Ratio Of CD4 Research Articles

Temporal patterns of gene expression in response to inoculation with a virulent Anaplasma phagocytophilum strain in sheep

The aim of this study was to characterize the gene expression of host immune- and cellular responses to a Norwegian virulent strain of Anaplasma phagocytophilum, the cause of tick-borne fever in sheep. Ten sheep were intravenously inoculated with a live virulent strain of A. phagocytophilum. Clinical-, observational-, hematological data as well as bacterial load, flow cytometric cell count data from peripheral blood mononuclear cells and host’s gene expression post infection was analysed. The transcriptomic data were assessed for pre-set time points over the course of 22 days following the inoculation. Briefly, all inoculated sheep responded with clinical signs of infection 3 days post inoculation and onwards with maximum bacterial load observed on day 6, consistent with tick-borne fever. On days, 3–8, the innate immune responses and effector processes such as IFN1 signaling pathways and cytokine mediated signaling pathways were observed. Several pathways associated with the adaptive immune responses, namely T-cell activation, humoral immune responses, B-cell activation, and T- and B-cell differentiation dominated on the days of 8, 10 and 14. Flow-cytometric analysis of the PBMCs showed a reduction in CD4+CD25+ cells on day 10 and 14 post-inoculation and a skewed CD4:CD8 ratio indicating a reduced activation and proliferation of CD4-T-cells. The genes of important co-stimulatory molecules such as CD28 and CD40LG, important in T- and B-cell activation and proliferation, did not significantly change or experienced downregulation throughout the study. The absence of upregulation of several co-stimulatory molecules might be one possible explanation for the low activation and proliferation of CD4-T-cells during A. phagocytophilum infection, indicating a suboptimal CD4-T-cell response. The upregulation of T-BET, EOMES and IFN-γ on days 8–14 post inoculation, indicates a favoured CD4 Th1- and CD8-response. The dynamics and interaction between CD4+CD25+ and co-stimulatory molecules such as CD28, CD80, CD40 and CD40LG during infection with A. phagocytophilum in sheep needs further investigation in the future.

Prolonged administration of total glucosides of paeony improves intestinal immune imbalance and epithelial barrier damage in collagen-induced arthritis rats based on metabolomics-network pharmacology integrated analysis.

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease characterized by synovial inflammation and joint damage with complex pathological mechanisms. In recent years, many studies have shown that the dysregulation of intestinal mucosal immunity and the damage of the epithelial barrier are closely related to the occurrence of RA. Total glucosides of paeony (TGP) have been used clinically for the treatment of RA in China for decades, while the pharmacological mechanism is still uncertain. The purpose of this study was to investigate the regulatory effect and mechanism of TGP on intestinal immunity and epithelial barrier in RA model rats. The results showed that TGP alleviated immune hyperfunction by regulating the ratio of CD3+, CD4+ and CD8+ in different lymphocyte synthesis sites of the small intestine, including Peyer's patches (PPs), intraepithelial lymphocytes (IELs), and lamina propria lymphocytes (LPLs). Specially, TGP first exhibited immunomodulatory effects on sites close to the intestinal lumen (IELs and LPLs), and then on PPs far away from the intestinal lumen as the administration time prolonged. Meanwhile, TGP restores the intestinal epithelial barrier by upregulating the ratio of villi height (V)/crypt depth (C) and expression of tight junction proteins (ZO-1, occludin). Finally, the integrated analysis of metabolomics-network pharmacology was also used to explore the possible regulation mechanism of TGP on the intestinal tract. Metabolomics analysis revealed that TGP reversed the intestinal metabolic profile disturbance in CIA rats, and identified 32 biomarkers and 163 corresponding targets; network pharmacology analysis identified 111 potential targets for TGP to treat RA. By intersecting the results of the two, three key targets such as ADA, PNP and TYR were determined. Pharmacological verification experiments showed that the levels of ADA and PNP in the small intestine of CIA rats were significantly increased, while TGP significantly decreased their ADA and PNP levels. In conclusion, purine metabolism may play an important role in the process of TGP improving RA-induced intestinal immune imbalance and impaired epithelial barrier.

CTIM-06. A PHASE 2 STUDY OF A NOVEL IMMUNOTHERAPY SL-701 IN ADULTS WITH RECURRENT GLIOBLASTOMA: EXPLORING THE PROGNOSTIC VALUE OF TREATMENT-INDUCED CD8+CD57+ T-CELLS AS A MARKER FOR SURVIVAL

Abstract Recurrent glioblastoma (GBM) remains a challenging disease with limited therapeutic options and poor prognosis. SL-701 is a novel immunotherapy composed of synthetic peptides designed to elicit an anti-tumor immune response against the overexpressed GBM antigens IL-13Rα2, ephrinA2, and survivin. In this study, we present updated findings from a phase 2 clinical trial (NCT02078648) evaluating SL-701+poly-ICLC+bevacizumab, where the 12-month overall survival (OS) was 50%. Using high-parameter flow cytometry, we compared the quality of the treatment induced T-cell response in patients with an OS < 12m (n = 15) versus OS >12m (n = 12). Among the patients assessed, 89% exhibited heterogeneous T-cell responses against SL-701, with no discernible correlation between the response to a specific peptide and survival. Consequently, we focused on identifying other characteristics of the pan-SL701-specific T-cell response with an association to survival. Assessing all time points collected, patients with an OS >12m generated a significantly higher frequency of SL-701-specific CD8 T-cells (79% increase, P < 0.005) and a lower frequency of CD4+ T-cells (36% decrease, P < 0.05) compared to patients with a lower OS. Moreover, the ratio of CD8:CD4 T-cells was 2-fold higher in patients with an OS >12m indicating a CD8-enriched response, whereas patients with an OS < 12m had a lower ratio of CD8:CD4 associated with CD4 enriched responses. Notably, patients with an OS >12m, expressed CD57 (identifying highly cytotoxic, differentiated memory T-cells) on 40% of their T-cells compared to 18% in patients with an OS < 12m (P < 0.05). Furthermore, the ratio of SL-701 specific CD57:CD107A expressing cells trended 20% lower in patients with an OS >12m, indicative of a replicating, cytotoxic T-cell response that is not terminally differentiated. These qualitative differences in the immune response, detectable as early as week 8 post treatment, may serve as biomarkers for monitoring and predicting survival. Deep sequencing of SL-701-specific T-cells is planned.

Spatial immune composition of tumor microenvironment in patients with pancreatic cancer

This study examined the composition of the immune microenvironment at different sites within resected pancreas specimens from patients with pancreatic ductal adenocarcinoma (PDAC). Therefore, single-cell suspensions were made from fresh tumor and non-tumorous tissue. Fourteen patients were included from whom twelve PDAC and five non-tumorous samples were obtained. These samples were analyzed with a nineteen marker panel on the Aurora spectral flow cytometer. Furthermore, slides from formalin-fixed paraffine PDACs of eight additional patients were stained with eight markers and analyzed by multispectral imaging. These corresponded to central tumor, periphery of the tumor, i.e., invasive front and resected lymph node and were divided into tumor and adjacent tissue. In the single-cell suspension, a decreased ratio between lymphoid and myeloid cells and between M1 and M2 macrophages was observed in the tumor tissue compared to non-tumorous tissue. Furthermore, an increase in CD169 + macrophages in patients undergoing neoadjuvant therapy was found. Using immunofluorescence, more macrophages compared to T cells were observed, as well as a lower ratio of CD8 to M2 macrophage, a higher ratio of CD4–CD8 T cells and a higher ratio of immune-suppressive cells to pro-inflammatory cells in the PDAC area compared to the adjacent non-tumorous tissue. Finally, there were more immune-suppressive cells in the central tumor area compared to the invasive front. In conclusion, we show a gradient in the immune-suppressive environment in PDAC from most suppressive in the central tumor to least suppressive in distant non-tumorous tissue.Graphical abstract

Paired Analysis from Patients with Myeloma Elucidates Favorable CAR-T Manufacture and Phenotype, Starting from Mobilized Pre-Transplant Apheresis Vs. Non-Mobilized Apheresis after Four Prior Lines

Introduction B-Cell Maturation Antigen (BCMA) targeting chimeric antigen receptor T-cells (CAR-T) have demonstrated unprecedented efficacy in patients with relapsed and refractory multiple myeloma, approved after 4 or more prior lines of therapy (PLOT). Given the known T-cell dysfunction that develops over the disease trajectory, autologous T-cells harvested after many PLOT may not be as biologically active as those collected earlier. Estimates suggest 93% of stored stem cell products collected from MM patients are never used (Liang et al, 2021). We sought to demonstrate the feasibility of using stored mobilized apheresis products to generate anti-BCMA CAR-T cells. Given the heterogeneity that can exist between MM patients, we evaluated the differences in starting T-cell phenotype, manufacturing characteristics, and CAR-T phenotype and function side by side using paired samples from the same patient, obtained prior to transplant or prior to commercial CAR-T. Methods We identified a cohort of paired samples from: 1) Mobilized unfractionated apheresis obtained prior to ASCT (preASCT); and 2) Apheresis for CAR-T manufacture after ≥4 PLOT (preCAR). The flow cytometry panel used for phenotyping included antibodies against: CD3, CD4, CD8, CCR7, CD45RO, CD39, TIGIT, PD1, LAG3 and TCF7. Second generation anti-aBCMA.4-1BB.CD3z (aBCMA.BBz) CAR-T cells were manufactured from CD3+ T cells negatively selected from either sample using a Pan T cell isolation kit (Miltenyi). Post manufacture flow cytometry was used to characterize immunophenotype and transduction efficiency. Doubling time was calculated using trypan blue exclusion on D+2 to D+5 after transduction. A BCMA-expressing myeloma cell line, MM.1S, was used to stimulate and evaluate cytotoxicity. Results In 10 patients with paired preASCT and preCAR samples, the median time from ASCT to CAR-T infusion was 4.2 years (range 2.5-12.5 years), all patients had received three drug induction regimens, and 50% had >1 line of combination therapy prior to ASCT. PreASCT samples ranged in collection from Nov 2008 - April 2019. All patients were triple class exposed prior to collection for CAR-T and treated with median 6 PLOT (range 4-13). Comparison of the T-cell composition in both apheresis samples, revealed a higher CD4:CD8 ratio in preASCT samples (p=0.006), and a higher proportion of stem central memory/SCM (CCR7+CD45RO-, p<0.05, Figure 1) in both CD4 and CD8 compartments. Additionally, in preASCT samples, patients with only 1 line of induction, exhibited a trend toward a higher proportion of SCM T-cells relative to more heavily treated patients. PreASCT CD4 (p=0.002) and CD8 (p=0.01) T-cells expressed more TCF7 compared with preCAR, however T-cells with an effector phenotype were proportionally higher in preCAR samples across both CD4 and CD8 compartments (CCR7-CD45RO-, p<0.05, Figure 1). PreCAR T-cells express higher levels of checkpoints (CP) PD1 and TIGIT, with dual positive CP more prevalent in CD4+ T-cells. CAR-T cells manufactured from preASCT samples expanded better (p=0.048) and also had increased CD4:CD8 ratios (p=0.011) as compared to preCAR, without significant differences in transduction efficiency, CAR density or viability. After 24h stimulation with BCMA+ MM.1S, CD8+ CAR-T cell phenotype was different between preASCT and preCAR products, with more SCM in preASCT (p=0.004) and more effector memory/EM (CCR7-CD45RO+) in preCAR (p=0.008); however, the killing capacity after 24h was similar for both CAR-T products at different E:T ratios. The CAR-T cells generated from T-cells preCAR (later in the disease course) expressed higher TIGIT, LAG3 and CD39 after stimulation, and more dual (p=0.027) or triple (p=0.027) positive CP than those from preASCT T-cells ( Figure 2). Conclusion This study suggests CAR-T cells manufactured from banked mobilized unfractionated stem cell product expand better during manufacture, yield an equally cytotoxic, less differentiated, and less exhausted product than those manufactured from later timepoints (preCAR samples). Importantly, we demonstrate two major findings: (1) that a possibly more efficacious anti-BCMA CAR-T product can be manufactured from cryopreserved, mobilized apheresis material regardless of storage time and (2) cryopreserved preASCT samples may expand autologous CAR-T cell therapy access to heavily treated patients with poor T cell quality. CLF & JAM contributed equally

Loss of p53 Impairs Death Receptor Expression and Confers Resistance to CD19-Directed CAR T-Cell Therapy in B-Cell Precursor Acute Lymphoblastic Leukemia

Background In recent years, new therapy strategies such as the antibody-drug conjugate inotuzumab ozogamicin, the bi-specific T-cell engager blinatumomab, and chimeric antigen receptor (CAR) T-cells have been introduced to salvage relapsed precursor B acute lymphoblastic leukemia (ALL). However, recent evidence indicates that these new therapies are ineffective in TP53-aberrant ALL, although the mechanism remains unclear. TP53 is the most frequently mutated gene in cancer, but in ALL, mutations or deletions affecting TP53 are rare, with an incidence of only 3% at diagnosis. However, at relapse, TP53 aberrations are found in approximately 12% of the patients and predict a very poor outcome. Consequently, relapsed TP53 deleted ALL is now classified as ‘very high risk’. Since p53-mediated apoptosis is an endpoint for many (cytotoxic) drugs, loss of p53 function induces therapy resistance to classical chemotherapeutics. Hence, there is a clear clinical need for more effective strategies to treat TP53 aberrant relapsed ALL. Methods Using CRISPR/Cas9, we introduced frameshift mutations in the TP53 gene to generate isogenic wild type and knockout models in TP53 WT ALL cell lines Nalm6 and RCH-ACV. Additionally, we prepared CD19-directed CAR T-cells with a 4-1BB co-stimulatory domain from three healthy donors by isolating, transducing, and expanding CD4+ and CD8+ T-cells separately. With flow cytometry, we assessed survival of p53 WT and p53 KO leukemic cells in the presence of CD4+ and CD8+ CAR T-cells at a 1:1 CD4:CD8 ratio. Moreover, we performed mRNA profiling by qRT-PCR and cell membrane profiling by flow cytometry to determine how loss of p53 affects CAR T mediated killing. Initial findings in cell lines were then validated in patient-derived xenografts. Results We observed that loss of p53 led to up to a 35% decrease in killing by overnight CAR T treatment in both cell line models (Figure 1A). Although loss of p53 has been linked to reduced cell surface expression of CD19 (Pan et al., 2020, Leukemia), no changes in CD19 expression were observed in response to p53 loss. In search for an explanation for the diminished killing, we performed mRNA profiling and found a reduced expression of death receptors CD95/Fas and DR5/TRAILR2 in p53-deficient cells, which was confirmed at the protein level with flow cytometry (Figure 1B). Indeed, CRISPR/Cas9 mediated loss of CD95/Fas expression in p53 wild type cells reduced CAR T mediated cell killing, consistent with a key role for CD95/Fas in T-cell mediated killing. Conversely, treatment of p53 proficient cells with MDM2 inhibitors induced expression of CD95/Fas and DR5/TRAILR2 (Figure 1B), which translated into enhanced killing of target cells. Most of these observations have already been recapitulated in a diagnosis-relapse pair of patient-derived cells in which the diagnosis was p53wild typeand the relapse was p53 deficient (Figure 1A-B). We observed lower baseline mRNA expression of CD95/Fas and DR5/TRAILR2 in p53-deficient cells, accompanied by a 50% reduction in killing relative to the p53 wild type controls. Conversely, MDM2 inhibition increased death receptor expression only in p53-proficient cells. The ongoing experiments validating enhanced killing after MDM2 inhibition in patient-derived cells will be presented during the meeting. Conclusions Loss of p53 impairs expression of CD95/Fas and DR5/TRAILR2, thereby protecting ALL cells from CAR T induced cell death. In addition, our results indicate that the p53-controlled expression of these receptors may be exploited to increase the efficacy of CAR T-cell therapy in p53-proficient patients.

Biomarker Correlates of Response to Ciltacabtagene Autoleucel in Patients with Relapsed or Refractory Multiple Myeloma from CARTITUDE-1, a Phase 1b/2 Open-Label Study, at the ~3 Year Follow-up

Ciltacabtagene autoleucel (cilta-cel) consists of autologous T cells genetically modified to express a two-binding domain chimeric antigen receptor (CAR). The target antigen of the CAR is B-cell maturation antigen (BCMA), which is highly expressed on malignant plasma cells (PC) from multiple myeloma subjects. In the Phase 1b/2 CARTITUDE-1 study, cilta-cel led to early, deep, and durable responses and was approved for the treatment of patients with relapsed or refractory multiple myeloma (RRMM) after four or more prior lines of therapy by FDA. Despite cilta-cel's outstanding overall response rate (97.9%) and duration of response (DOR) in RRMM patients, relapse still occurs. Biomarkers associated with this durable response and acquired resistance remain to be elucidated. We present updated correlative data from the CARTITUDE-1 study. At the ~3 year follow-up (October 2022 data cutoff), 97 patients had received a single infusion of cilta-cel (median 0.71 x10 6 cells/kg [range 0.52 x10 6-0.94 x10 6]); the median follow up was 33.4 months (range 1.5-45.2), median DOR was 33.9 months (95% CI, 25.5-not estimable [NE]) and median PFS was 34.9 months (95% CI, 25.2-NE) (Lin Y et al, ASCO 2023). Drug product (DP), baseline and on-treatment whole blood and bone marrow samples from enrolled patients were analyzed by methods including flow cytometry, MSD immunoassays, Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) and TCR sequencing. In CARTITUDE-1, the DP contained a mixture of transduced and non-transduced T cells; the median transduction efficiency was 16% (range 5-32%), with a balanced distribution of CAR+CD4+ and CAR+CD8+ cells, median frequency 12% (range 2-28%) and 6% (range 2-20%), respectively. Further, the DP T cell subset composition was variable but balanced between central (Tcm) and effector memory (Tem) phenotypes. CAR+ T cells expanded reaching a median peak concentration (C max of 730 cells/µL; range 3-13805 cells/µL) in blood between days 12-14 post-infusion and persisted in circulation for a median of 100 days (range 20-912 days). High efficacy (best response) and DOR were achieved despite variable CAR-T cell expansion and lack of detectable CAR-T cell persistence over time. At C max, the CD4:CD8 ratio (based on % of CAR+ cells) was 0.29, demonstrating a CAR+CD8+ T cell preferential expansion, and both CAR+CD4+ and CAR+CD8+ T cells were predominantly of a Tcm phenotype, median 95% (range 62-99.5%) and 96% (range 33-99.7%), respectively. Higher frequency of early-memory T cell phenotypes has been associated with clinical responses to CAR-T therapies in MM (Dancy et al., Blood 2018). CITE-seq analyses showed that DP characteristics such as a high CD8+ stem-like phenotype and a low CD4+ Treg-like phenotype in the CAR+ T cell compartment associated with longer DOR. Some of the most abundant clones in the DP single cell TCR data were CAR+CD8+ T cells expressing granzyme B, suggesting high cytotoxic potential. Additional bulk TCR sequencing data suggest both diversity and individual clonal expansion longitudinally after cilta-cel infusion. We also evaluated tumor and patient intrinsic characteristics at baseline. BCMA expression on bone marrow PC was prevalent and highly variable among RRMM patients; however, expression did not associate with best response or DOR. Further, high ORR was observed in patients with high-risk cytogenetics, high tumor burden (>= 60% bone marrow PC), or baseline plasmacytomas, although with a non-statistically significant shorter PFS. Sustained MRD negativity strongly associated with longer DOR. Conversely, patient-intrinsic characteristics associated with inflammation (CRP, ferritin, neutrophils, pro-inflammatory cytokines) correlated with shorter DOR. Treatment benefit of the magnitude seen with cilta-cel had not been reported with any other approved therapy in this setting. Correlative analyses suggest an emerging profile to help understand such outstanding clinical activity: a balanced CD4/CD8 ratio and Tcm/Tem phenotype of the manufactured DP and preferential expansion of CAR+CD8+ Tcm cells. Further, certain baseline patient-intrinsic and tumor characteristics may explain the longer DOR in the RRMM setting. These investigations help identify markers of response to cilta-cel and may lead to CAR-T cell design or manufacturing strategies that enhance drug product characteristics and thus clinical efficacy.

Accomplices in Cure: Blinatumomab + Dasatinib Reduces TCR Signaling and Effector Function Stronger Than Ponatinib and Ameliorates T-Cell Exhaustion

The tyrosine kinase inhibitors (TKIs) dasatinib (dasa) and ponatinib (pona) are being tested in clinical trials in philadelphia chromosome-positive (Ph +) B-cell precursor acute lymphoblastic leukemia (BCP-ALL) patients in combination with the CD19xCD3 bispecific blinatumomab (blina). Pona but not dasa is able to bind the BCR-ABL T315I mutation, which frequently occurs in BCP-ALL patients. Therefore, clinical studies are moving away from dasa and favor pona. Nevertheless, both TKIs in combination with blina have led to striking remission rates in Ph + BCP-ALL (complete response dasa: 98%, Foá 2020; pona: 92%, Jabbour 2023). Interestingly, increased lymphocyte frequencies were reported in patients under blina + dasa therapy (Puzzolo 2021), indicating that the effect of dasa is not limited to toxicity towards ALL cells conferred by BCR-ABL inhibition. Indeed, dasa modulates T-cell function by interfering with TCR signaling (Mestermann 2019). Also, we have shown that resting of T cells by high-dose dasa treatment ameliorates bispecific-induced T-cell exhaustion (Philipp 2022). In contrast, less is known about the effect of pona on T-cell function. Based on these data, we hypothesize that TKIs fine-tune TCR signaling, which ameliorates T-cell exhaustion during continuous blina infusion. Here, we systematically compared the influence of dasa and pona on blina-mediated TCR signaling and T-cell function. Inhibition of T-cell activation and function by dasa or pona at 0.3 - 100 nM was tested in vitro over 3 days: (1) CD69 expression, (2) blina-mediated killing as specific lysis of CD19 + tumor cells (3) T-cell expansion analyzed by dye dilution or CD2+ fold change (FC), (4) cytokine secretion measured by cytokine catch or cytometric bead array. Inhibition of TCR signaling was evaluated using a Jurkat-NFAT T-cell line stimulated for 6h with blina and CD19 + tumor cells. Phosphoflow of pAKT and pZAP70 was performed on CD3/CD28 activated primary human T cells. Toxicity of the TKIs against BCR-ABL +/- cell lines was evaluated after 3 days. T-cell exhaustion upon continuous blina + dasa stimulation was assessed in an in vitro long-term culture system for continuous bispecific exposure (Philipp 2022). Functional assays as described in (1)-(4) were performed every 7 days with isolated T cells from long-term cultures. Both TKIs inhibited T-cell function in a dose-dependent manner. Surprisingly, dasa was more potent than pona at low-intermediate doses in reducing T-cell function, shown by CD69 expression (mean % at TKI = 3 nM: basal = 70, dasa = 25, pona = 64, p<0.0001), cytotoxic function (mean % specific lysis at TKI = 12.5 nM: basal = 55, dasa = 0, pona = 54, p<0.0001), T-cell proliferation (mean % proliferated at TKI = 1.5 nM: basal = 62, dasa = 25, pona = 51, p<0.0001), as well as IFN secretion (normalized mean fluorescence intensity (MFI) at TKI = 12.5 nM: basal = 655, dasa = 0, pona = 924, p<0.0001). We also observed that NFAT engagement was significantly lower in dasa-treated Jurkat-NFAT cells (relative luminescence units at TKI 12.5 nM: basal = 1620, dasa = 392, pona = 996, p<0.001). Similarly, phosphoflow analyses revealed lower levels of pAKT and pZAP70 upon CD3/CD28 + dasa versus pona treatment (MFI of pAKT at TKI = 25 nM: basal = 2347, dasa = 1642, pona = 2045). Importantly, both TKIs induced cell death of BCR-ABL + but not BCR-ABL - cell lines at concentrations >3 nM. Interestingly, 14 days continuously stimulated T cells with blina + dasa (12.5 nM) did not lead to co-expression of inhibitory checkpoints (mean % of PD-1 +Tim-3 +LAG-3 + T cells on day 14: blina = 31, +dasa = 0.6, p<0.01). Furthermore, when isolated from long-term cultures and tested in functional assays, blina + dasa treated T cells maintained high cytokine secretion (mean IL-2 in pg/ml on day 14: blina = 730, blina + dasa = 5638, p<0.01), granzyme B expression (mean MFI ratio of CD8 + on day 14: blina = 96, blina + dasa = 335, p<0.01) and cytotoxic activity (mean % specific lysis on day 14: blina = 61, blina + dasa = 80). Together, these findings indicate that while both TKIs confer comparable toxicity to BCR-ABL + leukemia cells, dasa dampens TCR signaling and T-cell effector function more potently than pona. Strikingly, dasa can ameliorate T-cell exhaustion upon continuous blina exposure. Our data suggest that low-dose TKIs can fine-tune T-cell activity and might play a considerable role for the clinical success of combinatorial bispecific and TKI therapy in Ph + BCP-ALL.

CD8+ chimeric antigen receptor T cells manufactured in absence of CD4+ cells exhibit hypofunctional phenotype

BackgroundCell culture conditions during manufacturing can impact the clinical efficacy of chimeric antigen receptor (CAR) T cell products. Production methods have not been standardized because the optimal approach remains unknown....

An atlas of immune cell transcriptomes in human immunodeficiency virus-infected immunological non-responders identified marker genes that control viral replication.

Previous studies have examined the bulk transcriptome of peripheral blood immune cells in acquired immunodeficiency syndrome patients experiencing immunological non-responsiveness. This study aimed to investigate the characteristics of specific immune cell subtypes in acquired immunodeficiency syndrome patients who exhibit immunological non-responsiveness. A single-cell transcriptome sequencing of peripheral blood mononuclear cells obtained from both immunological responders (IRs) (CD4 + T-cell count >500) and immunological non-responders (INRs) (CD4 + T-cell count <300) was conducted. The transcriptomic profiles were used to identify distinct cell subpopulations, marker genes, and differentially expressed genes aiming to uncover potential genetic factors associated with immunological non-responsiveness. Among the cellular subpopulations analyzed, the ratios of monocytes, CD16 + monocytes, and exhausted B cells demonstrated the most substantial differences between INRs and IRs, with fold changes of 39.79, 11.08, and 2.71, respectively. In contrast, the CD4 + T cell ratio was significantly decreased (0.39-fold change) in INRs compared with that in IRs. Similarly, the ratios of natural killer cells and terminal effector CD8 + T cells were also lower (0.37-fold and 0.27-fold, respectively) in the INRs group. In addition to several well-characterized immune cell-specific markers, we identified a set of 181 marker genes that were enriched in biological pathways associated with human immunodeficiency virus (HIV) replication. Notably, ISG15 , IFITM3 , PLSCR1 , HLA-DQB1 , CCL3L1 , and DDX5 , which have been demonstrated to influence HIV replication through their interaction with viral proteins, emerged as significant monocyte marker genes. Furthermore, the differentially expressed genes in natural killer cells were also enriched in biological pathways associated with HIV replication. We generated an atlas of immune cell transcriptomes in HIV-infected IRs and INRs. Host genes associated with HIV replication were identified as markers of, and were found to be differentially expressed in, different types of immune cells.

Monophosphoryl lipid A-assembled nanovaccines enhance tumor immunotherapy.

Therapeutic cancer nanovaccines can induce strong antitumor immunity and establish long-term immune memory and have shown potential for curing tumors in some clinical trials. However, weak immunogenicity and safety concerns of nanocarriers limit the clinical translation of some therapeutic nanovaccines. Here, we developed minimal-component cancer nanovaccines, monophosphoryl lipid A (MPLA)-assembled nanovaccines (MANs), that could facilitate the clinical application of nanovaccines. The MANs were formed by protein antigens extracted from chemotherapy-induced tumor cell cultures and the amphiphilic immune adjuvant MPLA. Compared with free chemotherapy-induced antigens, MANs can activate the Toll-like receptor 4 (TLR4)-mediated signalling pathway and promote adaptive immunity against tumor antigens. Mechanistic analysis indicated that MANs induced antigen capture of DCs and promoted the activation of DCs and T cells, thereby optimizing the ratio of CD8+ T/Tregs in tumors and facilitating the transformation of the tumor immune microenvironment (TIME) from "cold" to "hot". In a CT26 colorectal cancer model, MANs+αPD-1 significantly improved the efficacy of αPD-1 treatment. Our work offers a strategy for designing minimal-component cancer nanovaccines with potential clinical benefits. STATEMENT OF SIGNIFICANCE: To address the weak immunogenicity of cancer vaccines and the safety concerns of nanocarriers, we prepared MPLA-assembled nanovaccines (MANs) using chemotherapy induced antigens and the immune adjuvant MPLA to promote cancer vaccines to clinical practice. MANs effectively internalized tumor antigens and induced DC maturation, indicating that the initial anti-tumor response had been activated. MANs+αPD-1 induced APCs, CD8+ T cells and memory T cells with positive anti-tumor effects to migrate to tumor tissue, thus leading to the transformation of the tumor immune microenvironment from "cold" to "hot". At the animal level, the combination of MANs and αPD-1 exerted synergistic effects and significantly enhanced tumor immunotherapy. Therefore, the treatment regimen of MANs+αPD-1 has potential clinical benefits.

Clinical and laboratory features in health care volunteers with inactivated SARS-CoV-2 vaccination.

To better optimize the inactivated vaccine-induced immune response and improve vaccine protection efficiency, a preliminary study was conducted on the influencing factors of producing neutralizing antibody (NAb) titers against the inactivated coronavirus disease 2019 (COVID-19) vaccine. A total of 91 health care volunteers were enrolled from the Immunology Division of the Laboratory Department of Chongqing General Hospital from February to March 2021. The study had a cross-sectional design. All of the volunteers were scheduled to receive a complete dose regimen of the inactivated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine and the vaccination interval between 2 doses was 14 days. Clinical and laboratory features were collected for further analysis. The NAb titers gradually increased after COVID-19 vaccination, and 72.53% (n = 66) of the volunteers had NAbs after the second dose. Eight variables, including CD16+CD56+ NK cell level before the first dose (HR = 0.94, p = 0.02), CD16+CD56+ NK cell level after the second dose (HR = 0.94, p = 0.03), interleukin (IL)-2 level before the first dose (HR = 2.09, p = 0.05), mean corpuscular volume (HR = 0.86, p = 0.02), serum urea level (HR = 0.69, p = 0.05), increment of CD19+ B cells (HR = 0.86, p = 0.03), increment of CD4+/CD8+ T cells (HR = 0.21, p = 0.03), and increment of the IL-6 level (HR = 0.75, p = 0.04) demonstrated a correlation with the NAb titers after COVID-19 vaccination. In the multivariate logistical regression analysis, the serum urea level (HR = 2.32, P = 0.03) and increment of CD19+ B cells (HR = 1.96, p = 0.03) were positively correlated with the NAb titers. The principal component analysis effectively distinguished the response after COVID-19 vaccination. The Pearson correlation analysis indicated that the CD19+ B cell level (r = 0.23, p < 0.001) and IL-2 (r = 0.24, p < 0.001) and IL-6 levels (r = 0.22, p < 0.001) were weakly positively correlated with the concentration of NAbs. The NAbs titers of the inactivated vaccines were positively correlated with the ratio of CD19+ B cell, IL-6, and IL-2 levels in the serum, which provide clinical guidance for inactivated SARS-CoV-2 vaccines.

Dupilumab-Associated Lymphoid Reactions in Patients With Atopic Dermatitis

Since the increased use of dupilumab for atopic dermatitis (AD) in daily practice, several cases have been reported on the development of cutaneous T-cell lymphomas (CTCL) and lymphoid infiltrates. To provide insight in the clinical and histopathologic features of patients with AD clinically suspected for CTCL during dupilumab treatment. This retrospective observational case series included adult (≥18 years) patients with AD treated with dupilumab between October 2017 and July 2022 at the University Medical Center Utrecht in the Netherlands. Relevant patient, disease, and treatment characteristics were evaluated. Skin biopsies before, during, and after treatment were collected and reassessed. Fourteen patients (54.5% male) with a median (IQR) age of 56 (36-66) years suspected for CTCL with deterioration of symptoms during dupilumab treatment were included. Of 14 patients, 3 were retrospectively diagnosed with preexistent mycosis fungoides (MF). Eleven patients with AD were eventually diagnosed with a lymphoid reaction (LR). These patients showed MF-like symptoms; however, histopathologic findings were different, and included sprinkled distribution of small hyperchromatic lymphocytes in the upper epidermal section, a dysregulated CD4:CD8 ratio, and CD30 overexpression, without loss of CD2/CD3/CD5. The median time to clinical worsening was 4.0 months (IQR, 1.4-10.0). Posttreatment biopsies showed complete clearance of the LR in all patients. This study found that dupilumab treatment can cause a reversible and benign LR, which mimics a CTCL, though has distinctive histopathologic features.

Features of the leukocyte pool of umbilical cell blood cell concentrate prepared for long-term cryostorage

Allogeneic hematopoietic cell transplantation is the standard of care for patients with highrisk acute leukemia. Cord blood (UCB) is an alternative source of allogeneic hematopoietic stem cells for patients who need transplantation but do not have a related donor. The purpose of our study was to evaluate the characteristics of leukocyte pool (cellular composition, immunophenotype) of umbilical cord blood (UCB) cell concentrates, which were harvested for public long-term cryostorage for the needs of transplantology. A study of 1096 samples of umbilical cord blood of full-term newborns before and after processing [isolation of hematopoietic stem cells (HSC) concentrate] was carried out. The number of cells in the pooled concentrate of HSC at a volume of 25 mL, after long-term cryostorage was as follows: leukocytes, 17.41±0.36 × 108; HSCs with CD34+ immunophenotype, 5.25±3.6 × 106; natural killer cells (CD3-CD16+CD56+), 1.65+0.7 × 108. Analysis of the NKT cell population revealed 6.22±2.1% of cells with CD3+CD16/CD56+ immunophenotype. The contents of CD3+CD56+ cells was 2.69±2.4%, the relative amount of CD3+CD16+ was 2.53±1.2%. In the pooled NKT cell preparations, 5.15±2.1% of CD56+CD16 cells were detected, and two minor subpopulations were also identified, which differ in CD56 and CD16 antigen expression: the level of CD56-CD16+ was 2.91±1.2%, and the ratio of CD56+CD16+ cells was 0.69±0.3%. The obtained data on relative amounts and characteristics of cellular immunophenotype as well as the heterogeneity of the NK and NKT cell population in the HSC UCB concentrate, subjected to long-term cryostorage must be taken into account when selecting the HSC UCB concentrates for the purpose of transplantation in oncohematological diseases.

Profibrotic COVID-19 subphenotype exhibits enhanced localized ER-dependent HSP47+ expression in cardiac myofibroblasts in situ

We recently described a subgroup of autopsied COVID-19 subjects (∼40%), termed ‘profibrotic phenotype,’ who exhibited clusters of myofibroblasts (Mfbs), which were positive for the collagen-specific chaperone heat shock protein 47 (HSP47+) in situ. This report identifies increased, localized (hot spot restricted) expression of αSMA, COLα1, POSTN and FAP supporting the identity of HSP47+ cells as myofibroblasts and characterizing a profibrotic extracellular matrix (ECM) phenotype. Coupled with increased GRP78 in COVID-19 subjects, these data could reflect induction of the unfolded protein response for mitigation of proteostasis (i.e., protein homeostasis) dysfunction in discrete clusters of cells. ECM shifts in selected COVID-19 subjects occur without significant increases in either global trichrome positive staining or myocardial injury based quantitively on standard H&E scoring. Our findings also suggest distinct mechanism(s) for ECM remodeling in the setting of SARS-CoV-2 infection. The ratio of CD163+/CD68+ cells is increased in hot spots of profibrotic hearts compared with either controls or outside of hot spots in COVID-19 subjects. In sum, matrix remodeling of human COVID-19 hearts in situ is characterized by site-restricted profibrotic mediated (e.g., HSP47+ Mfbs, CD163+ Mφs) modifications in ECM (i.e., COLα1, POSTN, FAP), with a strong correlation between COLα1 and HSP47+cells within hot spots. Given the established associations of viral infection (e.g., human immunodeficiency virus; HIV), myocardial fibrosis and sudden cardiac death, early screening tools (e.g., plasma biomarkers, noninvasive cardiac magnetic resonance imaging) for diagnosis, monitoring and treatment of fibrotic ECM remodeling are warranted for COVID-19 high-risk populations.

Characterization of CAR T Cells Manufactured Using Genetically Engineered Artificial Antigen Presenting Cells.

Chimeric antigen receptor (CAR) T cell therapy has recently emerged as a promising approach for the treatment of different types of cancer. Improving CAR T cell manufacturing in terms of costs and product quality is an important concern for expanding the accessibility of this therapy. One proposed strategy for improving T cell expansion is to use genetically engineered artificial antigen presenting cells (aAPC) expressing a membrane-bound anti-CD3 for T cell activation. The aim of this study was to characterize CAR T cells generated using this aAPC-mediated approach in terms of expansion efficiency, immunophenotype, and cytotoxicity. In this experimental study, we generated an aAPC line by engineering K562 cells to express a membrane-bound anti-CD3 (mOKT3). T cell activation was performed by co-culturing PBMCs with either mitomycin C-treated aAPCs or surface-immobilized anti-CD3 and anti-CD28 antibodies. Untransduced and CD19-CARtransduced T cells were characterized in terms of expansion, activation markers, interferon gamma (IFN-γ) secretion, CD4/CD8 ratio, memory phenotype, and exhaustion markers. Cytotoxicity of CD19-CAR T cells generated by aAPCs and antibodies were also investigated using a bioluminescence-based co-culture assay. Our findings showed that the engineered aAPC line has the potential to expand CAR T cells similar to that using the antibody-based method. Although activation with aAPCs leads to a higher ratio of CD8+ and effector memory T cells in the final product, we did not observe a significant difference in IFN-γ secretion, cytotoxic activity or exhaustion between CAR T cells generated with aAPC or antibodies. Our results show that despite the differences in the immunophenotypes of aAPC and antibody-based CAR T cells, both methods can be used to manufacture potent CAR T cells. These findings are instrumental for the improvement of the CAR T cell manufacturing process and future applications of aAPC-mediated expansion of CAR T cells.

Expression of Tim-3/Galectin-9 pathway and CD8+T cells and related factors in patients with cystic echinococcosis

ObjectiveOne of the primary reasons for the successful patriotization of Echinococcus multilocularis in patients is its ability to induce host immune tolerance. This study examined the expression of the immunosuppressive Tim-3/Galectin-9 pathway, CD8+T cells, and related factors in AE patients. The aim was to analyze the relationship between the Tim-3/Galectin-9 pathway and CD8+T cells in this disease and further understand the mechanism of immune tolerance induced by cystic echinococcosis. MethodsUsing flow cytometry, we evaluated the expression of CTL, CD8+CD28-T cells, CD8+CD28 + IFN-γ + T cells, CD8+CD28+perforin + T cells, CD8+CD28+granzyme B + T cells, CD8+CD28-IL-10 + T cells, CD8+CD28-TGF-β+T cells, and Tim-3 expression on CD8+T cells in the peripheral blood of control (n = 30) and AE patients (n = 33). qRT-PCR was used to measure CD107a and Tim-3/Galectin-9 mRNA levels in PBMCs from the control and AE groups. Immunohistochemistry was employed to detect IL-10, TGF-β, and Tim-3/Galectin-9 expressions in the infected livers of AE patients. ResultsAE patients exhibited a significant decrease in peripheral blood CTL ratio (P < 0.001) and an increase in CD8+CD28+IFN-γ+T cell ratio (P < 0.001). No significant changes were observed in the ratios of CD8+CD28+perforin + T cells (P = 0.720) and CD8+CD28+granzyme B + T cells (P = 0.051). The proportions of CD8+CD28-T cells (P < 0.001), CD8+CD28-IL-10 + T cells (P < 0.001), and CD8+CD28-TGF-β+T cells (P < 0.001) were notably higher than in the control group. The expression of Tim-3 on CTL and CD8+CD28-T cells in AE patients was significantly upregulated (P < 0.001, P < 0.001). AE patients displayed a substantial decrease in peripheral blood PBMC CD107a mRNA levels (P < 0.001) and significant elevations in Tim-3/Galectin-9 mRNA levels (P < 0.001, P < 0.001). A negative correlation was observed between CD107a mRNA levels and both Tim-3 (r^2 = 0.411, P < 0.001) and Galectin-9 (r2 = 0.180, P = 0.019) mRNA levels. Expressions of IL-10 (P < 0.001), TGF-β (P < 0.001), and Tim-3/Galectin-9 (P < 0.001, P < 0.001) in AE patient-infected livers were significantly higher than in uninfected regions. IL-10 and TGF-β expressions showed a positive correlation with Tim-3/Galectin-9. ConclusionThis study suggests that the high expression of Tim-3 on CD8+T cell surfaces in AE patients might promote an increase in CD8+CD28-T cells and related factors, while suppressing CTL and related factor expressions. This potentially induces the onset of immune tolerance, which is unfavorable for the clearance of Echinococcus multilocularis in patients, leading to the exacerbation of persistent infections.

Clinical Outcomes and Immunotoxicity in People with HIV (PWH) after Definitive Chemoradiation (CRT) for Anal Squamous Cell Carcinoma

Anal cancer disproportionately affects PWH despite antiretroviral therapy. Data on CRT outcomes are limited. Modern IMRT CRT decreases acute toxicity but may exacerbate immune dysregulation from chronic HIV. Although historical CRT has been associated with prolonged CD4 count suppression, little is known about late immunotoxicity in PWH after contemporary CRT. We report clinical outcomes and long term immunotoxicity. Single-center retrospective review of all PWH confirmed on chart review with anal squamous cell carcinoma without prior pelvic irradiation treated with definitive IMRT CRT. Patient and CRT factors including HIV suppression (<200 copies/mL), mean CD4 count (cells/µL), and receipt of capecitabine (C) or 5-fluorouracil (F) +/- mitomycin (M) were summarized with n (%) or median (interquartile range). Progression-free and overall survival (PFS; OS) were estimated per Kaplan-Meier with 95% confidence intervals and compared with log-rank tests. Mean CD4 count and CD4:CD8 were compared by HIV suppression status (Welch's t-test); longitudinal changes in median CD4 count and CD4:CD8 were compared between baseline vs. nadir (within 6 months of CRT start) and 1-year follow-up for patients with complete data (Wilcoxon signed-rank test). A total of 23 PWH were treated between 2010-2022, median age 52, median 16 (13 - 19) years after HIV diagnosis; 4 had unsuppressed HIV; AJCC 8th stage I/II/III/IV 5/5/12/1. Radiation dose was median 54 Gy in 30 fractions over 42 (40 - 44) days. Most had C+M (57%); only 43% had 2×M with either C or F. One had neoadjuvant carboplatin/paclitaxel/pembrolizumab. With 2.9 (1.03 - 3.3) years follow-up, median OS was 6.6 (6.2 - unreached [UR]) years. With 2.2 (0.67 - 2.7) years follow-up, median PFS was UR. OS and PFS were similar regardless of HIV suppression status (both P ≥ 0.09). Overall baseline CD4 count was 458 (226 - 484), and CD4:CD8 was 0.54 (0.2 - 0.7). Nadir CD4 was 100 (59 - 126) and CD4:CD8 was 0.3 (0.2 - 0.4). Baseline and nadir CD4 count and CD4:CD8 were lower if HIV-unsuppressed (each P ≤ 0.04). One year after CRT, CD4 count was 252 (102 - 276), while CD4:CD8 was 0.5 (0.2 - 0.7). For 7 patients with repeated values the change in median from baseline to nadir, 6-, and 12-months post-CRT was -282, -549 (both P = 0.02), -480 (P = 0.9) for CD4 counts, and -0.7, -0.5, -0.4 (each P > 0.5) for CD4:CD8 ratios; none had unsuppressed HIV. Definitive IMRT CRT with guideline-concordant doublet chemotherapy for anal cancer in PWH is effective despite unsuppressed HIV. Treatment leads to prolonged immunological changes that may increase the risk of HIV-related morbidity and mortality. Modifiable treatment-related causes of hematoimmunologic toxicity should be investigated further, and immune surveillance after CRT should be considered to better understand impact on quality of life.

Molecular profiles of different PD-L1 expression in patients with esophageal squamous cell carcinoma

ABSTRACT Background PD-1/PD-L1 inhibitors are approved treatments for patients with esophageal squamous cell carcinoma (ESCC). The present investigation aspired to explore the interrelation between molecular phenotype and PD-L1 expression in ESCC. Methods PD-L1 testing and targeted next-generation sequencing (NGS) were performed on tumoral tissues from 139 ESCC patients. Tumor-infiltrating lymphocytes (TILs) were scrutinized using a tyramide signal amplification system combined with immunohistochemistry. Results Among enrolled patients, 36.7% displayed high PD-L1 expression (combined positive score [CPS] ≥10). BRCA1 and NF1 gene mutations were significantly associated with high PD-L1 expression (p < .05) while TGFβ pathway alterations were linked to low PD-L1 expression (p = .02). High copy number instability (CNI) and copy number alterations (CNA) were correlated with low PD-L1 expression. Patients with CDKN2A deletion exhibited higher PD-L1 expression. Varying types of TILs were observed across different PD-L1 expression groups. The ratio of CD8+PD-L1+ T cells and CD8+PD-1+ T cells to CD8+ T cells remained comparable in both tumoral and stromal regions, but the ratio of CD68+PD-L1+ macrophages to CD68+ macrophages was higher than the ratio of CD68+PD-1+ macrophages to CD68+ macrophages. CPS was significantly correlated with PD-L1+ lymphocytes and CD68+ macrophages in the tumoral region. CD8+ T cell infiltration was positively correlated with PD-1+ cells in both tumoral and stromal regions. Conclusion In this study, we presented the prevalence rates of PD-L1 expression in Chinese ESCC patients. The association of genetic profiles with PD-L1 expression levels also provide the clue that genomic phenotype may interact with the immunologic phenotype in ESCC.

Mesenchymal stromal cells ameliorate chronic GVHD by boosting thymic regeneration in a CCR9-dependent manner in mice.

Chronic graft-versus-host disease (cGVHD) is a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation. Mature donor T cells within the graft contribute to severe damage of thymic epithelial cells (TECs), which are known as key mediators in the continuum of acute GVHD (aGVHD) and cGVHD pathology. Mesenchymal stromal cells (MSCs) are reportedly effective in the prevention and treatment of cGVHD. In our previous pilot clinical trial in patients with refractory aGVHD, the incidence and severity of cGVHD were decreased, along with an increase in levels of blood signal joint T-cell receptor excision DNA circles after MSCs treatment, which indicated an improvement in thymus function of patients with GVHD, but the mechanisms leading to these effects remain unknown. Here, we show in a murine GVHD model that MSCs promoted the quantity and maturity of TECs as well as elevated the proportion of Aire-positive medullary TECs, improving both CD4+CD8+ double-positive thymocytes and thymic regulatory T cells, balancing the CD4:CD8 ratio in the blood. In addition, CCL25-CCR9 signaling axis was found to play an important role in guiding MSC homing to the thymus. These studies reveal mechanisms through which MSCs ameliorate cGVHD by boosting thymic regeneration and offer innovative strategies for improving thymus function in patients with GVHD.