Natural Killer Cell Alloreactivity Research Articles

#2461 KIR-HLA-I genetic mismatch and the development of antibody-mediated rejection and microvascular inflammation after renal transplantation

Abstract Background and Aims Antibody-mediated rejection (ABMR) and microvascular inflammation (MVI) contribute to kidney transplant (KT) loss of function. The contribution of Natural Killer (NK) cells to the development of ABMR/MVI through inhibitory (iKIR) and activating (aKIR) HLA-I KIR (Killer-cell Immunoglobulin-like Receptors) has been proposed. The presence of an iKIR gene in the KT recipient (KTR) recognizing a self HLA-I ligand absent in the donor is defined as an iKIR-HLA-I mismatch (MM), and the presence of an aKIR gene in the KTR with HLA-I ligand in the graft absent in the KTR is defined as an aKIR-HLA-I MM. These MMs indicate the presence of potentially alloreactive NK cells that could contribute to the graft rejection, especially in the presence of NK receptor activating ligands or DSA. Our aim was to study the relationship between the number of KIR-HLA-I mismatches and ABMR/MVI. Method We selected 56 first KT, not treated with thymoglobulin or rituximab: 24 cases with ABMR/MVI and 32 controls without rejection in biopsies at 12-36 months. Donor and recipient HLA-I genotypes were determined by NGS, and recipient KIR genotypes by PCR-SSO (Luminex). The interactions considered were: KIR2DL1/HLA-C2, KIR2DL2&KIR2DL3/HLA-C1, KIR3DL1/HLA-Bw4, KIR3DL2/HLA-A*03, A*11, KIR2DS1/HLA-C2, KIR2DS2/HLA-C1, KIR3DS1/HLA-Bw4-I80 and KIR2DS4/HLA-A*11, C*02, C*04, C*05, C*16:01. Results KT recipients had a mean age of 54.55 years, 30.36% female and 8.92% living donor with no significant differences between ABMR/MVI and controls. The proportion of patients with ≥1 iKIR-HLA-I MM was higher in the ABMR/MVI group compared to controls (75% vs 56.25%, P = .1473), with a significant difference in the percentage of KIR3DL1 MM (29.17% vs 6.25%, P = .0296). The proportion of patients with at least one aKIR-HLA-I MM was non-significantly lower in ABMR/MVI compared to the control group (20.83% vs 28.13%, P = .5329). Overall, the proportion of KT recipients with ≥1 MM, either iKIR or aKIR, was 21% higher in the ABMR/MVI group (83.33% vs 68.75%, P = .2123). Conclusion In a population of immunologically low-risk KT recipients, we found a significantly greater proportion of patients with genetic iKIR-HLA-I MM in those with ABMR/MVI, but not of aKIR-HLA-I MM. NK cells with iKIR-HLA-I MM could contribute to antibody-mediated allograft damage together with other mechanisms.

Donor KIR genotype based outcome prediction after allogeneic stem cell transplantation: no land in sight.

Optimizing natural killer (NK) cell alloreactivity could further improve outcome after allogeneic hematopoietic cell transplantation (alloHCT). The donor's Killer-cell Immunoglobulin-like Receptor (KIR) genotype may provide important information in this regard. In the past decade, different models have been proposed aiming at maximizing NK cell activation by activating KIR-ligand interactions or minimizing inhibitory KIR-ligand interactions. Alternative classifications intended predicting outcome after alloHCT by donor KIR-haplotypes. In the present study, we aimed at validating proposed models and exploring more classification approaches. To this end, we analyzed samples stored at the Collaborative Biobank from HLA-compatible unrelated stem cell donors who had donated for patients with acute myeloid leukemia (AML) or myelodysplastic neoplasm (MDS) and whose outcome data had been reported to EBMT or CIBMTR. The donor KIR genotype was determined by high resolution amplicon-based next generation sequencing. We analyzed data from 5,017 transplants. The median patient age at alloHCT was 56 years. Patients were transplanted for AML between 2013 and 2018. Donor-recipient pairs were matched for HLA-A, -B, -C, -DRB1, and -DQB1 (79%) or had single HLA mismatches. Myeloablative conditioning was given to 56% of patients. Fifty-two percent of patients received anti-thymocyte-globulin-based graft-versus-host disease prophylaxis, 32% calcineurin-inhibitor-based prophylaxis, and 7% post-transplant cyclophosphamide-based prophylaxis. We tested several previously reported classifications in multivariable regression analyses but could not confirm outcome associations. Exploratory analyses in 1,939 patients (39%) who were transplanted from donors with homozygous centromeric (cen) or telomeric (tel) A or B motifs, showed that the donor cen B/B-tel A/A diplotype was associated with a trend to better event-free survival (HR 0.84, p=.08) and reduced risk of non-relapse mortality (NRM) (HR 0.65, p=.01). When we further dissected the contribution of B subtypes, we found that only the cen B01/B01-telA/A diplotype was associated with a reduced risk of relapse (HR 0.40, p=.04) while all subtype combinations contributed to a reduced risk of NRM. This exploratory finding has to be validated in an independent data set. In summary, the existing body of evidence is not (yet) consistent enough to recommend use of donor KIR genotype information for donor selection in routine clinical practice.

Phase I / II Study on Infusion of Alloreactive or Ex Vivo il-15 Stimulated Natural Killer Cells after Haploidentical Stem Cell Transplantation in Pediatric Patients with Acute Leukemia (PHINK): A Study of the Spanish Hematopoietic Stem Cell Transplantation Group (GETH)

Background: Haploidentical hematopoietic stem cell transplantation (haplo-HSCT) is a highly complicated but effective procedure for high-risk pediatric leukemia in the absence of an HLA-identical donor. Post-transplant relapse is the main problem for patient survival. According to literature, Natural Killer (NK) cells play a potent role as inducers of the graft-versus-leukemia (GvL) effect in haplo-HSCT. The presence of alloreactive NK cells correlates with a lower relapse rate, improved engraftment, decreasing graft-versus-recipient disease (GvHD) and viral infection risk. In the absence of donor-recipient NK cell alloreactivity, other NK cell activation strategies are necessary. We propose a phase I/II clinical trial with dose escalation, multicenter, framed in the GETH, to determine dose-limiting toxicity (DLT) and the maximum tolerated dose (MTD) of a single infusion of alloreactive NK cells (Allo-NK) or NK cells stimulated ex vivo with IL-15 (IL15-NK) after haplo-HSCT in pediatric patients with high-risk leukemias. Methods: Eighteen pediatric patients with high-risk leukemia were recruited between September 16, 2020, and May 12, 2023, at 5 hospitals in Spain, Hospital Universitario La Paz, Hospital Universitario Gregorio Marañón, Hospital Universitario Niño Jesús, Hospital Universitario Central of Asturias and Hospital Santa Creu and Sant Pau. Patients were stratified in 2 treatment groups: Allo-NK (n=9) or IL15-NK (n=9), depending on the KIR disparity between donor and recipient. Our phase I/II clinical trial presents a dose-escalation design (3+3). The treatment cohorts are divided into cohort 1 (up to 2x10 7 cells/kg), cohort 2 (2-5x10 7 cells/kg) and cohort 3 (5-10x10 7 cells/kg). We have monitored infections reactivation, as well as, chimerism and engraftment after transplantation. In Allo-NK group, the KIR clone that provides alloreactivity in the NK cell was also tracked. The phenotypic profile and functional capacity of NK cells present in the patient up to 360 days after transplantation have also been analyzed. Results: Nine patients (4 in Allo-NK and 5 in IL-15NK) were included in cohort 1, 7 patients (4 in Allo-NK and 3 in IL-15NK) in cohort 2 and 2 patients (1 in Allo-NK and 1 in IL-15NK) in cohort 3. We were unable to complete the dose escalation design initially proposed due to the high weight of some patients and the unavailability of ex vivo expanded NK cells. In IL15-NK group patients presented a higher prevalence of B-ALL (p=0.018) and had received a previous transplant (p=0.082). NK cell infusions of IL15-NK group showed higher expression of CD25 and CD69 receptors and higher functional capacity. In the Allo-NK group, the most prevalent KIR-reactive clone was KIR2DL1 (55.6%). We also observed a tendency for KIR2DL1 alloreactive clone to increase from 30 (8,18%) to 360 (25,63%) days after transplantation. Regarding clinical outcomes in 2 study arms, the incidence of severe (Grades III-IV) acute GvHD was 22.2% and severe chronic GvHD was only observed in 1 (5.6%) patient (Table 1). The incidence any infection or endothelial vascular complications were 72% and 33% respectively. No statistical differences were observed in GvHD, infections and endothelial vascular complications. We found 2 relapses, 1 in each study arm (11.1%). Five patients died, 1 (5.6%) because of disease progression and 4 (22.2%) because transplant related complications (TRM) (Table 1). Dividing patients into the 3 dose-escalation cohorts, we found no significant differences between cohorts in the primary clinical outcomes analysed. One-years overall survival (OS) and disease-free survival (DFS) was 72.22% (CI95% 51.93-100) and GvHD-free/relapse-free survival (GRSF) was 64.1% (CI95% 44.62-92.1), finding no significant differences between either 2 study arms or the 3 dose-escalation cohorts. Regarding the analysis of the NK phenotype, we observed a tendency to increase the expression of CD16 and CD57 receptors between 30 to 360 days after transplantation in both groups. Conclusions: Two study arms shows similar data respect to clinical outcome. Patients infused with IL-15-stimulated NK cells showed similar toxicity to the alloreactive NK group. According to dose escalation, we observed no association between increased toxicity and increased number of infused NK cells. We were unfortunately unable to complete cohort 3 because of the cells/kg ratio in some patients.

Donor selection for adoptive immunotherapy with NK cells in AML patients: Comparison between analysis of lytic NK cell clones and phenotypical identification of alloreactive NK cell repertoire.

Natural killer (NK) cell-based adoptive immunotherapy in leukemia patients is an emerging field of interest based on clinical evidence of efficacy and safety. Elderly acute myeloid leukemia (AML) patients have been successfully treated with NK cells from HLA-haploidentical donors, especially when high amounts of alloreactive NK cells were infused. The aim of this study was comparing two approaches to define the size of alloreactive NK cells in haploidentical donors for AML patients recruited in two clinical trials with the acronym "NK-AML" (NCT03955848), and "MRD-NK". The standard methodology was based on the frequency of NK cell clones capable of lysing the related patient-derived cells. The alternative approach consisted of the phenotypic identification of freshly derived NK cells expressing, as inhibitory receptors, only the inhibitory KIR(s) specific for the mismatched KIR-Ligand(s) (HLA-C1, HLA-C2, HLA-Bw4). However, in KIR2DS2+ donors and HLA-C1+ patients, the unavailability of reagents staining only the inhibitory counterpart (KIR2DL2/L3) may lead to an underestimated identification of the alloreactive NK cell subset. Conversely, in the case of HLA-C1 mismatch, the alloreactive NK cell subset could be overestimated due to the ability of KIR2DL2/L3 to recognize with low-affinity also HLA-C2. Especially in this context, the additional exclusion of LIR1-expressing cells might be relevant to refine the size of the alloreactive NK cell subset. We could also associate degranulation assays, using as effector cells IL-2 activated donor peripheral blood mononuclear cells (PBMC) or NK cells upon co-culture with the related patient target cells. The donor alloreactive NK cell subset always displayed the highest functional activity, confirming its identification accuracy by flow cytometry. Despite the phenotypic limitations and considering the proposed corrective actions, a good correlation was shown by the comparison of the two investigated approaches. In addition, the characterization of receptor expression on a fraction of NK cell clones revealed expected but also few unexpected patterns. Thus, in most instances, the quantification of phenotypically defined alloreactive NK cells from PBMC can provide data similar to the analysis of lytic clones, with several advantages, such as a shorter time to achieve the results and, perhaps, higher reproducibility/feasibility in many laboratories.

KIR genotype and haplotype frequencies in the multi-ethnic population of Malaysia

Killer cell immunoglobulin-like receptors (KIR) genotype and haplotype frequencies have been reported to vary distinctly between populations, which in turn contributes to variation in the alloreactivity of natural killer (NK) cells. Utilizing the diverse KIR genes to identify suitable transplant donors would prove challenging in multi-ethnic countries, even more in resource-limited countries where KIR genotyping has not been established. In this study, we determined the KIR genotypes from 124 unrelated Malaysians consisting of the Malays, Chinese, Indians, and aboriginal people through polymerase chain reaction sequence-specific primer (PCR-SSP) genotyping and employing an expectation–maximization (EM) algorithm to assign haplotypes based on pre-established reference haplotypes. A total of 27 distinct KIR haplotypes were discerned with higher frequencies of haplotype A (55.2%) than haplotype B (44.8%). The most frequent haplotypes were cA01:tA01 (55.2%), cB01:tB01 (18.1%), and cB02:tA01 (13.3%), while the least frequent haplotypes were cB03:tB01 (1.2%), cB04:tB03 (0.4%), and cB03:tA01 (0.4%). Several haplotypes were identified to be unique to a specific ethnic group. The genotype with the highest frequency was genotype AB (71.8%), followed by AA (19.4%), and BB (8.9%). The Indians exhibited the lowest genotype AA but the highest genotype BB, whereas genotype BB was absent in the aboriginal people. Despite the limitations, the genotype and haplotypes in the Malaysian population were successfully highlighted. The identification of ethnic-specific KIR genotypes and haplotypes provides the first step to utilizing KIR in identifying suitable transplant donors to further improve the transplant outcome in the Malaysian population.

Haploidentical Hematopoietic Stem Cell Transplant Plus Add-Back NK Cells in Pediatric High-Risk ALL

Introduction: Natural killer (NK) lymphocytes are cells of innate immunity that protect against tumor and infections. NK cells recognize self and non-self HLA class I ligands, are regulated by a balance between activator and inhibitory signals through surface receptors, and can interact with HLA class I molecules on target cells. Killer-cell Immunoglobulin-like Receptors (KIRs) are inhibitory molecules expressed on NK cells and belong to the immunoglobulin superfamily. KIRs function to "educate" NK cells in effective defense against unhealthy self/non-self cells and tolerance against healthy self cells. NKs are thought to play a key role in preventing early relapse of hematologic malignancies, virus infection and graft versus host disease (GvHD) after haploidentical hematopoietic stem cell transplantation (haplo-HSCT).In B-/T-cell alpha/beta depleted haplo-HSCT, donor-versus-recipient allo-reactive NK cells can exert an efficient graft versus leukemia (GvL) effect, mediated by inhibitory KIRs where the HLA ligand is missing on recipient cells. Donor NK cells after haplo-HSCT are "immature" and not effective as the mature NKs. These immature NK cells are CD56bright, are derived from CD34+ stem cells, and mature to a CD56dim phenotype. The mature CD56dim NK cells are characterized by acquisition of KIRs, CD16 and CD57 in mature stages, which have functional significance in targeting and medicating their cytolytic activity. Based on these considerations, we performed haplo-HSCT with donor NK cell add-back to provide mature, functional immunological surveillance against tumor (MRD) and virus reactivation, which can frequently occur in manipulated haplo-HSCT for pediatric acute lymphoblastic leukemia (ALL). Methods: We evaluated the alloreactive effect of donor-derived NK cells in three pediatric high-risk ALL who underwent B-/T-cell alpha/beta depleted haplo-HSCT followed by donor NK infusion. Patients' characteristics are summarized in the Table. All 3 patients were male; median age was 13 months. Patients 1 and 2 had infant ALL, and patient 3 had refractory-relapsed (r/r) ALL. All patients received a median of 3 lines chemotherapy. Patient 1 received a prior allo-HSCT. Patients 1 and 2 received chemo-based conditioning regimens consisting of thyotepa, and fludarabine; In addition, patient1received treosulfan, and patient 2 received busulfan. Treosulfan was given to patient 1, due to less toxicity profile given prior allo-HSCT. Patient 3 received Total Body Irradiation (TBI), thyotepa, and fludarabine. All patients received rituximab and letermovirfor EBV and CMV prophylaxis, respectively, for all patients. Mother was Donor for all patients. Median CD34+, CD19+, CD3+-alpha/beta cell dose was 8.8, 0.05, 0.41 x 106/kg, respectively. NK cell allo-reactivity was tested by evaluating KIR genotyping and KIR/KIR-ligand mismatch in graft-versus-host direction. All three patients present KIR/KIR-ligand mismatch. Results: Engraftment was day +13, +14 and +19 for ANC, and +14, +18 and +17 for PLT (for both >20K and >50K), for patients 1, 2 and 3, respectively. NK cell number were monitored on day +7, +14, +21 and +28 (data pending). Patients received NK cell infusion on day 30 +/- 2 days. No infusion reactions were observed. MedianNK infused were 256x106/kg. None of the patients developed CMV, EBV or ADV reactivation. Patient 2 developed Grade 2 acute GvHD (skin) treated with steroids. No Grade GvHD 3-4 was observed. Median follow up was 58 days. All patients had full donor chimerism at day 30 ±2. All patients are currently in hematological CR; patient 1 converted to positive MRD at nine months from transplant. Conclusion: Alloreactive NK cell add-back infusion after B-/T-cell alpha/beta depleted haplo-HSCT could represent an effective option to improve transplant outcome and prevent virus reactivation. These NK cells may improve efficacy by enhancing GvL and could contribute to maintain the "disease control" through immunologic surveillance in high-risk ALL. Indeed, NK killing, which is higher in mature NK cells, depends on acquisition of sequential phenotypic maturation. Thus, infusion of mature donor NK cells may help to maintain control of MRD and virus reactivation through immunologic surveillance. Although our preliminary observations seem to be promising, this hypothesis should be confirmed in clinical trial of a larger number of patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Phase II Clinical Trial of "Off-the-Shelf" NK Cells with Allogeneic Stem Cell Transplantation to Decrease Disease Relapse in Patients with High-Risk Myeloid Malignancies

Background Acute myeloid leukemia, myelodysplastic syndrome, and TKI resistant CML continue to be among the most aggressive forms of blood cancer. Allogeneic hematopoietic stem cell transplantation (alloSCT) remains one of the most effective treatments available. Much of the benefit of AlloSCT is due to the immune-mediated graft-versus-leukemia effect to prevent relapse. Nevertheless, disease relapse remains the most important cause of treatment failure after alloSCT. A potential solution to reduce the risk of relapse may be found in Natural Killer (NK) cells, which have a demonstrable ability to eliminate leukemic and virally infected cells. However, naturally produced NK cells seen in patients early post-transplant are functionally impaired. A potential solution would be to provide donor-derived, expanded and activated NK cells in the peri-transplant setting. Natural killer cells are a unique class of lymphocytes, with cytotoxic, and immunoregulatory function which can mediate potent anti-leukemia effects. NK cells are regulated by Killer-cell Immunoglobulin-like Receptors (KIR) receptor-ligand interactions and mediate cytotoxicity. Alloreactive NK cells have been reported to enhance engraftment, reduce GVHD and prevent relapse of leukemia post transplant. This study will utilize NK cells from third party donors as adjuvant treatment to eradicate leukemia and prevent disease relapse after transplantation. Study Design and Methods Our clinical trial is registered as NCT05115630. Inclusion criteria includes age 18 to 65 years old, weighing at least 42 kg, with High risk AML, MDS, or TKI-resistant/intolerant CML who have either an HLA matched related donor, HLA matched unrelated donor, a haploidentical related donor, or a one antigen mismatched unrelated donor. HLA matching includes HLA A, B, C, and DR-B1. Exclusion criteria include patients who are HIV positive or has active hepatitis B or C, uncontrolled infections, liver cirrhosis, CNS involvement within 3 months prior to the transplant, or positive pregnancy test in a woman with child bearing potential. We will also exclude any patient with inability to comply with medical therapy or follow-up, patient with a known history of allergic reactions to any constituents of the product, including a known history of allergic reactions to cellular products or DMSO, other malignancy/cancer diagnosis <2 years ago. We will also exclude any patient requiring systemic corticosteroids with prednisone dose >10 mg or equivalent or who has KDS-1001 Donor specific antibodies (dsa) >5000 MFI units, or has KDS-1001 Donor anti-C1q positive. Statistical considerations and exploratory endpoints This is a single-arm Phase II trial of 3rd party natural killer (NK) cells at a fixed dose added to an allogeneic stem cell transplant for AML/MDS/CML. The goal is to reduce the risk of relapse of the malignancy post transplant, and to improve relapse free survival. The primary endpoint is the adverse event Failure = [graft failure, grade 3-4 GVHD, relapse or non-relapse death within 100 days post-transplant]. The stopping rule for time-to-failure (defined as graft failure, gr 3-4 GHVD, or non-relapse death) will be implemented to avoid waiting 100 days (3 months) to evaluate each patient to perform safety monitoring. The design will be based on T with 3 months follow-up, applying the design of Thall et al. Possibly right-censored values of T for patients treated in the trial will be monitored continuously throughout the trial. The accrual rate is expected to be approximately 2 patients per month, with each patient's follow-up continued until all patients have been followed for 3 months from their date of first NK cell administration. The distributions of time to failure and RFS time will be estimated using the method of Kaplan and Meier. Exploratory analyses with immunophenotyping for NK cell chimerism and persistence will be done as feasible. Immune reconstitution studies or additional future research may be performed.

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.

Selective Expansion of NKG2C+ Adaptive NK Cells Using K562 Cells Expressing HLA-E.

Adaptive natural killer (NK) cells expressing self-specific inhibitory killer-cell immunoglobulin-like receptors (KIRs) can be expanded in vivo in response to human cytomegalovirus (HCMV) infection. Developing a method to preferentially expand this subset is essential for effective targeting of allogeneic cancer cells. A previous study developed an in vitro method to generate single KIR+ NK cells for enhanced targeting of the primary acute lymphoblastic leukemia cells; however, the expansion rate was quite low. Here, we present an effective expansion method using genetically modified K562-HLA-E feeder cells for long-term proliferation of adaptive NK cells displaying highly differentiated phenotype and comparable cytotoxicity, CD107a, and interferon-γ (IFN-γ) production. More importantly, our expansion method achieved more than a 10,000-fold expansion of adaptive NK cells after 6 weeks of culture, providing a high yield of alloreactive NK cells for cell therapy against cancer.

Donor natural killer cells trigger production of β-2-microglobulin to enhance post–bone marrow transplant immunity

AbstractAllogeneic hematopoietic transplantation is a powerful treatment for hematologic malignancies. Posttransplant immune incompetence exposes patients to disease relapse and infections. We previously demonstrated that donor alloreactive natural killer (NK) cells ablate recipient hematopoietic targets, including leukemia. Here, in murine models, we show that infusion of donor alloreactive NK cells triggers recipient dendritic cells (DCs) to synthesize β-2-microglobulin (B2M) that elicits the release of c-KIT ligand and interleukin-7 that greatly accelerate posttransplant immune reconstitution. An identical chain of events was reproduced by infusing supernatants of alloreactive NK/DC cocultures. Similarly, human alloreactive NK cells triggered human DCs to synthesize B2M that induced interleukin-7 production by thymic epithelial cells and thereby supported thymocyte cellularity in vitro. Chromatography fractionation of murine and human alloreactive NK/DC coculture supernatants identified a protein with molecular weight and isoelectric point of B2M, and mass spectrometry identified amino acid sequences specific of B2M. Anti-B2M antibody depletion of NK/DC coculture supernatants abrogated their immune-rebuilding effect. B2M knock-out mice were unable to undergo accelerated immune reconstitution, but infusion of (wild-type) NK/DC coculture supernatants restored their ability to undergo accelerated immune reconstitution. Similarly, silencing the B2M gene in human DCs, before coculture with alloreactive NK cells, prevented the increase in thymocyte cellularity in vitro. Finally, human recombinant B2M increased thymocyte cellularity in a thymic epithelial cells/thymocyte culture system. Our studies uncover a novel therapeutic principle for treating posttransplant immune incompetence and suggest that, upon its translation to the clinic, patients may benefit from adoptive transfer of large numbers of cytokine-activated, ex vivo–expanded donor alloreactive NK cells.

Natural Killer Cell Alloreactivity Predicted By Killer Cell Immunoglobulin-Like Receptor Ligand Mismatch Does Not Impact Engraftment in Umbilical Cord Blood and Haploidentical Stem Cell Transplantation

Natural killer cell alloreactivity is determined by killer cell immunoglobulin-like receptor (KIR) ligands in donor and recipient pairs. A small, single institution study suggested that the risk of primary graft failure after cord blood hematopoietic cell transplantation (CBT) can be predicted by host-versus-graft (HvG)–directed natural killer cell alloreactivity. In the haploidentical transplantation (Haplo HCT) cohort, graft failures were observed only in graft-versus-host (GvH) KIR ligand mismatched pairs. A subsequent study was designed to explore the association between HvG and GvH KIR ligand mismatching and engraftment in both CBT and Haplo HCT using the large, multicenter transplant population of the Center for International Blood and Transplant Research database. Nine hundred single CBT (sCBT), 954 double CBT (dCBT), and 671 Haplo HCT performed between 2008 and 2017 for acute leukemias and myelodysplastic syndrome were examined. Several models of KIR-L interactions were analyzed by multiple regression analyses for their association with engraftment, overall survival (OS), and transplant-related mortality (TRM). In sCBT, although HvG or bidirectional KIR ligand mismatch (KIR-L-MM) was initially associated with higher TRM in the first 6 months after transplantation, this effect was nullified after 6 months such that long-term survival was not different compared to GvH KIR-L-MM or KIR-L matched (KIR-L-M) pairs. There was no significant difference in neutrophil and platelet engraftment. In dCBT, no significant differences were seen in engraftment, OS and TRM. In the Haplo cohort there was faster platelet recovery in the GvH KIR-L-MM/KIR-L-M pairs versus HvG KIR-L-MM or bidirectional mismatch (HR 1.23, P= .0116). There was no significant association with OS, TRM, or neutrophil engraftment. In this large registry study, KIR-L mismatching did not significantly impact engraftment, TRM, or survival in CBT and Haplo HCT, although an association with platelet engraftment in Haplo HCT was demonstrated.

Feeder Cells at the Interface of Natural Killer Cell Activation, Expansion and Gene Editing.

Genome engineered natural killer (NK) cell therapies are emerging as a promising cancer immunotherapy platform with potential advantages and remaining uncertainties. Feeder cells induce activation and proliferation of NK cells via cell surface receptor-ligand interactions, supported by cytokines. Feeder cell expanded NK cell products have supported several NK cell adoptive transfer clinical trials over the past decade. Genome engineered NK cell therapies, including CAR-NK cells, seek to combine innate and alloreactive NK cell anti-tumor activity with antigen specific targeting or additional modifications aimed at improving NK cell persistence, homing or effector function. The profound activating and expansion stimulus provided by feeder cells is integral to current applications of clinical-scale genome engineering approaches in donor-derived, primary NK cells. Herein we explore the complex interactions that exist between feeder cells and both viral and emerging non-viral genome editing technologies in NK cell engineering. We focus on two established clinical-grade feeder systems; Epstein-Barr virus transformed lymphoblastoid cell lines and genetically engineered K562.mbIL21.4-1BBL feeder cells.

Long-Term Outcome After Adoptive Immunotherapy With Natural Killer Cells: Alloreactive NK Cell Dose Still Matters.

Recently, many reports were published supporting the clinical use of adoptively transferred natural killer (NK) cells as a therapeutic tool against cancer, including acute myeloid leukemia (AML). Our group demonstrated promising clinical response using adoptive immunotherapy with donor-derived alloreactive KIR-ligand-mismatched NK cells in AML patients. Moreover, the antileukemic effect was correlated with the dose of infused alloreactive NK cells (“functional NK cell dose”). Herein, we update the results of our previous study on a cohort of adult AML patients (median age at enrollment 64) in first morphological complete remission (CR), not eligible for allogeneic stem cell transplantation. After an extended median follow-up of 55.5 months, 8/16 evaluable patients (50%) are still off-therapy and alive disease-free. Overall survival (OS) and disease-free survival (DFS) are related with the dose of infused alloreactive NK cells (≥2 × 105/kg).

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.

Recipient HLA-B Leader Genotype, and Its Relationship to Total KIR Missing Ligand, Informs Relapse and Survival Following Haploidentical Transplantation Using Post-Transplant Cyclophosphamide

▪In addition to donor T cells, natural killer (NK) cells are proposed to play a significant role in the graft-versus-leukemia (GVL) effect following haploidentical donor transplantation (HIDT). Following HIDT, donor NK cells express activating (NKG2C) receptors (Ciurea, Leukemia 2021), whose ligand is the non-classical human leukocyte antigen, HLA-E. For surface expression, HLA-E requires binding of leader peptides derived from class I HLA molecules. The rs1050458C/T dimorphism at position -21 of exon 1 of HLA-B gives rise to leader peptides with either methionine (M) or threonine (T) at the second residue of the processed leader peptide. M-containing HLA-B leader peptides promote higher HLA-E expression than T-leaders, potentially favoring more robust natural NK cell recognition of HLA-E-expressing tumor cells. Alternatively, donor NK cells can be activated through inhibitory killer cell immunoglobulin-like receptors (iKIR: 2DL1, 2DL23, 3DL1, 3DL2), if they fail to engage a corresponding class I HLA ligand on recipient leukemia cells (missing ligand (ML) paradigm). We hypothesized that M-containing B-leaders (either MM or MT genotype), and potentially its association with KIR ML, may inform relapse and survival after HIDT using post-transplant cyclophosphamide (PTCy). A total of 322 patients with acute leukemia, MDS, lymphoma, CLL or CML, receiving a HIDT-PTCy from a single institution were evaluated with a median follow-up time of 45 months [range 6, 184]. Baseline characteristics included a median age of 50 years [19, 80], 47% non-white, HCT-CI ≥3 in 50%, PBSC graft in 80%, and myeloablative conditioning in 49%. M-containing B-leader genotype (either MM or MT) was seen in 42% and 44% of recipients and donors, respectively. The B-leader on the unshared donor-recipient haplotypes was matched (either T or M) in 61% of transplants. ML for iKIR 2DL1, 2DL23, 3DL1, and 3DL2 was noted in 29%, 20%, 24% and 72% respectively. Total ML was 0, 1, 2 and 3 in 10%, 43%, 40% and 7% respectively. In univariate analysis, an M-containing recipient B-leader genotype [R(M+)] improved OS and DFS compared to a recipient TT genotype [R(M-)] (75 vs. 53%, p<0.001; 66 vs. 47%, p<0.001), which was primarily due to a lower risk of relapse/progression (24% vs. 38%, p=0.012). In regard to total iKIR ML, the presence of 2-3 ML was associated with better overall (OS) and disease-free (DFS) survival compared with 0-1 (67% vs. 57%, p=0.08; 62% vs. 48%, p=0.019), which was due to lower relapse/progression (25% vs. 38%, p=0.015). There was no association of either recipient B-leader or total iKIR ML with the incidence of NRM, acute or chronic GVHD. When recipient B-leader and ML were combined, the detrimental effect of a R(M-) genotype was seen exclusively in patients with ML 0-1 (see figure). DFS for R(M-)/ML(0-1), R(M+)/ML(0-1), R(M-)/ML(2-3), R(M+)/ML(2-3) was 36%, 68%, 65% and 60%, respectively (p<0.001). Corresponding relapse/progression rates were 49%, 22%, 26% and 25%, respectively (P<0.001). In multivariate analysis, controlling for patient age/sex/race, disease risk index, donor age, HLA-DR mm, HLA-DP npmm and year of transplantation, both recipient B leader and total iKIR ML were independently associated with DFS (HR 0.57, p=0.002 and HR 0.67, p=0.026) and relapse/progression (HR 0.55, p=0.006 and HR 0.55, p=0.007). In summary, a recipient M-containing B-leader genotype (MM or MT) reduces relapse and improves survival following HIDT-PTCy, presumably through HLA-E-mediated effector cell engagement. Furthermore, the negative consequences of low total ML for iKIR, in terms of increased relapse risk and lower DFS, can be mitigated when a R(M+) B-leader is present. In addition to the clinical importance of this novel finding for optimizing HIDT-PTCy, it further supports the role of alloreactive donor NK cells for optimal GVL in this context. [Display omitted] DisclosuresSolh: Partner Therapeutics: Research Funding; Jazz Pharmaceuticals: Consultancy; ADCT Therapeutics: Consultancy, Research Funding; BMS: Consultancy.

Association of Ikir-Mismatching and Donor Akirs with Better Outcomes in Haploidentical Hematopoietic Stem Cell Transplantation for Acute Myeloid Leukemia

Objectives: Natural killer (NK) cells are the first donor-derived lymphocytes to be reconstituted after transplantation and play a critical role in improving transplantation outcomes. Their surface receptor, killer cell immunoglobulin like receptor (KIR), can trigger the alloreactivity of NK cells and have been shown to be protective for acute and chronic graft-versus-host disease (aGVHD, cGVHD) while retaining graft-versus-leukemia (GVL) effect. However, different results have been reported about KIR matching models and KIR alleles based on patient, donor and transplant characteristics, resulting in significant controversy about the best donor selection strategy. Here, we investigated the potential influence of KIR matching and KIR alleles on GVHD prophylaxis, overall survival (OS) and relapse rate (RR) of haploidentical hematopoietic stem cell transplantation (haplo-HSCT) in acute myeloid leukemia (AML) patients. Methods：Data from 79 patients with AML treated with haplo-HSCT between May 2015 and May 2021 in the transplantation unit of the Fujian Medical University Union Hospital were retrospectively analyzed. The cohort included 49 male patients (62.0%) and 30 female (38.0%), with a median age of 25 years (1-68 years). KIR genotyping was performed by the PCR-SSO method. The amplicons were quantified on the Luminex 200 flow analyzer and analyzed using the Quick-Type for Lifecodes software for generating KIR data. HLA-A and -B alleles were typed by polymerase chain reaction using sequence-specific primers (PCR-SSP) (TBG, Taipei, Taiwan). HLA-C genotyping was determined by reverse hybridization with sequence specific oligonucleotides probes (rSSO) Line probe assay. Cox proportional hazards model and Kaplan-Meier survival curves were used for analysis. Results：At the time of transplantation, 49 cases (62.0%) were at CR1, while 30 (38.0%) were not. aGVHD occurred in 16 patients (20.3%) and recurrence arose in 10 patients (12.7%), 4 (5.1%) cases cGVHD was observed. After adjusting for age, disease-risk, disease-status, HLA-match, donor gender, conditioning regimen intensity and type of post-grafting GVHD prophylaxis, Cox regression analysis revealed that both KIR ligand-ligand mismatching (KLM) and KIR receptor-ligand matching (RLM) was associated with an decreased risk of aGVHD and relapse compared to KIR ligand-ligand matching and receptor-ligand matching group, respectively (aGVHD: KLM: p=0.047, RLM: p＜0.001; RR: KLM: p=0.049, RLM: p=0.017). Furthermore, RLM shows more accurate in prediction of relapse and aGVHD compared with KLM in both aGVHD and relapse. (aGHVD: p=0.009; RR: p=0.039). After taking activating KIR (aKIR) into consideration to compensate the defect of only inhibitory KIR (iKIR) dominant model, we found that patients with more donor activating KIRs can reach the lower incidence of aGVHD and relapse, and the benefit can gradually increase parallel with the increase in donor activating KIRs. (aGVHD：p=0.019；RR：p=0.037). Patients with both receptor-ligand mismatch and the most donor aKIRs can reach the lowest relapse, lowest incidence of aGVHD and best overall survival (OS). Conclusions: KIR-mismatch, both KLM and RLM significantly reduced the risk of aGVHD and relapse after halpo-HSCT in AML patients and RLM show more superiority in the prediction of HSCT outcome. The synergistic effects of receptor-ligand mismatch and more donor aKIRs can provide a better clinically applicable donor selection strategy to improve haplo-HSCT outcome in AML patients. DisclosuresHu: Astellas Pharma, Inc.: Research Funding.

Influence of Killer Immunoglobulin-Like Receptors and Somatic Mutations on Transplant Outcomes in Acute Myeloid Leukemia

Natural killer (NK) cells are regulated by killer immunoglobulin-like receptor (KIR) interactions with human leukocyte antigen class I ligands. Various models of NK cell alloreactivity have been associated with outcomes after allogeneic hematopoietic cell transplant (alloHCT), but results have varied widely. We hypothesized that somatic mutations in acute myeloid leukemia (AML) in the context of KIR profiles may further refine their association with transplant outcomes. In this single-center, retrospective, observational study, 81 AML patients who underwent matched-related donor alloHCT were included. Post-HCT outcomes were assessed based on mutational status and KIR profiles with the Kaplan-Meier method and log-rank test. On multivariable analysis those with any somatic mutations and C1/C2 heterozygosity had less acute graft-versus-host disease (GvHD) (hazard ratio [HR], 0.32; 95% confidence interval [CI], 0.14-0.75; P = .009), more relapse (HR, 3.02; 95% CI, 1.30-7.01; P = .010), inferior relapse-free survival (RFS; HR, 2.22; 95% CI, 1.17-4.20; P = .014), and overall survival (OS; HR, 2.21; 95% CI, 1.17-4.20; P = .015), whereas those with a missing KIR ligand had superior RFS (HR, 0.53; 95% CI, 0.30-0.94; P = .031). The presence of a somatic mutation and donor haplotype A was also associated with less acute GvHD (HR, 0.38; 95% CI, 0.16-0.92; P = .032), more relapse (HR, 2.72; 95% CI, 1.13-6.52; P = .025), inferior RFS (HR, 2.11; 95% CI, 1.07-4.14; P = .030), and OS (HR, 2.20; 95% CI, 1.11-4.38; P = .024). Enhanced NK cell alloreactivity from more KIR activating signals (donor B haplotype) and fewer inhibitory signals (recipient missing KIR ligand or C1 or C2 homozygosity) may help mitigate the adverse prognosis associated with some AML somatic mutations. These results may have implications for improving patient risk stratification prior to transplant and optimizing donor selection.

Relevance of Polymorphic KIR and HLA Class I Genes in NK-Cell-Based Immunotherapies for Adult Leukemic Patients

Simple SummaryImmunotherapies are promising approaches to curing different acute leukemias. Natural killer (NK) cells are lymphocytes that are efficient in the elimination of leukemic cells. NK-cell-based immunotherapies are particularly attractive, but the landscape of the heterogeneity of NK cells must be deciphered. This review provides an overview of the polymorphic KIR and HLA class I genes that modulate the NK cell repertoire and how these markers can improve the outcomes of patients with acute leukemia. A better knowledge of these genetic markers that are linked to NK cell subsets that are efficient against hematological diseases will optimize hematopoietic stem-cell donor selection and NK immunotherapy design.Since the mid-1990s, the biology and functions of natural killer (NK) cells have been deeply investigated in healthy individuals and in people with diseases. These effector cells play a particularly crucial role after allogeneic hematopoietic stem-cell transplantation (HSCT) through their graft-versus-leukemia (GvL) effect, which is mainly mediated through polymorphic killer-cell immunoglobulin-like receptors (KIRs) and their cognates, HLA class I ligands. In this review, we present how KIRs and HLA class I ligands modulate the structural formation and the functional education of NK cells. In particular, we decipher the current knowledge about the extent of KIR and HLA class I gene polymorphisms, as well as their expression, interaction, and functional impact on the KIR+ NK cell repertoire in a physiological context and in a leukemic context. In addition, we present the impact of NK cell alloreactivity on the outcomes of HSCT in adult patients with acute leukemia, as well as a description of genetic models of KIRs and NK cell reconstitution, with a focus on emergent T-cell-repleted haplo-identical HSCT using cyclosphosphamide post-grafting (haplo-PTCy). Then, we document how the immunogenetics of KIR/HLA and the immunobiology of NK cells could improve the relapse incidence after haplo-PTCy. Ultimately, we review the emerging NK-cell-based immunotherapies for leukemic patients in addition to HSCT.

Differentiation of natural killer cells from induced pluripotent stem cells under defined, serum- and feeder-free conditions

Background aimsTraditionally, natural killer (NK) cells are sourced from the peripheral blood of donors―a laborious and highly donor-specific process. Processes for generating NK cells from induced pluripotent stem cells (iPSCs) have demonstrated that it is possible to successfully generate renewable alloreactive NK cells that are not only functional in vivo but can also be genetically engineered for enhanced function. However, poor standardization and cumbersome differentiation procedures suggest that further improvements in the control of the differentiation process are necessary. MethodsHere the authors evaluated the potential of differentiating NK cells from centrally authenticated iPSCs under entirely chemically defined and serum-free conditions as well as their immunotherapeutic potential, after expansion in feeder-free media, against solid tumors targets. To address limitations of current differentiation approaches, the authors did not utilize feeder or stromal cell layers, TrypLE adaptation or peripheral blood during the differentiation process. The authors also evaluated the feasibility of utilizing centrally authenticated iPSC lines, thus circumventing protocol- and donor-induced variability associated with reprogramming approaches, and characterized these iPSC-NK cells in terms of cytotoxicity, cytokine production and degranulation potential against solid tumor cell lines and patient-derived targets. ResultsDifferentiation of iPSCs generated NK cells that were predominantly CD56+/CD16+/CD3− and expressed NK activation markers NKG2D, NKp30, NKp44, NKp46 and DNAM-1. These iPSC-NK cells mediated effector functions, including cytotoxicity, degranulation and IFN-γ production, in response to solid tumor targets, including patient-derived cancer cells, and could be cryopreserved and expanded in culture. ConclusionsThe ability to produce NK cells under defined conditions and the functional responses elicited by these iPSC-NK cells suggest that they could represent promising effectors in clinical adoptive transfer settings as a renewable source of donor-independent NK cells for immunotherapy of solid tumors.

HLA Class I Molecules as Immune Checkpoints for NK Cell Alloreactivity and Anti-Viral Immunity in Kidney Transplantation.

Natural killer (NK) cells are innate lymphocytes that can kill diseased- or virally-infected cells, mediate antibody dependent cytotoxicity and produce type I immune-associated cytokines upon activation. NK cells also contribute to the allo-immune response upon kidney transplantation either by promoting allograft rejection through lysis of cells of the transplanted organ or by promoting alloreactive T cells. In addition, they protect against viral infections upon transplantation which may be especially relevant in patients receiving high dose immune suppression. NK cell activation is tightly regulated through the integrated balance of signaling via inhibitory- and activating receptors. HLA class I molecules are critical regulators of NK cell activation through the interaction with inhibitory- as well as activating NK cell receptors, hence, HLA molecules act as critical immune checkpoints for NK cells. In the current review, we evaluate how NK cell alloreactivity and anti-viral immunity are regulated by NK cell receptors belonging to the KIR family and interacting with classical HLA class I molecules, or by NKG2A/C and LILRB1/KIR2DL4 engaging non-classical HLA-E or -G. In addition, we provide an overview of the methods to determine genetic variation in these receptors and their HLA ligands.