Adoptive Immunotherapy Research Articles

Efficacy and limitations of combined A2.CAR Treg and anti-CD154 therapy in a mouse model of haplo-mismatched heart transplantation.

The ability to induce allograft specific tolerance would reduce the need for daily pharmacological immunosuppression, improve post-transplant quality of life and transplant outcomes. Adoptive cell therapy with regulatory T cells expressing donor-specific Chimeric Antigen Receptors (CAR-Tregs) is a promising strategy, but monotherapy has not resulted in prolonged survival of grafts with multiple MHC mismatches. We used a clinically-relevant, haplo-mismatched model of heart transplantation in immune-competent C57BL/6 recipients to test the ability of HLA-A2-specific (A2) CAR Tregs to synergize with CD154 blockade to enhance graft survival. We found that in combination with a single low dose of anti-CD154, A2.CAR Tregs significantly prolonged heart allograft survival. Through use of grafts expressing the 2W-OVA transgene, tetramer tracking of 2W- and OVA-specific cells revealed that in mice with accepted grafts, the effects of A2.CAR Tregs included inhibition of endogenous donor-specific CD4 + and CD8 + T cell expansion, and B cell and antibody responses. Moreover, within the 2W-specific CD4 + T cell population, there was a significant increase in the proportion of FoxP3 pos cells, suggestive of infectious tolerance. In mice where CD154 blockade and A2.CAR Tregs failed to prolong graft survival, there was preferential expansion of FoxP3 neg A2.CAR T cells within the rejecting allograft. Thus, this study provides the first evidence for a synergistic effect between CAR Tregs and CD40-pathway blockade and supports the further refinement of this strategy as a promising future direction towards the goal of transplantation tolerance.

Feeder cell training shapes the phenotype and function of in vitro expanded natural killer cells.

Natural killer (NK) cells are candidates for adoptive cell therapy, and the protocols for their activation and expansion profoundly influence their function and fate. The complexity of NK cell origin and feeder cell cues impacts the heterogeneity of expanded NK (eNK) cells. To explore this, we compared the phenotype and function of peripheral blood-derived NK (PB-NK) and umbilical cord blood-derived NK (UCB-NK) cells activated by common feeder cell lines, including K562, PLH, and 221.AEH. After first encounter, most PB-NK cells showed degranulation independently of cytokines production. Meanwhile, most UCB-NK cells did both. We observed that each feeder cell line uniquely influenced the activation, expansion, and ultimate fate of PB eNK and UCB eNK cells, determining whether they became cytokine producers or killer cells. In addition, they also affected the functional performance of NK cell subsets after expansion, that is, expanded conventional NK (ecNK) and expanded FcRγ- NK (eg-NK)cells. Hence, the regulation of eNK cell function largely depends on the NK cell source and the chosen expansion system. These results underscore the significance of selecting feeder cells for NK cell expansion from various sources, notably for customized adoptive cell therapies to yield cytokine-producing or cytotoxic eNK cells.

Optimizing Adoptive Cell Therapy for Solid Tumors via Epigenetic Regulation of T‐cell Destiny

Optimizing Adoptive Cell Therapy for Solid Tumors via Epigenetic Regulation of T‐cell Destiny

Novel immunotherapeutic approaches in gastric cancer.

Gastric cancer is a malignant tumor that ranks third in cancer-related deaths worldwide. Early-stage gastric cancer can often be effectively managed through surgical resection. However, the majority of cases are diagnosed in advanced stages, where outcomes with conventional radiotherapy and chemotherapy remain unsatisfactory. Immunotherapy offers a novel approach to treating molecularly heterogeneous gastric cancer by modifying the immunosuppressive tumor microenvironment. Immune checkpoint inhibitors and adoptive cell therapy are regarded as promising modalities in cancer immunotherapy. Food and Drug Administration-approved programmed death-receptor inhibitors, such as pembrolizumab, in combination with chemotherapy, have significantly extended overall survival in gastric cancer patients and is recommended as a first-line treatment. Despite challenges in solid tumor applications, adoptive cell therapy has demonstrated efficacy against various targets in gastric cancer treatment. Among these approaches, chimeric antigen receptor-T cell therapy research is the most widely explored and chimeric antigen receptor-T cell therapy targeting claudin18.2 has shown acceptable safety and robust anti-tumor capabilities. However, these advancements primarily remain in preclinical stages and further investigation should be made to promote their clinical application. This review summarizes the latest research on immune checkpoint inhibitors and adoptive cell therapy and their limitations, as well as the role of nanoparticles in enhancing immunotherapy.

Mesothelin- and nucleolin-specific T cells from combined short peptides effectively kill triple-negative breast cancer cells

BackgroundTriple-negative breast cancer (TNBC), known for its aggressiveness and limited treatment options, presents a significant challenge. Adoptive cell transfer, involving the ex vivo generation of antigen-specific T cells from peripheral blood mononuclear cells (PBMCs), emerges as a promising approach. The overexpression of mesothelin (MSLN) and nucleolin (NCL) in TNBC samples underscores their potential as targets for T cell therapy. This study explored the efficacy of multi-peptide pulsing of PBMCs to generate MSLN/NCL-specific T cells targeting MSLN+/NCL+ TNBC cells.MethodsTNBC patient samples were confirmed for both MSLN and NCL expression via immunohistochemistry. Synthesized MSLN and NCL peptides were combined and administered to activate PBMCs from healthy donors. The cancer-killing ability of the resultant T cells was assessed using crystal violet staining, and their subtypes and cytotoxic cytokines were characterized through flow cytometry and cytokine bead array.ResultsFindings showed that 85.3% (127/149) of TNBC cases were positive for either MSLN or NCL, or both; with single positivity rates for MSLN and NCL of 14.1% and 28.9%, respectively. MSLN and NCL peptides, with high binding affinity for HLA-A*02, were combined and introduced to activated PBMCs from healthy donors. The co-pulsed PBMCs significantly induced TEM and TEMRA CD3+/CD8+ T cells and IFN-γ production, compared to single-peptide pulsed or unpulsed conditions. Notably, MSLN/NCL-specific T cells successfully induced cell death in MSLN+/NCL+ MDA-MB-231 cells, releasing key cytotoxic factors such as perforin, granzymes A and B, Fas ligand, IFN-γ, and granulysin.ConclusionsThese findings serve as a proof-of-concept for using multiple immunogenic peptides as a novel therapeutic approach in TNBC patients.

Full-Length Immune Repertoire Reconstruction and Profiling at the Transcriptome Level Using Long-Read Sequencing.

Due to the diversity of the immune repertoire (IR), reconstructing full-length IR using traditional short-read sequencing has proven challenging. A full-length IR sequencing (FLIRseq) work flow was developed with linear rolling circle amplification and nanopore sequencing. Its accuracy and quantification ability were verified by plasmid mixtures and commercial B-cell receptor/T-cell receptor sequencing (BCR/TCR-seq) based on short reads. IRs in tissues and the peripheral blood from 8 patients with acute lymphoblastic leukemia, 3 patients with allergic diseases, 4 patients with psoriasis, and 5 patients with prostate cancer were analyzed using FLIRseq. FLIRseq reads had lower mismatch rates and gap rates, and higher identify rates than nanopore reads (all P < 2.2 × -16). The relative quantification of components by FLIRseq was consistent with the actual quantification (P > 0.05). FLIRseq had superiority over BCR/TCR-seq, providing the long complementarity-determining region 3, B-cell isotype, and the rarely used V gene sequence. FLIRseq observed an increase in clonotype diversity (P < 0.05) and a decrease in the percentage of abnormal BCRs/TCRs in patients with leukemia in remission. For patients with allergic diseases or psoriasis, FLIRseq provided direct insights into V(D)J recombination and specific immunoglobulin classes. Compared with that in prostate cancer tissues, the full-length V segment of the biased T-cell receptor β chain from lymphocytes in psoriatic tissues showed a more consistent AlphaFold2-predicted protein structure (P < 0.05). FLIRseq enables unbiased and comprehensive analyses of direct V(D)J recombination and immunoglobulin classes, thereby contributing to characterizing pathogenic mechanisms, monitoring minimal residual disease, and customizing adoptive cell therapy.

Abstract C020: Identification of KRAS-specific TCRs from human peripheral blood

Abstract Mutant KRAS is the predominant driver oncogene in pancreatic cancer accounting for approximately 90% of KRAS mutations. However, these mutant KRAS peptides exhibit weak binding affinity to most human leukocyte antigens (HLAs), impeding their recognition by the immune system and the subsequent generation of a corresponding T cell response. Identification of KRAS-mutant specific T cells would be useful to track tumor-specific T cell responses in patients and could also be used for adoptive transfer of TCR-transduced cells. Current efforts to identify KRAS-specific T cells have been limited by their restriction to specific HLA haplotypes or the provision of only a few T cell receptor (TCR) sequences. In this study, we developed a facile means of detecting KRAS-specific T cells from peripheral blood at frequencies as low as 1 in 1,000,000. We employ an α41BB agonist-selective lentivirus system combined with a KRAS peptide pool to facilitate the high-throughput discovery of KRAS-specific TCRs in peripheral blood mononuclear cells (PBMCs) derived from both patients with pancreatic cancer and healthy donors. Through in vitro stimulation and single-cell TCR analysis, we identified a series of T cell expansions sharing the same clonotype, indicating a response to KRAS-mutant peptide stimulation. We have quantified CD4 and CD8 KRAS-specific TCRs from patients with a diverse range of HLA haplotypes. Citation Format: Jiao Shen, Blake E. Smith, Winiffer Conce Alberto, Ellen J. Kim, Michael L. Dougan, Harshabad Singh, Michael E. Birnbaum, Stephanie K. Dougan. Identification of KRAS-specific TCRs from human peripheral blood [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C020.

Cell therapy using ex vivo reprogrammed macrophages enhances antitumor immune responses in melanoma

BackgroundMacrophage-based cell therapies have shown modest success in clinical trials, which can be attributed to their phenotypic plasticity, where transplanted macrophages get reprogrammed towards a pro-tumor phenotype. In most tumor types, including melanoma, the balance between antitumor M1-like and tumor-promoting M2-like macrophages is critical in defining the local immune response with a higher M1/M2 ratio favoring antitumor immunity. Therefore, designing novel strategies to increase the M1/M2 ratio in the TME has high clinical significance and benefits macrophage-based cell therapies.MethodsIn this study, we reprogrammed antitumor and proinflammatory macrophages ex-vivo with HDAC6 inhibitors (HDAC6i). We administered the reprogrammed macrophages intratumorally as an adoptive cell therapy (ACT) in the syngeneic SM1 murine melanoma model and patient-derived xenograft bearing NSG-SGM3 humanized mouse models. We phenotyped the tumor-infiltrated immune cells by flow cytometry and histological analysis of tumor sections for macrophage markers. We performed bulk RNA-seq profiling of murine bone marrow-derived macrophages treated with vehicle or HDAC6i and single-cell RNA-seq profiling of SM1 tumor-infiltrated immune cells to determine the effect of intratumor macrophage ACT on the tumor microenvironment (TME). We further analyzed the single-cell data to identify key cell-cell interactions and trajectory analysis to determine the fate of tumor-associated macrophages post-ACT.ResultsMacrophage ACT resulted in diminished tumor growth in both mouse models. We also demonstrated that HDAC6 inhibition in macrophages suppressed the polarization toward tumor-promoting phenotype by attenuating STAT3-mediated M2 reprogramming. Two weeks post-transplantation, ACT macrophages were viable, and inhibition of HDAC6 rendered intratumor transplanted M1 macrophages resistant to repolarization towards protumor M2 phenotype in-vivo. Further characterization of tumors by flow cytometry, single-cell transcriptomics, and single-cell secretome analyses revealed a significant enrichment of antitumor M1-like macrophages, resulting in increased M1/M2 ratio and infiltration of CD8 effector T-cells. Computational analysis of single-cell RNA-seq data for cell-cell interactions and trajectory analyses indicated activation of monocytes and T-cells in the TME.ConclusionsIn summary, for the first time, we demonstrated the potential of reprogramming macrophages ex-vivo with HDAC6 inhibitors as a viable macrophage cell therapy to treat solid tumors.

Tumor-reactive T cell clonotype dynamics underlying clinical response to TIL therapy in melanoma

Adoptive cell therapy (ACT) using invitro expanded tumor-infiltrating lymphocytes (TILs) has inconsistent clinical responses. To better understand determinants of therapeutic success, we tracked TIL clonotypes from baseline tumors to ACT products and post-ACT blood and tumor samples in melanoma patients using single-cell RNA and Tcell receptor (TCR) sequencing. Patients with clinical responses had baseline tumors enriched in tumor-reactive TILs, and these were more effectively mobilized upon invitro expansion, yielding products enriched in tumor-specific CD8+ cells that preferentially infiltrated tumors post-ACT. Conversely, lack of clinical responses was associated with tumors devoid of tumor-reactive resident clonotypes and with cell products mostly composed of blood-borne clonotypes that persisted in blood but not in tumors post-ACT. Upon expansion, tumor-specific TILs lost tumor-associated transcriptional signatures, including exhaustion, and responders exhibited an intermediate exhausted effector state after TIL engraftment in the tumor, suggesting functional reinvigoration. Our findings provide insight into the nature and dynamics of tumor-specific clonotypes associated with clinical response to TIL-ACT, with implications for treatment optimization.

SKAP1 Expression in Cancer Cells Enhances Colon Tumor Growth and Impairs Cytotoxic Immunity by Promoting Neutrophil Extracellular Trap Formation via the NFATc1/CXCL8 Axis.

The mechanisms underlying the development and progression of colon cancer are not fully understood. Herein, Src kinase associated phosphoprotein 1 (SKAP1), an immune cell adaptor, is identified as a novel colon cancer-related gene. SKAP1 expression is significantly increased in colon cancer cells. High SKAP1 levels are independently predictive of poor survival in patients with colon cancer. Notably, SKAP1 expression in colon cancer cells exerted a significant tumor-promoting effect in vivo rather than in vitro. Screening of tumor-infiltrating immune cells revealed the involvement of neutrophils in SKAP1-induced colon tumor promotion. Enhanced formation of neutrophil extracellular traps (NETs) is found to be a key downstream event that contributed to the pro-tumor role of SKAP1. In colon cancer cells, SKAP1 increased the expression of C-X-C motif chemokine ligand 8 (CXCL8) via nuclear factor of activated T cells c1 (NFATc1). The blockade of CXCL8 or NFATc1 largely attenuated neutrophil infiltration, NET formation, and tumor promotion induced by SKAP1. Furthermore, inhibiting SKAP1-induced NET significantly enhanced the antitumor efficiency of adoptive natural killer cell therapy in colon tumor models. In conclusion, SKAP1 significantly promotes colon cancer growth via the cancer cell/neutrophil NFATc1/CXCL8/NET axis, suggesting that SKAP1 is a potential target for colon cancer therapy.

Multi-field coupling analysis of photovoltaic cells under long distance and multi-mode laser transmission

Most electronic devices are powered by electricity, and the transfer of energy to related electronic devices is a critical issue. Laser wireless power transmission (LWPT) has a broad prospect in the field of wireless energy transmission, such as distributed charging system (DLC), spacecraft sensor network, satellite-to-satellite communication and medium and long distance power transmission, ground to unmanned aerial vehicle (UAV), and so on. In this paper, a multi-field coupled model of LWPT system for laser transmission at medium and long distances is established. Through the existing LWPT experimental platform and test, the test obtained the correlation coefficient of the I-V relationship formula with the change of light intensity and temperature. On the basis of the modified parameters, the attenuation efficiency of laser power with transmission distance is calculated, and the heat transfer and electrical characteristics of photovoltaic cells are solved by finite element method. It is found that different atmospheric environment and laser transmission distance have obvious effects on the voltage, current and temperature variation of photovoltaic cells. The temperature of a photovoltaic cell has a huge impact on its output efficiency. The temperature can be controlled effectively by setting the laser interval of pulse mode reasonably. The cooling capacity of photovoltaic cells is the key to improve the electrical conversion efficiency of LWPT systems.

Efficient Capture and Traceless Release of Functional CD8+ T Cells with a Microfluidic Chip for Enhanced In Vitro and In Vivo CD4-CAR Transduction.

The chimeric antigen receptor (CAR) T cells targeting CD4 expressed cells in acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML) could reduce the risk of off target effects in normal tissues. However, the efficacy of adoptive cell therapy is predominantly attributed to CD8+ T cells, necessitating their purification before lentivirus transfection to enhance the production of CD4-CAR-T cells. In this study, we developed a microfluidic chip functionalized with an optimized CD8 aptamer, A3t-MU, to facilitate the enrichment and purification of CD8+ T cells. The presented chip showed efficient capture and seamless release of CD8+ T cells from cultured T cells and peripheral blood mononuclear cells (PBMCs). The purity of the released CD8+ T cells reached 98.1%, representing a 13% improvement over the conventional magnetic bead separation method. CD4-CAR was efficiently transduced into the purified CD8+ T cells to construct CAR-T cells. We evaluated the antitumor capability of the CD4-CAR transduced CD8+ T cells (anti-CD4 CD8-CAR T cells) both in vitro and in vivo. The anti-CD4 CD8-CAR T cells exhibited significant cancer-cell-killing capacity across multiple tumor cell lines, including CEM, Jurkat, and MV4-11. Meanwhile, anti-CD4 CD8-CAR T cells significantly inhibited tumor growth in vivo. In conclusion, the presented microfluidic chip offers a cost-effective and high-purity approach for CD8+ T cell separation, enhancing CD4-CAR transduction and achieving efficient antitumor capability both in vitro and in vivo.

Application of magnetic nanoparticles in adoptive cell therapy of cancer; training, guiding and imaging cells.

Adoptive cell therapy (ACT) is on the horizon as a thrilling therapeutic plan for cancer. However, widespread application of ACT is often restricted by several challenges, including complexity of priming tumor-specific T cells and poor trafficking in solid tumors. The convergence of nanotechnology and cancer immunotherapy is coming of age and could address the limitations of ACT. Recent studies have provided evidence on the application of magnetic nanoparticles (MNPs) to generate smart immune cells and to bypass problems associated with conventional ACT. Herein, we review current progress in the application of MNPs to improve preparing, guiding and tracking immune cells in cancer ACT. Besides, we comment on the challenges ahead and strategies to optimize MNPs for clinical settings.

BATF is a major driver of NK cell epigenetic reprogramming and dysfunction in AML.

Myelodysplastic syndrome and acute myeloid leukemia (AML) belong to a continuous disease spectrum of myeloid malignancies with poor prognosis in the relapsed/refractory setting necessitating novel therapies. Natural killer (NK) cells from patients with myeloid malignancies display global dysfunction with impaired killing capacity, altered metabolism, and an exhausted phenotype at the single-cell transcriptomic and proteomic levels. In this study, we identified that this dysfunction was mediated through a cross-talk between NK cells and myeloid blasts necessitating cell-cell contact. NK cell dysfunction could be prevented by targeting the αvβ-integrin/TGF-β/SMAD pathway but, once established, was persistent because of profound epigenetic reprogramming. We identified BATF as a core transcription factor and the main mediator of this NK cell dysfunction in AML. Mechanistically, we found that BATF was directly regulated and induced by SMAD2/3 and, in turn, bound to key genes related to NK cell exhaustion, such as HAVCR2, LAG3, TIGIT, and CTLA4. BATF deletion enhanced NK cell function against AML in vitro and in vivo. Collectively, our findings reveal a previously unidentified mechanism of NK immune evasion in AML manifested by epigenetic rewiring and inactivation of NK cells by myeloid blasts. This work highlights the importance of using healthy allogeneic NK cells as an adoptive cell therapy to treat patients with myeloid malignancies combined with strategies aimed at preventing the dysfunction by targeting the TGF-β pathway or BATF.

Batroxase promotes the effect of NK cell adoptive therapy on lung cancer by enhancing immune cell infiltration.

Batroxobin, isolated from Bothrops moojeni, is a defibrinogenating agent used as a thrombin-like serine protease against fibrinogen for improving microcirculation. Here, we investigated whether, and if so, how batroxobin acts in concert with NK cells in terms of anti-tumor effects. CD3+/CD56+ NK cells were isolated and cultured from C57BL/6 mouse spleen. NK cells' viability was tested via Lactate dehydrogenase (LDH) assay. Lewis lung cancer cell (1*107 cell/ml) was used to build animal models. All animals were divided into five groups and treated with Batroxobin and NK cells respectively. HE staining was used to detect the pathological morphology of tumor tissue. The contents of fibrinogen and TNF-α in serum were determined by ELISA. The protein expression levels of MMP2, MMP9, VEGF and CD44 in tumor tissues were detected by Western Blot or immunohistochemistry. Compared with Control group, Tumor growth was not significantly affected in the group treated with Batroxobin or NK cells alone, However, tumor growth was significantly inhibited in the NK cell combined with the Batroxobin group. Serum levels of Fbg and TNF-αin mice treated with Batroxobin combined with NK cells dropped significantly, bringing them closer to normal levels. WB results showed that the expression levels of MMP2/9, VEGF and CD44 in Batroxobin combined with NK cell group also significantly decreased. Batroxobin combined with adoptive immunotherapy with NK cells significantly inhibited the growth of Lewis lung cancer in mice.

Study on the impact of active intake air fluctuation on the performance and mass transfer of PEM Fuel Cell

Enhancing mass transfer is imperative for mitigating concentration polarization and thereby optimizing fuel cell performance. This study presents numerical investigation to explore the performance of a proton exchange membrane fuel cell, which involves implementation of actively controlled fluctuating mass fluxes at cathode inlet in order to achieve desirable mass transfer enhancement. The trajectories of liquid droplets within channel were predicted by employing volume of fluid method. The results revealed that the current density exhibits frequency-dependent fluctuations, aligning with input oscillations. A marginal increase in mean current density was observed with frequency amplified and the maximum power density with fluctuated flow increased by 3.9 % compared to that with steady flow inlet. A noteworthy and linear augmentation was evident with amplitude escalation. The mean current density surpassed 34.7 % of the steady current at the amplitude of 10. Beyond a frequency threshold of 1000 Hz, the fluctuating current density surpassed that of steady flow throughout the entire period, attributed to the time constant of gas transport within the gas diffusion layer. Moreover, fluctuation phenomenon of the pressure field within the fuel cell channel, which contributed to reinforced mass transfer and accelerated product alleviation in the region facing bipolar rib was identified during gas transport in the gas diffusion layer. Numerical simulations adopting the volume of fluid method revealed a heightened tendency for the disintegration and expulsion of liquid droplets in the channel attributable to fluctuating flow.

Accelerating accessibility of CAR-T/NK therapies – Are AlloCARs and rapid manufacturing platforms the road ahead in improving access in multiple myeloma?

While the advent of CAR-T therapies has heralded a new era of efficacious therapies in relapsed/refractory Multiple Myeloma, access continues to be a major limiting factor due to prolonged manufacturing times of autologous products and apheresis and/or manufacturing failures. Allogeneic adoptive cellular therapy products (CAR-T, CAR-NK), currently investigational, are “off-the-shelf” products that may address availability and manufacturing bottlenecks. Novel rapid manufacturing platforms that decrease adoptive cell therapy product development time by weeks are currently being tested in clinical trials and may additionally help bridge the demand-supply chasm. This review provides a comprehensive overview of allogeneic adoptive cellular therapies and rapid manufacturing platforms in development.

Enhanced tumor response to adoptive T cell therapy with PHD2/3-deficient CD8 T cells

While adoptive cell therapy has shown success in hematological malignancies, its potential against solid tumors is hindered by an immunosuppressive tumor microenvironment (TME). In recent years, members of the hypoxia-inducible factor (HIF) family have gained recognition as important regulators of T-cell metabolism and function. The role of HIF signalling in activated CD8 T cell function in the context of adoptive cell transfer, however, has not been explored in full depth. Here we utilize CRISPR-Cas9 technology to delete prolyl hydroxylase domain-containing enzymes (PHD) 2 and 3, thereby stabilizing HIF-1 signalling, in CD8 T cells that have already undergone differentiation and activation, modelling the T cell phenotype utilized in clinical settings. We observe a significant boost in T-cell activation and effector functions following PHD2/3 deletion, which is dependent on HIF-1α, and is accompanied by an increased glycolytic flux. This improvement in CD8 T cell performance translates into an enhancement in tumor response to adoptive T cell therapy in mice, across various tumor models, even including those reported to be extremely resistant to immunotherapeutic interventions. These findings hold promise for advancing CD8 T-cell based therapies and overcoming the immune suppression barriers within challenging tumor microenvironments.

Identifying ADGRG1 as a specific marker for tumor-reactive T cells in acute myeloid leukemia

Besides chemotherapy and hematopoietic stem cell transplantation (HSCT), autologous T cells can also serve as a new treatment approach for AML patients. However, the features of tumor-reactive T cells and their distinctive markers still lack full description. To evaluate the characteristics of tumor-reactive T cells, we collected bone marrow (BM) T cells from newly diagnosed AML patients with RUNX1::RUNX1T1 as examples for paired single-cell RNA sequencing and single-cell V(D)J sequencing. Based on the STARTRAC-like algorithm, we defined bystander T cells and tumor-reactive T cells. Compared with bystander T cells, tumor-reactive T cells presented as senescent-like cytotoxic terminally differentiated T cells (Temra) with upregulated NK-related markers. Additionally, we found ADGRG1 could serve as the specific marker of CD8+ T tumor-reactive T cell and validated it through the Runx1Runx1t1/+; Mx1-Cre mouse model. In chimeric antigen receptor (CAR)-T and target cell system, ADGRG1 was selectively upregulated upon antigen-TCR encounter. Moreover, ADGRG1+CD8+ T cells released a higher level of IFN-γ and showed higher cell-killing ability when exposed to matched AML blasts. Together, our findings depict the single-cell profile of tumor-reactive T cells in AML BM and propose that ADGRG1 can act as an indicator of T cell tumor reactivity in AML, which may be further harnessed for adoptive cell therapy and tumor-reactive TCR enrichment.

Enhanced osteogenic differentiation in 3D hydrogel scaffold via macrophage mitochondrial transfer

To assess the efficacy of a novel 3D biomimetic hydrogel scaffold with immunomodulatory properties in promoting fracture healing. Immunomodulatory scaffolds were used in cell experiments, osteotomy mice treatment, and single-cell transcriptomic sequencing. In vitro, fluorescence tracing examined macrophage mitochondrial transfer and osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs). Scaffold efficacy was assessed through alkaline phosphatase (ALP), Alizarin Red S (ARS) staining, and in vivo experiments. The scaffold demonstrated excellent biocompatibility and antioxidant-immune regulation. Single-cell sequencing revealed a shift in macrophage distribution towards the M2 phenotype. In vitro experiments showed that macrophage mitochondria promoted BMSCs’ osteogenic differentiation. In vivo experiments confirmed accelerated fracture healing. The GAD/Ag-pIO scaffold enhances osteogenic differentiation and fracture healing through immunomodulation and promotion of macrophage mitochondrial transfer.