KIR Genotype Research Articles

Patterns of KIR-genotypes in women with preimplantation losses after assisted reproductive technologies

Aim. To analyze the frequency and distribution of KIR genotypes in women with repeated preimplantation losses during ARTs compared to women with control group. Methods. DNA isolation from leukocytes by the salting method, PCR-SSP, agarose gel electrophoresis; methods of statistical analysis. Results. The molecular genetic determination of KIR genes in the group of women with recurrent implantation failures and in the control group of women was performed. The frequency and distribution of KIR genotypes in the study groups were determined. In women with RIF, a slightly shifted distribution of the frequency of KIR genotypes was found, towards the homozygous genotype by haplotype "A", the frequency of which was almost half of all identified genotypes (42.86 %). The distribution and frequency of KIR genotypes in the control group was normal and described by the Hardy-Weinberg principle. A comparative analysis of the study groups using statistical methods showed a significantly higher frequency of the KIR-AA genotype (χ2=8.875; p<0.005) in the group of women with RIF compared to the control group of women. The calculation of the odds ratio indicates a 2-fold increase of risk for recurrent implantation failures after ARTs (OR=1.94; CI 95 %: 1.25–3.00) in women with the KIR-AA genotype. Conclusions. We regard the KIR-AA genotype as a significant risk factor for preimplantation loss. KIR genotyping is considered an important molecular genetic test for determining the risk of fetal rejection by the maternal immune system, which will help to correctly direct treatment interventions to keep the pregnancy alive.

Subcutaneous G-CSF administration improves IVF outcomes in patients with recurrent implantation failure presenting a KIR/HLA-C mismatch

Research questionDespite advances in assisted reproductive technologies, many blastocysts are lost unexpectedly during implantation. Alterations in maternal immune tolerance towards fetal antigens may contribute to adverse IVF outcomes. The purpose of this study is to evaluate whether administering Granulocyte Colony-Stimulating Factor (G-CSF) to couples with a Human Leukocyte Antigen/Killer-Cell Immunoglobulin-Like Receptor (HLA/KIR) mismatch could positively modulate the implantation process in patients with recurrent implantation failure (RIF). A KIR/HLA-C mismatch occurs when the interaction between KIRs and HLA-C causes an inhibition of NK cells, which may result in reduced G-CSF secretion leading to impaired placentation and increased risk of miscarriage, pre-eclampsia and fetal growth restriction. DesignA retrospective monocentric cohort study conducted at the IVI Clinic in Rome, including women with a history of at least two failed blastocyst transfers. Couples underwent KIR and HLA-C testing. Couples with a KIR/HLA-C mismatch received G-CSF subcutaneously up to week nine of gestation. The mismatch included cases with inhibitory KIR genotypes and HLA-C2C2 females with HLA-C1C1, or C1C2 males or HLA-C1C2 females with male HLA-C2C2. The reproductive outcomes were assessed, and the logistic regression models controlled for potential confounders affecting IVF outcomes. Results79 patients with RIF and a KIR/HLA-C mismatch were included in the study. 30 patients were administered G-CSF, and 49 received no treatment. In the univariate analysis, no statistically significant differences were reported in the reproductive outcomes after IVF between the women treated with G-CSF and the control group. However, the logistic regression analysis that controlled for confounding factors showed that patients treated with subcutaneous G-CSF had statistically significant higher ongoing-pregnancy (aOR=3.808) and live-birth (aOR=4.998) rates, and a lower miscarriage rate (aOR=0.057). No statistically significant differences were found in other reproductive outcomes. ConclusionThe use of subcutaneous G-CSF in patients with a KIR/HLA-C mismatch undergoing IVF may reduce miscarriage and improve live-birth rates. G-CSF may modulate NK-mediated immune mechanisms and improve trophoblast invasion and development. Randomized trials are warranted to validate these findings and enhance the chances of successful pregnancies in couples with an immunological mismatch.

Multifactorial determinants of NK cell repertoire organization: insights into age, sex, KIR genotype, HLA typing, and CMV influence.

Polymorphisms in the KIR and HLA genes contribute to the diversity of the NK cell repertoire. Extrinsic factors also play a role in modifying this repertoire. The best example is cytomegalovirus, which promotes the expansion of memory-like NK cells. However, the mechanisms governing this phenotypic structure are poorly understood. Furthermore, the influence of age and sex has been understudied. In this study, we examined these parameters in a cohort of 200 healthy volunteer blood donors, focusing on the major inhibitory KIR receptors and CD94/NKG2A, as well as the differentiation marker CD57 and the memory-like population marker NKG2C. Flow cytometry and two joint analyses, unsupervised and semi-supervised, helped define the impact of various intrinsic and extrinsic markers on the phenotypic structure of the NK cell repertoire. In the KIR NK cell compartment, the KIR3DL1 gene is crucial, as unexpressed alleles lead to a repertoire dominated by KIR2D interacting only with HLA-C ligands, whereas an expressed KIR3DL1 gene allows for a greater diversity of NK cell subpopulations interacting with all HLA class I ligands. KIR2DL2 subsequently favors the KIR2D NK cell repertoire specific to C1/C2 ligands, whereas its absence promotes the expression of KIR2DL1 specific to the C2 ligand. The C2C2Bw4+ environment, marked by strong -21T motifs, favors the expansion of the NK cell population expressing only CD57, whereas the absence of HLA-A3/A11 ligands favors the population expressing only NKG2A, a population highly represented within the repertoire. The AA KIR genotype favors NK cell populations without KIR and NKG2A receptors, whereas the KIR B+ genotypes favor populations expressing KIR and NKG2A. Interestingly, we showed that women have a repertoire enriched in CD57- NK cell populations, while men have more CD57+ NK cell subpopulations. Overall, our data demonstrate that the phenotypic structure of the NK cell repertoire follows well-defined genetic rules and that immunological history, sex, and age contribute to shaping this NK cell diversity. These elements can contribute to the better selection of hematopoietic stem cell donors and the definition of allogeneic NK cells for cell engineering in NK cell-based immunotherapy approaches.cters are displayed correctly.

The Role of NK Cells in Allogeneic Hematopoietic Stem Cell Micro-Transplantation for Acute Myeloid leukemia

To explore the role of NK cells in allogeneic hematopoietic stem cell micro-transplantation(MST) in the treatment of patients with acute myeloid leukemia(AML). Data from 93 AML patients treated with MST at our center from 2013-2018 were retrospectively analyzed. The induction regimen was anthracycline and cytarabine combined with peripheral blood stem cells transplantation mobilization by granulocyte colony stimulating factor (GPBSC), followed by 2-4 courses of intensive treatment with medium to high doses of cytarabine combined with GPBSC after achieving complete remission (CR). The therapeutic effects of one and two courses of MST induction therapy on 42 patients who did not reach CR before transplantation were evaluated. Cox proportional hazards regression analysis was used to analyze the impact of donor NK cell dose and KIR genotype, including KIR ligand mismatch, 2DS1, haplotype, and HLA-Cw ligands on survival prognosis of patients. Forty-two patients received MST induction therapy, and the CR rate was 57.1% after 1 course and 73.7% after 2 courses. Multivariate analysis showed that, medium and high doses of NK cells was significantly associated with improved disease-free survival (DFS) of patients (HR=0.27, P =0.005; HR=0.21, P =0.001), and high doses of NK cells was significantly associated with improved overall survival (OS) of patients (HR=0.15, P =0.000). Donor 2DS1 positive significantly increases OS of patients (HR=0.25, P =0.011). For high-risk patients under 60 years old, patients of the donor-recipient KIR ligand mismatch group had longer DFS compared to the nonmismatch group (P =0.036); donor 2DS1 positive significantly prolonged OS of patients (P =0.009). NK cell dose, KIR ligand mismatch and 2DS1 influence the therapeutic effect of MST, improve the survival of AML patients.

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.

Fresh vs. frozen embryo transfers in patients with KIR Bx haplotype: impact on reproductive outcomes.

A controversy persists over whether or not the type of embryo transfer (ET) influences reproductive outcomes. This study aimed to evaluate the reproductive outcomes of pregnant patients undergoing their first in vitro fertilization procedure and explore the influence of various KIR genotypes on these reproductive outcomes. Prospective enrollment of patients with infertility who sought treatment at Origyn Fertility Center in Iasi, Romania, was conducted between January 2019 and March 2023. Descriptive statistics and average treatment effects (ATE) using propensity-score matching were employed to analyze our data. Our results indicated that both groups were homogenous regarding baseline characteristics. When we evaluated the ATE of fresh vs. frozen ET on the main outcomes, we discovered that only frozen ET significantly improved the pregnancy rates (ATE: 0.17, 95% CI: 0.04-0.30, p=0.011) and live birth rates (ATE: 0.36, 95% CI: 0.02-1.19, p=0.03). The miscarriage rates were similar between the two groups. None of the evaluated KIR genotypes had a significant influence on the ATE corresponding to fresh and frozen ET. KIR screening is not necessary before an IVF cycle, except for specific situations such as recurrent pregnancy loss or recurrent implantation failure.

Abatacept and Ptcy Based Gvhd Prophylaxis with CNI/Sirolimus for Haploidentical HCT: Donor KLRC2 Genotype Impacts Outcome

Background: Abatacept (Abata, CTLA4Ig), an inhibitor of a key T cell costimulation pathway has recently been approved for GVHD prophylaxis following unrelated donor HCT in combination with CNI/MTX. In 2015, we had developed a novel protocol combining Abata and PTCy for HCT from haploidentical family donors (HFD). We analysed the impact of donor immunogenetics and immune reconstitution on the long-term outcome of 182 patients receiving HFD-HCT on this protocol. Methods: In patients with nonmalignant diseases (NMD), Abata was administered on days 0, +5, +20, +35 and every 4 weeks thereafter until day +180. PTCy was administered at 50 mg/kg on days +3 and +4 and sirolimus was given from day -7 to 9 months post-HCT (AbaCyS protocol). In those with malignant diseases (MD), PTCy was administered as usual and Abata was administered on day 0 followed by PBSC infusion and on days+7, +21 and +35, followed 6 hours later by donor lymphocyte infusions (DLI). Cyclosporin was administered from day +5 to day +60 (AbaDCyC protocol). NK cell related immunogenetic parameters (KIR genotype/ KIR-ligand/ HLA-B leader peptide dimorphism/ KLRC2 genotype encoding NKG2C receptor, [KLRC2 deletion (del) vs wildtype (wt]) and T and NK cell subset reconstitution were analysed with respect to primary (OS, NRM, Relapse) and secondary (acute & chronic GVHD, CMV reactivation and GVHD & Event-free survival (GVHEFS) endpoints. Results: Primary graft failure was seen in 2/46 with NMD and 1/136 with MD, with median time to engraftment being 16 days (12-26). No secondary graft failure was observed. Incidence of aGVHD 2-4 was 8.5%, 4.7% in NMD and 9.8% in MD (p=0.3). Donor KLRC2del (D-KLRC2del) genotype was the only risk factor (33.8%{12/38} vs 2.2%{3/144} in dKLRC2wt, p<0.001, Figure-1) for aGVHD. This translated to a significantly improved NRM (overall 9.5%) in dKLRC2wt group (5.2% vs 28.9% in dKLRC2del, p<0.001). Incidence of cGVHD (mostly mild-moderate) was 15.3% and was not impacted by NMD vs MD or KLRC2 genotype. Relapse incidence (RI) was 21.4%, with no impact of pretransplant disease status (DRI low vs high) or any immunogenetic factors apart from KIR haplotype (lower RI in donor KIR B haplotype, p=0.01). OS was 84.3% at a median follow-up of 4 years, with a significant impact of dKLRC2 genotype (90% in wt vs 63% in del, p<0.001, Figure-2). The overall GVHEFS was 70% and this was also impacted by dKLRC2 genotype (38.2% vs 77.8%in dKLRC2wt, p<0.001). None of the other immunogenetic parameters had any impact on the outcome. T cell subset reconstitution was robust in both groups, predominantly of memory phenotype, which tended to be more rapid with AbaDCyC protocol in MD. However, recovery of Tregs was more rapid with AbaCyS protocol in NMD and the kinetics of Treg recovery positively correlated with reduced cGVHD in this group. Even though NK cell maturation was brisk in both groups, this was prompter at day+30 in the AbaDCyC protocol group. More importantly, kinetics of NKG2C+adaptive NK cell (ANK) recovery was the only determinant of RI (p<0.01). Conclusion: The combination of Abata and PTCy was extremely effective in reducing the incidence of aGVHD, which was strongly influenced by dKLRCdel genotype, which in turn adversely impacted NRM, OS and GVHEFS. AbaCyS protocol was associated with better recovery of Tregs vis-à-vis reduced cGVHD. Abata-primed DLI as in AbaDCyC was not associated with increase in GVHD and seemed to moderate the RI in patients with advanced malignancy, which was influenced by ANK recovery. Thus, immunogenetics and immune reconstitution related to ANK subset of NK cells strongly influenced the outcome with Aba-PTCy based GVHD prophylaxis. DLI could be safely administered with Abata priming without increase in GVHD and yet result in attenuation of RI, suggesting the potential of this approach to dissociate GVHD from a NK-mediated GVL effect.

Analysis of KIR genotyping results in women with recurrent implantation failure

Aim. To analyze the frequency and distribution of KIR genotypes in women with recurrent implantation failure after cycles of in vitro fertilization. Methods. DNA extraction and purification, PCR-SSP, agarose gel electrophoresis. Results. The spectrum of KIR genes and the frequency of KIR genotypes in women with recurrent implantation failure were determined. The study group was divided according to history, in particular, a group of 84 women whose diagnosis was outlined by the concept of "repeated failed implantations" was singled out. Statistical analysis of the obtained results using the Pearson test (χ2) indicates a significantly lower frequency of the KIR-AB genotype (χ2 = 9.7; p < 0.005) and a significantly higher frequency of the KIR-AA genotype (χ2 = 7.35; p < 0.01) in women with repeated failed implantations compared to the general group of women with reproductive failures. Conclusions. Taking into account the results of statistical data processing (OR = 2.05; CI 95 %: 1.21-3.45), we consider the KIR-AA genotype to be a significant risk factor for recurrent implantation failure after IVF. We define KIR genotyping as a genetic test to assess the risk of the embryo being rejected by the maternal immune system, and thus to direct medical interventions in order to achieve a successful pregnancy.

KIR Genotypes Impact Progression to Hepatocellular Carcinoma in Patients with Chronic Hepatitis C Infection

In Egypt, hepatocellular carcinoma (HCC) is the most prevalent cancer in men and the second most prevalent cancer in women. In addition, Egypt has one of the highest prevalences of hepatitis C infection in the world. The aim of the present work was to study the potential role of the 16 KIR genes in the outcome of individuals with chronic hepatitis C virus (HCV) infection in Egypt. The study was carried out under an IRB-approved protocol. Sequence-Specific-Primer-PCR (SSP-PCR) was used for KIR genotyping of germline DNA extracted from peripheral blood leukocytes or from the non-tumor liver of 83 HCC patients, 100 patients with chronic HCV infection without HCC, and 120 matched healthy controls. Out of the 83 HCC patients, only 7 (8.4%) were treated by interferon and/or interferon Ribavirin combination, while for the remaining patients 50 (60.2%) received no prior HCV therapy and 26 (31.3%) were treated with direct-acting antiviral (DAA). Our results showed that KIR haplotype AA that contains more inhibitory KIR genes and fewer activating genes was observed with a significantly lower frequency in HCC patients (6/83, 7.2%) compared to chronic HCV (27/100, 27.0%) (p = 0.0005, OR = 0.21 [0.08–0.53]) and healthy controls (29/119, 24.4%) (p = 0.001, OR = 0.24 [0.09–0.61]). In addition, the frequency of genotype 6 (G6) which contains all the KIR genes was significantly high in the HCC patients (16/83, 19.3%) compared to chronic HCV (8/100, 8.0%) (p = 0.02, OR = 2.7 [1.11–6.79]) and healthy controls (8/119, 6.7%) (p = 0.006, OR = 3.31 [1.35–8.16]). Activating KIR genes 2DS1 and 3DS1 were significantly higher in HCC patients (48/83, 57.83% and 45/83, 54.22%) compared to the chronic HCV patients (36/100, 36% and 34/100, 34%), p = 0.028, 0.027, respectively. Our results are contrary to a prior work on HCC from patients with HCV who were mostly treated by interferon-based therapies. In conclusion, KIR haplotype AA has an important role in host defense against HCC progression especially in patients treated by DAA, suggesting an important role of the KIR genotype status on the outcome of chronic HCV infection.

Establishment of a PCR-SSP method for the simultaneous amplification and identification of the presence of KIR genes

To develop a polymerase chain reaction-sequence specific primer (PCR-SSP) method for simultaneous amplification and identification of the KIR genes among Chinese population. Peripheral blood samples from 132 healthy donors who had given blood at Shenzhen Blood Center from January 2015 to November 2015 were selected as the study subjects. Based on the polymorphism and single nucleotide polymorphism (SNP) information of high-resolution KIR alleles in the Chinese population and the IPD-KIR database, specific primers were designed to amplify all the 16 KIR genes and the 2DS4-Normal and 2DS4-Deleted subtypes. The specificity of each pair of PCR primers was verified by using samples with known KIR genotypes. During PCR amplification of the KIR gene, co-amplification the fragment of human growth hormone (HGH) gene by multiplex PCR was used as the internal control to prevent false negative results. A total of 132 samples with known KIR genotypes were randomly selected for blind inspection to verify the reliability of the developed method. The designed primers can specifically amplify the corresponding KIR genes, with clear and bright bands for the internal control and KIR genes. The results of detection are fully consistent with the known results. The KIR PCR-SSP method established in this study can yield accurate results for the identification of the presence of KIR genes.

Association of KIR Genes Polymorphism and its HLA Ligands in Diffuse Large B-cell Lymphoma.

Non-Hodgkin lymphoma (NHL) is a heterogeneous disease, with each subtype associated with different risk factors. Within this group, diffuse large B-cell lymphoma (DLBCL) can be highlighted, the most common type of NHL.NK cells are key components of the innate immune response and may play an important antitumor role. The objective of the present work was to determine the polymorphism of KIR genes in Brazilian patients with DLBCL. Furthermore, we evaluated the association between the polymorphism of these genes and their ligands with the clinical course of the disease. For the study, 112 patients with DLBCL and 222 voluntary blood and bone marrow donors. The genetic material of these samples were extracted for KIR and HLA typing, determination of HLA ligands, determination of the KIR haplotype and search for the deletion of 22 bp in the KIR2DS4 gene. KIR genotype distributions were made by direct counting using 2 × 2 contingency tables using Fisher's exact test. The magnitude of the association was measured by odds ratio (OR) and 95% confidence interval. P values <.05 were considered significant. Overall survival and progression-free survival were assessed with a Kaplan-Meier estimator. In the present study, an association of HLA-Bw4 and HLA-Bw480I ligand was found with more advanced stages of the disease. Also, an association of the KIR2DL3 gene with a better response to treatment was found. With this, we can conclude that the polymorphism of KIR genes and the association with HLA ligands can influence the prognosis of DLBCL, as well as the response to treatment was found. With this, we can conclude that the polymorphism of KIR genes and the association with HLA ligands can influence the prognosis of DLBCL, as well as the response to treatment.Non-Hodgkin lymphoma (NHL) is a heterogeneous disease, with each subtype associated with different risk factors. Within this group, diffuse large B-cell lymphoma (DLBCL) can be highlighted, the most common type of NHL.NK cells are key components of the innate immune response and may play an important antitumor role. The objective of the present work was to determine the polymorphism of KIR genes in Brazilian patients with DLBCL. Furthermore, we evaluated the association between the polymorphism of these genes and their ligands with the clinical course of the disease. For the study, 112 patients with DLBCL and 222 voluntary blood and bone marrow donors. The genetic material of these samples were extracted for KIR and HLA typing, determination of HLA ligands, determination of the KIR haplotype and search for the deletion of 22 bp in the KIR2DS4 gene. KIR genotype distributions were made by direct counting using 2×2 contingency tables using Fisher's exact test. The magnitude of the association was measured by odds ratio (OR) and 95% confidence interval. P values <.05 were considered significant. Overall survival and progression-free survival were assessed with a Kaplan-Meier estimator. In the present study, an association of HLA-Bw4 and HLA-Bw480I ligand was found with more advanced stages of the disease. Also, an association of the KIR2DL3 gene with a better response to treatment was found. With this, we can conclude that the polymorphism of KIR genes and the association with HLA ligands can influence the prognosis of DLBCL, as well as the response to treatment was found. With this, we can conclude that the polymorphism of KIR genes and the association with HLA ligands can influence the prognosis of DLBCL, as well as the response to treatment.

Trans-population graph-based coverage optimization of allogeneic cellular therapy.

Pre-clinical development and in-human trials of 'off-the-shelf' immune effector cell therapy (IECT) are burgeoning. IECT offers many potential advantages over autologous products. The relevant HLA matching criteria vary from product to product and depend on the strategies employed to reduce the risk of GvHD or to improve allo-IEC persistence, as warranted by different clinical indications, disease kinetics, on-target/off-tumor effects, and therapeutic cell type (T cell subtype, NK, etc.). The optimal choice of candidate donors to maximize target patient population coverage and minimize cost and redundant effort in creating off-the-shelf IECT product banks is still an open problem. We propose here a solution to this problem, and test whether it would be more expensive to recruit additional donors or to prevent class I or class II HLA expression through gene editing. We developed an optimal coverage problem, combined with a graph-based algorithm to solve the donor selection problem under different, clinically plausible scenarios (having different HLA matching priorities). We then compared the efficiency of different optimization algorithms - a greedy solution, a linear programming (LP) solution, and integer linear programming (ILP) -- as well as random donor selection (average of 5 random trials) to show that an optimization can be performed at the entire population level. The average additional population coverage per donor decrease with the number of donors, and varies with the scenario. The Greedy, LP and ILP algorithms consistently achieve the optimal coverage with far fewer donors than the random choice. In all cases, the number of randomly-selected donors required to achieve a desired coverage increases with increasing population. However, when optimal donors are selected, the number of donors required may counter-intuitively decrease with increasing population size. When comparing recruiting more donors vs gene editing, the latter was generally more expensive. When choosing donors and patients from different populations, the number of random donors required drastically increases, while the number of optimal donors does not change. Random donors fail to cover populations different from their original populations, while a small number of optimal donors from one population can cover a different population. Graph-based coverage optimization algorithms can flexibly handle various HLA matching criteria and accommodate additional information such as KIR genotype, when such information becomes routinely available. These algorithms offer a more efficient way to develop off-the-shelf IECT product banks compared to random donor selection and offer some possibility of improved transparency and standardization in product design.

Influence of killer immunoglobulin-like receptors genes on the recurrence rate of ocular toxoplasmosis in Brazil.

Recurrence is a hallmark of ocular toxoplasmosis (OT), and conditions that influence its occurrence remain a challenge. Natural killer cells (NK) are effectors cells whose primary is cytotoxic function against many parasites, including Toxoplasma gondii. Among the NK cell receptors, immunoglobulin-like receptors (KIR) deserve attention due to their high polymorphism. This study aimed to analyse the influence of KIR gene polymorphism in the course of OT infection and its association with recurrences after an active episode. Ninety-six patients from the Ophthalmologic Clinic of the National Institute of Infectology Evandro Chagas were followed for up to five years. After DNA extraction, genotyping of the patients was performed by polymerase chain reaction sequence-specific oligonucleotide (PCR-SSO) utilising Luminex equipment for reading. During follow-up, 60.4% had a recurrence. We identified 25 KIR genotypes and found a higher frequency of genotype 1 (31.7%) with worldwide distribution. We note that the KIR2DL2 inhibitor gene and the gene activator KIR2DS2 were more frequent in patients without recurrence. Additionally, we observed that individuals who carry these genes progressed recurrence episodes slowly compared to individuals who do not carry these genes. The KIR2DL2 and KIR2DS2 are associated as possible protection markers against ocular toxoplasmosis recurrence (OTR).

Killer Cell Immunoglobulin-like Receptors (KIR) and Human Leucocyte Antigen C (HLA-C) Increase the Risk of Long-Term Chronic Liver Graft Rejection.

Chronic liver rejection (CR) represents a complex clinical situation because many patients do not respond to increased immunosuppression. Killer cell immunoglobulin-like receptors/Class I Human Leukocyte Antigens (KIR/HLA-I) interactions allow for predicting Natural Killer (NK) cell alloreactivity and influence the acute rejection of liver allograft. However, its meaning in CR liver graft remains controversial. KIR and HLA genotypes were studied in 513 liver transplants using sequence-specific oligonucleotides (PCR-SSO) methods. KIRs, human leucocyte antigen C (HLA-C) genotypes, KIR gene mismatches, and the KIR/HLA-ligand were analyzed and compared in overall transplants with CR (n = 35) and no-chronic rejection (NCR = 478). Activating KIR (aKIR) genes in recipients (rKIR2DS2+ and rKIR2DS3+) increased CR compared with NCR groups (p = 0.013 and p = 0.038). The inhibitory KIR (iKIR) genes in recipients rKIR2DL2+ significantly increased the CR rate compared with their absence (9.1% vs. 3.7%, p = 0.020). KIR2DL3 significantly increases CR (13.1% vs. 5.2%; p = 0.008). There was no influence on NCR. CR was observed in HLA-I mismatches (MM). The absence of donor (d) HLA-C2 ligand (dC2−) ligand increases CR concerning their presence (13.1% vs. 5.6%; p = 0.018). A significant increase of CR was observed in rKIR2DL3+/dC1− (p = 0.015), rKIR2DS4/dC1− (p = 0.014) and rKIR2DL3+/rKIR2DS4+/dC1− (p = 0.006). Long-term patient survival was significantly lower in rKIR2DS1+rKIR2DS4+/dC1− at 5–10 years post-transplant. This study shows the influence of rKIR/dHLA-C combinations and aKIR gene-gene mismatches in increasing CR and KIR2DS1+/C1-ligands and the influence of KIR2DS4+/C1-ligands in long-term graft survival.

P-367 Centromeric motif AA of the KIR: the best subrogate marker that defines Alloimmune Reproductive Failure

Abstract Study question Should the centromeric and telomeric motifs been studied separately and taked them in to account in women with recurrent reproductive failure? Summary answer The results show that when the classic KIR study is performed, there is a 12,5% of AA centromeric KIR patients with reproductive failure unidentified. What is known already During human pregnancy, the decidua is mainly populated by NK lymphocytes that express immunoglobulin-type receptors (KIR) recognizing their HLA-C ligands from trophoblast. The objective of this study was to analyze the association of KIR-HLAC phenotypes in couples with sterility, recurrent pregnancy loss (RPL), recurrent implantation failure (RIF), and fetal death (FD) in comparison with reference population and fertile control. Study design, size, duration It is a retrospective real-life observational study from 2015 to 2019 Participants/materials, setting, methods The frequency of the KIR and HLA-C genes were analyzed in 397 women and their partners with reproductive failure. We compare them with 33 fertile controls and reference Spanish population. KIR and HLA-C genotypes of the patients and controls were determined by Lifecodes Sequence-Specific Oligonucleotides (SSO) Typing KIT (Diagnostica Longwood®) performed at Centro de Transfusiones de la Comunidad de Madrid and those of controls at the Hospital Universitario Clínico San Carlos. Main results and the role of chance Centromeric (Cen) AA was the most frequent KIR genotype in women with unexplained RPL and RIG with respect to reference Spanish population (p = 0.001 and 0.02, respectively). Moreover, cenAA was associated with 1.51-fold risk of RPL and 1.2-fold risk of RIF. On the other hand, BB KIR showed lower risk of any reproductive failure tan non-BB KIR (0.12; p &amp;lt; 0.001). HLA-C1C1/C1C1 women and partner were significantly lower in RPL-Group (p &amp;lt; 0.001) and RIF-Group (p = 0.04). Indeed, the combination cenAA/C1C1 women with C1C1 partners were significantly higher in the control group compared with RPL (p = 0.009) and RIF (0.04) with 5-fold of successful pregnancy outcome. In our cohort, cenAA KIR was a more accurate biomarker tan classic AA KIR haplotype for RPL- and RIF-associated risk. Limitations, reasons for caution The fact of having stratified a lot by groups means that a small number of patients is obtained in each subgroup, which makes it more difficult to conclude the results. Wider implications of the findings Our study demonstrates the importance of the complete KIR genotype study, which would give us more comprehensive information on the behavior of NK cells, moreover we propose that centromeric KIR classification is superior to the KIR classical haplotypes, better identifying those patients with potential KIR-HLA-C mismatch. Trial registration number Not applicable

T-Cell Depleted Haploidentical Transplantation in Children With Hematological Malignancies: A Comparison Between CD3+/CD19+ and TCRαβ+/CD19+ Depletion Platforms.

BackgroundT-cell depleted (TCD) haploidentical transplantation using CD3+/CD19+ and TCRαβ+/CD19+ depletion techniques has been increasingly used in children with hematological malignancies. We present a retrospective study aimed to compare transplant outcomes in children with leukemia receiving a TCD haploidentical transplant using either CD3+/CD19+ or TCRαβ+/CD19+ platforms.MethodsA total of 159 children with leukemia (ALL=80) (AML=79) that received a TCD haploidentical transplantation using either CD3+/CD19+ (n=79) or TCRαβ+/CD19+ (n=80) platforms between 2005 and 2020 were included. Median age was 9 years in both groups. There were no differences in patient, donor, and transplant characteristics between groups except for donor KIR B genotype more frequent in the TCRαβ+/CD19+ group (91%) than in the CD3+/CD19+ group (76%) (p=0.009) and a high number of NK+ cells and lower CD19+ cells infused in the TCRαβ+/CD19+ group (35.32x106/kg and 0.06 x106/Kg) than in the CD3+/CD19 group (24.6x106/Kg and 0.25 x106/Kg) (p=0.04 and p=0.0001), respectively. Conditioning was based on TBF. Median follow-up for survivors was 11 years (range; 8-16 y) in CD3+/CD19+ group and 5 years (range; 2-9 y) in the TCRαβ+/CD19+ group.ResultsEngraftment kinetics were similar in both groups (13 days for neutrophils and 10 days for platelets). There was no difference in the incidence of acute GvHD II-IV (29 ± 5% in the CD3+/CD19+ group vs 38 ± 5% in the TCRαβ+/CD19+ group) and chronic GvHD (32 ± 5% vs 23 ± 4%, respectively). NRM was 23 ± 5% in the CD3+/CD19+group vs 21 ± 4% in the TCRαβ+/CD19+group. Relapse incidence was also similar, 32 ± 5% vs 34 ± 6%, respectively. DFS and OS were not different (45 ± 5% vs 45 ± 6% and 53 ± 6% vs 58 ± 6% respectively). As there were no differences on transplant outcomes between groups, we further analyzed all patients together for risk factors associated with transplant outcomes. On multivariate analysis, we identified that early disease status at transplant (HR: 0.16; 95%CI (0.07-0.35) (p=0.0001), presence of cGvHD (HR: 0.38; 95%CI (0.20-0.70) (p= 0.002), and donor KIR-B genotype (HR: 0.50; 95%CI (0.32-0.90) (p=0.04) were associated with better DFS.ConclusionsOur data suggest that there are no advantages in transplant outcomes between TCD platforms. Risk factors for survival are dependent on disease characteristic, donor KIR genotype, and chronic GvHD rather than the TCD platform used.

Abstract 1220: Accurately genotyping HLA and KIR alleles using cfDNA assay and k-mer based algorithm for immunotherapy

Abstract Background: HLA and KIR genotypes show great promise as emerging biomarkers for immune checkpoint inhibitors (ICIs) and understanding patient prognosis. Multiple studies have shown that HLA-I heterozygosity and high sequence divergence across alleles positively correlates with response to ICIs. However the high degree of polymorphism and allele sequence similarities in HLA and KIR present a challenge to accurate allele calling. To address these difficulties we developed kmerizer, a novel allele caller optimized for short fragments, such as reads from a cfDNA assay. Methods: We tested kmerizer on MHC class1 and class2 genes, and on KIR genes, on both simulated datasets and real samples (cell lines and plasma samples). To assess the capability of the algorithm to distinguish highly homologous allele pairs, we simulated cfDNA-like fragments with errors on randomly selected allele pairs, and on allele pairs with a high level of homology. Twelve plasma samples and 19 reference cell lines fragmented to cfDNA size were analyzed using a NGS cfDNA assay. For plasma samples, paired buffy coats were sent to an external vendor for HLA typing using multiplex PCR-based amplicon and sequenced by 300bp paired-end reads. Results: Of the 19 cell lines and 12 plasma samples for HLA typing (A, B, C, and DQB1 loci), kmerizer delivered 100% sensitivity with 98% specificity. On the simulated dataset, kmerizer achieved 99% sensitivity and specificity on all the MHC class1 and class 2 loci, and 90% sensitivity and specificity on all KIR loci, for both homozygous and heterozygous pairs. The novel allele caller kmerizer also demonstrated a lighter footprint on computational resource need: one deep-sequencing plasma sample on average can be processed in less than 2 minutes which is about 15 times faster than the most commonly used HLA typing tool HISAT21, which does not support KIR typing. Conclusions: As utilization of ICIs increases, the use of genetic and genomic information to accurately identify patients more likely to respond to ICIs will be critical. kmerizer is a fast and highly sensitive and specific allele caller, and it can effectively call alleles on both HLA and KIR.

Characterization of KIR+ NK cell subsets with a monoclonal antibody selectively recognizing KIR2DL1 and blocking the specific interaction with HLA-C.

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.

Influence of Fetomaternal Microchimerism on Maternal NK Cell Reactivity against the Child's Leukemic Blasts.

Persistence of fetal cells in the circulation of the mother (fetal microchimerism, FM) is associated with increased survival and reduced relapse of children with leukemia receiving a haploidentical hematopoietic stem cell transplantation (hHSCT). NK cells play an important role in maternal tolerance towards the unborn child. In this study, 70 mother–child pairs were prospectively analyzed for the occurrence of FM, KIR genotype and HLA-C type. We found that occurrence and level of FM were influenced by three maternal genetic factors: presence of an HLA-C1 allele, absence of KIR2DL3 and presence of a cen-B/B motif. Furthermore, an HLA-C match between mother and child favored persistence of FM. NK cells from FM+ mothers showed a 40% higher specific degranulation against their filial leukemic blasts than NK cells from FM− mothers, suggesting the presence of educated maternal NK cells. Nevertheless, cytotoxicity of parental NK cells against filial leukemic blasts was independent of KIR genetics (haplotype, B content score, centromeric and telomeric KIR gene regions) and independent of FM, indicating that additional immune effector mechanisms contribute to the beneficial effect of persisting FM in hHSCT.

P-068: Haplotypes rich in activating killer-cell immnuglobulin-like receptor genes are associated with delay on age of myeloma onset

<h3>Background</h3> Natural killer (NK) cells are known for their anti-tumoral cytotoxic effects. Effector NK-cell functions are controlled by interactions between inhibitory and activating killer-cell immnuglobulin-like receptors (iKIRs and aKIRs) on NK cells in the presence of human leukocyte antigen (HLA) class I ligands on target cells. The aim of this study was to investigate the frequency of KIR genotypes with/without their cognate ligands among myeloma (MM) patients compared to a healthy population. <h3>Methods</h3> 178 MM patients diagnosed between 2007-2018 enrolled into the study. The median age of patients was 63 (range, 33-95) with ISS:I/II/III:49/36/37; IgG/IgA/Light chain: 58/22/40 and median lines of treatment of three (range, 1-6) (38% history of transplant). As a control group, 449 healthy subjects screened for HLA and KIR genotyping aged median: 42 (7-82) as related/unrelated hematopoietic stem cell donors were included. The Olerup SSP KIR Genotyping Kit (Olerup, Stockholm, Sweden) and Olerup KIR HLA Ligand Detection Kit was used to type KIR and KIR ligands: For KIR genotype comparisons total AA, AB and BB, their telomeric and centromeric motifs were chosen. <h3>Results</h3> Among aKIR genotypes 2DL5B and 2DS3 were found to be less frequent among MM patients compared to healthy subjects (p=0.001; p=0.04). When KIR receptor genes were evaluated along with their cognate ligands, the frequencies of KIR2DL2 and C1, KIR2DL3 and C1 as well as KIR2DS2 and C1 were found to be less frequent among MM patients (p=0.002; p=0.03; p=0.002). The frequency of patients with aKIR ≥5 was significantly lower among MM patients (P<0.0001). When the total haplotype was compared,AB is more and BB less frequent among patients with similar inheritance of AA (p=0.001). Among MM patients, haplotype AA (with ligands C2+Bw4+) frequency is significantly higher compared to healthy controls (p=0.005). BB haplotype carrying male patients had a four years delay in onset age of diagnosis. This effect was in the opposite direction for females. AA haplotype carrying males had MM diagnosed seven years earlier than negative patients, an effect not visible among females. When the predictors for age at diagnosis was assessed, tAB1 haplotype appears to be a stronger factor (p=0.06) than gender (p=0.76). When impact on early relapse was analyzed cAB1 (n=7) was the only motif to have a minor effect. <h3>Conclusion</h3> This study is the first to demonstrate activating KIR containing haplotypes to be less frequent among myeloma patients when compared to healthy subjects. Furthermore specifically this effect was confirmed in the presence of their ligands and the haplotypes rich in activating KIRs. Haplotype BB which includes the highest and haplotype AA which has the least number of a KIRs were found to influence the age of onset mainly among males delaying (haplotype BB) or earlier onset (Haplotype AA). Similar analysis among smoldering MM is warranted to provide further evidence.