuous inhibitory receptor expression, decreased cytokine production, and diminished proliferative capacity. Other cell subsets, such as CD4+ T cells, innate-like T lymphocytes, NKT cells, and cord blood-derived hematopoietic stem cells, offer unique advantages in developing the next-generation CAR-T cell-based therapies. Future research should focus on optimizing T-cell-enhancing approaches and developing strategies to potentially cure patients with hematological diseases and solid tumors.

Safety and immunogenicity of a synthetic nanoparticle-based, T cell priming peptide vaccine against dengue in healthy adults in Switzerland: a double-blind, randomized, vehicle-controlled, phase 1 study

Vaccines that minimize the risk of vaccine-induced antibody-dependent enhancement and severe dengue are needed to address the global health threat posed by dengue. This study assessed the safety and immunogenicity of a gold nanoparticle (GNP)-based, multi-valent, synthetic peptide dengue vaccine candidate (PepGNP-Dengue), designed to provide protective CD8+ T cell immunity, without inducing antibodies. In this randomized, double-blind, vehicle-controlled, phase 1 trial (NCT04935801), healthy naïve individuals aged 18-45 years recruited at the Centre for primary care and public health, Lausanne, Switzerland, were randomly assigned to receive PepGNP-Dengue or comparator (GNP without peptides [vehicle-GNP]). Randomization was stratified into four groups (low dose [LD] and high dose [HD]), allocation was double-blind from participants and investigators. Two doses were administered by intradermal microneedle injection 21 days apart. Primary outcome was safety, secondary outcome immunogenicity. Analysis was by intention-to-treat for safety, intention-to-treat and per protocol for immunogenicity. 26 participants were enrolled (August-September 2021) to receive PepGNP-Dengue (LD or HD, n=10 each) or vehicle-GNP (LD or HD, n=3 each). No vaccine-related serious adverse events occurred. Most (90%) related adverse events were mild; injection site pain and transient discoloration were most frequently reported. Injection site erythema occurred in 58% of participants. As expected, PepGNP-Dengue did not elicit anti-DENV antibodies of significance. Significant increases were observed in specific CD8+ T cells and dengue dextramer+memory cell subsets in the LD PepGNP-Dengue but not in the HD PepGNP-Dengue or vehicle-GNP groups, specifically PepGNP-activated CD137+CD69+CD8+ T cells (day 90,+0.0318%, 95% CI: 0.0088-0.1723, p=0.046), differentiated effector memory (TemRA) and central memory (Tcm) CD8+ T cells (day 35,+0.8/105 CD8+, 95% CI: 0.19-5.13, p=0.014 and +1.34/105 CD8+, 95% CI: 0.1-7.34, p=0.024, respectively). Results provide proof of concept that a synthetic nanoparticle-based peptide vaccine can successfully induce virus-specific CD8+ T cells. The favourable safety profile and cellular responses observed support further development of PepGNP-Dengue. Emergex Vaccines Holding Limited.

Initial Clinical Results and Immune Correlates of a Phase 2 Study of Daratumumab, Bortezomib, Dexamethasone Followed By a Proteasome Inhibitor in-Class Transition ( iCT) to Daratumumab, Ixazomib, Dexamethasone in Relapsed Refractory Multiple Myeloma

Background: The regimen of daratumumab, bortezomib, and dexamethasone (DVd) was approved in relapsed refractory multiple myeloma (RRMM) patients (pts) with >1 line of therapy based on the pivotal phase 3 CASTOR study. Notably in CASTOR, bortezomib was administered for a fixed duration of 8 cycles, and both the parenteral administration route and treatment-related peripheral neuropathy (PN) may limit continuous bortezomib exposure in clinical practice. As an alternative, we conducted a phase 2 study of DVd, followed by daratumumab, ixazomib, dexamethasone (DId) utilizing a proteasome inhibitor (PI) in-class transition ( iCT) with the hypothesis that oral ixazomib may provide a more tolerable and convenient way to deliver continuous PI- and anti-CD38-based therapy to improve efficacy (NCT03763162). Methods: This phase 2 single-center study enrolled RRMM pts with 1-3 lines of prior therapy with disease progression on or following the last line of therapy. Key exclusion criteria included prior ixazomib exposure, refractoriness to anti-CD38 therapy, and refractoriness to bortezomib or carfilzomib therapy at last exposure. Enrolled pts received DVd for 3 cycles on a 21-day (D) cycle with daratumumab on D1, D8, and D15 and subcutaneous (SC) bortezomib 1.3 mg/m 2 on D1, D4, D8, and D11. Starting cycle (C) 4, pts transitioned to DId on a 28-day cycle, with daratumumab on D1 and D15 for C3-C7 and on D1 starting C8 and oral ixazomib 4 mg on D1, D8 and D15. Daratumumab was initially administered intravenously (16 mg/kg) but later changed to SC injection (1800 mg) with a protocol amendment. Bone marrow (BM) aspirates were performed pre-treatment, post-C3, post-C6 (optional), and at progression to evaluate immune correlates of therapeutic response and resistance using 4 16-color multiparameter flow cytometry panels (2 T-cell, 1 NK/myeloid, and 1 B-cell). The primary endpoint of the study was progression free survival (PFS) with planned enrollment of 40 pts. Results: As of 15 May 2023, 36 pts have enrolled with a median of 1 prior line of therapy. Median age was 66, 67% were male, 28% were Black, 97% were lenalidomide-refractory, and 42% had high-risk cytogenetics (Cg; del 17p, t(4;14), t(14;16), and/or +1q21 gain/amplification). Overall response rate was 83%, ≥very good partial response (VGPR) rate was 53%, and ≥complete response rate was 19%. At a median follow-up of 24.8 months, median PFS was 21.1 months and median response duration was 26.2 months. Median PFS among pts with high-risk Cg, 1 line of prior therapy, and 2-3 lines of prior therapy was 21.1 months, 22.8 months, and 14.3 months, respectively. The most common treatment emergent adverse events (TEAEs) of any grade were diarrhea (69%), thrombocytopenia (69%), fatigue (67%), and lymphopenia (67%). The most common ≥grade 3 TEAEs were lymphopenia (42%), thrombocytopenia (42%), and hypertension (31%). Treatment emergent PN occurred in 61% pts (11% ≥ grade 3). The most common reason for treatment discontinuation was disease progression (42%), and 16 (44%) pts remain on study with ongoing responses. Efficacy and safety stopping rules for the study have not been met. Multiparameter flow cytometry immune correlates from BM aspirates demonstrated significantly lower Ki67 + cell frequency and significantly higher PD-1 + cell frequency within the CD8 + T-cell population at baseline in pts with no response versus those with optimal responses (≥VGPR). Among pts with short PFS (<12 months), CD4 +/CD8 + ratio and CD4 + non-T-reg naïve T-cells were significantly higher post-C3 and post-C6 compared to pts with longer PFS (>12 months). Among initial responders who later progressed on therapy, T-regulatory cells were significant increased at progression compared to pre-C4, and NK cell CD16 expression was significantly reduced at progression compared to baseline. Finally, distinct patterns of CD4 + T effector memory (EM) cell subsets were observed with significantly decreased EM1 and increased EM3 subsets at progression compared to baseline. Conclusion: Treatment with DVd followed by DId with a PI iCT from bortezomib to ixazomib is a feasible approach for continuous PI and anti-CD38 therapy, leading to durable responses in RRMM pts, among whom nearly all (97%) were lenalidomide refractory. Immune correlates revealed distinct patterns of T-cell and NK-cell phenotypes when profiled serially in responding and non-responding pts. Additional correlative analyses are ongoing.

Disturbance in the reconstitution of distinct T-cell subsets and the incidence of GvHD following allo-HSCT in pediatric patients with non-malignant hematological disorders

BackgroundThe reconstitution of different T-cell subsets following allogeneic hematopoietic stem cell transplantation (allo-HSCT) is critical for efficient pathogen protection and the prevention of graft-versus-host disease (GvHD). In particular, studies have highlighted the importance of balanced ratios of regulatory T-cells (Tregs) and distinct functionally T-cells in preventing acute and chronic GvHD. MethodsWe evaluated the regeneration of CD4 and CD8 T-cell subpopulations in nine pediatric patients with non-malignant disorders following allo-HSCT from a fully HLA-identical donor. ResultsCD4 and CD8 T-cells were higher 12 months after the transplant but still lower than in healthy controls and pre-transplant. However, we found after allo-HSCT, central memory and effector memory cell subsets were the predominant phenotypes in the CD4 and CD8 T-cell populations, respectively. In patients who had developed acute GvHD, there was an increase in the frequency of TEMRA (effector memory T cells that re-express CD45RA) cells within the CD4 T-cell population. Meanwhile, in patients with chronic GvHD, we observed a decrease in Th1 cells in CD4 T-cells and effector memory cells within the CD8 T-cell population. In addition, we found decreased TEMRA cell subsets in CD4 and CD8 T-cell populations in chronic GvHD. ConclusionOur findings suggest a possible relationship between the influence of acute GvHD and its prevention on delayed CD4 T-cell reconstitution and, reciprocally, unbalanced regeneration of CD4 and CD8 T-cell subsets in the development of chronic GvHD.

Dynamics of circulating lymphocytes responding to human experimental enterotoxigenic Escherichia coli infection.

Enterotoxigenic Escherichia coli (ETEC) is an important cause of children's and travelers' diarrhea, with no licensed vaccine. This study aimed to explore the role of cellular immunity in protection against human ETEC infection. Nine volunteers were experimentally infected with ETEC, of which six developed diarrhea. Lymphocytes were collected from peripheral blood buffy coats, before and 3, 5, 6, 7, 10, and 28 days after dose ingestion, and 34 phenotypic and functional markers were examined by mass cytometry. Thirty-three cell populations, derived by manually merging 139 cell clusters from the X-shift unsupervised clustering algorithm, were analyzed. Initially, the diarrhea group responded with increased CD56dim CD16+ natural killer cells, dendritic cells tended to rise, and mucosal-associated invariant T cells decreased. On day 5-7, an increase in plasmablasts was paralleled by a consistent rise in CD4+ Th17-like effector memory and regulatory cell subsets. CD4+ Th17-like central memory cells peaked on day 10. All Th17-like cell populations showed increased expression of activation, gut-homing, and proliferation markers. Interestingly, in the nondiarrhea group, these same CD4+ Th17-like cell populations expanded earlier, normalizing around day 7. Earlier development of these CD4+ Th17-like cell populations in the nondiarrhea group may suggest a recall response and a potential role in controlling ETEC infections.

Canonical BAF complex activity shapes the enhancer landscape that licenses CD8+ T cell effector and memory fates

CD8+ Tcells provide host protection against pathogens by differentiating into distinct effector and memory cell subsets, but how chromatin is site-specifically remodeled during their differentiation is unclear. Due to its critical role in regulating chromatin and enhancer accessibility through its nucleosome remodeling activities, we investigated the role of the canonical BAF (cBAF) chromatin remodeling complex in antiviral CD8+ Tcells during infection. ARID1A, a subunit of cBAF, was recruited early after activation and established de novo open chromatin regions (OCRs) at enhancers. Arid1a deficiency impaired the opening of thousands of activation-induced enhancers, leading to loss of TF binding, dysregulated proliferation and gene expression, and failure to undergo terminal effector differentiation. Although Arid1a was dispensable for circulating memory cell formation, tissue-resident memory (Trm) formation was strongly impaired. Thus, cBAF governs the enhancer landscape of activated CD8+ Tcells that orchestrates TF recruitment and activity and the acquisition of specific effector and memory differentiation states.

IgG-based B7-H3xCD3 bispecific antibody for treatment of pancreatic, hepatic and gastric cancer.

T cell-based immunotherapy has significantly improved treatment options for many malignancies. However, despite these and other therapeutic improvements over the last decades, gastrointestinal cancers, in particular pancreatic, hepatic and gastric cancer, are still characterized by high relapse rates and dismal prognosis, with an accordingly high unmet medical need for novel treatment strategies. We here report on the preclinical characterization of a novel bispecific antibody in an IgG-based format termed CC-3 with B7-H3xCD3 specificity. In many cancer entities including pancreatic, hepatic and gastric cancers, B7-H3 (CD276) is overexpressed on tumor cells and also on the tumor vasculature, the latter allowing for improved access of immune effector cells into the tumor site upon therapeutic targeting. We demonstrate that CC-3 induces profound T cell reactivity against various pancreatic, hepatic and gastric cancer cell lines as revealed by analysis of activation, degranulation and secretion of IL2, IFNγ as well as perforin, resulting in potent target cell lysis. Moreover, CC-3 induced efficient T cell proliferation and formation of T cell memory subsets. Together, our results emphasize the potential of CC-3, which is presently being GMP-produced to enable clinical evaluation for treatment of pancreatic, hepatic and gastric cancer.

Mitochondrial Dysfunction in CD4+ T Effector Memory RA+ Cells.

Human ageing is accompanied by poor responses to infection and decreased vaccine efficacy. While the causes of this can be attributed to defects in the immune system that increase with age, it is unknown whether mitochondrial dysfunction may also contribute to these phenomena. This study aims to assess mitochondrial dysfunction in CD4+ terminal effector memory T cells re-expressing CD45RA (TEMRA) cells and other CD4+ memory T cell subtypes, which are increased in number in the elderly population, with respect to how their metabolic responses to stimulation are altered compared to CD4+ naïve T cells. In this study, we show that CD4+ TEMRA cells exhibit altered mitochondrial dynamics compared to CD4+ naïve cells and CD4+ central and effector memory cells, with a 25% reduction in OPA1 expression. CD4+ TEMRA and memory cells show increased upregulation of Glucose transporter 1 following stimulation and higher levels of mitochondrial mass compared to CD4+ naïve T cells. Additionally, TEMRA cells exhibit a decrease in mitochondrial membrane potential compared to other CD4+ memory cell subsets by up to 50%. By comparing young to aged individuals, more significant mitochondria mass and lower membrane potential were observed in CD4+ TEMRA of young individuals. In conclusion, we suggest that CD4+ TEMRA cells may be impaired with respect to their metabolic response to stimulation, possibly contributing to impaired responses to infection and vaccination.

Naive T cells inhibit the outgrowth of intractable antigen-activated memory T cells: implications for T-cell immunotherapy

BackgroundThe wider application of T cells targeting viral tumor-antigens via their native receptors is hampered by the failure to expand potent tumor-specific T cells from patients. Here, we examine reasons...

Abstract P3-08-07: Self-renewal signatures of peripheral blood T cells are associated with successful engraftment to establish a humanized mouse model of breast cancer

Abstract Background: Stable humanized immune mice transplanted with peripheral blood mononuclear cells (PBMCs) derived from breast cancer patients are important models for assessing the tumor immune responses and the tumor immune microenvironment in breast cancer, helping to advance both pre-clinical and clinical research. The PBMCs of breast cancer patients exhibit various differences from those of healthy individuals depending on the stage of cancer progression, subtype, and type of treatment. Recent studies indicate an association of the self-renewal signatures of T cells with successful generation of a humanized mouse model; however, the optimal T cell signature for the successful generation of a humanized mouse model derived from cancer patient cells is poorly understood. Therefore, we examined the relationship between the signature of T cell subsets, focusing on the self-renewal signatures, in PBMCs derived from patients with breast cancer and the successful generation of a humanized immune mouse model. Materials and Methods: We collected PBMCs from 12 patients with breast cancer. All samples were stimulated with interleukin-2 and beads coated with CD3 and CD28 agonist antibodies to expand T cells. After washing, 1 × 10^7 cells/mouse were intraperitoneally injected into NOD/Shi-scid IL2rgamma (null) (NOG) mice. Transplants were performed on three mice per case. Successful engraftment of immune cells into NOG mice was defined by the presence of human CD45+ cells in one or more mice. Self-renewal signatures of in vitro expanded T cells before injection into NOG mice, including the markers T cell factor-1 (TCF-1), CD45RA, CCR7, CD95, and CXCR3, were determined using flow cytometry, mass cytometry and quantitative RT-PCR. Comparisons between groups of data were evaluated by t-test. Results: The success rate of engraftment of immune cells derived from breast cancer patients into NOG mice was 66.7% (8 out of 12 patients). After expansion, the magnitude of the CD8+ stem cell memory subset and the TCF-1 expression level on the CD4+ and CD8+ T cells in the engrafted group were significantly higher than those in non-engrafted group. TCF-1 and CCR7 mRNA levels in the engrafted group were upregulated compared with those in non-engrafted group. The success of engraftment was not associated with clinical characteristics such as breast cancer progression, subtype, or prior chemotherapy treatment. Conclusion: The self-renewal signatures of T cells from breast cancer patients were associated with successful engraftment of a humanized mouse model. These results suggest that prior identification of the self-renewal signatures of the T-cell population is of direct relevance to the appropriate design of a pre-clinical model for testing immunotherapies and for elucidating the characteristics of the tumor immune microenvironment. Citation Format: Yukiko Fukui, Kosuke Kawaguchi, Ryuji Murakami, Wei Li, Yuki Nakamura, Yurina Maeshima, Sunao Tanaka, Shinpei Kawaoka, Eiji Suzuki, Masakazu Toi. Self-renewal signatures of peripheral blood T cells are associated with successful engraftment to establish a humanized mouse model of breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-08-07.

2130. Neonatal Immune Phenotypes and Neurodevelopmental Outcomes in Infants with Congenital Cytomegalovirus Infection (cCMV)

Abstract Background Infants with congenital cytomegalovirus infection (cCMV) are at risk for neurodevelopmental impairment (NDI). Antiviral therapy has been shown to improve neurodevelopmental outcomes but is not recommended for all infected infants. There are currently no predictive markers for NDI in infants with cCMV. Our objective was to compare neonatal T cell differentiation and memory phenotypes in CMV-infected infants with and without subsequent NDI. Methods Blood samples were collected from infants with cCMV and age-matched healthy controls. Peripheral blood mononuclear cells were analyzed by flow cytometry for CD4 and CD8 T cells expressing CD28, CD57, PD, CD45RA/RO, and CCR7. Demographic and clinical data were collected. NDI was defined as Bayley III/IV testing below the average range in at least one domain in infants for whom this developmental testing was available or based on diagnosis by infectious disease physician or primary care provider. Outcomes for infants with follow-up at least 6 months of age were included. Infants with isolated speech delay associated with hearing loss were not considered to have NDI. Results Samples were analyzed from 18 infants (Table 1) at time of diagnosis (≤ 60 days of age) and 5 uninfected age-matched controls. The frequencies of CD8+ T cells expressing CD28low (differentiation), CD57+ (terminal differentiation/senescence), PD1hi (inhibitory or exhausted phenotype) and CD45RO± (memory) phenotypes were higher for the cCMV group without NDI compared to that with NDI (Figure 1). The cCMV group without NDI had lower proportions of naïve (CCR7+/CD45RA+) effector memory, TEM (CCR7-/CD45RA-), and effector memory RA, TEMRA (CCR7-/CD45RA+) CD8+ T cells than those with NDI (Figure 2). There was no difference in memory distribution or surface marker expression of CD4+ T cells. Table 1 Demographic and clinical characteristics of the infants with congenital cytomegalovirus infection (cCMV) with and without neurodevelopmental impairment (NI) and age-matched controls. Continuous variables are compared using the Mann Whitney U test and categorical variables with the Fischer’s Exact test. There were no significant differences between infants with and without NI. Figure 1.CD8+ T cell phenotypes in cCMV groups with and without Neurodevelopmental Impairment (NDI) Frequencies of CD8+ T cells lacking expression of CD28 (A), or expressing CD57 (B), PD-1 (C), or CD45RO (D) are shown at age ≤ 60 days. Infants with congenital CMV (cCMV) and normal neurodevelopmental outcome are shown in blue, infants with neurodevelopmental impairment are shown in red, and uninfected age-matched controls are shown in black. Data are shown as medians [interquartile range]. Groups were compared using the Mann Whitney U Test. Infants with no NDI had significantly higher frequencies of CD28- (p = 0.002), CD57+ (p = 0.001), PD-1+ (p = 0.01) and CD45RO+ (p = 0.01) CD8 T cells. * p ≤ 0.05; ** p ≤ 0.01. Figure 2.T cell memory subset distributions among cCMV groups and uninfected controls Proportions of CD4+ (A) and CD8+ (B) naïve (CCR7+/CD45RA+), central memory, TCM (CCR7+/CD45RA-), effector memory, TEM (CCR7-, CD45RA-), and effector memory RA, TEMRA (CCR7-/CD45RA+) T cells. Infants with congenital CMV (cCMV) and normal neurodevelopmental outcome are shown in blue, infants with neurodevelopmental impairment (NI) are shown in red while uninfected age-matched controls are shown in black and gray. Comparisons between T cell groups were made using the Mann Whitney U test. Infants with cCMV without NI had significantly fewer naïve (p = 0.0001), greater TEM (p = 0.0001), and TEMRA (p = 0.005) CD8+ T cells than those with NI. The distributions of CD4+ T cells were not significantly different. Conclusion In this small cohort of infants infected with CMV, these data suggest that a more differentiated memory CD8+ T cell phenotype in the neonatal period may correlate with normal neurodevelopmental outcomes. Disclosures Alexandra K. Medoro, MD, Merck: Grant/Research Support.