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Purpose:Acute myeloid leukemia (AML) is a highly aggressive form of leukemia, which results in poor survival outcomes. Currently, diagnosis and prognosis are based on invasive single-point bone marrow biopsies (iliac crest). There is currently no AML-specific noninvasive imaging method to detect disease, including in extramedullary organs, representing an unmet clinical need. About 85% to 90% of human myeloid leukemia cells express CD33 cell surface receptors, highlighting CD33 as an ideal candidate for AML immunoPET.Experimental Design:We evaluated whether [64Cu]Cu-DOTA-anti-CD33 murine mAb can be used for immunoPET imaging of AML in a preclinical model. MicroCT was adjusted to detect spatial/anatomical details of PET activity. For translational purposes, a humanized anti-CD33 antibody was produced; we confirmed its ability to detect disease and its distribution. We reconfirmed/validated CD33 antibody-specific targeting with an antibody–drug conjugate (ADC) and radioimmunotherapy (RIT).Results:[64Cu]Cu-DOTA-anti-CD33–based PET-CT imaging detected CD33+ AML in mice with high sensitivity (95.65%) and specificity (100%). The CD33+ PET activity was significantly higher in specific skeletal niches [femur (P < 0.00001), tibia (P = 0.0001), humerus (P = 0.0014), and lumber spine (P < 0.00001)] in AML-bearing mice (over nonleukemic control mice). Interestingly, the hybrid PET-CT imaging showed high disease activity in the epiphysis/metaphysis of the femur, indicating regional spatial heterogeneity. Anti-CD33 therapy using newly developed humanized anti-CD33 mAb as an ADC (P = 0.02) and [225Ac]Ac-anti-CD33-RIT (P < 0.00001) significantly reduced disease burden over that of respective controls.Conclusions:We have successfully developed a novel anti-CD33 immunoPET-CT–based noninvasive modality for AML and its spatial distribution, indicating a preferential skeletal niche.

Increasing knowledge of cancer immunology has led to the design of therapies using immune cells directly or manipulating their activity, collectively termed immunotherapy. In the field of immuno-oncology, research on adaptive immune T cells has led to the development of CAR-T cells. Innate immune cells such as NK cells can also eliminate oncogenically transformed cells and regulate cells of the immune system. Considering NK cells as a live drug, numerous methods for the isolation and activation of NK cells have been shown to be clinically and therapeutically relevant. In such processes, various cytokines and antibodies present a source of stimulation of NK cells and enhance the efficacy of such treatments. The ex vivo expansion and activation of NK cells, along with genetic modification with CAR, enhance their antitumor activity. Recent preclinical studies have shown an antitumor effect through extracellular vesicles (EVs) derived from NK cells. Work with autologous NK cells has provided insights for clinical applications. In this review, we outline the recent advances of NK-cell-based immunotherapies, summarizing CAR-NK cells, BiKEs, and TriKEs as treatment options against cancer. This review also discusses the challenges of NK cell immunotherapy.

Purpose Although vascular alterations in solid tumor malignancies are known to decrease therapeutic delivery, the effects of leukemia-induced bone marrow vasculature (BMV) alterations on therapeutic delivery are not well known. Additionally, functional quantitative measurements of the leukemic BMV during chemotherapy and radiation therapy are limited, largely due to a lack of high-resolution imaging techniques available preclinically. This study develops a murine model using compartmental modeling for quantitative multiphoton microscopy (QMPM) to characterize the malignant BMV before and during treatment. Methods and Materials Using QMPM, live time-lapsed images of dextran leakage from the local BMV to the surrounding bone marrow of mice bearing acute lymphoblastic leukemia (ALL) were taken and fit to a 2-compartment model to measure the transfer rate (Ktrans), fractional extracellular extravascular space (νec), and vascular permeability parameters, as well as functional single-vessel characteristics. In response to leukemia-induced BMV alterations, the effects of 2 to 4 Gy low-dose radiation therapy (LDRT) on the BMV, drug delivery, and mouse survival were assessed post-treatment to determine whether neoadjuvant LDRT before chemotherapy improves treatment outcome. Results Mice bearing ALL had significantly altered Ktrans, increased νec, and increased permeability compared with healthy mice. Angiogenesis, decreased single-vessel perfusion, and decreased vessel diameter were observed. BMV alterations resulted in disease-dependent reductions in cellular uptake of Hoechst dye. LDRT to mice bearing ALL dilated BMV, increased single-vessel perfusion, and increased daunorubicin uptake by ALL cells. Consequently, LDRT administered to mice before receiving nilotinib significantly increased survival compared with mice receiving LDRT after nilotinib, demonstrating the importance of LDRT conditioning before therapeutic administration. Conclusion The developed QMPM enables single-platform assessments of the pharmacokinetics of fluorescent agents and characterization of the BMV. Initial results suggest BMV alterations after neoadjuvant LDRT may contribute to enhanced drug delivery and increased treatment efficacy for ALL. The developed QMPM enables observations of the BMV for use in ALL treatment optimization.

Background A major obstacle to the success of organ transplantation from pigs to humans, necessitated by the shortage of human organs, is robust humoral immune rejection by pig-reactive human antibodies. Mixed xenogeneic hematopoietic chimerism induces xenoreactive B cell tolerance in rodents, but whether mixed pig/human chimerism could induce tolerance of human B cells to pig xenoantigens is unknown. Methods We investigated this question using a humanized mouse model in which durable mixed (pig-human) xenogeneic chimerism can be established. Results Human natural anti-pig cytotoxic antibodies, predominantly IgM, are detectable in non-chimeric humanized mouse serum, and pig-reactive antibodies were reduced in mixed chimeric versus non-chimeric humanized mice. This difference required persistent mixed chimerism and was not due to the adsorption of antibodies on pig cells in vivo. Furthermore, human B cells from spleens of mixed chimeric mice produced lower levels of anti-pig antibodies when stimulated in vitro compared with those from non-chimeric mice. Conclusions Our findings demonstrate that mixed chimerism reduces human natural antibodies to pig xenoantigens, providing the first in vivo evidence of human B cell tolerance induction by mixed xenogeneic chimerism and supporting further evaluation of this approach for inducing human B cell tolerance to xenografts.

Engineered T cells expressing chimeric antigen receptors (CARs) on their cell surface can redirect antigen specificity. This ability makes CARs one of the most promising cancer therapeutic agents. CAR-T cells for treating patients with B cell hematological malignancies have shown impressive results. Clinical manifestation has yielded several trials, so far five CAR-T cell therapies have received US Food and Drug Administration (FDA) approval. However, emerging clinical data and recent findings have identified some immune-related toxicities due to CAR-T cell therapy. Given the outcome and utilization of the same proof of concept, further investigation in other hematological malignancies, such as leukemias, is warranted. This review discusses the previous findings from the pre-clinical and human experience with CAR-T cell therapy. Additionally, we describe recent developments of novel targets for adoptive immunotherapy. Here we present some of the early findings from the pre-clinical studies of CAR-T cell modification through advances in genetic engineering, gene editing, cellular programming, and formats of synthetic biology, along with the ongoing efforts to restore the function of exhausted CAR-T cells through epigenetic remodeling. We aim to shed light on the new targets focusing on acute myeloid leukemia (AML).

Relapse of leukemic cells that do not express the antigen targeted by chimeric antigen receptor (CAR) is still a risk. As is the potential for targeting hematopoietic stem cells (HSCs) that share the same antigen expression, off-tumor on-target toxicity. Further, CAR T cells that bind different epitopes of the same antigen can have different tumor-killing efficacies. Therefore, we screen murine single chain variable fragment (scFv) based for indirect affinity to identify a CAR that targets Acute myeloid leukemia (AML), while minimizing toxicities. Also, recent advances in CAR design have demonstrated that the requirement of two separate tumor antigens to be ligated by CARs can increase the specificity for tumor targets. So designing a CAR that only activates a T cell when it binds two separate AML antigens will allow T cells to enhance safety. Therefore, we set out to develop a affinity based multi-antigen CAR T cell therapy that targets well described antigens for AML, including CD33 and CD123. Mice were immunized with these antigens, spleens collected, and fused with myeloma cell lines. The antibodies of fused hybridomas were screened for binding and activation against antigens by high throughput flow cytometry. After screening, we derived multiple de novo CD33, and CD123 scFvs by sequencing. We incorporated CD33 and CD123 scFvs into standard mono-specific CARs utilizing a 41BB co-stimulatory domain to validate antigen-specificity. Gene transfer assessment of CAR T cells demonstrated about 50-80% transduction efficiency for CD33 and CD123 scFvs. There were no differences in CD4 and CD8 proportions in these CAR T cells. We next examined the CARs for their cytotoxic ability using a Real-Time Cell Analysis (RTCA) system. For the CD33 CARs, 2 (6A11-1 and 27A3-1) out of 5, and for the CD123 CAR, 2 (15A12-11 and 15 A12-12) out of 8, scFv sequences transduced into T cells were highly efficacious at killing target cells and generated significant amounts of cytokines such as IFN-g, TNF- a, and IL-6. CAR T cells with these same scFv sequences were able to proliferate better in response to targeted antigen. To find the best possible combination of CD33 and CD123 scFvs we double transduced T cells with four selected CD33 or CD123 scFvs each with only one co-stimulation domain either CD3z or 4-1BB in "AND" gate fashion. Clear differences in cytotoxic ability and cytokine production were observed. We selected 12 combination of CD33/123 CARs bi-specific CARs to evaluate in vitro efficacy, polyfunctionality, and safety. Finally, to find the best combination of CARs that would be less toxic to HSCs, we performed a Colony Forming Unit (CFU) assay with CD34+ bone marrow stem cells and found 5 bi-specific pairs that were less toxic to HSCs . Based on the CFU assay and PSI index, we were able to select the combination of CD33/123 scFvs that would target AML but minimize the killing of HSCs.

Purpose: Acute myeloid leukemia (AML) is a highly aggressive form of leukemia, which results in poor survival outcomes. Currently, diagnosis and prognosis are based on invasive single-point bone marrow biopsies (iliac crest). There is currently no AML-specific noninvasive imaging method to detect disease, including in extramedullary organs, representing an unmet clinical need. About 85% to 90% of human myeloid leukemia cells express CD33 cell surface receptors, highlighting CD33 as an ideal candidate for AML immunoPET. Experimental Design: We evaluated whether [64Cu]Cu-DOTA-anti-CD33 murine mAb can be used for immunoPET imaging of AML in a preclinical model. MicroCT was adjusted to detect spatial/anatomical details of PET activity. For translational purposes, a humanized anti-CD33 antibody was produced; we confirmed its ability to detect disease and its distribution. We reconfirmed/validated CD33 antibody-specific targeting with an antibody–drug conjugate (ADC) and radioimmunotherapy (RIT). Results: [64Cu]Cu-DOTA-anti-CD33–based PET-CT imaging detected CD33+ AML in mice with high sensitivity (95.65%) and specificity (100%). The CD33+ PET activity was significantly higher in specific skeletal niches [femur (P < 0.00001), tibia (P = 0.0001), humerus (P = 0.0014), and lumber spine (P < 0.00001)] in AML-bearing mice (over nonleukemic control mice). Interestingly, the hybrid PET-CT imaging showed high disease activity in the epiphysis/metaphysis of the femur, indicating regional spatial heterogeneity. Anti-CD33 therapy using newly developed humanized anti-CD33 mAb as an ADC (P = 0.02) and [225Ac]Ac-anti-CD33-RIT (P < 0.00001) significantly reduced disease burden over that of respective controls. Conclusions: We have successfully developed a novel anti-CD33 immunoPET-CT–based noninvasive modality for AML and its spatial distribution, indicating a preferential skeletal niche.

Vascular permeability, tissue transfer rate (Ktrans), fractional extracellular tissue space (νec) and blood perfusion are crucial parameters to assess bone marrow vasculature (BMV) function. However, quantitative measurements of these parameters in a mouse model are difficult because of limited resolution of standard macroscopic imaging modalities. Using intravital multiphoton microscopy (MPM), live imaging of dextran transfer from BMV to calvarium tissue of mice bearing acute lymphoblastic leukemia (ALL) was performed to obtain BMV parameters. Mice bearing ALL had increased BMV permeability, altered Ktrans, increased νec, decreased blood perfusion, and increased BMV permeability resulting in reduced drug uptake. Targeted 2 Gy radiation therapy (RT) to mice bearing ALL increased local BMV perfusion and ALL chemotherapy uptake (P

Mixed chimerism is a promising approach to inducing allograft and xenograft tolerance. Mixed allogeneic and xenogeneic chimerism in mouse models induced specific tolerance and global hyporesponsiveness, respectively, of host mouse natural killer (NK) cells. In this study, we investigated whether pig/human mixed chimerism could tolerize human NK cells in a humanized mouse model. Our results showed no impact of induced human NK cell reconstitution on porcine chimerism. NK cells from most pig/human mixed chimeric mice showed either specifically decreased cytotoxicity to pig cells or global hyporesponsiveness in an in vitro cytotoxicity assay. Mixed xenogeneic chimerism did not hamper the maturation of human NK cells but was associated with an alteration in NK cell subset distribution and interferon gamma (IFN-gamma) production in the bone marrow. In summary, we demonstrate that mixed xenogeneic chimerism induces human NK cell hyporesponsiveness to pig cells. Our results support the use of this approach to inducing xenogeneic tolerance in the clinical setting. However, additional approaches are required to improve the efficacy of tolerance induction while ensuring adequate NK cell functions.

Introduction Acute myeloid leukemia (AML) is a highly aggressive form of leukemia with poor long-term survival. Our clinical development of a targeted radiation treatment for relapsed/refractory AML disease led to an impressive two-year overall survival (OS) rate of 41% (Stein et al., 2019) in contrast to Methods We first developed a TMI treatment methodology using a Precision X-RAD small animal irradiator. A whole body CT image was acquired, and three-dimensional (3D) dose calculations were performed using a Monte Carlo dose engine-based SmART-Plan treatment system (van Hoof et al, 2013; Downes et al., 2009; Faddegon et al., 2009). Before treatment, mice were further imaged to verify target anatomy prior to delivering precise radiation doses to the entire skeletal system and spleen while sparing vital organs (lungs, liver, gut). We used a Cbfb-MYH11/Mpl (CMM)-induced mice leukemia model. At 5-10% CMM-GFP cells in peripheral blood (~20-30% in bone marrow; high disease burden), the mice were treated with three doses of CSI (2.5mg/kg) on alternate days with or without 4 Gy radiation (TMI or total body irradiation [TBI]). Cy3 labeled CSI uptake studies were carried out 48h post RT by flow cytometry, and immune cell trafficking and activation studies were conducted by harvesting bone marrow and spleen cells on day 8 post RT. Results Dose volume histogram (DVH) and radiation dose painting show that the TMI treatment plan significantly reduced doses to critical organs (lungs, liver, gut) while maintianing the same dose to the skeleton and spleen, unlike TBI (same dose to entire body) (Figure 1A-D). In TMI-treated mice, the CSI-Cy3 uptake was significantly higher in CMM cells than in the CMM cells of mice treated with CSI alone, indicating improved delivery post RT (Figure 1E, F). The trafficking of T helper cells (CD4+) and cytotoxic T lymphocytes (CD8+; CTLs) as well as effector (CD62L-CD44+) and effector memory T cells (CD62L+CD44+) is significantly augmented in the bone marrow of TMI+CSI treated mice compared to levels in mice treated with TBI+CSI or CSI alone (Figure 1G, H). An increased total number of IFNγ secreting CTLs, and a higher CD8:Treg (CD4+CD25+FOXP3+) ratio indicate enhanced anti-tumerogenic activity in TMI+CSI treated mice over that of mice treated with TBI+CSI or CSI alone (Figure 1I, J). Similarly, myeloid cell trafficking and activation was augmented in TMI-treated bone marrow (data not shown). The benefit of augmented immune modulation in TMI+CSI combinatorial therapy is reflected in a significantly increased survival (~39 median survival days) over untreated mice and those treated with CSI or TMI alone ( ~8-9 days median survival) (Figure 1K). Conclusion This is the first report of a novel radio-immunotherapy using a systemic targeted precise RT (TMI) in combination with STAT3 down-regulation (CSI) in AML. As hypothesized, low-dose targeted RT in combination with blocking of STAT3 signaling improved immune cell trafficking and activation, thereby enhancing the efficacy of this combinatorial therapy in high-disease burden. Further, newly developed low-dose TMI shows enhanced immune modulation over conventional TBI, suggesting the benefits of localized targeted RT in hematological malignancies. Disclosures Hui: Janssen Research & Development, LLC: Consultancy, Honoraria.

The therapeutic promise of chimeric antigen receptor (CAR) T cells was realized when complete remission rates of 90% were reported after treating B cell acute lymphoblastic leukemia (B-ALL) with CD19-targeted CAR T cells. However, with increasing numbers of patients treated, challenges have become evident, especially regarding poor CAR T cell responses, which might be caused by exhaustion. Detailed understanding of the mechanism of CD28-dependent exhaustion in CAR T cells will allow the design of a CAR less prone to exhaustion and reduce relapse rates. We hypothesized that by mutating the CD28 subdomains YMNM and PRRP and leaving only the PYAP CD28 subdomain active we would optimize CAR T cell signaling and reduce exhaustion. We found mutated CAR T cells with only a functional PYAP subdomain secrete 50% less IFNγ, IL6, and TNFα after 24hr stimulation as well as having significantly less TCR signaling as measured by Nur77 compared to non-mutated CD28 CAR T cells. Additionally, mutated CAR T cells injected into mice had decreased expression of PD1 (82% to 62%) in the BM after 2 months. Studies in Rag−/− mice also showed mutated CAR T cells were increased 35% in the BM and 92% in the spleen compared to non-mutated CD28 CAR T cells. After challenge with tumor, exhausted mutant CAR T cells had increased IFNγ (42%), TNFα (62%) and IL2 (73%) secretion compared to exhausted non-mutated CD28 CAR T cells. This suggests that CAR T cells with only the PYAP subdomain are able to persist and be effective longer because they have optimized CD28 signaling. This work allows for development of an enhanced 2nd generation CAR T cell therapy for B cell malignancies by optimizing CAR T cell activation and persistence which may reduce relapse rates and severe toxicities.

CD19 targeted 2nd generation chimeric antigen receptor T (CAR T) cells have been successful against relapsed and/or refractory B cell malignancies. The pending FDA-approval of 2 separate CD19 targeted CAR T products highlight the need to understand the biology behind this novel therapy. CAR design includes a single-chain variable fragment, which encodes antigen-binding, fused to a transmembrane domain, co-stimulatory domain, and CD3ζ activation domain. The two CARs likely to be approved as standard of care include a 41BB or CD28 co-stimulatory domain. CD28 is a critical co-stimulatory receptor required for full T cell activation and persistence, while 4-1BB is a member of the tumor necrosis factor receptor family and also a critical T cell co-stimulatory factor. Early evaluation of the co-stimulatory domains role in CAR design confirmed that they are required to enhance T cell function, but lacked insight regarding their mechanism for this enhancement. Furthermore, clinical outcomes suggest that the co-stimulatory domains in CARs support different T cell functions in patients. For example, while overall outcomes are similar between 41BB (19BBz) and CD28-containing CARs (1928z), 19BBz CAR T cells can persist for years in patients, but functional 1928z CAR T cells rarely persist longer than a month. Recent studies are providing insight to these differences and have demonstrated that 4-1BB-containing CARs reduce T cell exhaustion, enhance persistence, and increase central memory differentiation and mitochondrial biogenesis, while CD28-containing CARs support robust T cell activation and exhaustion, and are associated with effector-like differentiation. However, these studies have been performed mostly in vitro or in immune deficient mice, which limits their ability to model complex immune biology. Therefore, we evaluated murine CD19-targeting CARs with a 4-1BB (m19BBz) or CD28- (m1928z) co-stimulatory domain in relevant animal models of immunity. We directly compared m19BBz and m1928z CAR T cell immune phenotype, cytotoxicity, cytokine production, gene expression, intracellular signaling, and in vivo persistence, expansion, and B cell acute lymphoblastic leukemia (B-ALL) eradication. In vitro assays revealed that m1928z CAR T cells had enhanced cytotoxicity and cytokine production compared to m19BBz CAR T cells. Also, evaluation of m1928z and m19BBz CAR T cells displayed similar immune phenotypes, but markedly different gene expression with m1928z CAR T cells upregulating genes related to effector function and exhaustion, while m19BBz CAR upregulated genes critical for NFkB regulation, T cell quiescence and memory. In vivo, both m1928z and m19BBz CAR T cells supported equivalent protection against B-ALL. Similar to patients, in our mouse models there are functional differences between the mouse CD19-targeted CAR T cells. At 1 week post-infusion m19BBz CAR T cells are present in the blood of mice at significantly greater levels than m1928z CAR T cells. Furthermore, m19BBz CAR T cells enhance proliferation and/or anti-apoptosis protein expression to enhance B cell killing, which is evidenced by our observation that irradiation significantly weakens the in vivo efficacy of m19BBz but not m1928z CAR T cells. Our results suggest that B cell killing by m1928z CAR T cells is not impacted by irradiation because of their efficacious cytotoxicity of B cells. In contrast, m19BBz CAR T cells have enhanced viability and anti-apoptosis protein expression, which allows them to compensate for reduced effector function. We investigated potential mechanisms for the enhanced viability and anti-apoptosis of m19BBz CAR T cells and determined that NFkB signaling is upregulated much greater by m19BBz than m1928z. We have observed this difference in both a reporter cell line and primary mouse T cells. We are now dissecting what cellular components mediate increased NFkB signaling by the m19BBz CAR. Our animal models recapitulate equivalent anti-leukemia efficacy of CD19-targeted CAR T cells regardless of co-stimulatory domain, but underscore that anti-leukemia killing is mediated by different methods depending on the co-stimulatory domain. Our work sheds light on how 4-1BB mechanistically regulates and impacts CAR T function and has implications for future CAR design and evaluation. Disclosures No relevant conflicts of interest to declare.

Chimeric antigen receptor (CAR) T cells have mediated exciting results for patients. The CAR is a novel antigen-receptor that includes an antigen-binding domain, derived from an antibody, linked to an intracellular T cell activation domain. This 1st generation CAR design worked well in vitro but mediated poor results in vivo. Modification of the CAR to include a co-stimulatory domain, classified as a 2nd generation CAR design, achieved optimal T cell persistence and tumor killing in mice, which served as the rationale for evaluation of CAR T cells in patients. The most common clinically evaluated CARs target CD19 and include CD28 or 4-1BB co-stimulatory domains. However, there has been no extensive comparison of 2nd generation CAR T cells in mice, which could serve as a model for how these domains impact CAR T cell function in patients. Therefore, we evaluated and compared the function of mouse CD19 targeted CARs modified with a CD28 or 4-1BB domain. In contrast to clinical results, mouse CD28 CAR T cells imparted better leukemia-killing and persistence than mouse 4-1BB CAR T cells. We identified a defect in CAR T cell signaling mediated by mouse 4-1BB and mutation of this domain restored CAR signaling and in vivo leukemia killing. Also, we determined that CAR T cells with the mutation-optimized 4-1BB co-stimulatory domain proliferated in response to antigen in the bone marrow (BM) after a > 1-month period of rest, while CAR T cells with the CD28 co-stimulatory domain did not. We determined that this is likely due to the acquisition of an exhausted phenotype in the CD28 CAR T cells. Our results illustrate how co-stimulation modulates CAR T cell function in mice and suggest mechanisms for its impact on persistence and cancer-killing in patients.

Mixed hematopoietic chimerism permits durable tolerance of T, B and NK cells to xenoantigens in a rat to mouse bone marrow transplant model. However, it is unclear whether tolerance of human NK cells to pig xenoantigens can be induced by mixed hematopoietic chimerism. We assessed the tolerance of human NK cells towards pig cells in a humanized mouse model with established pig and human mixed xenogeneic chimerism. Pig and human mixed chimeras (MCs) were generated by injection of pig bone marrow cells to irradiated pig cytokine (IL3, GMCSF and SCF) transgenic NOD-scid common gamma chain knockout (NSG) mice followed by injection of human fetal liver CD34+ cells 3 day later. In the control group, only human CD34+ cells were transplanted. 12 weeks post-transplant, hydrodynamic injection of plasmid encoding human Flt3L followed by injection of three rounds of recombinant IL15/IL-15 receptor alpha Fc complex was given to promote human NK cell reconstitution. The control non–mixed chimeric group (Non-MC) received the same treatment without pig cells. 12 days following induction of human NK cell reconstitution, human NK cells from both MC and Non-MC mice were isolated from the spleen and their cytotoxic responses in vitro to pig cells were determined in a chromium release assay. In addition, the presence of pig cells in various tissues of the chimeric mice was studied. While human NK cells were usually undetectable in peripheral blood prior to the injection of human Flt3L plasmid and IL-15/IL-15 receptor alpha-Fc complex, they were detected by 5 days post-injection of IL-15 protein. 12 days post-induction of human NK cell reconstitution, pig cells remained detectable in peripheral blood, spleen, bone marrow and liver in the chimeric mice together with human NK cells. The co-existence of human NK cells and pig cells suggested that human NK cells in MCs might be tolerant to pig cells. Consistent with this notion, cytotoxicity assays showed that human NK cells from MCs demonstrated decreased killing of pig PBMC blasts compared to NK cells from Non-MC mice. Importantly, killing of K562 cells by NK cells from MCs mice was also decreased compared to that of Non-MC mice, suggesting that human NK cell tolerance to pig cells induced by mixed chimerism was associated with global hyporesponsiveness, as we have previously observed in a rat-to-mouse bone marrow transplantation model. Moreover, higher percentages of CD56highCD16low and lower percentages of CD56lowCD16high human NK cell subsets were observed in bone marrow of chimeric mice than in non-chimeric mice, indicating that the development of human NK cells in bone marrow might be altered by the presence of pig cells. In summary, our data suggest that mixed xenogeneic chimerism may induce tolerance of human NK cells towards porcine cells, but the tolerance may be associated with global hyporesponsiveness. Disclosures No relevant conflicts of interest to declare.