Successful transfer of alloreactive haploidentical KIR ligand-mismatched natural killer cells after infusion in elderly high risk acute myeloid leukemia patients

Purpose: In acute myeloid leukemia (AML), alloreactive natural killer (NK) cells are crucial mediators of immune responses after haploidentical stem cell transplantation. Allogeneic NK cell infusions have been adoptively transferred with promising clinical results. We aimed at determining whether the composition of NK graft in terms of frequency of alloreactive NK cells influence the clinical response in a group of elderly AML patients undergoing NK immunotherapy. Experimental Design: Seventeen AML patients, in first complete remission (CR; median age 64 years, range 53–73) received NK cells from haploidentical KIR-ligand–mismatched donors after fludarabine/cyclophosphamide chemotherapy, followed by IL2. To correlate donor NK cell activity with clinical response, donor NK cells were assessed before and after infusion. Results: Toxicity was moderate, although 1 patient died due to bacterial pneumonia and was censored for clinical follow-up. With a median follow-up of 22.5 months (range, 6–68 months), 9 of 16 evaluable patients (0.56) are alive disease-free, whereas 7 of 16 (0.44) relapsed with a median time to relapse of 9 months (range, 3–51 months). All patients treated with molecular disease achieved molecular CR. A significantly higher number of donor alloreactive NK cell clones was observed in responders over nonresponders. The infusion of higher number of alloreactive NK cells was associated with prolonged disease-free survival (0.81 vs. 0.14, respectively; P = 0.03). Conclusions: Infusion of purified NK cells is feasible in elderly AML patients as post-CR consolidation strategy. The clinical efficacy of adoptively transferred haploidentical NK cells may be improved by infusing high numbers of alloreactive NK cells. Clin Cancer Res; 22(8); 1914–21. ©2016 AACR . See related commentary by Muntasell and Lopez-Botet, p. 1831

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Larger Size of Donor Alloreactive NK Cell Repertoire Correlates with Better Response to NK Cell Immunotherapy in Elderly Acute Myeloid Leukemia Patients.

Lenalidomide, idelalisib, and rituximab are unacceptably toxic in patients with relapsed/refractory indolent lymphoma.

Using flow cytometry, fluorescent microscopy and examination of receptor glycosylation status, we demonstrate that an entire killer cell immunoglobulin-like receptor (KIR) locus (KIR2DS3)--assumed earlier to be surface expressed--appears to have little appreciable surface expression in transfected cells. This phenotype was noted for receptors encoded by three allelic variants including the common KIR2DS3*001 allele. Comparing the surface expression of KIR2DS3 with that of the better-studied KIR2DS1 molecule in two different cell lines, mutational analysis identified multiple polymorphic amino-acid residues that significantly alter the proportion of molecules present on the cell surface. A simultaneous substitution of five residues localized to the leader peptide (residues -18 and -7), second domain (residues 123 and 150) and transmembrane region (residue 234) was required to restore KIR2DS3 to the expression level of KIR2DS1. Corresponding simultaneous substitutions of KIR2DS1 to the KIR2DS3 residues resulted in a dramatically decreased surface expression. Molecular modeling was used to predict how these substitutions contribute to this phenotype. Alterations in receptor surface expression are likely to affect the balance of immune cell signaling impacting the characteristics of the response to pathogens or malignancy.

Dramatically reduced surface expression of NK cell receptor KIR2DS3 is attributed to multiple residues throughout the molecule.

BACKGROUND Kidney transplantation is a life-saving treatment for patients with end-stage renal disease. However, despite progress in surgical techniques and patient management, immunological rejection continues to have a negative impact on graft function and overall survival. Incompatibility between donors and recipients for human leukocyte antigens (HLA) of the major histocompatibility complex (MHC) generates a series of complex cellular and humoral immune response mechanisms that are largely responsible for rejection and loss of graft function. Within this context, a growing amount of evidence shows that alloreactive natural killer (NK) cells play a critical role in the immune response mechanisms elicited by the allograft. Killer immunoglobulin-like receptors (KIRs) are prominent mediators of NK cell alloreactivity. METHODS AND FINDINGS A cohort of 174 first cadaveric kidney allograft recipients and their donors were selected from a total cohort of 657 transplanted patients for retrospective immunogenetic analyses. Patients with HLA Class II mismatches were excluded. HLA Class I allele frequencies were compared among patients with chronic rejection, patients with stable graft function and a group of 2388 healthy controls. Activating and inhibitory KIR gene frequencies, KIR haplotypes, KIR-HLA ligand matches/mismatches and combinations of recipient KIRs and donor HLA Class I ligands were compared among patients with and without chronic rejection and a group of 221 healthy controls. Patients transplanted from donors homozygous for HLA-C1 antigens had a significantly higher risk for chronic rejection than patients transplanted from donors homozygous or heterozygous for HLA-C2 antigens or with epitopes belonging to the HLA-Bw4 ligand group. The Kaplan-Meier curves obtained by dividing the patients into 3 groups according to the presence or absence of one or both of the combinations of recipient KIRs and donor HLA ligands (rKIR2DL1/dHLA-C2 and rKIR3DL1/dHLA-Bw4) showed a significantly higher cumulative incidence of chronic rejection in the group of patients completely lacking these functional units. These patients showed a progressively stronger decline in modification of diet in renal disease-estimated glomerular filtration rate. CONCLUSIONS KIR genotyping should be performed at the time of enrolment of patients on the waiting list for organ transplantation. In our study, a significantly higher risk of chronic rejection after kidney transplantation was observed when recipient (r) and donor (d) pairs completely lacked the two functional rKIR-dHLA ligand combinations rKIR2DL1/dHLA-C2 and rKIR3DL1/dHLA-Bw4. This immunogenetic profile corresponds to low levels of NK cell inhibition. Therefore, patients with this high risk profile could benefit from immunosuppressive therapy aimed at reducing NK-cell cytotoxicity.

KIR and their HLA Class I ligands: Two more pieces towards completing the puzzle of chronic rejection and graft loss in kidney transplantation.

Killer cell immunoglobulin-like receptors (KIRs) are a family of inhibitory and activatory receptors that are expressed by most natural killer (NK) cells. The KIR gene family is polymorphic: genomic diversity is achieved through differences in gene content and allelic polymorphism. The number of KIR loci has been reported to vary among individuals, resulting in different KIR haplotypes. In this study we report the genotypic structure of KIRs in 217 unrelated healthy Italian individuals from 22 immunogenetics laboratories, located in the northern, central and southern regions of Italy. Two hundred and seventeen DNA samples were studied by a low resolution PCR-SSP kit designed to identify all KIR genes. All 17 KIR genes were observed in the population with different frequencies than other Caucasian and non-Caucasian populations; framework genes KIR3DL3, KIR3DP1, KIR2DL4 and KIR3DL2 were present in all individuals. Sixty-five different profiles were found in this Italian population study. Haplotype A remains the most prevalent and genotype 1, with a frequency of 28.5%, is the most commonly observed in the Italian population. The Italian Caucasian population shows polymorphism of the KIR gene family like other Caucasian and non-Caucasian populations. Although 64 genotypes have been observed, genotype 1 remains the most frequent as already observed in other populations. Such knowledge of the KIR gene distribution in populations is very useful in the study of associations with diseases and in selection of donors for haploidentical bone marrow transplantation.

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Distribution of killer cell immunoglobulin-like receptors genes in the Italian Caucasian population

The killer cell immunoglobulin-like receptor (KIR)-human leukocyte antigen (HLA) interaction represents an example of genetic epistasis, where the concomitant presence of specific genes or alleles encoding receptor-ligand units is necessary for the activity of natural killer (NK) cells Although KIR and HLA genes segregate independently, they co-evolved under environmental pressures to maintain particular KIR-HLA functional blocks for species survival We investigated, in 270 Italian healthy individuals, the distribution of KIR and HLA polymorphisms in three climatic areas (from cold north to warm south), to verify their possible geographical stratification We analyzed the presence of 13 KIR genes and genotyped KIR ligands belonging to HLA class I: HLA-C, HLA-B and HLA-A We did not observe any genetic stratification for KIR genes and HLA-C ligands in Italy By contrast, in a north-to-south direction, we found a decreasing trend for the HLA-A3 and HLA-A11 ligands (P = 0012) and an increasing trend for the HLA-B ligands carrying the Bw4 epitope (P = 00003) and the Bw4 Ile80 epitope (P = 00005) The HLA-A and HLA-B KIR ligands were in negative linkage disequilibrium (correlation coefficient -01211), possibly as a consequence of their similar function in inhibiting NK cells The distribution of the KIR-HLA functional blocks was different along Italy, as we observed a north-to-south ascending trend for KIR3DL1, when coupled with HLA-B Bw4 ligands (P = 00067) and with HLA-B Bw4 Ile80 (P = 00027), and a descending trend for KIR3DL2 when coupled with HLA-A3 and HLA-A11 ligands (P = 00044) Overall, people from South Italy preferentially use the KIR3DL1-HLA-B Bw4 functional unit, while those from the North Italy equally use both the KIR3DL2-HLA-A3/A11 and the KIR3DL1-HLA-B Bw4 functional units to fight infections Thus, only KIR3DL receptors, which exert the unique role of microbial sensors through the specific D0 domain, and their cognate HLA-A and HLA-B ligands are selectively pressured in Italy according to geographical north-to-south distribution

The distribution of KIR-HLA functional blocks is different from north to south of Italy

The novel allele human leukocyte antigen(HLA)-DQB1*06:04:04 differs from HLA-DQB1*06:04:01 by a silent nucleotide substitution at codon 75 (TTG → CTG).

E. Cosentini

Papers

Distribution of killer cell immunoglobulin-like receptors genes in the Italian Caucasian population

The distribution of KIR-HLA functional blocks is different from north to south of Italy

Characterization of a novel HLA-DQB106 allele, HLA-DQB106:04:04.

E. Cosentini

Papers

Distribution of killer cell immunoglobulin-like receptors genes in the Italian Caucasian population

The distribution of KIR-HLA functional blocks is different from north to south of Italy

Characterization of a novel HLA-DQB1*06 allele, HLA-DQB1*06:04:04.

Characterization of a novel HLA-DQB106 allele, HLA-DQB106:04:04.