Killer Immunoglobulin Receptor Research Articles

Polymorphic residues in HLA-B that mediate HIV control distinctly modulate peptide interactions with both TCR and KIR molecules.

Immunogenetic studies have shown that specific HLA-B residues (67, 70, 97, and 156) mediate the impact of HLA class I on HIV infection, but the molecular basis is not well understood. Here we evaluate the function of these residues within the protective HLA-B∗5701 allele. While mutation of Met67, Ser70, and Leu156 disrupt CD8+ Tcell recognition, substitution of Val97 had no significant impact. Thermal denaturation of HLA-B∗5701-peptide complexes revealed that Met67 and Leu156 maintain HLA-peptide stability, while Ser70 and Leu156 facilitate Tcell receptor (TCR) interactions. Analyses of existing structures and structural models suggested that Val97 mediates HLA-peptide binding to inhibitory KIR3DL1 molecules, which was confirmed by experimental assays. These data thereby demonstrate that the genetic basis by which host immunity impacts HIV outcomes occurs by modulating HLA-B-peptide stability and conformation for interaction with TCR and killer immunoglobulin receptor (KIR) molecules. Moreover, they indicate a key role for epitope specificity and HLA-KIR interactions to HIV control.

Abstract 6332: Evaluating the relationship of affinity, functional avidity, and in vivo potency in KIR-CAR T cells

Abstract Chimeric antigen receptors (CARs) targeting CD19 demonstrate remarkable clinical outcomes in patients (pts) with B-cell malignancies. Importantly, all four FDA-approved CD3ζ-based CAR T cell therapies use the same FMC63-derived single-chain variable fragment (scFv) to target CD19+ tumors. Although response rates are high in diffuse large B cell lymphoma, only ~40% of pts achieve durable, long-term remissions. Improved CAR T cell therapies are therefore needed. We have previously shown that a novel killer immunoglobulin receptor (KIR)-based CAR expressed in T cells as a multichain receptor with DAP12 (KIR-CAR T) has enhanced anti-tumor activity in several preclinical solid tumor models suggesting that this platform may show better activity in lymphoma. Some recent studies report that lower-affinity CD19 binders compared to the benchmark FMC63-scFv yield equivalent or increased anti-tumor activity of CAR T in pts, raising the question of optimal affinity for a KIR-based CAR. In this study, we aimed to evaluate the relationship of affinity, functional avidity, and in vivo potency of CD19 binders in a KIR-based CAR T cell. Using a highly-diverse 40-billion member canine scFv phage display library, we generated 19 unique human CD19-specific binders that compete with FMC63 binder supporting a similar binding epitope. Different KIR-CAR T cells were generated, and relative affinities were compared via binding to a recombinant (rec) CD19 protein. We found that although CAR receptor expression was similarly expressed, the concentration of soluble rec CD19 protein required to saturate the surface CAR varied more than 2-log among the different scFv binders suggesting a range of affinities relative to FMC63. We selected several scFvs with lower, and equivalent CD19 binding MFI compared to the FMC63-scFv and found that all scFvs could mediate similarly robust in vitro potency. In vivo anti-tumor efficacy was next assessed using the NSG xenograft mouse model of B-cell leukemia NALM6 cells. Interestingly, despite their comparable in vitro functions, some binders with relatively similar binding MFI failed to control the tumors. In contrast, two binders with an ~30-fold apparent affinity relative to FMC63 exhibited robust anti-tumor activity comparable to the benchmark scFv. These preliminary data suggest that the in vivo functional activity of chimeric KIR-based immunoreceptors is insensitive to binder affinity over a wide range of affinities. The failure of some scFvs with comparable affinity and binding epitope suggest that other, yet to be characterized attributes of the binder may be critical for optimal function. Further characterization of these binders is being conducted to understand the relationship between affinity, functional avidity, antigen density, and in vivo potency. This study paves the way for developing an improved CD19-specific KIR-CAR T cell therapy for treating pts with B-cell malignancies. Citation Format: Jun Xu, Selene Nunez-Cruz, John M. Leferovich, Gayathri Gulendran, Chune Zhang, Nora D. Yucel, Megan C. Blair, William S. Stanley, Laura A. Johnson, Don L. Siegel, Michael C. Milone. Evaluating the relationship of affinity, functional avidity, and in vivo potency in KIR-CAR T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6332.

Analysis of KIR gene variants in The Cancer Genome Atlas and UK Biobank using KIRCLE

BackgroundNatural killer (NK) cells represent a critical component of the innate immune system’s response against cancer and viral infections, among other diseases. To distinguish healthy host cells from infected or tumor cells, killer immunoglobulin receptors (KIR) on NK cells bind and recognize Human Leukocyte Antigen (HLA) complexes on their target cells. However, NK cells exhibit great diversity in their mechanism of activation, and the outcomes of their activation are not yet understood fully. Just like the HLAs they bind, KIR receptors exhibit high allelic diversity in the human population. Here we provide a method to identify KIR allele variants from whole exome sequencing data and uncover novel associations between these variants and various molecular and clinical correlates.ResultsIn order to better understand KIRs, we have developed KIRCLE, a novel method for genotyping individual KIR genes from whole exome sequencing data, and used it to analyze approximately sixty-thousand patient samples in The Cancer Genome Atlas (TCGA) and UK Biobank. We were able to assess population frequencies for different KIR alleles and demonstrate that, similar to HLA alleles, individuals’ KIR alleles correlate strongly with their ethnicities. In addition, we observed associations between different KIR alleles and HLA alleles, including HLA-B*53 with KIR3DL2*013 (Fisher’s exact FDR = 7.64e−51). Finally, we showcased statistically significant associations between KIR alleles and various clinical correlates, including peptic ulcer disease (Fisher’s exact FDR = 0.0429) and age of onset of atopy (Mann-Whitney U FDR = 0.0751).ConclusionsWe show that KIRCLE is able to infer KIR variants accurately and consistently, and we demonstrate its utility using data from approximately sixty-thousand individuals from TCGA and UK Biobank to discover novel molecular and clinical correlations with KIR germline variants. Peptic ulcer disease and atopy are just two diseases in which NK cells may play a role beyond their “classical” realm of anti-tumor and anti-viral responses. This tool may be used both as a benchmark for future KIR-variant-inference algorithms, and to better understand the immunogenomics of and disease processes involving KIRs.

Expression of Killer Immunoglobulin Receptor Genes among HIV-Infected Individuals with Non-AIDS Comorbidities

Combined antiretroviral therapy (cART) increased the life expectancy of people living with HIV (PLHIV) and remarkably reduced the morbidity and mortality associated with HIV infection. However, non-AIDS associated comorbidities including diabetes, hypertension, hyperlipidemia, and cardiovascular diseases (CVD) are increasingly reported among PLHIV receiving cART. Killer cell immunoglobulin receptors (KIRs) expressed on the surface of natural killer (NK) cells have been previously implicated in controlling HIV disease progression. The aim of this study is to investigate the role of KIRs in developing non-AIDS associated comorbidities among PLHIV. Demographic and behavioral data were collected from voluntary participants using a standardized questionnaire. Whole blood samples were collected for KIR genotyping. Hypertension (29.5%) and hyperlipidemia (29.5%) followed by diabetes (23.7%) and CVD (9.7%) were mainly reported among our study participants with higher rate of comorbid conditions observed among participants > 40 years old. The observed KIR frequency (OF) was ≥90% for inhibitory KIR2DL1 and KIR3DL1, activating KIR2DS4 and the pseudogene KIR2DP1 among study participants. We detected significant differences in the expression of KIR3DS4 and KIR3DL1 (p = 0.038) between diabetic and nondiabetic and in the expression of KIR2DL3 between hypertensive and normotensive HIV-infected individuals (p = 0.047). Moreover, KIR2DL1 and KIR2DP1 were associated with significantly reduced odds of having CVD (OR 0.08; 95% CI: 0.01-0.69; p = 0.022). Our study suggests the potential role of KIR in predisposition to non-AIDS comorbidities among PLHIV and underscores the need for more studies to further elucidate the role of KIRs in this population.

A Phase I Clinical Trial Testing the Safety of IL-21-Expanded, Universally Alloreactive Donor-Derived Natural Killer Cells for Relapsed/Refractory Acute Myeloid Leukemia and Myelodysplastic Syndrome

Background: Natural Killer (NK) cells are cytotoxic lymphocytes that are able to exert an anti-tumor effect in an MHC-I independent manner, but are dysfunctional and reduced in number in patients with leukemia. Several studies have shown therapeutic potential for related donor NK cells expanded and/or activated ex vivo when administered to cancer patients, including patients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). However, personalized, donor-derived cell therapies require time for donor identification, manufacturing and product release, resulting in patient attrition. As NK alloreactivity and NKG2C expression play critical roles in mediating anti-tumor effects, we identified ‘ideal’ NK cell donors (in partnership with “Be the Match Biotherapies”) for collection. Human leukocyte antigen (HLA) and Killer immunoglobulin receptor (KIR) genotyping were done to screen and select donors. Following this, a sample was collected from donors to expand NK cells under small-scale conditions. If donor NK cells showed robust expansion, they proceeded with apheresis. To ensure these therapeutic cells would be readily-available, we established a third-party NK cell bank through scalable, affordable mass-production with membrane-bound IL-21 feeder cells (FC21), and cryopreserved large numbers of these NK cells for immediate ‘off-the-shelf’ administration to recipients. Here, we report our initial experience in a Phase I trial of these off-the-shelf (OTS) alloreactive NK cells as immunotherapy for patients with relapsed/refractory AML and MDS.Methods: Patients were treated at Dose level 1, to allow infusion at a dose of 1 x 10e7 NK cells/ kg. Each dose level had two cohorts, stratified by age. Patients were enrolled into Cohort 1 (patients <60 yrs. received Fludarabine (Flu) 30 mg/m2/day & Cytarabine 2 g/m2/day on days -6 to -2) and Cohort 2 (patients ≥ 60 yrs. received Flu 30 mg/m2/day (days -5 to -2) & Decitabine 20 mg/m2/day (days -6 to -2). Patients were evaluated to see if they had any donor-directed antibodies. After chemotherapy, NK cells were infused thrice weekly for 6 doses, to be given over a period of 2 weeks (Clinicaltrials.gov: NCT04220684). Persistence of donor NK cells was determined by flow cytometry using haplotype-discriminating anti-HLA antibodies (Figure 1, 2). NK cells were completely HLA-mismatched between donor and recipients.Results: 6 patients (3/cohort) were treated at the first dose level of 1x10 7 NK cells/kg/dose. All 6 patients had relapsed/refractory AML and had received at least two prior lines of treatment. One patient (005) withdrew from the study prior to infusion of NK cells. 6 patients received the first NK cell dose as an inpatient and were discharged to receive the remaining 5 doses as an outpatient. 5 patients tolerated infusion of all 6 doses of NK cells administered over 2 weeks. One patient (006) developed a Cytokine response (CRS) - like syndrome in the context of streptococcus bacteremia and received only one dose of NK cells. Symptoms suggestive of CRS responded fully with steroids and patient was able to successfully wean off steroids and discharged to the outpatient setting. One patient proceeded to allogeneic hematopoietic cell transplant. No infusion-related reactions, neurotoxicity or graft versus host disease was observed. In vivo persistence/expansion NK cells were superior with FLU/CY lymphodepletion (cohort 1).Conclusions: Cryopreserved, third-party donor-derived NK cells are safe and feasible in relapsed/refractory AML patients, and even at this first dose level, completely HLA-mismatched NK cells can persist and expand to high levels in vivo. [Display omitted] DisclosuresVasu: Kiadis, Inc.: Research Funding; Seattle Genetics: Other: travel support; Boehringer Ingelheim: Other: Travel support; Omeros, Inc.: Membership on an entity's Board of Directors or advisory committees. Walker: Newave: Other: clinical trial support; Geron: Other: clinical trial support; Novartis: Other: clinical trial support. Blachly: INNATE: Consultancy, Honoraria; KITE: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; AstraZeneca: Consultancy, Honoraria. de Lima: Incyte: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Miltenyi Biotec: Research Funding. Lee: Kiadis Pharma: Divested equity in a private or publicly-traded company in the past 24 months, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Courier Therapeutics: Current holder of individual stocks in a privately-held company.

Donor Killer Immunoglobulin Receptor Gene Content and Ligand Matching and Outcomes of Pediatric Patients with Juvenile Myelomonocytic Leukemia Following Unrelated Donor Transplantation

Natural killer (NK) cell determinants predict relapse-free survival after allogeneic hematopoietic cell transplantation (HCT) for acute myelogenous leukemia, and previous studies have shown a beneficial graft-versus-leukemia effect in patients with juvenile myelomonocytic leukemia (JMML). However, whether NK cell determinants predict protection against relapse for JMML patients undergoing HCT is unknown. Therefore, we investigated NK cell-related donor and recipient immunogenetics as determinants of HCT outcomes in patients with JMML. Patients with JMML (age 0 to <19 years) who underwent a first allogeneic HCT from an unrelated donor between 2000 and 2017 and had available donor samples from the Center for International Blood and Marrow Transplant Research Repository were included. Donor killer immunoglobulin receptor (KIR) typing was performed on pre-HCT samples. The primary endpoint was disease-free survival (DFS); secondary endpoints included relapse, grade II-IV acute graft versus-host-disease (aGVHD), chronic GVHD (cGVHD), GVHD-free relapse-free survival, transplantation-related mortality, and overall survival (OS). Donor KIR models tested included KIR genotype (AA versus Bx), B content (0-1 versus ≥2), centromeric and telomeric region score (AA versus AB versus BB), B content score (best, better, or neutral), composite score (2 versus 3 versus 4), activating KIR content, and the presence of KIR2DS4. Ligand-ligand and KIR-ligand mismatch effects on outcomes were analyzed in HLA-mismatched donors (≤7/8; n=74) only. Univariate analyses were performed for primary and secondary outcomes of interest, with a P value <.05 considered significant. One hundred sixty-five patients (113 males), with a median follow-up of 85 months (range, 6 to 216 months) met the study criteria. Of these, 111 underwent an unrelated donor HCT and 54 underwent a UCB HCT. Almost all (n=161; 98%) received a myeloablative conditioning regimen. After exclusion of recipients of reduced-intensity/nonmyeloablative conditioning regimens and ex vivo T cell-depleted grafts (n=8), there were 42 AA donors and 115 Bx donors, respectively. Three-year DFS, OS, relapse, and GRFS for the entire cohort were 58% (95% confidence interval [CI], 50% to 66%), 67% (95% CI, 59% to 74%), 26% (95% CI, 19% to 33%), and 27% (95% CI, 19% to 35%), respectively. The cumulative incidence of grade II-IV aGVHD at 100 days was 36% (95% CI, 27% to 44%), and that of cGVHD at 1 year was 23% (95% CI, 17% to 30%). There were no differences between AA donors and Bx donors for any recipient survival outcomes. The risk of grade II-IV aGVHD was lower in patients with donors with a B content score of ≥2 (hazard ratio [HR], 0.46; 95% CI, 0.26 to 0.83; P=.01), an activating KIR content score of >3 (HR, 0.52; 95% CI, 0.29 to 0.95; P=.032), centromeric A/B score (HR, 0.57; 95% CI, 033 to 0.98; P=.041), and telomeric A/B score (HR, 0.58; 95% CI, 0.34 to 1.00; P=.048). To our knowledge, this is the first study analyzing the association of NK cell determinants and outcomes in JMML HCT recipients. This study identifies potential benefits of donor KIR-B genotypes in reducing aGVHD. Our findings warrant further study of the role of NK cells in enhancing the graft-versus-leukemia effect via recognition of JMML blasts.

Unconventional Peptide Presentation by Classical MHC Class I and Implications for T and NK Cell Activation.

T cell-mediated immune recognition of peptides is initiated upon binding of the antigen receptor on T cells (TCR) to the peptide-MHC complex. TCRs are typically restricted by a particular MHC allele, while polymorphism within the MHC molecule can affect the spectrum of peptides that are bound and presented to the TCR. Classical MHC Class I molecules have a confined binding groove that restricts the length of the presented peptides to typically 8–11 amino acids. Both N- and C-termini of the peptide are bound within binding pockets, allowing the TCR to dock in a diagonal orientation above the MHC-peptide complex. Longer peptides have been observed to bind either in a bulged or zig-zag orientation within the binding groove. More recently, unconventional peptide presentation has been reported for different MHC I molecules. Here, either N- or C-terminal amino acid additions to conventionally presented peptides induced a structural change either within the MHC I molecule that opened the confined binding groove or within the peptide itself, allowing the peptide ends to protrude into the solvent. Since both TCRs on T cells and killer immunoglobulin receptors on Natural Killer (NK) cells contact the MHC I molecule above or at the periphery of the peptide binding groove, unconventionally presented peptides could modulate both T cell and NK cell responses. We will highlight recent advances in our understanding of the functional consequences of unconventional peptide presentation in cellular immunity.

Altered oxidative stress markers in relation to T cells, NK cells & killer immunoglobulin receptors that are associated with disease activity in SLE patients.

Systemic Lupus Erythematosus is an autoimmune disease with symptoms pervasive to all organ systems. It affects more females as compared to males (in the ratio 9:1). Oxidative stress plays a major role in the pathogenesis of SLE and other autoimmune diseases. In order to understand the relationship between cell specific oxidative stress and the severity of SLE, this research study involving the estimation of intracellular ROS accumulation in T and NK cell was conducted on SLE patients of North Indian Population. At the same time, to estimate anti-oxidant defense, Keap1 and Nrf2 levels were estimated in these cell types. The relationship between the expression of Killer immunoglobulin receptors i.e., KIR2DL4 & KIR3DL1 and oxidative stress was also evaluated as these receptors are imperative for the function and self-tolerance of NK cells.Oxidative stress was raised along with Keap1 and Nrf2 in T and NK cell subsets in SLE patients. The expression of KIR2DL4 was raised and that of KIR3DL1 was reduced in the NK cells of patients. The intensity of change in expression and its significance varied among the subsets. Nrf2 expression was raised in these species against oxidative stress as the antioxidant defense mechanism pertaining to Keap1-Nrf2 pathway, but the adequacy of response needs to be understood in further studies. The expression of KIR2DL4 and KIR3DL1 varied among the patient and healthy controls and the expression of the latter was found to have a significant positive relationship with plasma Glutathione(reduced) concentration.

Investigation of donor KIR content and matching in children undergoing hematopoietic cell transplantation for acute leukemia

Multiple models of donor killer immunoglobulin receptor (KIR) alloreactivity or KIR genotype have been reported to be protective against leukemia relapse after allogeneic transplantation. However, few studies have addressed this topic in the pediatric population. Here, we assessed the outcomes of allogeneic transplantation in children with acute lymphoblastic leukemia (ALL; n = 372) or acute myeloid leukemia (AML; n = 344) who received unrelated donor (URD) transplantation and were reported to the Center for International Blood and Marrow Transplant Research (CIBMTR) from 2005 to 2016. As expected in this pediatric population, most patients underwent myeloablative conditioning while in remission and with bone marrow as a stem cell source. We tested KIR ligand mismatch, KIR gene content (centromeric [Cen] B), KIR2DS1 mismatching, and Cen B/telomeric A using Cox regression models and found that none were significantly associated with either relapse or disease-free survival when considering the entire cohort of patients (ALL and AML), AML, or ALL separately. Moreover, there was no significant association with outcomes in the in vivo T-cell-depleted (ie, serotherapy) cohort. This study, which is the largest analysis of donor KIR in the pediatric acute leukemia population, does not support the use of KIR in the selection of URDs for children undergoing T-replete transplantation.

The Role of Natural Killer Cells and Regulatory T Cells While Aging with Human Immunodeficiency Virus.

Combined antiretroviral therapy (cART) has increased the quality of life of people living with HIV (PLHIV). Consequently, the number of PLHIV >50 years is increasing worldwide. Patients on cART are known to remain in a proinflammatory state. The latter is linked to the development of non-AIDS-related chronic conditions. Although the number of aging PLHIV is increasing, the effect of HIV infection on the process of aging is not fully understood. Understanding the complexity of aging with HIV by investigating the effect of the latter on different components of the innate and adaptive immune systems is important to reduce the impact of these comorbid conditions and improve the quality of life of PLHIV. The role of killer immunoglobulin receptors (KIRs), expressed on the surface of natural killer (NK) cells, and their human leukocyte antigen (HLA) ligands in the clearance, susceptibility to or disease progression following HIV infection is well established. However, data on the effect of KIR-HLA interaction in aging HIV-infected population and the development of non-AIDS-related comorbid conditions are lacking. Moreover, conflicting data exist on the role of regulatory T cells (Tregs) during HIV infection. The purpose of this review is to advance the current knowledge on the role of NK cells and Tregs while aging with HIV infection.

Optimized FVIII-Domain-Based Chimeric Antigen Receptors to Specifically Target FVIII Inhibitor-Producing B Cells in Hemophilia a

Hemophilia A (HA) is a X-linked bleeding disorder caused by deficiency of coagulation factor VIII (FVIII). Optimal clinical management centers on FVIII protein concentrate replacement. However, up to 30% of patients with severe HA develop neutralizing antibodies to FVIII (inhibitors) upon exposure to therapeutic FVIII. Inhibitors neutralize the infused FVIII and, thus, pose a significant challenge in the management of these patients. Immune tolerance induction (ITI) using high-dose FVIII infusions can eliminate inhibitors but is not effective at generating long-term eradication in all patients. Thus, we developed an immunotherapy to more effectively eliminate FVIII-specific B cells to induce lasting eradication of inhibitors.Adoptive T cell immunotherapies using chimeric antigen receptor (CAR)-modified T cells (CARTs) are showing encouraging results in the treatment of cancer. We previously reported a proof-of-concept CART approach to eliminate autoantibodies to desmoglein-3, implicated in pemphigus vulgaris, by targeting non-malignant B cells. Here, using a similar strategy, we constructed two FVIII-based CARs using isolated FVIII A2 and C2 domains, which are commonly targeted by inhibitorsBoth FVIII fragments were used as the CAR extracellular domain followed by CD8a-derived hinge and transmembrane domains and intracellular signaling domains derived from 4-1BB and CD3z (BBz), as has been used in a recently FDA-approved CD19-targeted CAR (CART19) that have shown efficacy in patients. A2- and C2-CARs were introduced into primary human T cells using lentivirus vectors and were found to express on the cell surface. Primary expansion following anti-CD3/CD28 activation and lentivirus transduction yielded comparable population doublings of CART19 and A2-CARTs over 9-11 days (mean, 4.3 vs. 3.7; p=0.16). However, C2-CARTs consistently achieved fewer doublings compared to CART19 (mean 4.3 vs. 2.6; p=0.01). Additionally, C2-CARTs maintained larger cell volumes following initial activation compared to A2 and CART19, reminiscent of other CARs that demonstrate a high-level of ligand-independent basal activation. Consistent with hyper-activation and activation-induced cell death, flow cytometric analysis of C2-CART cultures showed prolonged expression of CD69, increased levels of cell death, and gradual loss of CAR+ cells compared to A2 and CART19 cultures. The FVIII C2 domain contains hydrophobic surfaces involved in binding to phospholipid membranes and von Willebrand Factor. We hypothesized that these hydrophobic regions may cause unfavorable interactions that result in CAR clustering and, in-turn, ligand-independent signaling. However, since these regions are also targeted by FVIII inhibitors, mutation of hydrophobic residues to improve CAR function would likely result in loss of binding to intended targets. Thus, we tested two independent strategies to improve function of C2-CARTs, both of which maintain native FVIII domain sequences. First, to block unfavorable interactions with C2 hydrophobic surfaces, we tested whether addition of C2 domain specific antibodies to the CART culture would improve expansion. Indeed, addition of anti-C2 antibodies improved C2-CART expansion by approximately one population doubling and reduced both cell volume and CD69 expression in C2-CARTs. As an alternate and perhaps more easily translatable approach, we replaced the BBz CAR signaling domain with a killer immunoglobulin receptor (KIR)-based multi-chain signaling system, which we previously utilized to improve function of a mesothelin-targeted CAR. KIR-based signaling effectively “rescued” the C2 CAR, and showed an expansion and activation profile comparable to CART19. Lastly, KIR-based FVIII CARs, demonstrated specific lysis of and cytokine response to target cells engineered to express surface anti-FVIII, even in the presence of soluble inhibitors.These data indicate that unfavorable interactions may be ameliorated directly by blocking agents or, indirectly, by altering intracellular signal transduction domains. These strategies have allowed us to construct optimized FVIII-based CARTs to target FVIII-specific B cells that are responsible for producing FVIII inhibitors in patients with HA. DisclosuresDoshi:Bayer Hemophilia Awards Program: Research Funding. Milone:Novartis: Patents & Royalties.

Cytomegalovirus-Specific T Cells Restricted by HLA-Cw*0702 Increase Markedly with Age and Dominate the CD8+ T-Cell Repertoire in Older People.

Cytomegalovirus (CMV) infection elicits a strong T-cell immune response, which increases further during aging in a process termed “memory inflation.” CMV downregulates the expression of HLA-A and HLA-B on the surface of infected cells to limit presentation of viral peptides to T-cells although HLA-C is relatively spared as it also engages with inhibitory killer immunoglobulin receptor receptors and therefore reduces lysis by natural killer cells. We investigated the magnitude and functional properties of CMV-specific CD8+ T-cells specific for 10 peptides restricted by HLA-C in a cohort of 53 donors between the age of 23 and 91 years. This was achieved via peptide stimulation of PBMCs followed by multicolor flow cytometry. Three peptides, derived from proteins generated in the immediate-early period of viral replication and restricted by HLA-Cw*0702, elicited strong immune responses, which increased substantially with age such that the average aggregate response represented 37% of the CD8+ T-cell pool within donors above 70 years of age. Remarkably, a single response represented 70% of the total CD8+ T-cell pool within a 91-year-old donor. HLA-Cw*0702-restricted CD8+ T-cell responses were immunodominant over HLA-A and HLA-B-restricted CMV-specific responses and did not show features of exhaustion such as PD-1 or CD39 expression. Indeed, such CTL exhibit a polyfunctional cytokine profile with co-expression of IFN-γ and TNF-α and a strong cytotoxic phenotype with intracellular expression of perforin and granzymeB. Functionally, HLA-Cw*0702-restricted CTL show exceptionally high avidity for cognate peptide-HLA and demonstrate very early and efficient recognition of virally infected cells. These observations indicate that CD8+ T-cells restricted by HLA-C play an important role in the control of persistent CMV infection and could represent a novel opportunity for CD8+ T-cell therapy of viral infection within immunosuppressed patients. In addition, the findings provide further evidence for the importance of HLA-C-restricted T-cells in the control of chronic viral infection.

Killing viral helicases

Natural Killer Cells Recognition of evolutionarily conserved pathogen-associated molecules drives innate immune responses. Naiyer et al. report that a killer immunoglobulin receptor (KIR) called KIR2DS2 promotes activation of natural killer (NK) cells. KIR2DS2 recognizes conserved peptides from flaviviral RNA helicases when they are presented by a particular human leukocyte antigen (HLA) allele called HLA-C*0102. Two distinct peptide motifs are sensed by KIR2DS2: LNPSVAATL and MCHAT. The former is conserved across hepatitis C virus isolates; the latter is conserved in several flaviviruses, including dengue, Zika, and yellow fever. Hence, a single KIR receptor has evolved to activate NK cells in response to multiple pathogenic viruses. Sci. Immunol. 2 , eaal5296 (2017).

Reduced Expression of Siglec-7, NKG2A, and CD57 on Terminally Differentiated CD56-CD16+ Natural Killer Cell Subset Is Associated with Natural Killer Cell Dysfunction in Chronic HIV-1 Clade C Infection.

HIV-1 viremia has been shown to induce several phenotypic and functional abnormalities in natural killer (NK) cells. To assess immune defects associated with HIV viremia, we examined NK cell function, differentiation status, and phenotypic alterations based on expression of inhibitory and activating receptors on NK cells in HIV-1 subtype C chronically infected participants from Durban, South Africa. NK cell phenotypic profiles were characterized by assessing sialic acid-binding immunoglobulin-like lectin-7 (Siglec-7), NKG2A, and NKG2C markers on frozen peripheral blood mononuclear cells from viremic, antiretroviral therapy (ART)-naive HIV-1 chronically infected participants (n = 23), HIV-1 chronically infected participants who had been on combination antiretroviral therapy (cART) for at least 12 months (n = 23) compared with healthy donors (n = 23). NK cell differentiation was assessed by measurement of killer immunoglobulin receptor (KIR) and NKG2A expression; CD57 and CD107a measurements were carried out in HIV viremic and healthy donors. All phenotypic and functional assessments were analyzed by using multicolor flow cytometry. HIV-1-infected participants displayed greater frequencies of the CD56-CD16+ (CD56negative) NK cell subset compared with healthy donors (p < .0001). Downregulation of Siglec-7 and NKG2A and upregulation of NKG2C were more pronounced in the CD56negative NK cell subset of viremic participants. The CD56negative subset demonstrated a differentiated (KIR+NKG2A-) phenotype with reduced CD57 expression and lower degranulation capacity in HIV-1-infected participants compared with healthy donors. HIV-1 infection induces the expansion of the CD56negative NK cell subset marked by altered receptor expression profiles that are indicative of impaired function and may explain the overall NK cell dysfunction observed in chronic HIV-1 infection.

Human leucocyte antigen (HLA)-DQB1*03:02 and HLA-A*02:01 have opposite patterns in their effects on susceptibility to HIV infection.

The objective of this study was to seek correlates of immune protection in HIV infection. We sought to elucidate the association between the presence of human leucocyte antigen (HLA) alleles, as well as killer immunoglobulin receptor (KIR) genotypes, and the susceptibility to HIV infection in a Spanish cohort of HIV-exposed seronegative (HESN) individuals. A total of 152 individuals were evaluated: 29 HESN individuals in stable heterosexual relationships with an HIV-infected partner admitting high-risk sexual intercourse for at least 12 months prior to inclusion in the study, 61 HIV-infected patients and 62 healthy controls. HLA class I and II alleles and KIR genotypes were assessed in genomic DNA from all individuals in the study by polymerase chain reaction-sequence-specific oligonucleotide (PCR-SSO) using bead array technology. HESN individuals showed a higher prevalence of HLA-A3 (62%) and HLA-B44 (83%) supertypes compared with HIV-infected individuals (42% and 66%, respectively). Regarding specific HLA alleles, HESN individuals had a higher prevalence of HLA-A*33:01, DRB1*04 and DQB1*03:02 alleles (14%, 34% and 31%, respectively) and a lower prevalence of the HLA-A*02:01 allele (27%) than HIV-infected patients (3%, 15%, 11% and 52%, respectively; P < 0.05). Interestingly, in a multivariate analysis, only the presence of DQB1*03:02 and the absence of A*02:01 alleles were independently associated with HESN status [odds ratio (OR) 3.4 (95% confidence interval (CI) 1.1-10.5) and 0.4 (95% CI: 0.1-0.9), respectively; P < 0.05]. No KIR genotype was associated with susceptibility to HIV infection. Our data showed that the presence of the HLA class II allele DQB1*03:02 was a correlate of immune protection against HIV infection, while the presence of the HLA class I allele A*02:01 was associated with being infected with HIV.

Activating Killer Immunoglobulin Receptors and HLA-C: a successful combination providing HIV-1 control

Several studies demonstrated a relevant role of polymorphisms located within the HLA-B and -C loci and the Killer Immunoglobulin Receptors (KIRs) 3DL1 and 3DS1 in controlling HIV-1 replication. KIRs are regulatory receptors expressed at the surface of NK and CD8+ T-cells that specifically bind HLA-A and -B alleles belonging to the Bw4 supratype and all the -C alleles expressing the C1 or C2 supratype. We here disclose a novel signature associated with the Elite Controller but not with the long-term nonprogressor status concerning 2DS activating KIRs and HLA-C2 alleles insensitive to miRNA148a regulation. Overall, our findings support a crucial role of NK cells in the control of HIV-1 viremia.

Assessment of Human Leukocyte Antigen (HLA) Type on Outcomes of Allogeneic Transplant for Chronic Lymphocytic Leukemia (CLL)

BACKGROUNDAllogeneic hematopoietic cell transplant (allo-HCT) can achieve prolonged disease free survival (DFS) in patients with high risk relapsed CLL through a graft-versus-leukemia effect. This is likely mediated through alloreactive donor T-cells that recognize and eliminate residual CLL targets expressing unique and potentially tumor-specific antigens within the context of the Class I Major Histocompatibility Complex (MHC). Histocompatibility Leukocyte Antigen (HLA) alleles encoding class I MHC can be protective or predisposing to the development of a variety of hematologic malignancies. Specifically, HLA-A*02:01, which is enriched in Caucasians, increases the risk of development of CLL, while HLA A1:01 is protective against the development of CLL. Previous single center data of reduced intensity conditioning (RIC) allo-HCT with post-transplant rituximab and donor lymphocyte infusion identified an improvement in complete remission (CR) rates and DFS in patients positive for HLA-A1, negative for HLA-A2 and HLA-B44 (HLA-A1, non-A2 and non-B44) compared to patients without these HLA alleles (Khouri, et al 2011). The impact of this combination of HLA alleles (HLA-A1, non-A2 and non-B44) on outcomes of allo-HCT for CLL has not been previously explored in a multi-center study. We therefore sought to compare outcomes of CLL patients undergoing fully-matched related or unrelated donor allo-HCT in CIBMTR centers, based on recipient HLA type.METHODSCLL patients ≥ 18 years old who received a fully matched allo-HCT (from matched sibling donor or 8 out of 8 matched unrelated donors) using myeloablative (MA), non-myeloablative (NMA), or reduced intensity conditioning (RIC) between 2000 and 2013 were included. Patients with Richter’s transformation were excluded. Patient outcomes were compared based on the presence of the HLA combination HLA-A1, non-A2 and non-B44 (n=83) vs. other (n=481). Patient-related variables included age, gender, coexistent disease or organ impairment, HCT comorbidity score, Karnofsky score, donor/recipient CMV status. Disease-related variables included presence/absence of 17p-, 11q-, 13q-, and trisomy 12, IGHV mutation status, Rai stage at diagnosis, elevated LDH or bulky adenopathy at the time of HCT, type and number of treatments, response to pre-transplant chemotherapy, and disease status at transplant. Transplant-related variables included time from diagnosis to HCT, preparative regimen: MA vs. RIC vs. NMA, graft source, donor age, and donor-recipient sex match. Cox proportional hazard analysis was used to identify significant risk factors. For power calculation, it was assumed that overall survival (OS) at 1 year, 2 years and 3 years were 70%, 57%, and 50%, respectively. Median censoring time was assumed to be 70 months. There was 80% power to detect hazard ratio (HR) of 1.65 at the significance level 0.05. This HR was based on prior publication by Khoury et al.RESULTSMedian age of patients was 56 (range 22-75). 73% were male. Baseline characteristics between the groups with and without A1, non-A2, non-B44 were well matched in terms of prior therapy, remission status at time of allo-HCT, use of RIC or NMA conditioning, and baseline cytogenetics. In multivariable analysis, the HLA combination HLA-A1, non-A2, non-B44 was not associated with an improvement in GVHD, relapse rates, or OS (see Table ). Kaplan Meier estimates of the OS of the groups are shown in the accompanying Figure. There was a non-statistically significant trend towards improvement in DFS in patients without the HLA combination of interest (P = 0.06). In univariable analysis HLA-A1, A2 and B44 were not individually associated with OS.CONCLUSIONSPreviously-identified HLA alleles were not associated with any measurable outcomes of allogeneic transplant from fully matched related or unrelated donors captured in the CIBMTR registry, either alone or in combination. Future study of the impact of HLA on transplant outcomes may require assessment of killer immunoglobulin receptor (KIR) subtypes for more robust exploration of the immunologic basis for differences in allo-HCT outcomes for CLL. [Display omitted] [Display omitted] DisclosuresCortes:ARIAD: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Teva: Research Funding.

Killer immunoglobulin receptor genes in spondyloarthritis.

We focus on the role of killer immunoglobulin receptor (KIR) interactions with the human leukocyte antigens (HLA)-B27 ligand and the potential contribution of KIR-expressing natural killer and T cells in spondyloarthritis, more specifically in ankylosing spondylitis (AS). In AS strong epidemiological evidence of significant genetic associations with the major histocompatibility complex was convincingly identified. HLA-B27-positive first-degree relatives of AS cases are 5-16 times more likely to develop disease than HLA-B27-positive carriers in the general community. The GWAS era has enabled rapid progress in identifying non-major histocompatibility complex associations of AS. These findings show a number of important pathways in AS pathogenesis, including the IL-23-IL-17 pathway, aminopeptidases, peptide presentation, and KIR-HLA-B27 interactions. Studies using genetic markers, including KIRs may be used for a risk assessment about whom may benefit most from the various treatment protocols in spondyloarthritis, now that alternative therapeutic options have become feasible.

Potential role of killer immunoglobulin receptor genes among individuals vaccinated against hepatitis B virus in Lebanon.

AIMTo explore the role of killer immunoglobulin receptor (KIR) genes in responsiveness or non-responsiveness to vaccination against hepatitis B virus.METHODSWe recruited 101 voluntary participants between March 2010 and December 2011. Sera samples from vaccinated and non-vaccinated participants were tested for the presence of anti-HBs antibodies as a measure of protection against hepatitis B, hepatitis B surface antigen and hepatitis B core antibody as indicators of infection by enzyme-linked immunosorbent assay. KIR gene frequencies were determined by polymerase chain reaction.RESULTSSera samples from 99 participants were tested for the levels of anti-HBs as an indicator of protection (≥ 10 mIU/mL) following vaccination as defined by the World Health Organization international reference standard. Among the vaccinated participants, 47% (35/74) had anti-HBs titers above 100 mIU/mL, 22% (16/74) had anti-HBs ranging between 10-100 mIU/mL, and 20% (15/74) had values of less than 10 mIU/mL. We report the lack of significant association between the number of vaccine dosages and the titer of antibodies among our vaccinated participants. The inhibitory KIR2DL1, KIR2DL4, KIR3DL1, KIR3DL2, and KIR3DL were detected in more than 95%, whereas KIR2DL2, KIR2DL3, KIR2DL5 (KR2DL5A and KIR2DL5B) were expressed in 56%, 84% and 42% (25% and 29%) of participants, respectively. The observed frequency of the activating KIR genes ranged between 35% and 55% except for KIR2DS4, detected in 95% of the study participants (40.6% 2DS4*001/002; 82.2% 2DS4*003/007). KIR2DP1 pseudogene was detected in 99% of our participants, whereas KIR3DP*001/02/04 and KIR3DP1*003 had frequencies of 17% and 100%, respectively. No association between the frequency of KIR genes and anti-HBs antibodies was detected. When we compared the frequency of KIR genes between vaccinated individuals with protective antibodies titers and those who lost their protective antibody levels, we did not detect a significant difference. KIR2DL5B was significantly different among different groups of vaccinated participants (group I > 100 mIU/mL, group II 10-100 mIU/mL, group III < 10 mIU/mL and group IV with undetectable levels of protective antibodies).CONCLUSIONTo our knowledge, this is the first study screening for the possible role of KIR genes among individuals vaccinated against hepatitis B virus (HBV). Our results can be used to design larger studies to better understand the role of KIR genes in protection against or susceptibility to HBV post vaccination.

Diversity in KIR gene repertoire in HIV-1 exposed infected and uninfected infants: A study from India.

Natural killer (NK) cells have antiviral activity mediated through killer immunoglobulin receptors (KIRs). Studies have shown the importance of KIR receptors in HIV infection. However reports on association of KIR genes in HIV infection from Indian population are limited, not a single study is reported in HIV exposed uninfected (EU) and infected infants. This study compared the KIR gene repertoire of HIV-1 positive (n = 29) with EU (n = 76) infants to elucidate its association with transmission. KIR genotyping was analysed using the PCR-SSP method. Viral load of mothers, CD4 count of both mothers and infected infants were done using commercial kits. The data was analysed using SPSS software. Results revealed presence of significantly high frequencies of activating gene KIR 2DS5 (P = 0.040) and inhibitory gene KIR 2DL3 (P = 0.013) in EU infants as compared to HIV-1 positive infants, confirmed with multivariable linear regression modelling. Fifty-nine KIR genotypes were identified in these 105 infants. Nine genotypes were unique, reported for the first time. Twenty six genotypes were shared with the World populations. Twenty four genotypes were reported for the first time from India. Specific KIR genotype combinations (GIDs) were exclusively present either in HIV-1 positive (n = 19) or in EU infants (n = 30). The Linkage disequilibrium (LD) analysis shows a strong linkage between four pairs of genes in HIV-1 positive and three pairs of genes in EU infants. In conclusion, this study revealed that, besides maternal confounding factors such as ART and viral load, specific KIR genes are associated independently with perinatal HIV infection.