Background: Survival of HER2-positive (HER2+) metastatic breast cancer (MBC) patients (pts) has improved with targeted therapies, but there is still a need of developing new therapeutic approaches. Double blockade alone with trastuzumab (T) and pertuzumab (P) showed significant clinical activity. T and P exert part of their activity based on antibody dependent cell mediated cytotoxicity (ADCC), mediated by natural killer cells (NK). Through the binding of the CD16 receptor of the NK cell to the Fc domain of T, NK cells can eliminate tumor cells covered by these antibodies. Furthermore, ADCC activation of an innate immune response induces cytokine secretion and antigen release, which may trigger an adaptive immune response against tumor antigens. Solid tumors usually present poor NK infiltration due to limited homing and immunosuppressive microenvironment. Our hypothesis is that the effect of T and P can be improved by regulating the efficiency of the ADCC activity through the infusion of ex-vivo activated allogenic NK cells. We propose a proof-of-concept phase I clinical trial for pts with HER2+ MBC refractory to antiHER2 therapies, to test the infusion of allogenic NK cells in order to enhance the ADCC of T and P to overcome this resistance and improve clinical outcome. Methods: This is a single arm, open label, multi-center, proof of concept investigator-initiated phase Ib trial to assess the safety and the tolerability of NK adoptive cell therapy (NK-ACT) and T+P when used in combination in refractory HER+ MBC. A total of 6 pts will be included in the safety lead-in phase. If signs of both clinical and biological activity are seen, and no more than 1 treatment-limiting toxicity (TLT) is observed, the study will expand with 14 additional pts. The NK investigational cell product comprises a live cell suspension of allogenic NK cells obtained from peripheral blood from a healthy donor, that will be infused on Day 2 at a minimum dose of 5x107 NK and a maximum dose limit of 5x108 NK. IL-2 will be administered on Days 2 (within 24h after NK infusion), 4 and 6 as a subcutaneous dose of 5x105 UI/m2. A preparative IV single-dose cyclophosphamide (600mg/m2) is given between Days -3 and -5 before NK cell infusion. On day 1, T is given at a dose of 8mg/kg IV for the loading dose, and 6mg/kg IV for the maintenance dose and P at a dose of 840mg IV for the loading dose, and 420 mg IV for the maintenance, both every 3 weeks, until disease progression, unacceptable toxicity or study termination. Major eligibility criteria: HER2+ pts as per ASCO/CAP guidelines; ECOG performance status ≤1; measurable disease; received at least two lines in the metastatic setting including trastuzumab/pertuzumab and an anti-HER2 ADC; progressed to previous therapy; normal organ and marrow function; patient has potential NK allogenic donors. Key exclusion criteria: Prior treatment with adoptive cellular therapy; Symptomatic or untreated primary or metastatic CNS malignancy. The primary objectives are to assess the safety and the tolerability of NK-ACT and T/P when used in combination. The secondary objective is to evaluate the initial clinical activity of NK-ACT concomitant with T/P. Exploratory objectives include describing the mechanisms of action and assessing biomarkers of the immunomodulatory effect and anti-tumor activity of the combination of NK-ACT and T/P. Statistical methods: no formal hypothesis testing and no formal sample size calculation. The safety lead-in phase will include the first 6 pts. If no more than 1 TLT is observed amount those pts, the study will expand with 14 additional pts. Citation Format: Santiago Escrivá-de-Romaní, Vladimir Galvao, Maria Castro-Henriques, Ascensión Lopez-Díaz de Cerio, Aura Muntasell, Carlos Vilches, Miguel López-Botet, Maria del Carmen Ochoa, Susana Inmaculada Inoges, Sara Santana, Marta Rotxes, Pere Barba, Julia Lostes, Gabriela Ene, Sonia Servitja, Guillermo Villacampa, Susana Muñoz, Darío López, Xenia Villalobos, Silvia Martin Lluesma, Cristina Saura, Joan Albanell, Elena Garralda, Ignacio Melero, Maria Martinez-Garcia. A phase Ib study of the safety, tolerability, biological effect, and efficacy of allogenic natural killer cells in combination with trastuzumab and pertuzumab in patients with refractory HER2-positive metastatic breast cancer [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P2-12-27.

HLA-E and -G class Ib molecules were considered unrelated to viral antigen presentation. HLA-E binds nonamers from the leader sequences of other HLA-I molecules and the human cytomegalovirus (HCMV) UL40 protein, interacting with CD94/NKG2 NK cell receptors. Yet, evidence that HLA-E may present some pathogen-derived peptides to CD8+ T lymphocytes has been reported. By contrast, HLA-G binds a broad spectrum of endogenous sequences but its role in antigen presentation is unknown. An experimental approach was set up to search for HCMV antigens displayed by HLA-G in infected cells. Among the analysed peptidome, 22 sequences corresponding to 16 HCMV molecules were identified; 17 peptides were confirmed to interact in vitro with HLA-G of which 10 displayed characteristic anchor residues. As compared to the response in short-term (6 h) assays to immunodominant IE-1 and pp65 antigens, none of the HLA-G-binding peptides stimulated cytokine production by CD8+ T cells from HCMV-seropositive blood donors (n = 15). Following a 14-day peptide stimulation of PBMC and expansion with IL-2, CD8+ T cells specifically responding to a subset of these viral antigens were detected in some individuals, yet were not restricted by HLA-G in functional assays. A subset of viral peptides did bind to both HLA-G and -E but were not recognised by CD94/NKG2 NK cell receptors. Our results provide the first evidence that HLA-G may display potentially immunogenic viral peptides in HCMV-infected cells, yet do not support their ability to promote HLA-G-restricted CD8+ T cell responses nor to modulate NK cell functions.

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. The authors show that knocking out SMAD4, human NK cells resist TGFβ and activin A suppression and demonstrate that SMAD4 knockout enhances the antitumor function of several NK cell products in clinical development.

Inhibitory killer cell immunoglobulin-like receptors (iKIRs) are randomly expressed by natural killer (NK) cell subsets and recognize motifs shared by HLA class-I (HLA-I) allotypes. Such interactions prevent NK cell autoreactivity while enhancing their response against cells lacking those HLA-I molecules (missing self), a situation defined in transplantation as iKIR-HLA-I mismatch (iKIR-MM), whose genotypic prediction has been associated with microvascular inflammation (MVI). Herein, we compared iKIR-MM in kidney transplant recipients with MVI ≥2 (n = 19) and controls with MVI ≤1 (n = 36). In parallel to genetic analysis of iKIR-MM, which was more frequent in MVI ≥2 patients, putative alloreactive iKIR-MM NK cells were defined by flow cytometry as NKG2A(-) cells bearing self-specific but lacking donor-specific iKIR. Although iKIR-MM NK cells were detected in both groups, their pretransplant numbers were higher in MVI ≥2 patients (median = 11.02, interquartile range = 0-58.31 vs median = 0, interquartile range = 0-9.46), especially in the presence of donor-specific antibodies or C4d, and correlated with MVI grade. Pretransplant, a subset of MVI ≥2 patients showed high proportions and numbers of oligoclonal iKIR-MM NK cells, which displayed an NKG2C(+) adaptive phenotype associated with cytomegalovirus infection. This pilot study provides a novel perspective on the contribution of iKIR-MM NK cells to MVI, with potential practical implications.

A subpopulation of NK cells with distinctive phenotype and function differentiates and expands specifically in response to infection by human cytomegalovirus (HCMV). A hallmark of these adaptive NK cells is their increased expression levels of the activating CD94/NKG2C receptor for HLA-E, and lack of expression of its inhibitory homologue CD94/NKG2A. Their frequency is highly variable in HCMV+ individuals, and the basis for such differences is only partially understood. Here, we explore the possible influence of sequence polymorphism of the NKG2C (or KLRC2) gene on the expansion of NKG2C+NKG2A- NK cells in healthy HCMV-seropositive donors. Our results show a significant association of greater proportions of adaptive NK cells with allele NKG2C*02. This is defined by two amino acid substitutions in comparison with the most prevalent allele, NKG2C*01, and associates with additional sequence polymorphisms in noncoding regions. Furthermore, we demonstrate consistently higher mRNA levels of NKG2C*02 in heterozygous individuals co-expressing this allele in combination with NKG2C*01 or *03. This predominance is independent of polymorphisms in the promoter and 3 ' UTRs and is appreciated also in HCMV-seronegative donors. In summary, although additional factors are most likely implicated in the variable expansion of NKG2C+NKG2A- NK cells in response to HCMV, our results demonstrate that host immunogenetics, in particular NKG2C diversity, influences the magnitude of such response.

Abstract Background and Aims Antibody-mediated rejection (ABMR) and microvascular inflammation (MVI) contribute to kidney transplant (KT) loss of function. The contribution of Natural Killer (NK) cells to the development of ABMR/MVI through inhibitory (iKIR) and activating (aKIR) HLA-I KIR (Killer-cell Immunoglobulin-like Receptors) has been proposed. The presence of an iKIR gene in the KT recipient (KTR) recognizing a self HLA-I ligand absent in the donor is defined as an iKIR-HLA-I mismatch (MM), and the presence of an aKIR gene in the KTR with HLA-I ligand in the graft absent in the KTR is defined as an aKIR-HLA-I MM. These MMs indicate the presence of potentially alloreactive NK cells that could contribute to the graft rejection, especially in the presence of NK receptor activating ligands or DSA. Our aim was to study the relationship between the number of KIR-HLA-I mismatches and ABMR/MVI. Method We selected 56 first KT, not treated with thymoglobulin or rituximab: 24 cases with ABMR/MVI and 32 controls without rejection in biopsies at 12-36 months. Donor and recipient HLA-I genotypes were determined by NGS, and recipient KIR genotypes by PCR-SSO (Luminex). The interactions considered were: KIR2DL1/HLA-C2, KIR2DL2&KIR2DL3/HLA-C1, KIR3DL1/HLA-Bw4, KIR3DL2/HLA-A*03, A*11, KIR2DS1/HLA-C2, KIR2DS2/HLA-C1, KIR3DS1/HLA-Bw4-I80 and KIR2DS4/HLA-A*11, C*02, C*04, C*05, C*16:01. Results KT recipients had a mean age of 54.55 years, 30.36% female and 8.92% living donor with no significant differences between ABMR/MVI and controls. The proportion of patients with ≥1 iKIR-HLA-I MM was higher in the ABMR/MVI group compared to controls (75% vs 56.25%, P = .1473), with a significant difference in the percentage of KIR3DL1 MM (29.17% vs 6.25%, P = .0296). The proportion of patients with at least one aKIR-HLA-I MM was non-significantly lower in ABMR/MVI compared to the control group (20.83% vs 28.13%, P = .5329). Overall, the proportion of KT recipients with ≥1 MM, either iKIR or aKIR, was 21% higher in the ABMR/MVI group (83.33% vs 68.75%, P = .2123). Conclusion In a population of immunologically low-risk KT recipients, we found a significantly greater proportion of patients with genetic iKIR-HLA-I MM in those with ABMR/MVI, but not of aKIR-HLA-I MM. NK cells with iKIR-HLA-I MM could contribute to antibody-mediated allograft damage together with other mechanisms.

Background The variability in responses to neoadjuvant treatment with anti-HER2 antibodies prompts to personalized clinical management and the development of innovative treatment strategies. Tumor-infiltrating Natural Killer (TI-NK) cells can predict the efficacy of HER2-targeted antibodies independently from clinicopathological factors in primary HER2-positive breast cancer patients. Understanding the mechanism/s underlying this association would contribute to optimizing patient stratification and provide the rationale for combinatorial approaches with immunotherapy. Methods We sought to uncover processes enriched in NK cell-infiltrated tumors as compared to NK cell-desert tumors by microarray analysis. Findings were validated in clinical trial-derived transcriptomic data. In vitro and in vivo preclinical models were used for mechanistic studies. Findings were analysed in clinical samples (tumor and serum) from breast cancer patients. Results NK cell-infiltrated tumors were enriched in CCL5/IFNG-CXCL9/10 transcripts. In multivariate logistic regression analysis, IFNG levels underlie the association between TI-NK cells and pathological complete response to neoadjuvant treatment with trastuzumab. Mechanistically, the production of IFN-ɣ by CD16 + NK cells triggered the secretion of CXCL9/10 from cancer cells. This effect was associated to tumor growth control and the conversion of CD16 into CD16 - CD103 + NK cells in humanized in vivo models. In human breast tumors, the CD16 and CD103 markers identified lineage-related NK cell subpopulations capable of producing CCL5 and IFN-ɣ, which correlated with tissue-resident CD8 + T cells. Finally, an early increase in serum CCL5/CXCL9 levels identified patients with NK cell-rich tumors showing good responses to anti-HER2 antibody-based neoadjuvant treatment. Conclusions This study identifies specialized NK cell subsets as the source of IFN-ɣ influencing the clinical efficacy of anti-HER2 antibodies. It also reveals the potential of serum CCL5/CXCL9 as biomarkers for identifying patients with NK cell-rich tumors and favorable responses to anti-HER2 antibody-based neoadjuvant treatment.

Isolated microvascular inflammation (iMVI) without HLA donor-specific antibodies or C4d deposition in peritubular capillaries remains an enigmatic phenotype that cannot be categorized as antibody-mediated rejection (ABMR) in recent Banff classifications. We included 221 kidney transplant recipients with biopsies with ABMR (n = 73), iMVI (n = 32), and normal (n = 116) diagnoses. We compared peripheral blood leukocyte distribution by flow cytometry and inflammatory infiltrates in kidney transplant biopsies among groups. Flow cytometry showed fewer lymphocytes and total, CD4+, and CD8+ peripheral T cells in iMVI compared with ABMR and normal cases. ABMR and iMVI had fewer total natural Killer (NK) cells but more NKG2A+ NK cells. Immunohistochemistry indicated that ABMR and iMVI had greater CD3+ and CD68+ glomerular infiltration than normal biopsies, whereas CD8+ and TIA1+ cells showed only increased iMVI, suggesting they are cytotoxic T cells. Peritubular capillaries displayed more CD3+, CD56+, TIA1+, and CD68+ cells in both ABMR and iMVI. In contrast, iMVI had less plasma cell infiltration in peritubular capillaries and interstitial aggregates than ABMR. iMVI displayed decreased circulating T and NK cells mirrored by T cell and NK cell infiltration in the renal allograft, similar to ABMR. However, the lesser plasma cell infiltration in iMVI may suggest an antibody-independent underlying stimulus.

The authors regret that the funding statement present in the original article was incomplete and missed funding details for Instituto de Salud Carlos III (ISCIII) the European Union. The full and complete funding statement is available here: Funding A.B. has support from a Rio Hortega contract (CM19/00004, ISCIII), an M-AES grant (MV20/00072, ISCIII), and a Spanish Society of Nephrology scholarship. This study was performed with funding from projects PI16/00619 and PI20/00090, funded by Instituto de Salud Carlos III (ISCIII) and co-funded by the European Union; RD16/0009/0013 (ISCIII FEDER REDinREN), and 201822-10 (Fundació la Marató de TV3). M.C. is partially supported by a grant from the Spanish Ministry of Health ISCIII FIS-FEDER INT21/0003. The authors would like to apologize for any inconvenience caused. Microvascular inflammation in the absence of human leukocyte antigen-donor-specific antibody and C4d: An orphan category in Banff classification with cytotoxic T and natural killer cell infiltrationAmerican Journal of TransplantationVol. 23Issue 4PreviewIsolated microvascular inflammation (iMVI) without HLA donor-specific antibodies or C4d deposition in peritubular capillaries remains an enigmatic phenotype that cannot be categorized as antibody-mediated rejection (ABMR) in recent Banff classifications. We included 221 kidney transplant recipients with biopsies with ABMR (n = 73), iMVI (n = 32), and normal (n = 116) diagnoses. We compared peripheral blood leukocyte distribution by flow cytometry and inflammatory infiltrates in kidney transplant biopsies among groups. Full-Text PDF

Human cytomegalovirus (HCMV) infection exerts broad effects on the immune system. These include the dif-ferentiation and persistent expansion of a mature NK cell subset which displays a characteristic phenotypic and functional profile hallmarked by expression of the HLA-E-specific CD94/NKG2C activating receptor. Based on our experience and recent advances in the field, we overview the adaptive features of the NKG2C+ NK cell response, discussing observations and open questions on: (a) the mechanisms and influence of viral and host factors; (b) the existence of other NKG2C-NK cell subsets sharing adaptive features; (c) the development and role of adaptive NKG2C+ NK cells in the response to HCMV in hematopoietic and solid organ transplant patients; (d) their relation with other viral infections, mainly HIV-1; and (e) current perspectives for their use in adoptive immunotherapy of cancer.

Figures S1, S2, S3, S4, S5, S6

Supplementary Figures 1-6 and Tables 1-3

Death receptor ligand TRAIL is a promising cancer therapy due to its ability to selectively trigger extrinsic apoptosis in cancer cells. However, TRAIL-based therapies in humans have shown limitations, mainly due inherent or acquired resistance of tumor cells. To address this issue, current efforts are focussed on dissecting the intracellular signaling pathways involved in resistance to TRAIL, to identify strategies that sensitize cancer cells to TRAIL-induced cytotoxicity. In this work, we describe the oncogenic MEK5-ERK5 pathway as a critical regulator of cancer cell resistance to the apoptosis induced by death receptor ligands. Using 2D and 3D cell cultures and transcriptomic analyses, we show that ERK5 controls the proteostasis of TP53INP2, a protein necessary for full activation of caspase-8 in response to TNFα, FasL or TRAIL. Mechanistically, ERK5 phosphorylates and induces ubiquitylation and proteasomal degradation of TP53INP2, resulting in cancer cell resistance to TRAIL. Concordantly, ERK5 inhibition or genetic deletion, by stabilizing TP53INP2, sensitizes cancer cells to the apoptosis induced by recombinant TRAIL and TRAIL/FasL expressed by Natural Killer cells. The MEK5-ERK5 pathway regulates cancer cell proliferation and survival, and ERK5 inhibitors have shown anticancer activity in preclinical models of solid tumors. Using endometrial cancer patient-derived xenograft organoids, we propose ERK5 inhibition as an effective strategy to sensitize cancer cells to TRAIL-based therapies.

NK cells monitor altered molecular patterns in tumors and infected cells through an ample array of receptors. Two families of evolutionarily distant receptors have converged to enable human NK cells to sense levels of HLA class I ligands, frequently abnormal in altered cells. Whilst different forms of polymorphism are a hallmark of killer‐cell immunoglobulin‐like receptors and their classic HLA‐A, B, and C ligands, genetic diversity of killer‐cell lectin‐like receptors for the non‐classical HLA‐E (CD94/NKG2 heterodimers) is less conspicuous and has attracted less attention. A common pattern of diversification in both receptor families is evolution of pairs of inhibitory and activating homologs for a common ligand, the genes encoding activating receptors being more frequently affected by copy number variation (CNV). This is exemplified by the gene encoding the activating NKG2C subunit ( KLRC2 or NKG2C ), which marks an NK‐cell subpopulation that differentiates or expands in response to cytomegalovirus. We have studied NKG2C diversity in 240 South European individuals, using polymerase chain reaction and sequencing methods to assess both gene CNV and single‐nucleotide polymorphisms (SNPs) affecting its promoter, coding and 3′‐untranslated (3′UT) regions. Sequence analysis revealed eight common SNPs—one in the promoter, two in the coding sequence, and five in the 3′UT region. These SNPs associate strongly with each other, forming three conserved extended haplotypes (frequencies: 0.456, 0.221, and 0.117). Homo‐ and heterozygous combination of these, together with complete gene deletion (0.175) and additional haplotypes with frequencies lower than 0.015, generate a diversity of NKG2C genotypes of potential immunological importance.

Killer immunoglobulin-like receptor (KIR) genes code for a family of inhibitory and activating receptors, finely tuning NK cell function. Numerous studies reported the relevance of KIR allelic polymorphism on KIR expression, ligand affinity, and strength in signal transduction. Although KIR variability, including gene copy number and allelic polymorphism, in combination with HLA class I polymorphism, impacts both KIR expression and NK cell education, only a precise phenotypic analysis can define the size of the different KIRpos NK cell subsets. In this context, reagents recognizing a limited number of KIRs is essential. In this study, we have characterized the specificity of an anti-KIR mAb termed HP-DM1. Testing its binding to HEK-293T cells transfected with plasmids coding for different KIRs, we demonstrated that HP-DM1 mAb exclusively reacts with KIR2DL1. Using site-directed mutagenesis, we identified the four amino acids relevant for HP-DM1 recognition: M44, S67, R68, and T70. HP-DM1 mAb binds to a conformational epitope including M44, the residue crucial for HLA-C K80 recognition by KIR2DL1. Based on the HP-DM1 epitope characterization, we could extend its reactivity to all KIR2DL1 allotypes identified except for KIR2DL1*022 and, most likely, KIR2DL1*020, predicting that it does not recognize any other KIR with the only exception of KIR2DS1*013. Moreover, by identifying the residues relevant for HP-DM1 binding, continuously updating of its reactivity will be facilitated.

The phenotypic identification of different NK cell subsets allows more in‐depth characterization of KIR repertoire and function, which are of potential interest in KIR and disease association studies. KIR genes are highly polymorphic, but a great homology exists among the various sequences and few monoclonal antibodies (mAbs) specifically recognize a single KIR. This is the case of HP‐DM1 which was demonstrated by analysis of cell transfectants and epitope mapping to be exclusively KIR2DL1‐specific, covering all allotypes identified to date, except for KIR2DL1*022 and *020, and also to react with KIR2DS1*013. Here, we compared in immunofluorescence analyses the staining of HP‐DM1 with other available mAbs to precisely identify KIR2DL1+ NK cells in potential donors for αβT/B‐depleted haplo‐HSCT, with known KIR genotype. HP‐DM1 mAb was used in combination with EB6 or 11PB6 (anti‐KIR2DL1/S1 and anti‐KIR2DL3*005), 143211 (anti‐KIR2DL1/S5), and HP‐MA4 (anti‐KIR2DL1/S1/S3/S5) mAbs, allowing the accurate identification of different KIR+ NK cell subsets. These phenotypic evaluations appeared useful to dissect the expression pattern of various KIR2D in NK cells from KIR2DL3*005+ individuals, particularly if KIR2DS1 is present. HP‐DM1 mAb remarkably refined NK cell phenotyping of donors carrying KIR2DS5, either in the centromeric or telomeric region. Functional assays with KIR2DL1+/S1+/S5+ NK cells confirmed that only HP‐DM1 exclusively reacts with KIR2DL1. Finally, we demonstrated that HP‐DM1 mAb blocked KIR2DL1 recognition of C2+ HLA‐C. Altogether, the data support that HP‐DM1 is a unique reagent valuable for characterizing KIR+ NK cell subsets.

The novel NKG2C*03 allele encodes a hybrid of the NKG2C*01 and NKG2C*02 primary structures.

Abstract Enhancing natural killer (NK) cell–based cancer immunotherapy by overcoming immunosuppression is an area of intensive research. Here, we have demonstrated that the anti-CD137 agonist urelumab can overcome TGFβ-mediated inhibition of human NK-cell proliferation and antitumor function. Transcriptomic, immunophenotypic, and functional analyses showed that CD137 costimulation modified the transcriptional program induced by TGFβ on human NK cells by rescuing their proliferation in response to IL2, preserving their expression of activating receptors (NKG2D) and effector molecules (granzyme B, IFNγ) while allowing the acquisition of tumor-homing/retention features (CXCR3, CD103). Activated NK cells cultured in the presence of TGFβ1 and CD137 agonist recovered CCL5 and IFNγ secretion and showed enhanced direct and antibody-dependent cytotoxicity upon restimulation with cancer cells. Trastuzumab treatment of fresh breast carcinoma–derived multicellular cultures induced CD137 expression on tumor-infiltrating CD16+ NK cells, enabling the action of urelumab, which fostered tumor-infiltrating NK cells and recapitulated the enhancement of CCL5 and IFNγ production. Bioinformatic analysis pointed to IFNG as the driver of the association between NK cells and clinical response to trastuzumab in patients with HER2-positive primary breast cancer, highlighting the translational relevance of the CD137 costimulatory axis for enhancing IFNγ production. Our data reveals CD137 as a targetable checkpoint for overturning TGFβ constraints on NK-cell antitumor responses.

Epstein Barr virus (EBV) causes a highly prevalent and lifelong infection contributing to the development of some malignancies. In addition to the key role played by T cells in controlling this pathogen, NK cells mediate cytotoxicity and IFNγ production in response to EBV-infected B cells in lytic cycle, both directly and through antibody (Ab)-dependent activation. We recently described that EBV-specific Ab-dependent NK cell interaction with viral particles (VP) bound to B cells triggered degranulation and TNFα secretion but not B cell lysis nor IFNγ production. In this report we show that NK cell activation under these conditions reduced B cell transformation by EBV. NK cells eliminated VP from the surface of B cells through a specific and active process which required tyrosine kinase activation, actin polymerization and Ca2+, being independent of proteolysis and perforin. VP were displayed at the NK cell surface before being internalized and partially shuttled to early endosomes and lysosomes. VP transfer was encompassed by a trogocytosis process including the EBV receptor CD21, together with CD19 and CD20. Our study reveals a novel facet of the antibody-dependent NK cell mediated response to this viral infection.

Antibody-mediated rejection (ABMR) is a frequent cause of renal allograft loss. There is increasing evidence of the role of Natural Killer (NK) cells in the establishment of ABMR damage. Our group described that patients with donor-specific antibodies (DSA) and ABMR present higher proportions of NKG2A+ NK cell subset in peripheral blood than those without HLA DSA or HLA antibodies. We selected 177 kidney transplant recipients (KT) with renal biopsies 2011-2017: 77 with ABMR (DSA+: 53, DSA-: 24) and 100 without ABMR (DSA+: 15, DSA-: 85). We assessed graft survival with a median time of follow-up since the renal biopsy of 53 months. In 138 KT we evaluated the peripheral blood NK cell immunophenotyping and its value as a prognostic biomarker. Graft survival was worse in ABMR-KT at the end of follow-up (p<0.001) independently of DSA detection (p=0.63). Regarding NK cell immunophenotyping, we observed a lower proportion and absolute NK cell count in ABMR+DSA+-KT and ABMR+DSA--KT compared with ABMR-DSA--KT (p=0.027, p=0.017). ABMR+DSA+-KT showed higher proportion of NKG2A+ NK cells compared with ABMR-DSA--KT (p=0.007). All ABMR+ patients, independently of DSA detection, presented lower absolute NKG2A- NK cell count in comparison with ABMR-DSA--KT (p=0.001, p=0.017). Finally, a proportion of NKG2A- <30% was associated with lower graft survival 36 months after graft biopsy with ABMR (p=0.067) (Figure). Graft survival is worse in ABMR+ compared with ABMR- KT independently of DSA detection. Kidney transplant recipients with ABMR show reduced peripheral absolute numbers of NK cells and NKG2A- NK cells regardless of undetectable DSA. This NK cell phenotype associated with a worse medium-term graft survival in cases with ABMR.