BackgroundPlasmodium infection has been proven to activate antitumor immune responses. This study comprehensively analyzes the immune cell populations in peripheral blood and tumor microenvironment to elucidate the potential immunological mechanisms by which Plasmodium infection inhibits tumor growth.MethodsWe established a subcutaneous Lewis lung cancer model in C57BL/6J mice and treated them with intraperitoneal injection of Plasmodium yoelii. The long and short diameters of tumors were measured. Then, high-dimensional flow cytometry was used to analyze the T cell subsets, macrophages and myeloid-derived suppressor cells (MDSCs) in peripheral blood and tumor tissues. Immunosuppression-related phosphorylated signal transducer and activator of transcription 3 (pSTAT3) and TGFβ in tumor tissues were also measured through Western blotting assay.ResultsPlasmodium infection inhibited the growth of Lewis lung cancer in mice. The infection increased in the numbers of CD3+ T cells, including CD4+ and CD8+ T, CD4+ central memory T (Tcm), CD4+ effector memory T (Tem), CD8+ Tcm, CD8+ Tem, CD8+ virtual memory T (Tvm), CD8+ short-lived effector cells (SLEC), and CD8+ memory precursor effector cells (CD8+ MPEC) in peripheral blood. Concurrently, the infection also increased the numbers of CD3+ T cells, including CD4+ and CD8+ T, CD4+ Tcm, CD4+ Tem, CD4+ tissue resident memory T (Trm), CD8+ Tcm, CD8+ Tem, CD8+ Trm and CD8+ SLEC in tumor tissues. In addition, Plasmodium infection reduced the expression levels of PD-1 on CD4+ and CD8+ T, the number of polymorphonuclear MDSCs, and increased the ratio of M1/M2 macrophages in the tumor tissues. The initial mechanism study revealed that Plasmodium infection significantly reduced the expression levels of pSTAT3 and TGFβ in tumor tissues, providing direct evidence that Plasmodium infection activated the antitumor immune responses.ConclusionBased on our past and current studies, we can draw the following conclusion: Plasmodium infection fully remodels and activates the immune system, targets and inhibits the entire tumor ecosystem through the key signals of pSTAT3 and TGFβ. This is completely different from the mechanisms of action of the current immune checkpoint blockade therapies, representing a new form of cancer immunotherapy, namely, the immune ecotherapy.

BackgroundIn previous studies, I established theoretical frameworks for immunodynamics and tumor ecodynamics, respectively, to quantitatively describe immune responses and the interactions between the immune system and the tumor ecosystem. Although these two theories are closely related, they are still distinct. This study aims to integrate these two theories into a unified theory termed tumor immuno-ecodynamics, and provide a simple standard mathematical model for medical researchers and clinicians.Methods and resultsAfter conducting an information-theoretical analysis on the fundamental equations of immunodynamics and tumor ecodynamics, and incorporating them into the framework of information theory, I standardized a series of equations by subtracting the background values from the measured values of the variables. At the same time, I mathematized the cancer immunoediting theory that had been included in this framework, and rigorously proved some important formulae mathematically. Thus, a standard mathematical model termed the theory of tumor immuno-ecodynamics was established to unify the above two different theories. This model containing ten standard conceptual equations focuses on the signal (information) connections and interactions between the immune system and the tumor ecosystem, which are redefined as two subsystems of the tumor immuno-ecosystem.ConclusionThe standard mathematical model composed of these ten conceptual equations unifies immunodynamics and tumor ecodynamics into the theory of tumor immuno-ecodynamics, which incorporates four existing important theories, namely the theories of immune equilibrium, the tumor ecosystem, the cancer hallmarks, and the cancer immunoediting. This theory can quantitatively describe the actions of the tumor immuno-ecosystem, laying a theoretical foundation for the development of immuno-oncology from qualitative to quantitative science.

Background While CD19-targeting chimeric antigen receptor T-cell (CAR-T) therapies have shown efficacy in B-cell hematological malignancies, treatment of antigen-negative or relapsed tumors remains challenging. The recombinant malaria protein VAR2CSA (rVAR2) binds specifically to placental-like chondroitin sulfate A (pl-CSA), which is expressed on many cancer types, enabling its use in various tumor-targeting platforms. However, rVAR2-based CAR-T strategies are limited by poor protein thermostability, short serum half-life, and safety concerns related to irreversible CAR activation. To address these limitations, we developed a navigable CAR-T cell system that uses an rVAR2-based navigator to direct CAR-T cells to pl-CSA+ tumors.Methods We overexpressed and purified rVAR2 and three catenation proteins in Escherichia coli, then produced and epitope-mapped the anti-rVAR2 monoclonal antibody 5H4VAR2omab following murine immunization. Using its single-chain fragment variable, we constructed a third-generation CAR-T with CD28 and 4-1BB co-stimulatory domains. An interlocking rVAR2 catenane, AXVB, was identified via orthogonal screening and employed as a navigator for directing CAR-T cells to pl-CSA+ tumors. The antitumor efficacy of this navigable CAR-T cell system was evaluated in vitro and in hematologic cancer cell line-derived xenograft models.Results We developed an orthogonal CAR-T cell system that employs a navigator protein (rVAR2 or AXVB) to target pl-CSA+ tumors. Through a reversible switch mechanism, this system enables indirect tumor recognition by CAR-T cells, thereby improving the safety profile of conventional CAR-T therapies. Compared with the rVAR2 monomer, the AXVB multimer exhibited enhanced thermostability and target affinity. Both in vitro and in vivo, AXVB-(navigation)-CAR T cells potently eliminated CD19-positive and CD19-negative pl-CSA+ tumor cells and significantly prolonged survival in tumor-bearing mice.Conclusions Protein catenation improved the thermostability and functional activity of rVAR2. In a murine model of hematologic malignancy, CAR-T cells utilizing the rVAR2 catenane (AXVB) as a targeting module exhibited superior antitumor activity.

Background: While the resident microbiome of tumors has been shown to be associated with the occurrence and progression of non-small cell lung cancer, there remains a significant knowledge gap in understanding the correlation between the microbial spectrum and immunity response to cancer therapy. In the case of lung adenocarcinoma (LUAD), the tumor microenvironment, encompassing a diverse array of microbes and immune cells, plays a crucial role in modulating therapeutic response. Towards comprehending the underlying mechanism, we present the microbe-immunity interactive networks to delineate the microbiota and immunity repertoires for two distinct molecular subtypes in LUAD. Methods: We obtained multi-omics data of LUAD patients from the publicly available database. In this study, we conducted a systematic exploration of the microbial and immunological etiology of cancer prognosis, by integrating the microbiome, genome, transcriptome, and clinic data. The mutational signature analysis, transcriptome analysis, gene set enrichment analysis, and microbiota-immunity network analysis were performed. Results: Based on the transcriptome repertories, we classified the patients into two molecular subtypes and observed that the overall survival of molecular subtype 2 (MS2) was notably shortened. We identified the microbial biomarkers in patients that distinguished between these molecular subtypes. The significant up-regulation of gamma 8T and neutrophil in MS2, suggesting the inflammation augmentation and stimulation of gamma 8T activation. What is more, the MS2 are characterized by a correlation network between microbiota biomarkers and gamma 8T cell, which may contribute to suppression of anti-tumor immunity and poor overall survival. Conclusions: Our findings not only display the repertoires of tumor microbiota and immune cells, but

Paradoxically, tumor development and progression can be inhibited and promoted by the immune system. After three stages of immune editing, namely, elimination, homeostasis and escape, tumor cells are no longer restricted by immune surveillance and thus develop into clinical tumors. The mechanisms of immune escape include abnormalities in antitumor-associated immune cells, selection for immune resistance to tumor cells, impaired transport of T cells, and the formation of an immunosuppressive tumor microenvironment. A population of distinct immature myeloid cells, myeloid-derived suppressor cells (MDSCs), mediate immune escape primarily by exerting immunosuppressive effects and participating in the constitution of an immunosuppressive microtumor environment. Clinical trials have found that the levels of MDSCs in the peripheral blood of cancer patients are strongly correlated with tumor stage, metastasis and prognosis. Moreover, animal experiments have confirmed that elimination of MDSCs inhibits tumor growth and metastasis to some extent. Therefore, MDSCs may become the target of immunotherapy for many cancers, and eliminating MDSCs can help improve the response rate to cancer treatment and patient survival. However, a clear definition of MDSCs and the specific mechanism involved in immune escape are lacking. In this paper, we review the role of the MDSCs population in tumor development and the mechanisms involved in immune escape in different tumor contexts. In addition, we discuss the use of these cells as targets for tumor immunotherapy. This review not only contributes to a systematic and comprehensive understanding of the essential role of MDSCs in immune system reactions against tumors but also provides information to guide the development of cancer therapies targeting MDSCs.

There are three types of dendritic cells (DCs) including CD1c+ conventional DCs (cDCs), CD141+cDCs and CD303+plasmacytoid DCs (pDCs) in human peripheral blood, however, more details of DC subsets are still obscure. Here we systemically investigated the subpopulations of human blood-derived DCs. Our data showed that there are three subsets of CD1c+cDCs and three subpopulations of CD141+cDCs expressing CD205 and/or CD103 in human peripheral blood. CD303+pDCs can be divided by two groups: CD303high(hi) and CD303lowpDCs expressing CD205 and/or CD103. There are six new subpopulations expressing CD205 and/or CD103 in CD303+pDCs. The protein expression of co-stimulatory molecules and the production of pro-/anti-inflammatory cytokines by these cDC and pDC subsets are different from those of each other. Our results imply that CD1c+CD205-DC, CD141+CD205- cDC and CD303+CD205-pDC subpopulations may be immune tolerogenic and immature DCs, but CD1c+CD205+cDC, CD141+CD205+cDC and CD303+CD205+pDC subsets are probably inflammatory and mature DCs due to their different levels of co-stimulatory molecule expression and pro-/anti-inflammatory cytokine production. The subpopulations of cDCs and pDCs expressing CD205 and/or CD103 in human peripheral blood may perform diverse immune functions according to their different biological features, therefore, these new subsets of cDCs and pDCs may be potential targets for immunotherapy to treat autoimmune diseases and tumors in the clinical trials in the future.

Objectives There is no theory to quantitatively describe the complex tumor ecosystem. At the same time, cancer immunotherapy is considered a revolution in oncology, but the methods used to describe tumors and the criteria used to evaluate efficacy are not keeping pace. The purpose of this study is to establish a new theory for quantitatively describing the tumor ecosystem, innovating the methods of tumor characterization, and establishing new efficacy evaluation criteria for cancer immunotherapy.Methods Based on the mathematization of immune equilibrium theory and the establishment of immunodynamics in a previous study, the method of reverse immunodynamics was used, namely, the immune braking force was regarded as the tumor ecological force and the immune force was regarded as the tumor ecological braking force, and the concept of momentum in physics was applied to the tumor ecosystem to establish a series of tumor ecodynamic equations. These equations were used to solve the fundamental and applied problems of the complex tumor ecosystem.Results A series of tumor ecodynamic equations were established. The tumor ecological momentum equations and their component factors could be used to distinguish disease progression, pseudoprogression, and hyperprogression in cancer immunotherapy. On this basis, the adjusted tumor momentum equations were established to achieve the equivalence of tumor activity (including immunosuppressive activity and metabolic activity) and tumor volume, which could be used to calculate individual disease remission rate and establish new efficacy evaluation criteria (ieRECIST) for immunotherapy of solid tumor based on tumor ecodynamics. At the same time, the concept of moving cube-to-force square ratio and its expression were proposed to calculate the area under the curve of tumor ecological braking force of blood required to achieve an individual disease remission rate when the adjusted tumor ecological momentum was known.Conclusions A new theory termed tumor ecodynamics emphasizing both tumor activity and tumor volume is established to solve a series of basic and applied problems in the complex tumor ecosystem. It can be predicted that the future will be the era of cancer immune ecotherapy that targets the entire tumor ecosystem.

ObjectiveOur previous studies have demonstrated that Plasmodium immunotherapy (infection) has antitumor effects in mice. However, as a new form of immunotherapy, this therapy has a weakness: its specific killing effect on tumor cells is relatively weak. Therefore, we tested whether Plasmodium immunotherapy combined with gemcitabine (Gem), a representative chemotherapy drug, has synergistic antitumor effects.MethodsWe designed subcutaneously and intravenously implanted murine Lewis lung cancer (LLC) models to test the antitumor effect of Plasmodium chabaudi ASS (Pc) infection in combination with Gem treatment and explored its underlying mechanisms.ResultsWe found that both Pc infection alone and Gem treatment alone significantly inhibited tumor growth in the subcutaneous model, and combination therapy was more effective than either monotherapy. Monotherapy only tended to prolong the survival of tumor-bearing mice, while the combination therapy significantly extended the survival of mice, indicating a significant synergistic effect of the combination. In the mechanistic experiments, we found that the combination therapy significantly upregulated E-cadherin and downregulated Snail protein expression levels, thus inhibiting epithelial-mesenchymal transition (EMT) of tumor cells, which may be due to the blockade of CXCR2/TGF-β-mediated PI3K/Akt/GSK-3β signaling pathway.ConclusionThe combination of Pc and Gem plays a synergistic role in inhibiting tumor growth and metastasis, and prolonging mice survival in murine lung cancer models. These effects are partially attributed to the inhibition of EMT of tumor cells, which is potentially due to the blockade of CXCR2/TGF-β-mediated PI3K/Akt/GSK-3β/Snail signaling pathway. The clinical transformation of Plasmodium immunotherapy combined with Gem for lung cancer is worthy of expectation.

CRISPR/Cas9 technology applied to Plasmodium falciparum offers the potential to greatly improve gene editing, but such expectations including large DNA fragment knock-ins and sequential gene editing have remained unfulfilled. Here, we achieved a major advance in addressing this challenge, especially for creating large DNA fragment knock-ins and sequential editing, by modifying our suicide-rescue-based system that has already been demonstrated to be highly efficient for conventional gene editing. This improved approach was confirmed to mediate efficient knock-ins of DNA fragments up to 6.3 kb, to produce "marker-free" genetically engineered parasites and to show potential for sequential gene editing. This represents an important advancement in establishing platforms for large-scale genome editing, which might gain a better understanding of gene function for the most lethal cause of malaria and contribute to adjusting synthetic biology strategies to live parasite malaria vaccine development. Site-directed knock-in of large DNA fragments is highly efficient using suicide-rescue-based CRISPR/Cas9 system, and sequential gene insertion is feasible but further confirmation is still needed.

Liver-stage Plasmodium in humans is an early stage of malarial infection. Decoquinate (DQ) has a potent multistage antimalarial activity.

Background Coinfection with HIV and Plasmodium parasites is fairly common, but the sequence of infection with these two pathogens and their impact on disease progression are poorly understood. Methods A Chinese rhesus macaque HIV and Plasmodium coinfection model was established to compare the impact of pre-existing and subsequent malaria on the progression of SIV infection. Results We found that a pre-existing malaria caused animals to produce a greater number of CD4 + CCR5 + T cells for SIV replication, resulting in higher viral loads. Conversely, subsequent malaria induced a substantially larger proportion of CD4 + CD28 high CD95 high central memory T cells and a stronger SIV-specific T cell response, maintained the repertoire diversity of SIV-specific T cell receptors, and generated new SIV-specific T cell clonotypes to trace SIV antigenic variation, resulting in improved survival of SIV-infected animals. Conclusion The complex outcomes of this study may have important implications for research on human HIV and malaria coinfection. The infection order of the two pathogens (HIV and malaria parasites) should be emphasized.

Objective The immunology field has long been short of a universally applicable theoretical model that can quantitatively describe the immune response, and the theory of immune equilibrium (balance) is usually limited to the interpretation of the philosophical significance of immune phenomena. Therefore, it is necessary to establish a new immunological theory, namely, immunodynamic theory, to reanalyze the immune response. Methods By quantifying the immune dynamic equilibrium as the ratio of positive and negative immune power, the immune dynamic equilibrium equation was established. Then, the area under the curve of the positive and negative immune power was assumed to be equal in the whole process of immune response (regardless of correct or not), and through thought experiments based on this key hypothesis, a series of new concepts and expressions were derived, to establish a series of immunodynamic equations. Results New concepts of immune force and immune braking force and their expression equations, namely, the theoretical equations of immunodynamics, were derived through thought experiments, and the theoretical curves of immunodynamics were obtained according to these equations. Via the equivalent transformation of the theoretical equations and practical calculation of functional data, and by the methods of curve comparison and fitting, some practical equations of immunodynamics were established, and these practical equations were used to solve theoretical and practical problems that are related to the immunotherapy of infectious diseases and cancers. Conclusion The traditional theory of immune equilibrium has been mathematized and transformed from a philosophical category into a new concrete scientific theory, namely the theory of immunodynamics, which solves the dilemma that the traditional theory cannot guide individualized medical practice for a long time. This new theory may develop into one of the core theories of immunology in the future.

Immune checkpoint blockade therapy (ICB) is ineffective against cold tumors and, although it is effective against some hot tumors, drug resistance can occur. We have developed a Plasmodium immunotherapy (PI) that can overcome these shortcomings. However, the specific killing effect of PI on tumor cells is relatively weak. Radiotherapy (RT) is known to have strong specific lethality to tumor cells. Therefore, we hypothesized that PI combined with RT could produce synergistic antitumor effects. We tested our hypothesis using orthotopic and subcutaneous models of mouse glioma (GL261, a cold tumor) and a subcutaneous model of mouse non-small cell lung cancer (NSCLC, LLC, a hot tumor). Our results showed that, compared with each monotherapy, the combination therapy more significantly inhibited tumor growth and extended the life span of tumor-bearing mice. More importantly, the combination therapy could cure approximately 70 percent of glioma. By analyzing the immune profile of the tumor tissues, we found that the combination therapy was more effective in upregulating the perforin-expressing effector CD8(+) T cells and downregulating the myeloid-derived suppressor cells (MDSCs), and was thus more effective in the treatment of cancer. The clinical transformation of PI combined with RT in the treatment of solid tumors, especially glioma, is worthy of expectation.

EDITORIAL article Front. Microbiol., 19 October 2022Sec. Infectious Agents and Disease Volume 13 - 2022 | https://doi.org/10.3389/fmicb.2022.1062904

Abstract Background Severe malaria caused by Plasmodium falciparum leads to most malaria-related deaths globally. Decoquinate (DQ) displays strong activity against multistage infection by Plasmodium parasites. However, the development of DQ as an oral dosage form for the treatment of malaria at the blood stage has not been successful. In this study, liposome formulations of DQ were created for intravenous (IV) injection to suppress Plasmodium berghei, a parasite that causes severe malaria in mice. Methods DQ liposomes were prepared by conventional ethanol injection method with slight modifications and encapsulation efficiency evaluated by the well-established centrifugation method. Potency of the DQ liposomes against P. falciparum was assessed in vitro using freshly isolated human red blood cells. The efficacy of the DQ liposomes was examined in the mouse model of severe malaria. Results The DQ liposomes were around 150 nm in size and had the encapsulation efficiency rates > 95%. The freshly prepared and lyophilized liposomes were stable after storage at − 20 °C for 6 months. The liposomes were shown to have excellent activity against P. falciparum in vitro with DQ IC50 0.91 ± 0.05 nM for 3D7 (chloroquine sensitive strain) and DQ IC50 1.33 ± 0.14 nM for Dd2 (multidrug resistant strain), which were 18- and 14-fold more potent than artemisinin, respectively. Mice did not have any signs of toxicity after receiving high dose of the liposomes (DQ 500 mg/kg per mouse) by IV injection. In the mouse model of severe malaria, the liposomes had impressive efficacy against P. berghei with DQ ED50 of 0.720 mg/kg. Conclusion The DQ liposomes prepared in this study were stable for long term storage and safe for IV injection in mammalian animals. The newly created liposome formulations had excellent activity against Plasmodium infection at the blood-stage, which encourages their application in the treatment of severe malaria.

Postoperative recurrence causes a high mortality rate among patients with hepatocellular carcinoma (HCC). The current study aimed to determine the effects of Plasmodium infection on HCC metastasis and recurrence. The antitumor effects of Plasmodium infection were determined using two murine orthotopic HCC models: The non-resection model and the resection model. Tumour tissues derived from tumour-bearing mice treated with or without Plasmodium infection were harvested 15 days post-tumour inoculation. The expression levels of biomarkers related to epithelial-mesenchymal transition (EMT) and molecules associated with CC-chemokine receptor 10 (CCR10)-mediated PI3K/Akt/GSK-3 beta/Snail signalling were identified using reverse transcription-quantitative PCR and western blotting. The results demonstrated that Plasmodium infection significantly suppressed the progression, recurrence and metastasis of HCC in the two mouse models. The expression levels of E-cadherin were significantly higher in the Plasmodium-treated group compared with that in the control group, whereas the expression levels of Vimentin and Snail were significantly lower in the Plasmodium-treated group. Furthermore, Plasmodium infection inhibited the activation of Akt and GSK-3 beta in the tumour tissues by downregulating the expression levels of CCR10 and subsequently suppressing the accumulation of Snail, which may contribute to the suppression of EMT and the prevention of tumour recurrence and metastasis. In conclusion, the results of the present study demonstrated that Plasmodium infection inhibited the recurrence and metastasis and improved the prognosis of HCC by suppressing CCR10-mediated PI3K/Akt/GSK-3 beta/Snail signalling and preventing the EMT. These results may be important for the development of novel therapies for HCC recurrence and metastasis, especially for patients in the perioperative period.

CD303+ plasmacytoid dendritic cells (pDCs) play an important role in the induction of immune tolerance and antitumor immunity. Here, we focused on the effect of NSCLC cells on the development of CD303+ pDC subsets expressing CD205 and/or CD103. The NSCLC cell line H1299 and primary NSCLC cells were incubated with DCs. The protein expression of costimulatory molecules on CD303+ pDCs, the production of pro-inflammatory and anti-inflammatory cytokines by CD303+ pDCs and the development of CD303+ pDC subsets were detected by using flow cytometry. Coculture with NSCLC cells modulates the protein expression of CD86 and HLA-DR on CD303+ pDCs. Moreover, NSCLC cells suppressed the production of IL-12 and IL-23 but facilitated the secretion of IL-27 and TGF-β by CD303+ pDCs. There were new CD303+ pDC subsets expressing CD205 and/or CD103 in healthy donors and NSCLC patients: CD303+CD205+CD103+, CD303+CD205+CD103-, CD303+CD205-CD103+ and CD303+CD205-CD103- pDCs. NSCLC cells modulated the differentiation of CD303+ pDC subpopulations by regulating the protein expression of CD205 and/or CD103 on CD303+ pDCs. NSCLC cells may regulate the immune functions of CD303+ pDCs by modulating the expression of costimulatory molecules on DCs and the production of pro-inflammatory/anti-inflammatory cytokines by DCs. NSCLC cells also regulate the development of CD303+ pDC subsets expressing CD205 and/or CD103. These outcomes may reveal a new cellular mechanism leading to the NSCLC-induced immune-suppressive microenvironment.

We previously reported that Plasmodium infection promotes antitumor immunity in a murine Lewis lung cancer. In this study, we investigated the effects of Plasmodium infection on the tumor inhibition and antitumor CD8+ T cell responses in a murine triple negative breast cancer (TNBCA) model. The results showed that Plasmodium infection significantly inhibited tumor growth, and increased the survival rate of the tumor-bearing mice. Both effector and memory CD8+ T cells were increased in peripheral blood and tumor-draining lymph node (DLN) in the infected mice. The co-stimulatory (CD40L, GITR and OX-40) and co-inhibitory (PD-1, CTLA-4, TIM-3, LAG3) immune checkpoints were up-regulated on CD8+ T cells in infected mice. Importantly, Py induced remarkable effects on the infiltration of CD8+ T cells in the tumor and granzym B+ CD8+ T cells in tumor-bearing mice while not in tumor-free mice. In summary, the results suggested that the effects of Plasmodium infection on murine 4T1 breast cancer might be related to the induction of CD8+ T cell-mediated antitumor immune responses. This finding may provide a novel strategy for the treatment of triple negative breast cancer.

Hematopoietic stem cell (HSC)-based gene therapy targeting CCR5 represents a promising way to cure human immunodeficiency virus type 1 (HIV-1) infection. Yet the preclinical animal model with transplantation of autologous CCR5-ablated HSCs remains to be optimized. In this study, four Chinese rhesus macaques of simian immunodeficiency virus (SIV) chronic infection were given long-term antiretroviral therapy (ART), during which peripheral CD34(+) hematopoietic stem and progenitor cells (HSPCs) were purified and infected with CCR5-specific CRISPR/Cas9 lentivirus (three monkeys) or GFP lentivirus (one monkey). After non-myeloablative conditioning, the CCR5-modified or GFP-labeled HSPCs were autotransplanted to four recipients, and ART was withdrawn following engraftment. All of the recipients survived the process of transplantation. The purified CD34(+) HSPCs harbored an undetectable level of integrated SIV DNA. The efficiency of CCR5 disruption in HSPCs ranges from 6.5% to 15.6%. Animals experienced a comparable level of hematopoietic reconstuction and displayed a similar physiological homeostasis Despite the low-level editing of CCR5 in vivo (0.3%-1%), the CCR5-disrupted cells in peripheral CD4(+) Effector Memory T cell (TEM) subsets were enriched 2- to 3-fold after cessation of ART. Moreover, two of the three treated monkeys displayed a delayed viral rebound and a moderately recovered immune function 6 months after ART withdrawal. This study highlights the importance of improving the CCR5-editing efficacy and augmenting the virus-specific immunity for effective treatment of HIV-1 infection.