Expansion of CD56dimCD16neg NK Cell Subset and Increased Inhibitory KIRs in Hospitalized COVID-19 Patients

Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-2) infection induces elevated levels of inflammatory cytokines, which are mainly produced by the innate response to the virus. The role of NK cells, which are potent producers of IFN-γ and cytotoxicity, has not been sufficiently studied in the setting of SARS-CoV-2 infection. We confirmed a different distribution of NK cell subsets in hospitalized COVID-19 patients despite their NK cell deficiency. The impairment of this innate defense is mainly focused on the cytotoxic capacity of the CD56dim NK cells. On the one hand, we found an expansion of the CD56dimCD16neg NK subset, lower cytotoxic capacities, and high frequencies of inhibitory 2DL1 and 2DL1/S1 KIR receptors in COVID-19 patients. On the other hand, the depletion of CD56dimCD16dim/bright NK cell subsets, high cytotoxic capacities, and high frequencies of inhibitory 2DL1 KIR receptors were found in COVID-19 patients. In contrast, no differences in the distribution of CD56bright NK cell subsets were found in this study. These alterations in the distribution and phenotype of NK cells might enhance the impairment of this crucial innate line of defense during COVID-19 infection.

KIR Receptors as Key Regulators of NK Cells Activity in Health and Disease

Natural killer (NK) cells are part of the cellular immune response. They target mainly cancer and virally infected cells. To a high extent cytotoxic activity of NK cells is regulated inter alia by signals from killer immunoglobulin-like receptors (KIR). The major histocompatibility complex (MHC) class I molecules are important ligands for KIR receptors. Binding of ligands (such as MHC I) to the KIR receptors has the important role in solid organ or hematopoietic cell transplantation. Of note, the understanding of the relationship between KIR and MHC receptors may contribute to the improvement of transplant results. Donor-recipient matching, which also includes the KIR typing, may improve monitoring, individualize the treatment and allow for predicting possible effects after transplantation, such as the graft-versus-leukemia effect (GvL) or viral re-infection. There are also less evident implications of KIR/MHC matching, such as with pregnancy and cancer. In this review, we present the most relevant literature reports on the importance of the KIR/MHC relationship on NK cell activity and hematopoietic stem cell transplantation (HSCT)/solid organ transplantation (SOT) effects, the risk of allograft rejection, protection against post-transplant cytomegalovirus (CMV) infection, pregnancy complications, cancer and adoptive therapy with NK cells.

Blocking LILRB and KIR receptors by B57 open conformers induces potent antitumor activity and acts synergistically with checkpoint blockade inhibition.

e14137 Background: HLA open conformers (OC) are defined as HLA class I molecules lacking beta-2-microglobulin (β2m) and peptide. OC can be derived from different HLA I molecules such as HLA-B27, -B57, and –Cw08. OC have a different three dimensional structure when compared to their respective HLA/β2m/peptide counterpart and induce distinct immunological processes by binding to LILRB and KIR molecules, both key receptors of the innate immune system. B57 OC expression is associated with enhanced immunity against viruses and can cause autoimmunity. Its potential anti-tumor activity has not been exploited so far. Methods: B57 OC and control molecules were expressed as IgG4 fusion proteins in CHO cells. The affinity for protein-ligand interaction was measured by surface plasmon resonance (SPR). Human macrophages M1/M2, phagocytosis and NK cytotoxicity were assessed by flow cytometry and cellular assays. Syngeneic pancreatic cancer (Pan02) or colon cancer (MC38) mouse models were used. Mice with tumors of 80mm3were treated twice weekly at 5 mg/kg. Tumor and blood samples were analysed. FDA cancer panels were assessed by IHC for LILRB1-5 expression. Results: B57 OC displays distinct protein-ligand interactions with high affinity binding to LILRB2 & 4, KIR2L1-3, and KIR3DL1. Therapeutic efficacy in pancreatic and colon cancer models was observed with monotherapy (p < 0.01), and combo therapy using PD1 and/or 41BB antibodies (p < 0.0001). Ex vivotumor sample analysis revealed a significant reduction of MDSC & Tregs, and an increase of M1 type macrophages. In addition, loss of MDSC functionality and enhanced CD8+ T cell expansion was noticed. IHC of human tissue demonstrated enhanced LILRB2 expression, notably in colon and lung cancer. Conclusions: B57 OC has a unique binding profile to LILRB and KIR receptors. B57 OC induces anti-tumor activity in diverse syngeneic mouse models and acts synergistically with PD1 or 41BB antibodies. Pre-clinical and human in vitroexperiments demonstrate that B57 OC mode of action is established with inhibition of MDSCs, macrophage polarization to M1 phenotype, activation of NK cells and LILRB blockade on tumor cells, which in turn support the adaptive immune system by increasing CD8+ effector T cells and reducing Treg numbers. B57 OC is a first-in-class therapeutic with robust anti-tumor activity.

Mismatches In Killer Immunoglobulin Receptor (KIR) Ligands and Inhibitory KIR Receptors Between Donor and Recipients Improve Survival After Non T Cell Depleted Haploidentical Transplantation

Introduction Alloreactivity triggered by interaction of Killer immunoglobulin-like receptors (KIRs) on donor Natural killer (NK) cells and their ligands on recipients plays a role in the graft-versus-tumour effect. Different predictive models have been postulated for measuring alloreactivity: ligand incompatibility model, receptor-ligand model, missing ligand model and KIR gene-gene model. Our aim in this study is to evaluate the importance of differences in KIR and HLA genotype between donor and recipient (missing ligand model and KIR gene-gene model) in the setting of non T cell depleted haploidentical haematopoietic stem cell transplantation (HSCT) with high-dose, post-infusion cyclophosphamide (Cy) Patients and Methods 33 consecutive patients with haematological malignancies who received haploidentical HSCT with high-dose Cy post-transplantation between 2007 and 2013, and their donors were included for analysis (Table 1). HLA typing was used to identify KIR ligands HLA-B and HLA-C. KIR genotype was analyzed by PCR (KIR Typing, Miltenyi Biotec) on genomic DNA (Maxwell 16 Blood DNA Kit, Promega) from peripheral blood samples, and revealed with ethidium bromide after agarose gel electrophoresis. Results Demographic data and KIR genotype characteristics of donors and recipients are listed in Table 1. We found that KIR ligands mismatch between donor and recipient is related with improve of OS (52% vs. 82%; p=0,041) and DFS (63% vs. 22%; p=0,037) a year after transplant (Figure 1). Mismatch of inhibitors KIR receptors (iKIR) gene content between donors and recipients is also related with an improvement of OS (94% vs. 75%; p=0,049) and DFS (13% vs. 61%; p= 0,028) compared with no mismatch pairs. In the multivariable analysis, both KIR ligands mismatches (HR=4,38 CI:0.9-21; p=0,061) as well as iKIR mismatches (HR=4,15 CI:1-16; p=0,047), show a tendency to be independent variables for the reduction of disease relapse rate. Cumulative incidence of relapse for both variables studied is shown in Figure 2. Conspicuously, a sub-analysis in Hodgkin Lymphoma patients group shows and improve in DFS a year after transplant in patients with KIR ligands mismatches ( 100% vs. 48% p: 0,006) and iKIR mismatches (100% vs. % 33% p: 0,049), despite the small number of patients. Contrary to data published by other groups, patients receiving donor’s progenitors with Bx haplotype with centromeric genes (Cen-BB) did not show a benefit in survival (SG 85% vs. 90%, p= 0,05 and DFS 66% vs. 10%, p=0,047) a year after transplant Conclusion Our data suggest that in the setting of non T cell depleted haploidentical HSCT with high dose Cy post-infusion, KIR ligands and iKIR mismatch are related with an improve in survival, specially in the sub-group of patients with Hodgkin disease Disclosures: No relevant conflicts of interest to declare.

Recovery of KIR Expression After Allogeneic Stem Cell Transplantation in Multiple Myeloma Patients

Abstract 4555 Introduction: Activating and inhibitory killer immunoglobulin like receptors (KIR) are predominantly expressed on natural killer (NK) cells. KIR mismatch allogeneic stem cell transplantation (alloSCT) has been reported to provide beneficial effects for Multiple Myeloma (MM). However, their recovery in MM patients remains poorly understood. We, therefore, analysed KIR recovery in 90 MM patients after alloSCT. Methods: KIR expression (CD158a/h, CD158b/b2, CD158e1/e2) on NK cells and T cell subsets was measured by flow cytometry at different time points after alloSCT. Results: During the first 90 days after alloSCT NK cells represent the largest lymphocyte subset. Activating receptors like NKp30 and NKp44 showed a fluctuating expression while members of the KIR family were expressed at a constant rate (20% of NK cells). There was no significant difference in the early post transplantation period (day 0–90) compared to later time points (day 360). In contrast, T cells showed increased KIR expression during the first 30 days after alloSCT, which was highly significant for CD158e (p=0,0001). After 30 days the expression declined to baseline. Furthermore, T cell activation marker HLA-DR reached its highest expression between days 60 and 90 when KIR receptors were expressed at their lowest level (27% vs. 8%, p < 0,0001). Conclusions: We conclude that KIR receptors were differentially expressed on NK and T cells. Because KIR receptors are constantly expressed by NK cells and NK cells are the most frequent lymphocyte populations early after alloSCT, NK cells may be useful for KIR mismatch cellular therapy. Disclosures: No relevant conflicts of interest to declare.

Increased Group B Killer Cell Immunoglobulin-Like Receptor (KIR) Haplotypes with Mismatched MHC Class I and Altered NK Repertoire Distribution in Bone Marrow Failure Syndromes

Abstract 2412 Background: Killer immunoglobulin like receptors (KIR) are expressed on NK cells and a subset of T cells where they bind HLA-C alleles to discriminate self from non-self during anti-tumor and anti-viral responses. KIR receptors exist in inhibitory and activating forms (KIRDL and KIRDS, respectively) that regulate effector functions. The KIR family consists of 14 genes inherited independently from MHC-class I ligands. An imbalance of KIR receptors tilted in favor of more activating KIR genotypes is associated with improved antiviral responses, but also an increase in autoimmunity. We reported previously that KIR/HLA mismatches occur at a higher frequency in bone marrow failure: Aplastic Anemia (AA), Myelodysplastic Syndrome (MDS), Large Granular Lymphocyte leukemia (LGLL), and Paroxysmal Nocturnal Hemaglobinuria (PNH) (Epling-Burnette et al, Blood (ASH Annual Meeting abstracts 2008 112: abstract 4121). We now present data on a larger cohort of patients along with phenotype analysis and individual KIR genotype analysis. Methods: KIR genotyping by bead array (One Lambda, Inc) was performed on 205 cases and resolved in 199 (97%). Samples from 93 unrelated individuals were used as controls. HLA genotyping was analyzed in 190 and 51 cases and controls, respectively. The diagnosis of MDS (n=72), AA (n=59), LGLL (n=46), and PNH (n=28) were confirmed by pathology review. Flow cytometry for KIR2DS1/DL1 was assessed by staining for CD158a (KIR2DS1/DL1) and CD158b (KIR2DS2/DL2/3) on (CD56+/CD16+) NK and (CD4/CD8/CD3) T cells by multiparameter flow cytometry. Antigens at the HLA-C locus were divided into two groups, the HLA-C1 with alleles encoding Ser-77-Asn80 and HLA-C2 alleles encoding Asn-77-Lys-80 that bind KIR2D2 and KIR2D1, respectively. A mismatch was defined as the presence of the KIR gene in the absence of its cognate ligand. Results: KIR2DL2 and KIR2DS2 gene frequency was increased in the combined BMF group (120/198, 60% DL2 and 117/199, 59%, DS2) compared to controls (43/93, 46% for both DL2 and DS2) (p=0.02 and p=0.058, respectively) in association with a higher frequency of C1 activating and inhibitory receptor mismatches in cases (20 of 190 cases vs. 0 of 51 controls, p=0.009). KIR genotypes can be separated into Group A and B haplotypes with Group A haplotypes containing only one activating KIR (KIR2DS4) and group B haplotypes containing two or more activating KIR. Compared to controls patients with BMF had significantly decreased group A (23%) and increased group B (77%) haplotypes (p0.04). We detected 31 different genotypes in BMF patients and 23 in controls representing both the A and B haplotypes. AA1 was the most common group A genotype in cases (32/35, 91.4%) and controls (49/50, 98%). Specific genotypes that contain KIR2DL2 and DS2 were overrepresented in the BMF cases with AB9 increased in AA (p=0.0054) and AB1 increased in LGLL (p=0.02). KIR phenotyping was performed on NK cells and T cells from 49 BMF patients and 15 controls. KIRs were variably expressed on T cells, and NKG2D had normal expression on NK and T cell populations. CD158a (KIR2DL1/DS1) positive NK cells were significantly reduced in BMF cases compared to controls (17% vs 35%, p0.00008), which was individually significant in AA (18%, p0.003), MDS (20%, p0.01), and LGLL (14%, p=0.0002). The percentage of CD158b (KIR2DS2/DL2/3) positive NK cells, which recognizes the receptors associated with the C1 mismatch, were also significantly reduced in cases compared to controls (30% vs. 43%, p0.02) indicating altered NK repertoire distribution. Conclusions: KIRs and HLA are inherited independently and have a high level of diversity, but previous results suggest that there is an epistatic relationship between these loci because matched KIR/HLA combinations have evolved through selection. In BMF syndromes, increased frequency of KIR2DL2/KIR2DS2 genotypes in the absence of the appropriate HLA C allele may alter NK and T cell effector responses in favor of autoimmunity. Two to 8 different KIR receptors are present on individual NK cells and these are expressed in a stochastic manner. In previous studies, the expression of a receptor did not depend on the presence of the cognate HLA. Our phenotyping data suggests that the genetic disparities or disease characteristics in BMF may influence NK repertoire distribution leading to fewer KIR positive NK cells with possible pathological consequences. Disclosures: No relevant conflicts of interest to declare.

Neonatal NK-cell repertoires are functionally, but not structurally, biased toward recognition of self HLA class I

Human natural killer (NK)–cell repertoires are biased toward more frequent expression of inhibitory killer cell Ig-like receptor (KIR) receptors for self-human leukocyte antigen (HLA) class I. Moreover, only those NK cells that express cognate receptors for self are fully functional in terms of cytotoxicity and cytokine production. It is so far unknown whether functional education and structural adaptation to HLA class I are implemented during NK-cell development and whether both processes are mechanistically connected. Here we show that NK-cell repertoires in cord blood are not yet shaped toward increased clonal frequencies of KIR for self-HLA class I as determined for the 3 major KIR ligands C1, C2, and Bw4. Nonetheless, neonatal NK cells expressing cognate KIR exhibited enhanced effector function on the level of degranulation and cytokine production. The study suggests that functional education of cognate KIR by self-HLA class I precedes structural adaptation of KIR repertoires and that both processes are not directly linked to each other.