Case report: Preventive infusion of donor-derived CD7 chimeric antigen receptor T cells after allogeneic hematopoietic stem cell transplantation.

Chimeric antigen receptor T cells (CAR T) targeting CD7 for T-cell acute lymphoblastic leukemia/lymphoma (T-ALL/LBL) showed promising efficacy and safety in some clinical trials. However, most of them were bridged with allogeneic hematopoietic stem cell transplantation (allo-HSCT). We described successful treatment with preventive donor-derived anti-CD7 CAR-T therapy in a case of refractory T lymphoblastic lymphoma following allo-HSCT, who could not receive autologous anti-CD7 CAR-T products due to the low-quality of T lymphocytes. To date, the patient's complete remission has persisted for 20 months after HSCT.

Wiskott Aldrich syndrome protein (WASp)-deficient Th1 cells promote R-loop-driven transcriptional insufficiency and transcription-coupled nucleotide excision repair factor (TC-NER)-driven genome-instability in the pathogenesis of T cell acute lymphoblastic leukemia.

BACKGROUND: T-ALL is an aggressive hematological tumor that develops as the result of a multi-step oncogenic process which causes expansion of hematopoietic progenitors that are primed for T cell development to undergo malignant transformation and growth. Even though first-line therapy has a significant response rate, 40% of adult patients and 20% of pediatric patients will relapse. Therefore, there is an unmet need for treatment for relapsed/refractory T-ALL to develop potential targeted therapies. METHODS: Pediatric T-ALL patient derived T cells were grown under either nonskewingTh0 or Th1-skewing conditions to further process for ChIP-qPCR, RDIP-qPCR and other RT-PCR assays. Endogenous WASp was knocked out using CRISPR-Cas9 and was confirmed using flow cytometry and western blotting. LC-MS/MS was performed to find out proteomic dataset of WASp-interactors generated from Th1-skewed, human primary Th-cells. DNA-damage was assessed by immunofluorescence confocal-imaging and single-cell gel electrophoresis (comet assay). Overexpression of RNaseH1 was also done to restore normal Th1-transcription in WASp-deficient Th1-skewed cells. RESULTS: We discovered that nuclear-WASp is required for suppressing R-loop production (RNA/DNA-hybrids) at Th1-network genes by ribonucleaseH2 (RNH2) and topoisomerase1. Nuclear-WASp is associated with the factors involved in preventing and dissolving R-loops in Th1 cells. In nuclear- WASp-reduced malignant Th1-cells, R-loops accumulate in vivo and are processed into DNA-breaks by transcription-coupled-nucleotide-excision repair (TC-NER). Several epigenetic modifications were also found to be involved at Th1 gene locus which are responsible for active/repressive marks of particular genes. By demonstrating WASp as a physiologic regulator of programmed versus unprogrammed R-loops, we suggest that the transcriptional role of WASp in vivo extends also to prevent transcription-linked DNA damage during malignancy and through modification of epigenetic dysregulations. CONCLUSION: Our findings present a provocative possibility of resetting R-loops as a therapeutic intervention to correct both immune deficiency and malignancy in T-cell acute lymphoblastic leukemia patients and a novel role of WASp in the epigenetic regulation of T helper cell differentiation in T-ALL patients, anticipating WASp's requirement for the suppression of T-ALL progression.

Oculocerebrorenal syndrome of Lowe (OCRL) controls leukemic T-cell survival by preventing excessive PI(4,5)P2 hydrolysis in the plasma membrane.

T-cell acute lymphoblastic leukemia (T-ALL) is one of the deadliest and most aggressive hematological malignancies, but its pathological mechanism in controlling cell survival is not fully understood. Oculocerebrorenal syndrome of Lowe is a rare X-linked recessive disorder characterized by cataracts, intellectual disability, and proteinuria. This disease has been shown to be caused by mutation of oculocerebrorenal syndrome of Lowe 1 (OCRL1; OCRL), encoding a phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] 5-phosphatase involved in regulating membrane trafficking; however, its function in cancer cells is unclear. Here, we uncovered that OCRL1 is overexpressed in T-ALL cells, and knockdown of OCRL1 results in cell death, indicating the essential role of OCRL in controlling T-ALL cell survival. We show OCRL is primarily localized in the Golgi and can translocate to plasma membrane (PM) upon ligand stimulation. We found OCRL interacts with oxysterol-binding protein-related protein 4L, which facilitates OCRL translocation from the Golgi to the PM upon cluster of differentiation 3 stimulation. Thus, OCRL represses the activity of oxysterol-binding protein-related protein 4L to prevent excessive PI(4,5)P2 hydrolysis by phosphoinositide phospholipase C ß3 and uncontrolled Ca2+ release from the endoplasmic reticulum. We propose OCRL1 deletion leads to accumulation of PI(4,5)P2 in the PM, disrupting the normal Ca2+ oscillation pattern in the cytosol and leading to mitochondrial Ca2+ overloading, ultimately causing T-ALL cell mitochondrial dysfunction and cell death. These results highlight a critical role for OCRL in maintaining moderate PI(4,5)P2 availability in T-ALL cells. Our findings also raise the possibility of targeting OCRL1 to treat T-ALL disease.

High-valency Anti-CD99 Antibodies Toward the Treatment of T Cell Acute Lymphoblastic Leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive form of leukemia that currently requires intensive chemotherapy. While childhood T-ALL is associated with high cure rates, adult T-ALL is not, and both are associated with significant short- and long-term morbidities. Thus, less toxic and effective strategies to treat T-ALL are needed. CD99 is overexpressed on T-ALL blasts at diagnosis and at relapse. Although targeting CD99 with cytotoxic antibodies has been proposed, the molecular features required for their activity are undefined. We identified human antibodies that selectively bound to the extracellular domain of human CD99, and the most potent clone, 10A1, shared an epitope with a previously described cytotoxic IgM antibody. We engineered clone 10A1 in bivalent, trivalent, tetravalent, and dodecavalent formats. Increasing the antibody valency beyond two had no effects on binding to T-ALL cells. In contrast, a valency of ≥3 was required for cytotoxicity, suggesting a mechanism of action in which an antibody clusters ≥3 CD99 molecules to induce cytotoxicity. We developed a human IgG-based tetravalent version of 10A1 that exhibited cytotoxic activity to T-ALL cells but not to healthy peripheral blood cells. The crystal structure of the 10A1 Fab in complex with a CD99 fragment revealed that the antibody primarily recognizes a proline-rich motif (PRM) of CD99 in a manner reminiscent of SH3-PRM interactions. This work further validates CD99 as a promising therapeutic target in T-ALL and defines a pathway toward the development of a selective therapy against T-ALL.

CD99 antibody disrupts T-cell acute lymphoblastic leukemia adhesion to meningeal cells and attenuates chemoresistance.

Central nervous system (CNS) relapse is a significant cause of treatment failure among patients with acute lymphoblastic leukemia. In prior work we found that the meninges, the thin layer of tissue that covers the brain and spinal cord, harbor leukemia cells in the CNS. Importantly, direct interactions between leukemia and meningeal cells enabled leukemia chemoresistance. Herein, we show that an antibody targeting CD99, a transmembrane protein expressed on meningeal cells and many leukemia cells, disrupts adhesion between leukemia and meningeal cells and restores sensitivity of the leukemia cells to chemotherapy. This work identifies a mechanism regulating critical intercellular interactions within the CNS leukemia niche and may lead to novel therapeutic approaches for overcoming niche-mediated chemoresistance.

CAR T cells targeting CD99 as an approach to eradicate T-cell acute lymphoblastic leukemia without normal blood cells toxicity.

CAR T cell therapy has shown dramatic clinical success in relapsed or refractory B-ALL and other hematological malignancies. However, the loss of specific antigens, cell fratricide, T cell aplasia, and normal T cell separation are challenges in treating T cell leukemia/lymphoma with CAR T therapy. CD99 is a promising antigen to target T-ALL and AML as it is strongly expressed on the majority of T-ALL and AML. Here, we isolated a low-affinity CD99 (12E7) antibody, which specifically recognizes leukemia cells over normal blood cells. Moreover, T cells transduced with an anti-CD99-specific CAR that contained the 12E7 scFv expanded with minor fratricide and without normal blood cells toxicity. We observed that our anti-CD99 CAR T cells showed robust cytotoxicity specifically against CD99+ T-ALL cell lines and primary tumor cells in vitro and significantly prolonged cell line-derived xenografts (CDXs) or patient-derived xenografts (PDXs) models survival in vivo. Together, our results demonstrate that anti-CD99 CAR T cells could specifically recognize and efficiently eliminate CD99+ leukemia cells.

Up-regulation of circPVT1 in T cell acute lymphoblastic leukemia promoted cell proliferation via miR-30e/DLL4 induced activating NOTCH signaling.

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic cancer with dismal prognosis. Recent studies disclosed that circPVT1 played an oncogene role in various cancers. But its role in T-ALL is still unclear. In this study, we found the expression levels of circPVT1 in bone marrows and cell lines of T-ALL were significantly up regulated and knock-down of circPVT1 in T-ALL cell lines could inhibit the cell proliferation and increase the cell apoptosis. Further analysis showed that circPVT1 could bind directly to miR-30e and contributed to the activate the Notch signaling by regulating miR-30e/DLL4 pathway. The levels of circPVT1 were obviously related to cumulative relapse rate and 5-year survival rate. In conclusion, our study reveals that circPVT1 participates in the progression of T-ALL through the miR-30e/DLL4 pathway and might represent a potential therapeutic target for T-ALL treatment.

The rational development of CD5-targeting biepitopic CARs with fully human heavy-chain-only antigen recognition domains.

T cell malignancies are a group of hematologic cancers with high recurrence and mortality rates. CD5 is highly expressed in ∼85% of T cell malignancies, although normal expression of CD5 is restricted to thymocytes, T cells, and B1 cells. However, CD5 expression on chimeric antigen receptor (CAR)-T cells leads to CAR-T cell fratricide. Once this limitation is overcome, CD5-targeting CAR-T therapy could be an attractive strategy to treat T cell malignancies. Here, we report the selection of novel CD5-targeting fully human heavy-chain variable (FHVH) domains for the development of a biepitopic CAR, termed FHVH3/VH1, containing FHVH1 and FHVH3, which were validated to bind different epitopes of the CD5 antigen. To prevent fratricide in CD5 CAR-T cells, we optimized the manufacturing procedures of a CRISPR-Cas9-based CD5 knockout (CD5KO) and lentiviral transduction of anti-CD5 CAR. In vitro and in vivo functional comparisons demonstrated that biepitopic CD5KO FHVH3/VH1 CAR-T cells exhibited enhanced and longer lasting efficacy; produced moderate levels of cytokine secretion; showed similar specificity profiles as either FHVH1, FHVH3, or the clinically tested H65; and is therefore suitable for further development.

Chimeric antigen receptor T cells targeting CD7 in a child with high-risk T-cell acute lymphoblastic leukemia.

Effective systemic treatments for relapsed or refractory T-cell acute lymphoblastic leukemia (T-ALL) are limited. Recent clinical application of chimeric antigen receptor (CAR) immunotherapy has demonstrated successful control of B-cell malignancies by CAR-T cells; however, designing CARs for T-ALL remains a challenge. CD7 overexpression in T-cell malignancies may be an attractive target for immunotherapy in T-ALL. This study aimed to describe the safe and effective use of autologous CD7-CAR T cells (4SCAR7) for the treatment of T-ALL with induction failure in an 11-year-old patient. Based on The Chinese Children's Cancer Group-ALL (CCCG-ALL) study protocol, minimal residual disease (MRD) by flow cytometry (FC) analysis was detected on days 19 and 46 of remission induction. At the end of remission-induction chemotherapy, the patient achieved morphologic complete remission, though with MRD 16.13% and RT-PCR of KMT2A-MLLT1 fusion positive, which indicated induction failure. The cerebrospinal fluid (CSF) was negative for blasts at diagnosed. CAR-T therapy and allogeneic transplant were recommended as the next treatment options. CD3+ lymphocytes were collected from the patient 18 days after the high-dose MTX chemotherapy through leukapheresis. The 4SCAR7 CD7-targeting CAR-T cells were generated thereafter. The patient received lymphodepleting chemotherapy prior to 4SCAR7 infusion. Oral administration of itraconazole and sulfamethoxazole was performed from day 0 after CAR-T cell infusion. The patient did not have hypotension, hypoxia, or serious biochemical change or abnormality, but had fever on day 9. Although grade 1 cytokine-release syndrome (CRS) was diagnosed, it was successfully treated with ibuprofen. Anti-CD7 CAR transgene copy numbers in peripheral blood were determined by qPCR, which showed effective expansion initially, then dropped quickly, and persisted at a low level. Although experienced cytopenia from days 14 to 21, the patient achieved remission on day 17. After complete remission, the patient received hematopoietic stem cell transplantation (HSCT) and has recovered well to thisdate. Overall, this report suggested that 4SCAR7 could be a safe and effective strategy for the treatment of pediatric patients with high-risk T-cell malignancies.

CD2 and CD7 are sensitive flow cytometry screening markers for T-lineage acute leukemia(s): a study of 465 acute leukemia cases.

T-lymphoblastic leukemia/lymphoma (T-ALL/LBL) is a rare acute leukemia that expresses cytoplasmic CD3 (cCD3) and frequently lacks surface CD3. Given that routine flow cytometric testing for cCD3 may not be feasible and cCD3 interpretation may be difficult, we investigate if surface CD2 and/or CD7 expression on blasts can be used by flow cytometry to screen for T-lineage acute leukemia. We retrospectively reviewed flow cytometric data from 233 acute leukemias (36 T-ALL/LBL, 8 mixed-phenotype acute leukemia T/myeloid, 80 acute myeloid leukemia, 97 B-ALL/LBL, 8 mixed-phenotype acute leukemia B/myeloid, and 4 acute undifferentiated leukemia cases). Uniform expression (≥75% of blasts) of CD2 and/or CD7 was seen in all 44 cCD3-positive cases but in only 11% (20/189) of cCD3-negative acute leukemias, thus demonstrating 100% sensitivity and 89% specificity in the identification of cCD3-positive (T-lineage) acute leukemia. To avoid selection bias, we prospectively studied 232 consecutive acute leukemias for which cCD3, CD2, and CD7 were automatically performed in all cases. Similar to the retrospective study, uniform expression of CD2 and/or CD7 on blasts showed 100% sensitivity and 88% specificity in the screening for cCD3-positive (T-lineage) acute leukemia. Therefore, acute leukemias with uniform expression of CD2 and/or CD7 warrant further testing for cCD3 to evaluate for T-lineage acute leukemia. Blasts that lack both uniform CD2 and CD7 expression do not require additional cCD3 testing. We propose that CD2 and CD7 could be utilized in a limited antibody flow cytometry panel as a sensitive, robust, and cost-effective way to screen for T-lineage acute leukemia.

The Transferrin Receptor-Directed CAR for the Therapy of Hematologic Malignancies.

As many patients ultimately relapse after chimeric antigen receptor (CAR) T-cell therapy, identification of alternative targets is currently being evaluated. Substantial research efforts are underway to develop new targets. The transferrin receptor (TfR) is prevalently expressed on rapidly proliferating tumor cells and holds the potential to be the alternative target. In order to investigate the efficacy and challenges of TfR-targeting on the CAR-based therapy strategy, we generated a TfR-specific CAR and established the TfR-CAR-modified T cells. To take the advantage of TfR being widely shared by multiple tumors, TfR-CAR T cells were assessed against several TfR+ hematological malignant cell lines. Data showed that TfR-CAR T cells were powerfully potent in killing all these types of cells in vitro and in killing T-ALL cells in vivo. These findings suggest that TfR could be a universal target to broaden and improve the therapeutic efficacy of CAR T cells and warrant further efforts to use these cells as an alternative CAR T cell product for the therapy of hematological malignancies.

Therapeutic afucosylated monoclonal antibody and bispecific T-cell engagers for T-cell acute lymphoblastic leukemia.

BACKGROUND: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease with a poor cure rate for relapsed/resistant patients. Due to the lack of T-cell restricted targetable antigens, effective immune-therapeutics are not presently available and the treatment of chemo-refractory T-ALL is still an unmet clinical need. To develop novel immune-therapy for T-ALL, we generated an afucosylated monoclonal antibody (mAb) (ahuUMG1) and two different bispecific T-cell engagers (BTCEs) against UMG1, a unique CD43-epitope highly and selectively expressed by T-ALL cells from pediatric and adult patients. METHODS: UMG1 expression was assessed by immunohistochemistry (IHC) on a wide panel of normal tissue microarrays (TMAs), and by flow cytometry on healthy peripheral blood/bone marrow-derived cells, on 10 different T-ALL cell lines, and on 110 T-ALL primary patient-derived cells. CD43-UMG1 binding site was defined through a peptide microarray scanning. ahuUMG1 was generated by Genetic Glyco-Engineering technology from a novel humanized mAb directed against UMG1 (huUMG1). BTCEs were generated as IgG1-(scFv)2 constructs with bivalent (2+2) or monovalent (2+1) CD3ε arms. Antibody dependent cellular cytotoxicity (ADCC), antibody dependent cellular phagocytosis (ADCP) and redirected T-cell cytotoxicity assays were analysed by flow cytometry. In vivo antitumor activity of ahUMG1 and UMG1-BTCEs was investigated in NSG mice against subcutaneous and orthotopic xenografts of human T-ALL. RESULTS: Among 110 T-ALL patient-derived samples, 53 (48.1%) stained positive (24% of TI/TII, 82% of TIII and 42.8% of TIV). Importantly, no expression of UMG1-epitope was found in normal tissues/cells, excluding cortical thymocytes and a minority (<5%) of peripheral blood T lymphocytes. ahUMG1 induced strong ADCC and ADCP on T-ALL cells in vitro, which translated in antitumor activity in vivo and significantly extended survival of treated mice. Both UMG1-BTCEs demonstrated highly effective killing activity against T-ALL cells in vitro. We demonstrated that this effect was specifically exerted by engaged activated T cells. Moreover, UMG1-BTCEs effectively antagonized tumor growth at concentrations >2 log lower as compared with ahuUMG1, with significant mice survival advantage in different T-ALL models in vivo. CONCLUSION: Altogether our findings, including the safe UMG1-epitope expression profile, provide a framework for the clinical development of these innovative immune-therapeutics for this still orphan disease.

Eradication of T-ALL Cells by CD7-targeted Universal CAR-T Cells and Initial Test of Ruxolitinib-based CRS Management.

PURPOSE: Although chimeric antigen receptor T-cell (CAR-T) therapy development for B-cell malignancies has made significant progress in the last decade, broadening the success to treating T-cell acute lymphoblastic leukemia (T-ALL) has been limited. We conducted two clinical trials to verify the safety and efficacy of GC027, an "off-the-shelf" allogeneic CAR-T product targeting T-cell antigen, CD7. Here, we report 2 patients as case reports with relapsed/refractory T-ALL who were treated with GC027. PATIENTS AND METHODS: Both the trials reported here were open-label and single-arm. A single infusion of GC027 was given to each patient after preconditioning therapy. RESULT: Robust expansion of CAR-T cells along with rapid eradication of CD7+ T lymphoblasts were observed in the peripheral blood, bone marrow, and cerebrospinal fluid. Both patients achieved complete remission with no detectable minimal residual disease. At data cutoff, 30 September 2020, 1 of the 2 patients remains in ongoing remission for over 1 year after CAR T-cell infusion. Grade 3 cytokine release syndrome (CRS) occurred in both patients and was managed by a novel approach with a ruxolitinib-based CRS management. Ruxolitinib showed promising activity in a preclinical study conducted at our center. No graft-versus-host disease was observed. CONCLUSIONS: The two case reports demonstrate that a standalone therapy with this novel CD7-targeted "off-the-shelf" allogeneic CAR-T therapy may provide deep and durable responses in select patients with relapsed/refractory T-ALL. GC027 might have a potential to be a promising new approach for treating refractory/relapsed T-ALL. Further studies are warranted.

Single-cell RNA-seq reveals developmental plasticity with coexisting oncogenic states and immune evasion programs in ETP-ALL.

Lineage plasticity and stemness have been invoked as causes of therapy resistance in cancer, because these flexible states allow cancer cells to dedifferentiate and alter their dependencies. We investigated such resistance mechanisms in relapsed/refractory early T-cell progenitor acute lymphoblastic leukemia (ETP-ALL) carrying activating NOTCH1 mutations via full-length single-cell RNA sequencing (scRNA-seq) of malignant and microenvironmental cells. We identified 2 highly distinct stem-like states that critically differed with regard to cell cycle and oncogenic signaling. Fast-cycling stem-like leukemia cells demonstrated Notch activation and were effectively eliminated in patients by Notch inhibition, whereas slow-cycling stem-like cells were Notch independent and rather relied on PI3K signaling, likely explaining the poor efficacy of Notch inhibition in this disease. Remarkably, we found that both stem-like states could differentiate into a more mature leukemia state with prominent immunomodulatory functions, including high expression of the LGALS9 checkpoint molecule. These cells promoted an immunosuppressive leukemia ecosystem with clonal accumulation of dysfunctional CD8+ T cells that expressed HAVCR2, the cognate receptor for LGALS9. Our study identified complex interactions between signaling programs, cellular plasticity, and immune programs that characterize ETP-ALL, illustrating the multidimensionality of tumor heterogeneity. In this scenario, combination therapies targeting diverse oncogenic states and the immune ecosystem seem most promising to successfully eliminate tumor cells that escape treatment through coexisting transcriptional programs.

T-Cell Lymphoblastic Lymphoma/Leukemia Presenting as a Diffuse Viral Exanthem-like Reaction: A Clinical and Histopathological Challenge.

Cutaneous involvement by leukemia, or leukemia cutis, is a rare manifestation of leukemic disorders, most frequently occurring in children. The skin findings, which usually include multiple violaceous or erythematous nodules on the face, most often follow the classic presenting signs and symptoms of leukemia and occur in patients with an established primary diagnosis. Patients with T-cell acute lymphoblastic leukemia and associated leukemia cutis typically present with a solitary firm red to bluish nodule, often with an accompanying mediastinal mass, that can produce respiratory symptoms. In this article, we report a case of a patient with primary T-cell acute lymphoblastic leukemia/lymphoma presenting with a diffuse exanthem mimicking a viral illness with an associated SET-NUP214 translocation.

Monocyte-Derived Leukemia-Associated Macrophages Facilitate Extramedullary Distribution of T-cell Acute Lymphoblastic Leukemia Cells.

Macrophages play important roles in both physiologic and pathologic processes and arise from successive waves of embryonic and adult hematopoiesis. Monocyte-derived macrophages (MOMF) exert distinct functions under pathologic conditions, and leukemia-associated macrophages (LAM) show considerable diversities in activation and functional phenotype. However, their origin and pathologic roles have not been well elucidated. Here we used wild-type and CCR2-/- mice to study the pathologic roles of monocyte-derived LAM in extramedullary tissues in models of Notch1-induced T-cell acute lymphoblastic leukemia (T-ALL). MOMF existed in the resting liver and spleen. In the spleen, Ly6C+ monocytes gave rise to the Ly6C+ macrophage subset. Furthermore, an increase of monocyte-derived LAM, including the Ly6C+ subset, was detected in the extramedullary tissues in leukemic mice. More monocyte-derived LAM, including Ly6C+ LAM, was detected in the spleens of leukemic mice transplanted with exogeneous mononuclear cells. Moreover, Ly6C+ LAM exhibited increased M1-related characteristics and contributed to sterile inflammation. In CCR2-/- leukemic mice, reduced Ly6C+ LAM, relieved sterile inflammation, and reduced distribution of leukemia cells were detected in extramedullary tissues. In addition, monocyte-derived Ly6C+ LAM expressed high levels of CCL8 and CCL9/10. Blocking CCR1 and CCR2 relieved hepatosplenomegaly and inhibited the extramedullary distribution of leukemia cells in T-ALL mice. Collectively, our findings reveal the multifaceted pathologic roles of monocyte-derived LAM in T-ALL progression. SIGNIFICANCE: This study links monocyte-derived leukemia-associated macrophages with noninfectious inflammation and extramedullary distribution of leukemia cells during leukemia progression, providing new insight into macrophage-based immunotherapy in leukemia.

New insight into 20(S)-ginsenoside Rh2 against T-cell acute lymphoblastic leukemia associated with the gut microbiota and the immune system.

T-cell acute lymphoblastic leukemia (T-ALL) is a hematopoietic malignancy associated with unfavorable factors including male gender and over nine years of age. Chemotherapy toxicity continues to present a major challenge. There is a need to develop novel natural agents to improve survival and quality of life in patients with T-ALL. 20(S)-ginsenoside Rh2 (GRh2) exhibits immune regulation and anti-tumor effects in both cellular and murine xenograft models. In the present study, the anti-cancer mechanisms of 20(S)-GRh2 involved in the immune system and intestinal microbiota were investigated in T-ALL mice. We revealed that 20(S)-Rh2 suppressed T-ALL by blocking the PI3K/Akt/mTOR signaling pathway, and enhanced immunity in the spleen by regulating immune factors. In addition, 20(S)-GRh2 altered the composition of the gut microbiota, and promoted intestinal homeostasis by elevating the levels of tight junction proteins, antimicrobial peptides and IgA. 20(S)-GRh2 ameliorated the LPS-induced inflammatory response in the intestine of T-ALL mice. Furthermore, Bacteroidetes, Verrucomicrobia, Akkermansia, Lactobacillus, and Lachnospiraceae_NK4A136_group were positively correlated with anti-tumor immune factors, intestinal barrier-related factors, and the anti-inflammatory response. Conversely, Firmicutes, Proteobacteria, Parabacteroides and Alistipes had the opposite correlation. Collectively, these results suggest that 20(S)-GRh2 is a safe and effective natural product, that shows promise for the prevention and treatment of T-ALL.