HLA class I NK-epitopes and KIR diversities in patients with multiple myeloma.

Multiple myeloma (MM) is a hematological malignancy caused by the clonal expansion of malignant plasma cells in the bone marrow. Myeloma cells are susceptible to killing by natural killer (NK) cells, but NK cells fail to control disease progression, suggesting immunosuppression. The activation threshold of NK-effector function is regulated by interaction between KIRs and self-HLA class I, during a process called "education" to ensure self-tolerance. NK cells can respond to diseased cells based on the absence of HLA class I expression ("Missing-self" hypothesis). The HLA and KIR repertoire is extremely diverse; thus, the present study aimed to characterize potential variances in genotypic composition of HLA Class I NK-epitopes and KIRs between MM patients and healthy controls. Genotypic expression of KIR and HLA (HLA-C group-C1/C2 and Bw4 motifs (including HLA-A*23, A*24, A*32) were analyzed in 172 MM patients and 195 healthy controls. Compared to healthy controls, we did not observe specific KIR genes or genotypes, or HLA NK-epitopes with higher prevalence among MM patients. The presence of all three HLA NK-epitopes (C1+C2+Bw4+) was not associated with MM occurrence. However, MM patients were more likely to be C1-/C2+/Bw4+ (p = 0.049, OR 1.996). In line with this, there was a trend of increased genetic co-occurrence of Bw4 and KIR3DL1 in MM patients (p = 0.05, OR 1.557). Furthermore, MM patients were more likely to genetically express both C2/KIR2DL1 and Bw4/KIR3DL1 (p = 0.019, OR 2.453). Our results reveal an HLA NK-epitope combination that is associated with the occurrence of MM. No specific KIR genotypes were associated with MM.

Resolving unknown nucleotides in the IPD-IMGT/HLA database by extended and full-length sequencing of HLA class I and II alleles.

In the past, identification of HLA alleles was limited to sequencing the region of the gene coding for the peptide binding groove, resulting in a lack of sequence information in the HLA database, challenging HLA allele assignment software programs. We investigated full-length sequences of 19 HLA class I and 7 HLA class II alleles, and we extended another 47 HLA class I alleles with sequences of 5' and 3' UTR regions that were all not yet available in the IPD-IMGT/HLA database. We resolved 8638 unknown nucleotides in the coding sequence of HLA class I and 2139 of HLA class II. Furthermore, with full-length sequencing of the 26 alleles, more than 90 kb of sequence information was added to the non-coding sequences, whereas extension of the 47 alleles resulted in the addition of 5.5 kb unknown nucleotides to the 5' UTR and > 31.7 kb to the 3' UTR region. With this information, some interesting features were observed, like possible recombination events and lineage evolutionary origins. The continuing increase in the availability of full-length sequences in the HLA database will enable the identification of the evolutionary origin and will help the community to improve the alignment and assignment accuracy of HLA alleles.

Ellipro scores of donor epitope specific HLA antibodies are not associated with kidney graft survival.

In kidney transplantation, donor HLA antibodies are a risk factor for graft loss. Accessibility of donor eplets for HLA antibodies is predicted by the ElliPro score. The clinical usefulness of those scores in relation to transplant outcome is unknown. In a large Dutch kidney transplant cohort, Ellipro scores of pretransplant donor antibodies that can be assigned to known eplets (donor epitope specific HLA antibodies [DESAs]) were compared between early graft failure and long surviving deceased donor transplants. We did not observe a significant Ellipro score difference between the two cohorts, nor significant differences in graft survival between transplants with DESAs having high versus low total Ellipro scores. We conclude that Ellipro scores cannot be used to identify DESAs associated with early versus late kidney graft loss in deceased donor transplants.

Determination of the clinical relevance of donor epitope-specific HLA-antibodies in kidney transplantation.

In kidney transplantation, survival rates are still partly impaired due to the deleterious effects of donor specific HLA antibodies (DSA). However, not all luminex-defined DSA appear to be clinically relevant. Further analysis of DSA recognizing polymorphic amino acid configurations, called eplets or functional epitopes, might improve the discrimination between clinically relevant vs. irrelevant HLA antibodies. To evaluate which donor epitope-specific HLA antibodies (DESAs) are clinically important in kidney graft survival, relevant and irrelevant DESAs were discerned in a Dutch cohort of 4690 patients using Kaplan-Meier analysis and tested in a cox proportional hazard (CPH) model including nonimmunological variables. Pre-transplant DESAs were detected in 439 patients (9.4%). The presence of certain clinically relevant DESAs was significantly associated with increased risk on graft loss in deceased donor transplantations (p < 0.0001). The antibodies recognized six epitopes of HLA Class I, 3 of HLA-DR, and 1 of HLA-DQ, and most antibodies were directed to HLA-B (47%). Fifty-three patients (69.7%) had DESA against one donor epitope (range 1-5). Long-term graft survival rate in patients with clinically relevant DESA was 32%, rendering DESA a superior parameter to classical DSA (60%). In the CPH model, the hazard ratio (95% CI) of clinically relevant DESAs was 2.45 (1.84-3.25) in deceased donation, and 2.22 (1.25-3.95) in living donation. In conclusion, the developed model shows the deleterious effect of clinically relevant DESAs on graft outcome which outperformed traditional DSA-based risk analysis on antigen level.

Radiotherapy to reinvigorate immunotherapy activity after acquired resistance in metastatic non-small-cell lung cancer: A pooled analysis of two institutions prospective phase II single arm trials.

AIM: The current work aimed to investigate the clinical benefit of radiotherapy in patients with metastatic non-small cell lung cancer (NSCLC) developing acquired resistance to immune checkpoint inhibitors. METHOD: We report on a pooled, two-institution, phase II single-arm prospective cohort study. The study included patients with stage IV NSCLC who showed progression of one or more measurable lesions under anti-PD-(L)1 inhibition alone, after initially having achieved at least stable disease. Hypofractionated radiotherapy (hRT) of one to four metastases was performed, while one or more lesions were kept untreated. Following hRT, treatment with immune checkpoint inhibitors was continued unchanged until further evidence of tumor progression or unacceptable toxicity. Primary endpoint of the pooled analysis was progression-free survival (PFS), secondary endpoints included overall survival (OS) and toxicity. RESULTS: A total of 48 patients were enrolled: mean age was 67.1 ± 9.3 years, 50 % were male and 72.9 % were PD-L1 positive. Immunotherapy was in 95.8 % of patients the first or second line therapy at time of enrollment. hRT was performed to one (93.8 % of cases) or more lesions (median total dose: 27.5 Gy, median 6.5 Gy/fraction). Forty-five patients (93.8 %) were able to continue immunotherapy for a median of 6.2 months following hRT. Median PFS was 4.4 months, with 62.5 % disease control at three months and 37.5 % at six months. Median OS was 14.9 months. Severe adverse events (grade ≥ 2) were reported in 12 cases (25 %), of which none were radiotherapy-related and four were immunotherapy-related. Salvage therapy consisted of chemotherapy (48.8 %) or repeated irradiation (21.9 %). No further tumor treatment was performed in 29.3 % of patients. CONCLUSIONS: The current pooled analysis is a prospective evaluation of the role of radiation therapy for metastatic NSCLC in the setting of newly acquired immunotherapy resistance. Hypofractionated radiotherapy can support the outcome of immune checkpoint inhibitors and thus allow continuation of treatment for a relevant amount of time despite initial tumor progression.

Low Levels of Serum and Intracellular Vitamin C in Hospitalized COVID-19 Patients.

Vitamin C is a crucial micronutrient for human immune cell function and has potent antioxidant properties. It is hypothesized that vitamin C serum levels decline during infection. However, the precise mechanisms remain unknown. To gain deeper insights into the true role of vitamin C during infections, we aimed to evaluate the body's vitamin C storage during a SARS-CoV-2 infection. In this single-center study, we examined serum and intracellular vitamin C levels in peripheral blood mononuclear cells (PBMCs) of 70 hospitalized COVID-19 patients on the first and fifth days of hospitalization. Also, clinical COVID-19 severity was evaluated at these timepoints. Our findings revealed a high prevalence of hypovitaminosis C and vitamin C deficiency in hospitalized COVID-19 patients (36% and 15%). Moreover, patients with severe or critical disease exhibited a higher prevalence of low serum vitamin C levels than those with moderate illness. Serum vitamin C levels had a weak negative correlation with clinical COVID-19 severity classification on the day of hospitalization; however, there was no correlation with intracellular vitamin C. Intracellular vitamin C levels were decreased in this cohort as compared to a healthy cohort and showed further decline during hospitalization, while serum levels showed no relevant change. Based on this observation, it can be suggested that the reduction of intracellular vitamin C may be attributed to its antioxidative function, the need for replenishing serum levels, or enhanced turnover by immune cells. These data give an incentive to further investigate the role of intracellular vitamin C in a larger and more heterogeneous cohort as well as the underlying mechanisms.

Antibody-dependent cellular cytotoxicity-inducing antibodies enhance the natural killer cell anti-cancer response against patient-derived pancreatic cancer organoids.

Introduction: Pancreatic cancer is associated with poor prognosis, and limited treatment options are available for the majority of patients. Natural killer (NK) cells in combination with antibodies inducing antibody-dependent cell-mediated cytotoxicity (ADCC) could be a highly effective new therapeutic option in pancreatic cancer. Accurate predictive preclinical models are needed to develop successful NK cell immunotherapy. Tumor organoids, in vitro 3D organ-like structures that retain important pathophysiological characteristics of the in vivo tumor, may provide such a model. In the current study, we assessed the cytotoxic potential of adoptive NK cells against human pancreatic cancer organoids. We hypothesized that NK cell anti-tumor responses could be enhanced by including ADCC-triggering antibodies. Methods: We performed cytotoxicity assays with healthy donor-derived IL-2-activated NK cells and pancreatic cancer organoids from four patients. A 3D cytotoxicity assay using live-cell-imaging was developed and enabled real-time assessment of the response. Results: We show that NK cells migrate to and target pancreatic cancer organoids, resulting in an increased organoid death, compared to the no NK cell controls (reaching an average fold change from baseline of 2.1±0.8 vs 1.4±0.6). After 24-hours of co-culture, organoid 2D growth increased. Organoids from 2 out of 4 patients were sensitive to NK cells, while organoids from the other two patients were relatively resistant, indicating patient-specific heterogeneity among organoid cultures. The ADCC-inducing antibodies avelumab (anti-PD-L1) and trastuzumab (anti-HER2) increased NK cell-induced organoid cell death (reaching an average fold change from baseline of 3.5±1.0 and 4.5±1.8, respectively). Moreover, combination therapy with avelumab or trastuzumab resulted in complete disintegration of organoids. Finally, inclusion of ADCC-inducing antibodies was able to overcome resistance in NK-organoid combinations with low or no kill. Discussion: These results support the use of organoids as a relevant and personalized model to study the anti-tumor response of NK cells in vitro and the potential of ADCC-inducing antibodies to enhance NK cell effector function.

Analysis of FCGR3A-p.176Val variants in women with recurrent pregnancy loss and the association with CD16a expression and anti-HLA antibody status.

Natural Killer (NK) cells have been implicated in recurrent pregnancy loss (RPL). The p.Val176Phe (or Val158Phe) Single Nucleotide Polymorphism (SNP) in the FCGR3A gene encoding the FcγRIIIA or CD16a receptor has been associated with an enhanced affinity for IgG and stronger NK-mediated antibody-dependent cellular cytotoxicity. We hypothesized that the presence of at least one p.176Val variant associates with RPL and increased CD16a expression and alloantibodies e.g., against paternal human leukocyte antigen (HLA). In 50 women with RPL, we studied frequencies of the p.Val176Phe FCGR3A polymorphisms. Additionally, CD16a expression and anti-HLA antibody status were analyzed by flowcytometry and Luminex Single Antigens. In woman with RPL, frequencies were: 20% (VV), 42% (VF) and 38% (FF). This was comparable to frequencies from the European population in the NCBI SNP database and in an independent Dutch cohort of healthy women. NK cells from RPL women with a VV (22,575 [18731-24607]) and VF (24,294 [20157-26637]) polymorphism showed a higher expression of the CD16a receptor than NK cells from RPL women with FF (17,367 [13257-19730]). No difference in frequencies of the FCGR3A-p.176 SNP were detected when comparing women with or without class I and class II anti-HLA antibodies. Our study does not provide strong evidence for an association between the p.Val176Phe FCGR3A SNP and RPL.

Exploring the potential of combining IL-2-activated NK cells with an anti-PDL1 monoclonal antibody to target multiple myeloma-associated macrophages.

Multiple myeloma (MM) is an incurable disease, characterized by malignant plasma cells in the bone marrow. MM growth is largely dependent on the tumor microenvironment (TME), consisting of complex cellular networks that shape a tumor-permissive environment. Within the TME, tumor-associated cells (TAC) comprise heterogeneous cell populations that collectively support immunosuppression. Reshaping the TME toward an immunostimulatory environment may enhance effectiveness of immunotherapies. Here, we investigated interactions between donor-derived natural killer (NK) cells and TAC, like tumor-associated macrophages (TAM) and M1 macrophages, and assessed whether anti-tumor effector functions of NK cells could be enhanced by an ADCC-triggering antibody targeting macrophages. Monocytes were polarized in vitro toward either M1 or TAM before co-culture with high-dose IL-2-activated NK cells. NK cell responses were assessed by measuring degranulation (CD107a) and IFN-γ production. We found that NK cells degranulated and produced IFN-γ upon interaction with both macrophage types. NK cell responses against PD-L1+ M1 macrophages could be further enhanced by Avelumab, an anti-PD-L1- and ADCC-inducing antibody. Additionally, NK cell responses were influenced by HLA class I, shown by stronger degranulation in NK cell subsets for which the corresponding HLA ligand was absent on the macrophage target cells (KIR-ligand mismatch) compared to degranulation in the presence of the HLA ligand (KIR-ligand match). Our results suggest that NK cells could, next to killing tumor cells, get activated upon interaction with TAC, like M1 macrophages and TAMs, and that NK cells combined with PD-L1 blocking antibodies with ADCC potential could, through IFN-γ secretion, promote a more immune-favorable TME.

An in vitro model to monitor natural killer cell effector functions against breast cancer cells derived from human tumor tissue.

Adoptive natural killer (NK) cell-based immunotherapy poses a promising treatment approach in cancer. Despite minimal toxicities associated with NK cell infusion, the potential of NK cell therapy is inhibited by the immunosuppressive tumor microenvironment (TME). Multiple approaches to improve anti-cancer NK cell effector functions are being investigated. While much of this preclinical research is currently performed with commercially available tumor cell lines, this approach lacks the influence of the TME and heterogeneity of the primary tumor in patients. Here, we describe a comprehensive protocol for NK cell cytotoxicity- and degranulation assays against tumor cells derived from primary breast cancer tissue. Treatments to boost NK cell anti-tumor effector functions can be implemented in this model. Moreover, by using culture supernatants in follow up assays or by including additional cell types in the co-culture system, other NK cell effector mechanisms that further orchestrate innate and adaptive immunity could be studied.

Inhibitory receptors for HLA class I as immune checkpoints for natural killer cell-mediated antibody-dependent cellular cytotoxicity in cancer immunotherapy.

Natural killer (NK) cells mediate potent anti-tumor responses, which makes them attractive targets for immunotherapy. The anti-tumor response of endogenous- or allogeneic NK cells can be enhanced through clinically available monoclonal antibodies that mediate antibody-dependent cellular cytotoxicity (ADCC). NK cell activation is regulated by interaction of inhibitory receptors with classical- and non-classical human leukocyte antigens (HLA) class I molecules. Inhibitory receptors of the killer immunoglobulin-like receptor (KIR) family interact with HLA-A, -B or -C epitopes, while NKG2A interacts with the non-classical HLA-E molecule. Both types of inhibitory interactions may influence the strength of the ADCC response. In the present review, we provide an overview of the effect of inhibitory KIRs and NKG2A on NK cell-mediated ADCC, which highlights the rationale for combination strategies with ADCC triggering antibodies and interference with the NK cell relevant inhibitory immune checkpoints, such as KIR and NKG2A.

No Effect of Vitamin C Administration on Neutrophil Recovery in Autologous Stem Cell Transplantation for Myeloma or Lymphoma: A Blinded, Randomized Placebo-Controlled Trial.

Vitamin C is an important micronutrient for various immune cells. It increases phagocytic cell function and is necessary for T and natural killer (NK) cell development. Patients in need of an autologous hematopoietic stem cell transplantation (HSCT) are often vitamin C-depleted. We therefore hypothesized that vitamin C supplementation could improve immune recovery in autologous HSCT patients. This blinded, placebo-controlled trial included 44 patients randomized to receive vitamin C or a placebo. The following outcome measures used were clinical and immunological parameters, among others: time to neutrophil recovery, serum, and intracellular vitamin C values. Twenty-one patients received vitamin C, and 23 received a placebo. The time to neutrophil recovery did not differ between the two groups at 11.2 days (p = 0.96). There were no differences in hospitalization time (19.7 vs. 19.1 days, p = 0.80), the incidence of neutropenic fever (57% vs. 78%, p = 0.20), or 3-month overall survival (90.5% vs. 100%, p = 0.13). Bacteremia seemed to occur less in the vitamin C group (10% vs. 35%, p = 0.07). Our study shows no benefit from vitamin C supplementation on neutrophil recovery and hospitalization, despite possible lower rates of bacteremia in the vitamin C group. Therefore, we do not advise vitamin C supplementation in this treatment group.

Natural killer cell profiles in recurrent pregnancy loss: Increased expression and positive associations with TACTILE and LILRB1.

PROBLEM: NK cells are important for healthy pregnancy and aberrant phenotypes or effector functions have been associated with RPL. We compared expression of a broad panel of NK cell receptors, including immune checkpoint receptors, and investigated their clinical association with RPL as this might improve patient stratification and prediction of RPL. METHOD OF STUDY: Peripheral blood mononuclear cells were isolated from 52 women with RPL and from 2 women with an uncomplicated pregnancy for flowcytometric analysis and plasma was used to determine anti-CMV IgG antibodies. RESULTS: Between RPL and controls, we observed no difference in frequencies of T-, NKT or NK cells, in CD56dimCD16+ or CD56brightCD16- NK cell subsets or in the expression of KIRs, NKG2A, NKG2C, NKG2D, NKp30, NKp44, NKp46 or DNAM1. NK cells from women with RPL had a higher expression of LILRB1 and TACTILE and this was associated with the number of losses. The immune checkpoint receptors PD1, TIM3 and LAG3 were not expressed on peripheral blood NK cells. In RPL patients, there was a large variation in NKG2C expression and higher levels could be explained by CMV seropositivity. CONCLUSION: Our study identified LILRB1 and TACTILE as NK cell receptors associated with RPL. Moreover, we provide first support for the potential role of CMV in RPL via its impact on the NK cell compartment. Thereby our study could guide future studies to confirm the clinical association of LILRB1, TACTILE and NKG2C with RPL in a larger cohort and to explore their functional relevance in reproductive success.

The Immunomodulatory Role of Hypoxic Tumor-Derived Extracellular Vesicles.

Tumor-associated immune cells frequently display tumor-supportive phenotypes. These phenotypes, induced by the tumor microenvironment (TME), are described for both the adaptive and the innate arms of the immune system. Furthermore, they occur at all stages of immune cell development, up to effector function. One major factor that contributes to the immunosuppressive nature of the TME is hypoxia. In addition to directly inhibiting immune cell function, hypoxia affects intercellular crosstalk between tumor cells and immune cells. Extracellular vesicles (EVs) play an important role in this intercellular crosstalk, and changes in both the number and content of hypoxic cancer-cell-derived EVs are linked to the transfer of hypoxia tolerance. Here, we review the current knowledge about the role of these hypoxic cancer-cell-derived EVs in immunosuppression. In addition, we provide an overview of hypoxia-induced factors (i.e., miRNA and proteins) in tumor-derived EVs, and their role in immunomodulation.

Polymorphic differences within HLA-C alleles contribute to alternatively spliced transcripts lacking exon 5.

The HLA genes are amongst the most polymorphic in the human genome. Alternative splicing could add an extra layer of complexity, but has not been studied extensively. Here, we applied an RNA based approach to study the influence of allele polymorphism on alternative splicing of HLA-C in peripheral blood. RNA was isolated from these peripheral cells, converted into cDNA and amplified specifically for 12 common HLA-C allele groups. Through subsequent sequencing of HLA-C, we observed alternative splicing variants of HLA-C*04 and *16 that resulted in exon 5 skipping and were co-expressed with the mature transcript. Investigation of intron 4 sequences of HLA-C*04 and *16 compared with other HLA-C alleles demonstrated no effect on predicted splice sites and branch point. To further investigate if the unique polymorphic positions in exon 5 of HLA-C*04 or *16 may facilitate alternative splicing by acting on splicing regulatory elements (SRE), in-silico splicing analysis was performed. While the HLA-C*04 specific SNP in exon 5 had no effect on predicted exonic SRE, the HLA-C*16 specific exon 5 SNP did alter exonic SRE. Our findings provide experimental and theoretical support for the concept that polymorphisms within the HLA-C alleles influence the alternative splicing of HLA-C.

Endometrial factors in the implantation failure spectrum: protocol of a MUltidisciplinary observational cohort study in women with Repeated Implantation failure and recurrent Miscarriage (MURIM Study).

INTRODUCTION: Women with repeated implantation failure (RIF) and unexplained recurrent miscarriage (RM) are proposed to be at opposite ends of the implantation spectrum, with RM representing an overly receptive endometrium (implantation of genetically aberrant or poor-quality embryos) versus RIF representing an overly selective endometrium (no implantation even with good quality embryos). In both cases, often no explanation for reproductive failure can be found and although promising add-on treatments have been introduced, therapeutic options are frequently limited to supportive care. Both RM and RIF are multifactorial and research indicates that the interplay between steroidogenesis, uterine natural killer (uNK) cells and the microbiome determine the capacity of the endometrium to be a biosensor for invading embryos. Our objective is to elucidate whether there is a difference in endometrial receptivity parameters (ie, steroid metabolism, uNK cells and the microbiome) between women aged 18-38 years with reproductive failure (RIF and RM), and fertile controls. METHODS AND ANALYSIS: Single-centre, observational cohort study. Endometrial biopsies, vaginal swabs and peripheral blood will be collected during the window of implantation and menstrual blood in the subsequent menstruation. The study parameters are the steroid profile (steroid levels and mRNA levels, protein expression and activity of steroid enzymes) in endometrial tissue and peripheral blood, as well as the activating or inhibitory phenotype of uNK cells based on receptor expression in menstrual blood and endometrial tissue and determination of the vaginal and endometrial microbiome using the inter spacer bacterial profiling technique. ETHICS AND DISSEMINATION: The protocol is approved by the local medical ethical review committee at the Maastricht University Medical Centre. Findings from this study will be shared with the academic and medical community and the patient organisations to optimise and individualise medical care of patients with implantation failure and miscarriages. TRIAL REGISTRATION NUMBER: NTR7571, registered 28 February 2019.

Intravenous immunoglobulins improve live birth rate among women with underlying immune conditions and recurrent pregnancy loss: a systematic review and meta-analysis.

Intravenous immunoglobulin (IVIG) is increasingly used as a treatment for recurrent pregnancy loss (RPL) despite lack of clear evidence on efficacy. Recent data suggest IVIG might be more effective in a subgroup of women with an aberrant immunological profile. Therefore, a systematic review and meta-analysis of studies on the effectiveness of IVIG treatment on pregnancy outcome among women with RPL and underlying immunological conditions (e.g., elevated NK cell percentage, elevated Th1/Th2 ratio, diagnosis with autoimmune disorders) was conducted. Eight non-randomized controlled trials, including 478 women (intervention: 284; control: 194), met eligibility criteria. Meta-analysis showed that treatment with IVIG was associated with a two-fold increase in live birth rate (RR 1.98, 95% CI 1.44-2.73, P < 0.0001). The effect of IVIG was particularly marked in the subgroup of studies including patients based on presence of elevated (> 12%) NK-cell percentage (RR 2.32, 95% CI 1.77-3.02, P < 0.0001) and when starting intervention prior to or during cycle of conception (RR 4.47, 95% CI 1.53-13.05, P = 0.006). In conclusion, treatment with IVIG may improve live birth rate in women with RPL and underlying immune conditions. However, these results should be interpreted with caution as studies are limited by low number of participants and the non-randomized design, which represent seriously biases. Future randomized controlled trials in women with RPL and underlying immune conditions are needed before using IVIG in a clinical setting.