Transforming growth factor beta (TGFβ) and activin A suppress natural killer (NK) cell function and proliferation, limiting the efficacy of adoptive NK cell therapies. Inspired by the partial resistance to TGFβ of NK cells with SMAD4 haploinsufficiency, we used CRISPR-Cas9 for knockout of SMAD4 in human NK cells. Here we show that SMAD4KO NK cells were resistant to TGFβ and activin A inhibition, retaining their cytotoxicity, cytokine secretion and interleukin-2/interleukin-15-driven proliferation. They showed enhanced tumor penetration and tumor growth control, both as monotherapy and in combination with tumor-targeted therapeutic antibodies. Notably, SMAD4KO NK cells outperformed control NK cells treated with a TGFβ inhibitor, underscoring the benefit of maintaining SMAD4-independent TGFβ signaling. SMAD4KO conferred TGFβ resistance across diverse NK cell platforms, including CD19-CAR NK cells, stem cell-derived NK cells and ADAPT-NK cells. These findings position SMAD4 knockout as a versatile and compelling strategy to enhance NK cell antitumor activity, providing a new avenue for improving NK cell-based cancer immunotherapies.
© 2025. The Author(s).

BACKGROUNDCow's milk (CM) allergy is the most common food allergy in young children. Treatment with oral immunotherapy (OIT) has shown efficacy, but high rates of adverse reactions. The aim of this study was to determine whether baked milk OIT (BMOIT) could reduce adverse reactions while still inducing desensitization, and to identify immunological correlates of successful BMOIT.METHODSThis phase II, randomized trial evaluated the safety and efficacy of BMOIT in milk-allergic children 3-18 years old. After the initial placebo-controlled first year of treatment, placebo-treated participants crossed over to active BMOIT. Double-blind, placebo-controlled oral food challenges (OFCs) were conducted with BM after year 1 and to both BM and unheated milk (UM) after year 2. IgG and IgE antibodies were measured along with CM-specific (CM+) CD4+ memory T cell populations, profiled using flow cytometry and scRNA-Seq.RESULTSTwenty-one of 30 (70%) reached the primary endpoint of tolerating 4044 mg of BM protein at month 24, and 11 of 30 tolerated 2000 mg or more of UM protein. Dosing symptoms were common, but more than 98% were mild, with no severe reactions. Immunological changes associated with desensitization included increased CM IgG4, CM+ FOXP3+ cells, and Tregs and corresponding decreases in CM IgE, CM+ Th2A cells, and CD154+ cells. T cell and antibody measurements were combined to build a model that predicted UM OFC outcomes.CONCLUSIONBMOIT was well tolerated and induced desensitization to BM and UM. This desensitization corresponded to redistribution within antigen-specific antibody and T cell compartments that provided insight into the mechanistic changes that occur with OIT treatment.TRIAL REGISTRATIONClinicalTrials.gov NCT03462030.FUNDING: Myra Reinhardt Family Foundation (grant number 128388), NIH/NIAID (U19AI135731, T32AI125179, S10OD025052).

NK cells are dysfunctional in myasthenia gravis (MG), but the mechanism is unclear. This study aims to measure associations and underlying mechanisms between the NK cells and the development of MG.
Twenty healthy controls (HCs) and 53 MG patients who did not receive glucocorticoids and immunosuppressants were collected. According to the Myasthenia Gravis Foundation of America (MGFA) classification, MG patients were categorized into MGFA I group (n = 18) and MGFA II-IV group (n = 35). Flow cytometry, cell sorting, ELISA, mRNA-sequencing, RT-qPCR, western blot, and cell culture experiments were performed to evaluate the regulatory mechanism of exhausted NK cells.
Peripheral NK cells in MGFA II-IV patients exhibit exhausted phenotypes than HCs, marked by the dramatic loss of total NK cells, CD56dimCD16- NK cells, elevated PD1 expression, reduced NKG2D expression, impaired cytotoxic activity (perforin, granzyme B, CD107a) and cytokine secretion (IFN-γ). Plasma IL-6 and IL-21 are elevated in MG patients and mainly derived from the aberrant expansion of monocytes and Tfh cells, respectively. IL-6/IL-21 cooperatively induced NK-cell exhausted signature via upregulating SOCS2 and inhibiting the phosphorylation of STAT5. SOCS2 siRNA and IL-2 supplement attenuated the IL-6/IL-21-mediated alteration of NK-cell phenotypes and function.
Inhibition of IL-6/IL-21/SOCS2/STAT5 pathway and recovery of NK-cell ability to inhibit autoimmunity may be a new direction in the treatment of MG.
Copyright © 2024 Zhang, Han, Bi, Yang, Lin, Li, Zhang and Bu.

The majority of patients with lung cancer exhibit drug resistance after anti-PD-1 immunotherapy, leading to shortened patient survival time. Previous studies have suggested an association between epigenetic abnormalities such as methylation and clinical response to anti-PD-1 immunotherapy, while the role of SUMOylation in resistance to anti-PD-1 antibody immunotherapy is still unclear.
Here, the mRNA expression of 15 SUMO machinery components in PBMC from lung cancer patients receiving anti-PD-1 immunotherapy were analyzed using real-time PCR. Base on the percentage change in mRNA levels, the relationship between the expression of SUMO machinery components and outcomes of anti-PD-1 immunotherapy, and the influencing factors of SUMOylation were evaluated. PBMC was treated with different concentrations of 2-D08 (a specific inhibitor of SUMOylation) in vitro, and analyzed the activation and the death rates of lymphocyte subsets by flow cytometry analysis.
A predictive method, base on the gene expression of three SUMO machinery components (SUMO1, SUMO3 and UBE2I), were developed to distinguish non-responders to PD-1 inhibitors. Furthermore, the number of lymphocytes in peripheral blood significantly reduced in the dysregulated SUMOylation groups (the percentage change >100 or -50 ~ -100 groups). In vitro studies confirmed that lightly low SUMOylation level improved the activation status of T and NK lymphocytes, but extremely low SUMOylation level lead to the increased death rates of lymphocytes.
Our findings implied that dysregulated gene expression of SUMO machinery components could induce the resistance of anti-PD-1 immunotherapy in lung cancer by upregulating the death of peripheral blood lymphocytes. These data might provide effective circulating biomarkers for predicting the efficacy of anti-PD-1 immunotherapy, and uncovered a novel regulatory mechanism of resistance to anti-PD-1 immunotherapy.
Copyright © 2024 Wang, Sun, Liu, Xu, Li, Zhang and Huang.

Arenaviral vaccine vectors encoding simian immunodeficiency virus (SIV) immunogens are capable of inducing efficacious humoral and cellular immune responses in nonhuman primates. Several studies have evaluated the use of immune modulators to further enhance vaccine-induced T-cell responses. The hematopoietic growth factor Flt3L drives the expansion of various bone marrow progenitor populations, and administration of Flt3L was shown to promote expansion of dendritic cell populations in spleen and blood, which are targets of arenaviral vectors. Therefore, we evaluated the potential of Flt3 signaling to enhance the immunogenicity of arenaviral vaccines encoding SIV immunogens (SIVSME543 Gag, Env, and Pol) in rhesus macaques, with a rhesus-specific engineered Flt3L-Fc fusion protein. In healthy animals, administration of Flt3L-Fc led to a 10- to 100-fold increase in type 1 dendritic cells 7 days after dosing, with no antidrug antibody (ADA) generation after repeated dosing. We observed that administration of Flt3L-Fc fusion protein 7 days before arenaviral vaccine increased the frequency and activation of innate immune cells and enhanced T-cell activation with no treatment-related adverse events. Flt3L-Fc administration induced early innate immune activation, leading to a significant enhancement in magnitude, breadth, and polyfunctionality of vaccine-induced T-cell responses. The Flt3L-Fc enhancement in vaccine immunogenicity was comparable to a combination with αCTLA-4 and supports the use of safe and effective variants of Flt3L to augment therapeutic vaccine-induced T-cell responses.IMPORTANCEInduction of a robust human immunodeficiency virus (HIV)-specific CD4+ and CD8+ T-cell response through therapeutic vaccination is considered essential for HIV cure. Arenaviral vaccine vectors encoding simian immunodeficiency virus (SIV) immunogens have demonstrated strong immunogenicity and efficacy in nonhuman primates. Here, we demonstrate that the immunogenicity of arenaviral vectors encoding SIV immunogens can be enhanced by administration of Flt3L-Fc fusion protein 7 days before vaccination. Flt3L-Fc-mediated increase in dendritic cells led to robust improvements in vaccine-induced T- and B-cell responses compared with vaccine alone, and Flt3L-Fc dosing was not associated with any treatment-related adverse events. Importantly, immune modulation by either Flt3L-Fc or αCTLA-4 led to comparable enhancement in vaccine response. These results indicate that the addition of Flt3L-Fc fusion protein before vaccine administration can significantly enhance vaccine immunogenicity. Thus, safe and effective Flt3L variants could be utilized as part of a combination therapy for HIV cure.