NK Clones Research Articles

Whole Exome Sequencing Analysis in Chronic Lymphoproliferative Disorder of NK Cells (CLPD-NK) Patients Fails to Detect Significant Viral Load

Background. Chronic Lymphoproliferative Disorder of Natural Killer cells (CLPD-NK) is a rare disease characterized by chronic NK cell lymphocytosis, typically equipped with a restricted pattern of activating killer immunoglobulin like receptors. Several lines of evidence point to a putative role of viral agents in the pathogenetic mechanisms accounting for NK cell proliferation. A role of Epstein Barr Virus (EBV) has been claimed in the aggressive form of NK cell proliferation [i.e. Aggressive NK Cell Leukemia (Kawa Ha et al, JCI 1989)]. At variance, in the indolent CLPD-NK, EBV DNA was seldom detected by PCR (Loughran TP Jr et al, Blood 1993), whereas we provided serologic evidence for previously experienced viral infections in the majority of CLPD-NK patients (Zambello R et al, Leukemia 1995). However, the role of infectious agents in the pathogenesis of chronic antigen stimulation of NK cells in CLPD-NK patients is still elusive. Methods. Several studies showed that whole exome sequencing (WES), as well as RNA-seq, can identify pathogens, particularly viruses, in cancer clones enabling the definition of possible integration sites in the host genome or their presence as episomal forms. In the present study, we searched viral sequences in WES data of a cohort of 10 CLPD-NK patients. High-depth (average 51 M reads) WES data of highly purified (>97% purity) leukemic NK clone and autologous granulocytes, as control, from CLPD-NK patients were analyzed for pathogen discovery. Three different methods for virus detection were used: i) Centrifuge (Kim et al, Genome Research 2016), a metagenomic tool performing a wide search against GenBank viral and bacterial sequences, which has been recently used to detect EBV load in ANKL and NK T cell Lymphoma (NKTCL) tumor clones (Dufva et al. Nat Comm 2018); ii) VirusSeq (Chen et al., Bioinformatics 2013), a software to identify viruses and eventually their integration sites, grounding on its custom and relatively wide viral sequence database; and iii) ViFi (Nguyen et al. Nucleic Acids Res, 2018), which can find viral integration in the human genome, with an analysis focused on a narrow set of viruses (EBV, HPV, HBV) with well-established oncogenic potential. Results. First, as positive control two datasets were used: RNA-seq data of an HBV positive sample with known site of integration (provided by VirusSeq) and WES data of three NKTCL tumor and control clones with high, moderate or low EBV load in the tumor samples previously detected by Dufva et al. (Dufva O et al, Nat Comm 2018), publicly available at NCBI SRA. HBV presence was detected in the positive sample by all the three methods and our reanalysis of NKTCL samples with Centrifuge confirmed published results, detecting EBV in all tumoral samples and in none of the matched control samples. Next, analysis of samples from the 10 CLPD-NK patients with the three methods did not identify a significant viral load neither in tumor nor in control specimens. Neither VirusSeq nor ViFi found viruses in CLPD-NK samples, whereas, according to Centrifuge analysis, the human disease virus with the highest load in most patients was Varicella Zoster (Human alphaherpesvirus 3) but with low read count and, most relevant, without specificity for the cancer clone. Conclusions. Our very thorough WES analysis did not identify a significant and meaningful viral load in CLPD-NK samples, neither in leukemic clones nor in control cell population. Although not ruling out that a viral infection might play a role as starting event for inciting NK cell proliferation, our data do not support a role of viral infections in maintaining the proliferation in CLPD-NK. Disclosures Zambello: Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

Clonal Tracking of the Geographic Distribution of Hematopoiesis in Nonhuman Primates Provides New Insights into HSPC Migration and Differentiation

Hematopoietic stem and progenitor cells (HSPC) primarily reside in bone marrow (BM) niches, but are also found in peripheral blood (PB) in small numbers. Large numbers of HSPC can be pharmacologically mobilized from the BM into the PB and home back to BM niches following transplantation. We used the rhesus macaque to study the process of hematopoiesis in both space and time following autologous transplantation, as a model with great relevance to humans. We labeled individual CD34+ HSPC and their progeny with genetic "barcodes" via lentiviral transduction with a very high diversity barcode library, allowing tracking of the output from individual HSPC clones in vivo following autologous transplantation, in a quantitative and sensitive manner (Wu et al., 2014). In this study we investigated the pattern of HSPC clonal output in various anatomic niches, sampling left (L) vs right (R) iliac crest BM, PB and lymph node (LN) over time post ablative autologous transplantation, for all major hematopoietic lineages and CD34+ HSPC.We tracked thousands of HSPC clones in 6 rhesus macaques from 3.5 months (m) to up to 18.5m post transplantation. L and R iliac crest BM and PB were serially collected and CD34+ HSPC along with CD3+CD4+ T, CD3+CD8+ T, CD20+ B, CD14+ monocytes (Mo), CD33+ granulocytes (Gr), and two NK cell subsets (CD3-CD20-CD14-CD16+/ or CD56+) were purified. In all animals, there was marked geographic segregation of CD34+ HSPC for at least 6m post-transplant, with individual clones localized only to the L or R side but not both (Pearson correlation at 3.5-6m: r=0.019±0.08 for CD34+ L vs R, n=6), despite rapid expansion of CD34+ HSPC during early engraftment. This result suggests that during this phase of recovery, HSPC spread contiguously in the BM, and there is little geographic "mixing" via egress into and re-entry from the PB. With time between 7m and 18.5m post-transplant, the distribution of CD34+ HSPC became more homogeneous, with clones detected on both L and R (r=0.52±0.22 at 12-18.5m, n=3). The geographic restriction of clones at earlier time points suggests that release and homing may be dependent on local contiguous niche occupancy status, with migration only occurring following hematopoietic recovery. We next examined the clonal distribution of the more mature lineage-committed cells in the L vs R BM and in PB. Gr, Mo and B cells were produced locally in BM, with the clonal pattern of each lineage matching the CD34+ cells collected from the same side BM time point through 6m (r>0.80 for each lineage vs same side CD34+ cells), and distinct from same lineages (r<0.16 for each lineage in L vs in R BM) and CD34+ cells on the other side (r<-0.01 for each lineage vs other side CD34+ cells). CD16+CD56-/dim NK clones were completely shared between PB and both L and R BM, suggesting they were not produced locally in BM, but instead in other sites with homogenous lodging or homing back to BM. Most surprisingly, we found population of CD3+CD8+ T cells that appeared to be produced locally, with barcodes matching the CD34+ HSPC at the same location, suggesting a novel T cell development pathway within the BM for this subset during early hematopoietic reconstitution. In contrast, CD3+CD4+ BM T cells had similar clonal constitution on the L vs R, and matched the PB, suggesting they had re-circulated back to the BM following maturation elsewhere, such as the thymus. T, B and NK cells from two LNs obtained simultaneously were also analyzed. Clonal contributions to T, B, and NK cells from L vs R LNs were highly correlated (r=0.95, r=0.88, and r=0.89 respectively). The clonal composition of T or B cells in LN were shared with circulating T or B cells (r=0.88, r=0.85 respectively), while LN NK cells (which are primarily CD56+/CD16-) shared barcodes with circulating CD16-CD56+ NK (r=0.81), not with PB CD16+CD56- cells(r=0.26), suggesting a non-precursor/progeny relationship.Our model for the first time documents the dynamics of HSPC geographic distribution and migration in primates following transplantation, findings with direct clinical relevance, and provides new insights into hematopoietic lineage development, including a potential novel T cell development pathway in the BM. Our findings may also help explain the extremely patchy distribution of hematopoiesis in humans following transplantation or in the setting of marrow failure or aging, and suggest that analysis of individual BM samples may not fully reflect ongoing global hematopoiesis. DisclosuresNo relevant conflicts of interest to declare.

Emergence of a STAT3 mutated NK clone in LGL leukemia

Large granular lymphocyte (LGL) leukemia is a chronic clonal lymphoproliferative disorder. Here, a T-LGL leukemia patient developed NK-LGL leukemia with residual leukemic T-LGL. TCRVβ usage and CDR3 sequence drifts were observed with disease progression. A STAT3 S614R mutation was identified in NK but not T-cells in the mixed leukemic stage. Multiple, non-dominant T-cell clones with distinct STAT3 mutations were present throughout. Our results suggest that T and NK-LGL leukemia may share common pathogenesis mechanisms and that STAT3 mutation alone is insufficient to bring about clonal expansion. Mutational and immunological monitoring may provide diagnostic and therapeutic significance in LGL leukemia.

Long-Term Hematopoietic Clonal Stability Tracked Using Molecular Barcoding in Non-Human Primates

By transplanting rhesus macaques with autologous CD34+ hematopoietic stem and progenitor cells (HSPCs) labeled with lentivirally delivered high diversity oligonucleotide barcodes, we are able to track complex clonal contributions to hematopoiesis over time and across lineages via low cycle PCR and high-throughput sequencing of the inserted barcode. We reported on patterns of reconstitution for the first 9 months following engraftment in a recent publication (Wu et al, Cell Stem Cell, 2014). For the first 1 to 3 months short lived unilineage progenitors predominated, followed by increased clonal diversity and the emergence of first shared myeloid (My)/B clones, and then My/B/T clones. A major novel finding was a distinct ontogeny for NK cells, with the major CD16+ blood NK population showing non-overlapping origin with My/B and T lineages through 6-9 months. We now report on hematopoietic clonal patterns up to 23 months post-transplant in four animals.In all four animals, a group of high contributing NK clones contributed minimally to other lineages until 9 to 12 months, at which point these unilineage NK clones extinguished and a new generation of NK high contributors appeared. In two animals, these emerging clones continued to be either unilineage or highly biased towards the NK lineage, while in two other animals, they began to contribute highly to all lineages, a discrepancy which possibly derives from differences between animals in barcode marking level. Over the 17 and 23 months since transplant tracked in the two highly marked animals, 5.6±1.4 and 9±0.9 out of the top 10 NK contributing clones, respectively, were highly biased towards NK and away from other lineages, as determined by unsupervised hierarchical clustering of correlations between high contributing clones. Though shared My/B/T progenitors emerged after 6 months in both animals, 3.1±1.3 and 6.4±0.9 out of the T cell top 10 contributing clones were highly biased towards T cells and away from all other lineages up to 17 and 23 months. In contrast to the highly biased NK contributors, these T biased clones did not extinguish in the time surveyed so far, and more frequently were detected at low levels in the My/B lineages. The degree of bias of these high contributing clones towards the NK and T lineages was steady over time, suggesting that strong clonal bias is stable. The top 10 clones in NK contributed a significantly greater fraction of hematopoiesis (20±4% and 38±10%) than the top 10 clones in the T lineage (15±2% and 13±2%) in both animals (p=0.01, p<0.0001).In all lineages at all time points from 1 to 23 months, a small subset (14±3%) of clones detected at each individual time point contributed at least 50% of barcoded hematopoiesis. Polyclonality increased following initial hematopoietic reconstitution but started to fall around 6 months, reflecting the increased contribution of certain high contributing, totipotent clones. These clones did not extinguish after beginning to contribute, and have characteristics of long-term repopulating HSCs.We observed novel instability in the lineage bias of some individual HSPC contributions. For instance, an early totipotent clone which was a top 10 producer of granulocytes halted B cell contribution almost completely at 4.5 months, before regaining it by 5 months later and becoming the highest contributing clone in the entire animal. The high degree of bias towards and away from certain lineages observed in many long term repopulating HSCs is not necessarily in violation of hypothesized progenitor hierarchies, but it indicates the presence of poorly understood nuances in clonal control. In addition to the long-term dominant unbiased HSCs whose hematopoietic contribution increased over time, we noticed a cohort of clones biased towards a combined myeloid/B cell lineage.The varied clonal patterns observed between animals could result from individual differences in transplant progenitor composition or dose, or degree of recipient endogenous HSPC depletion. Understanding the lifespan and population dynamics of repopulating clones in macaques after transplantation is relevant for optimizing human hematopoietic stem cell transplants and also provides an approach for identifying progenitor populations, inferring mechanisms of cell fate control, and calculating rates of differentiation. DisclosuresNo relevant conflicts of interest to declare.

T Large Granular Lymphocytes Leukemia (T-LGLL) and Natural Killer Chronic Lymphoproliferative Disorder (NK-CLPD): Two Diseases With a Common Etiopathogenetic Mechanism?

IntroductionThe large granular lymphocytes (LGL) disorders are characterized by expansions of T or NK cells with cytotoxic activity. WHO classification considers these disorders as separate entities, referred to as T-LGL Leukemia (T-LGLL) or NK-Chronic Lymphoproliferative Disease (NK-CLPD). The marker of clonality is the rearrangement of TCR in T-LGLL and a restricted pattern of Killer Immunoglobulin-like Receptor (KIR) expression in NK-CLPD. Although these disorders are characterized by the expansion of different cells types, compelling evidence supports the hypothesis that a common pathogenetic mechanism would be involved in both these disorders. Accordingly, the transcription factor STAT3 has been found mutated in a consistent percentage in both T-LGLL and NK-CLPD, suggesting a similar molecular dysregulation. Here, we evaluated whether clonal T and KIR restricted NK cell populations were detectable in patients with LGL, to support the hypothesis that a common pathogenetic mechanism takes place in both T-LGLL and NK-CLPD disorders. MethodsWe enrolled 370 therapy-free patients with LGL, 133 of which were available for serial measurements over 2 to 15 years. Peripheral blood sample and data collection from patients were performed at clinically indicated testing after informed consent, according to the protocols approved by the Institutional Review Board of the Experimentation Ethic Committee of Padua and in accordance with the Declaration of Helsinki. Based on World Health Organization guidelines, the diagnosis of T-LGLL was made according to the presence of monoclonal TCRγ-chain rearrangement, flow cytometric evidence of an abnormal CTL population characterized by expression of CD2, CD3, TCRαβ (or γδ), CD4, CD5dim, CD8, CD16/56 and CD57, TCR Vβ expansions (when detectable) with negativity of CD28. The diagnosis of NK-CLPD was made according to flow cytometric evidence of a KIR restricted CD16+/CD3- NK population. For both disorders, a LGL count by peripheral blood smear of > 2,000 LGL/μL (this is not a critical criteria, since patients who met all other criteria but with an LGL count < 2,000 cells/μL were included) and persistence of these conditions for more than 6 months were confirmed. ResultsBased on World Health Organization guidelines for T-LGLL and NK-CLPD diagnosis, we classified our patients in 3 group: Group 1 (n=247), with a T clonal expansion characteristic of T-LGLL, Group 2 (n=79), with a KIR restricted NK expansion characteristic of NK-CLPD, and Group 3 (n=44), characterized by patients in whom the presence of both KIR restricted NK population and T clonal expansion were present. Moreover, during the follow-up, we observed that 15 patients underwent a clonal switch of pathologic clones as follows. Five patients switched from NK-CLPD to T-LGLL. Seven patients developed additional clone(s) during follow-up, leading to the coexistence of both NK and T clonal populations; 3 patients demonstrated either a gradual emergence of a dominant T or NK clone from an oligoclonal background observed at diagnosis. ConclusionsThese results support the hypothesis that an antigenic pressure present in LGL disorders sustains the proliferation of both NK and T clones, triggering in some circumstances the gradual switch from a disorder to the other one. This observation challenges the classification of these disorders as two separate entities, questioning the accuracy of T-LGLL and NK-CLPD classification made by WHO in 2008. Disclosures:No relevant conflicts of interest to declare.

NK Cell Tolerance of Self-Specific Activating Receptor KIR2DS1 in Individuals with Cognate HLA-C2 Ligand

NK cells are regulated by inhibiting and activating cell surface receptors. Most inhibitory receptors recognize MHC class I Ags and protect healthy cells from NK cell-mediated autoaggression. However, certain activating receptors, including the human activating killer cell Ig-like receptor (KIR) 2DS1, also recognize MHC class I. This fact raises the question of how NK cells expressing such activating receptors are tolerized to host tissues. We investigated whether the presence of HLA-C2, the cognate ligand for 2DS1, induces tolerance in 2DS1-expressing NK cells. Anti-HLA-C2 activity could be detected in vitro in some 2DS1 positive NK clones irrespective of the presence or absence of HLA-C2 ligand in the donor. The frequency of anti-HLA-C2 reactivity was high in donors homozygous for HLA-C1. Surprisingly, no significant difference was seen in the frequency of anti-HLA-C2 cytotoxicity in donors heterozygous for HLA-C2 and donors without HLA-C2 ligand. However, donors homozygous for HLA-C2, compared with all other donors, had significantly reduced frequency of anti-HLA-C2 reactive clones. The 2DS1 positive clones that express inhibitory KIR for self-HLA class I were commonly noncytotoxic, and anti-HLA-C2 cytotoxicity was nearly exclusively restricted to 2DS1 single positive clones lacking inhibitory KIR. 2DS1 single positive NK clones with anti-HLA-C2 reactivity were also present posttransplantation in HLA-C2 positive recipients of hematopoietic stem cell transplants from 2DS1 positive donors. These results demonstrate that many NK cells with anti-HLA-C2 reactivity are present in HLA-C1 homozygous and heterozygous donors with 2DS1. In contrast, 2DS1 positive clones from HLA-C2 homozygous donors are frequently tolerant to HLA-C2.

Expression and function of CD300 in NK cells.

The killing activity of NK cells is regulated by signals derived from inhibitory and activating NK cell receptors, including the CD300 family of proteins. CD300a was reported to be expressed on all NK cells and to deliver an inhibitory signal upon binding to a yet unknown ligand/s. The CD300a protein contains four ITIMs and is highly homologous to CD300c. Little is known about the function and distribution of these two receptors and the identity of their ligand/s. In this article, we show that CD300a is indeed an inhibitory receptor expressed by human NK cells, but surprisingly, we show that not all NK clones are inhibited in a CD300a-dependent manner. We demonstrate, using a panel of 13 new anti-CD300a and CD300c Abs that we generated, that CD300a and CD300c are indistinguishable on the surface of NK cells. Using mutational-analysis survey, we show that tyrosine 267 located in the third ITIM motif of the CD300a protein is important for the inhibitory function of CD300a.

KIR2DS1 positive Natural Killer cells from individuals expressing cognate HLA ligand are tolerant. (89.58)

Abstract Inhibitory KIR-HLA interactions are critical for NK tolerance to self. KIR2DS1 is the only activating counterpart of inhibitory KIRs to have ligand specificity for HLA class I, recognizing HLA-C group 2. Since KIR and HLA genes segregate independently, individuals that carry KIR2DS1 may have HLA-C antigens of the HLA-KIR ligand group C2. We here report that 2DS1 positive NK cells from such individuals are tolerant to such self HLA antigens. We demonstrate that the mechanism for tolerance is different for individuals homozygous or heterozygous for the HLA ligand. NK clones have been generated from KIR2DS1-positive and HLA-C2 homozygous, HLA-C1/C2 heterozygous or HLA-C1 homozygous individuals. Their response against HLA-KIR ligand group C2-expressing targets and their KIR repertoire have been assessed. NK clones expressing KIR2DS1 display anti-HLA-C2 alloreactivity but are tolerant to HLA-C2 if this is a self antigen. This hyporesponsiveness to C2 in C2/C2 homozygous donors is also observed for NK clones lacking 2DL1. In turn, the co-expression of inhibitory KIRs with ligand specificity for self HLA class I including C2 is an important mechanism for tolerance in HLA-C1/C2 heterozygous individuals. This is the first demonstration of activating NK receptor tolerance to cognate self MHC ligand. NK clones expressing only the activating receptor and not the homologous inhibitory receptor are readily identified. Remarkably, NK clones with self HLA ligand specificity are not deleted.

Selecting Haploidentical Transplant Donors for Deploying Optimal NK Cell Allotherapy.

Abstract 511Donor-versus-recipient NK cell alloreactivity has been established as a key therapeutic element in HLA haplotype mismatched hematopoietic transplants in adult AML (Ruggeri et al. Blood 1999; Science 2002; Blood 2007; Stern et al., Blood 2008) and paediatric ALL (Pende et al. Blood 2009). It is effected by donor NK cells which express inhibitory KIRs for self class I KIR ligands (HLA-C1, C2, Bw4). In KIR ligand mismatched recipients, they sense missing expression of donor KIR ligand(s) and mediate alloreactions. Recent studies have proposed refinements to this mechanism and have emphasized the role of donor activating KIR variants in the control of leukemia relapse after haploidentical (Pende et al. Blood 2008) and unrelated (Cooley et al. Blood 2009) hematopoietic cell transplantation. ∼25% of Caucasians are homozygous for A haplotypes which contain inhibitory KIR genes and the KIR2DS4 activating KIR (non-functional in 2/3 of individuals). 75% of Caucasians are either heterozygous or homozygous for B haplotypes which carry not only inhibitory KIRs but also various combinations of activating KIRs (KIR2DS1-2-3-5 and KIR3DS1). Our extensive functional assessments of donor NK clone repertoires from representative sets of A (homozygous) or B (homozygous and heterozygous) haplotype donors, involving functional analyses of >5,000 NK clones from 70 donors, revealed that frequencies of alloreactive NK cell clones did not differ significantly in A vs B haplotype donors, that NK alloreactive repertoires in B haplotype positive individuals were largely composed of clones which did not express activating KIR(s) and, finally, that activating KIR-negative clones killed as efficiently as the positive. In contrast, dramatic differences emerged when inflammatory cytokine production was assessed in alloreactive NK clones with possessed or did not possess activating KIR variants. Upon incubation with KIR ligand-mismatched LPS or Aspergillus or CMV antigen-treated DCs, NK clones expressing activating KIR receptors produced several-fold more IFN-gamma and TNF-alfa than NK clones not expressing activating KIRs. The clinical role of donor activating KIR genetics was evaluated in 86 haploidentical transplants for AML. 49 recipients were transplanted from NK alloreactive (KIR ligand-mismatched) donors (12 with group A KIR gene haplotypes vs 37 with B haplotypes) and 37 recipients from non-NK alloreactive (KIR ligand-matched) donors (8 with group A KIR gene haplotypes vs 29 with B haplotypes). The remarkable GvL effect of NK alloreactive transplants (Ruggeri et al. Blood 2007) was unaffected by donor A vs B KIR gene haplotypes. In contrast, in transplants from NK alloreactive donors, presence of group B haplotype KIR genes in the donors was associated with reduced incidence of TRM (largely infection-related) (B vs A haplotypes: 20% vs 67% TRM, p<0.01). In multivariate analyses it was the only significant variable predicting protection from TRM (RR: 0.24; 95% CI:0.14-0.42; p<0.01). When the number of activating KIR genes in the donor was taken into account, donors carrying ≥ 3 activating KIR genes provided significant protection from TRM and significantly better EFS compared with A haplotype donors (TRM: 12% vs 67%, p<0.003) (EFS: 71% vs 33%, p=0.02). In multivariate analysis, transplantation from alloreactive donors carrying ≥3 group B haplotype activating KIR genes was the only variable predicting protection from TRM (RR: 0.36; 95% CI: 0.25-0.54; p<0.01) and tended to improve EFS (RR: 0.64; 95% CI: 0.38-1.07; p<0.1). We conclude that NK alloreactive, KIR ligand-mismatched donors who possess activating KIRs may confer protection against infections through their enhanced NK cell cytokine secretion upon interaction with recipient pathogen-infected DCs. Disclosures:No relevant conflicts of interest to declare.

IL-12-Dependent Inducible Expression of the CD94/NKG2A Inhibitory Receptor Regulates CD94/NKG2C+ NK Cell Function

The inhibitory CD94/NKG2A and activating CD94/NKG2C killer lectin-like receptors specific for HLA-E have been reported to be selectively expressed by discrete NK and T cell subsets. In the present study, minor proportions of NK and T cells coexpressing both CD94/NKG2A and CD94/NKG2C were found in fresh peripheral blood from adult blood donors. Moreover, CD94/NKG2A surface expression was transiently detected upon in vitro stimulation of CD94/NKG2C+ NK cells in the presence of irradiated allogeneic PBMC or rIL-12. A similar effect was observed upon coculture of NKG2C+ NK clones with human CMV-infected autologous dendritic cell cultures, and it was prevented by an anti-IL-12 mAb. NKG2A inhibited the cytolytic activity of NKG2C+ NK clones upon engagement either by a specific mAb or upon interaction with a transfectant of the HLA class I-deficient 721.221 cell line expressing HLA-E. These data indicate that beyond its constitutive expression by an NK cell subset, NKG2A may be also transiently displayed by CD94/NKG2C+ NK cells under the influence of IL-12, providing a potential negative regulatory feedback mechanism.

Donor's NK Cells May Influence the Engraftment in Pediatrics Patients after T-Cell Depleted Haploidentical Stem Cell Transplant for Thalassemia

In haploidentical hematopoietic transplantation, donor-versus-recipient NK cell alloreactivity derives from a mismatch between donor NK clones bearing inhibitory Killer Cell Ig-like Receptors (KIRs) for self HLA class I molecules and their HLA class I ligands (KIR ligands) on recipient cells. The mechanism whereby alloreactive NK cells exert their benefits in transplantation has been elucidated. The infusion of alloreactive NK cellsablates recipient T cells which reject the graft, andablates recipient dendritic cells (DCs) which trigger GvHD, thus protecting from GvHD (Ruggeri et al., Science 2002).NK cell alloreactivity also boosts very rapid rebuilding of donor adaptive immunity to infections. In this study we analysed the potential role of NK cells after haploidentical transplant in b-thalassemia patients.T and B cell depletion was carried out with CD34+ coated magnetic microbeads and the CliniMACS device (Miltenyi Biotec©) from peripheral blood and bone marrow of donors (the mothers) and resulted in grafts consisting of stem cells and effector cells (NK cells, monocytes) with the addition of bone marrow mononuclear cells (BMMNCs 3 × 105/kg of the recipient). A total of 11 pediatric patients with b-thalassemia received T and B cell depleted transplants from their haploidentical mothers with a median number of 15 ×106 CD34 stem cells. To analyse the mechanisms involved in immunological reconstitution post transplant, we analysed T cell subsets by flow cytometry, particularly NK sets (CD3- CD56+, CD3− CD16+ and CD56+CD16+ NK cells) at day + 20 and + 60 post transplant.Day + 20 post transplant, the patients had significantly lower CD4+ T cells in comparison to the controls (1.9 ± 1.4% vs. 47.5 ± 6% respectively), whereas CD8+ T cells numbers did not statistically differ between patients and controls (24.2 ± 33.7% vs. 20 ± 7%). NK cells were among the first lymphocytes to repopulate the peripheral blood, and up to 70% of these cells were CD3-CD56+bright cells. Interestingly, a direct correlation has been observed between the percentages of CD56+CD16+ NK subset and the BM engraftment (in mean 71 ± 21% CD56+CD16+ in the four patients with full engraftment, 27 ± 28% in the three patients with a stable mixed chimerism after BM transplant (70–80% of donor cells) and 1.4 ± 1% in the four patients with rejection). In all the patients the origin of the NK subsets was from the mothers. Day + 60 post transplant an increase in the percentages of CD4+ T cells, naïve CD4+ cells and in thymic naïve Th cells were observed (3 ± 1.2%, 2.9 ± 2.1%, 2.7 ± 1%, respectively). CD8+ T cells were also increased (in mean 35 ± 27.5%), in parallel with the increase of the CD3-CD16+ NK cells (potent cytotoxic effector cells) especially in the patients with full engraftment (in mean 47 ± 20% vs. 28 ± 31% in mixed chimerism)NK CD56+bright cells develop more rapidly than other lymphocytes, but CD16+ NK cells (with cytotoxic potential) require more prolonged exposure to maturation factor (IL-2) in the bone marrow. Interestingly we observed higher percentages of NK subsets just twenty days post transplant in the patients with full engraftment respect the mixed chimerism and the rejection, suggesting a role of donor NK cells on improved engraftment and on prevention of the rejection with the attack of the host lympho-hematopoietic cells. These observations may suggest the importance of NK subsets analyses at the first time of the transplant as an useful parameter for the outcome of the transplant and/or the use of donor's alloreactive NK cells especially in haploidentical recipients.

The reactivity of Bw4+ HLA-B and HLA-A alleles with KIR3DL1: implications for patient and donor suitability for haploidentical stem cell transplantations

AbstractNatural killer (NK)–cell alloreactivity can be exploited in haploidentical hematopoietic stem cell transplantation (HSCT). NK cells from donors whose HLA type includes Bw4, a public epitope present on a subset of HLA-B alleles, can be alloreactive toward recipients whose cells lack Bw4. Serologically detectable epitopes related to Bw4 also exist on a subset of HLA-A alleles, but the interaction of these alleles with KIR3DL1 is controversial. We therefore undertook a systematic analysis of the ability of most common HLA-B alleles and HLA-A alleles with Bw4 serologic reactivity to protect target cells from lysis by KIR3DL1-dependent NK cells. All Bw4− HLA-B alleles failed to protect target cells from lysis. All Bw4+ HLA-B alleles with the exception of HLA-B*1301 and -B*1302 protected targets from lysis. HLA-A*2402 and HLA-A*3201 unequivocally protected target cells from lysis, whereas HLA-A*2501 and HLA-A*2301 provided only weak protection from lysis. KIR3DL1-dependent alloreactive NK clones were identified in donors with HLA-A*2402 but not in donors with HLA-B*1301 or -B*1302. These findings clarify the HLA types that donors and recipients need in haploidentical HSCT and other NK allotherapies in order to benefit from NK alloreactivity.

KIR2DS1-mediated activation overrides NKG2A-mediated inhibition in HLA-C C2-negative individuals

NK cell cytotoxicity is controlled through a balance of both activating and inhibitory signals. The HLA specificity of alloreactive NK cells has been previously shown to be controlled by inhibitory killer immunoglobulin-like receptors (KIRs). Alloreactive NK cells lyse targets that lack the HLA ligand for their inhibitory KIR. We have characterized in detail an alloreactive NK clone in which the specificity is controlled by an activating receptor, KIR2DS1. Only target cells expressing the HLA-C group 2 (C2) epitope were lysed by this clone and homozygous C2 targets were lysed more strongly than heterozygous C1/C2 targets. Anti-CD158a (KIR2DS1) blocked lysis of targets confirming KIR2DS1 was responsible. Although this NK clone expressed NKG2A, an inhibitory receptor whose ligand is HLA-E, targets with ligands for both KIR2DS1 and NKG2A were lysed by this clone indicating that the KIR2DS1-mediated activation signal overrides the NKG2A-mediated inhibitory signal. KIR2DS1 activated NK clones in polyclonally expanded NK cultures from a donor that lacked the C2 epitope accounted for approximately 1% of all NK cells. This study highlights a potential role for NK cells controlled by activating KIR in mediating NK alloreactivity.

The Activating KIR2DS2 Gene Influences NK Alloreactivity and NK Repertoire.

The hematopoietic stem cell transplant (HCT) donor KIR genotype has been correlated with disease-free survival in patients with acute myelogenous leukemia. The Killer Cell Immunoglobulin-like Receptor (KIR) gene family encodes highly homologous pairs of activating and inhibiting receptors, 2DL1–2DS1; 2DL2/3–2DS2; and 3DL1–3DS1. Inhibitory members are known to regulate NK cell function through interactions with HLA Class I antigens. The role of activating KIRs and their ligand specificity is, however, not well defined. The activating receptor, KIR2DS1, is known to bind the HLA-Cw C2 group antigens and we have recently demonstrated a role for this receptor in NK cell allorecognition. In contrast, KIR2DS2 does not bind HLA-Cw C1 group antigens, and a functional role of this receptor even in NK allorecognition has not been established. We now demonstrate, that presence of the activating KIR2DS2 gene in NK donors homozygous for the HLA-KIR ligand group C2 is associated with significant alloreactivity against C1 homozygous target cells (polyclonal NK cells, p=0.006; NK clones, p=0.001). This alloreactivity is mediated by “missing self” on the target and is dominated by “lack of C2 group on target”. The “missing C2” effect was absent, however, in C2 homozygous donors lacking 2DS2 (p=0.99). Only very rare cytotoxic NK clones expressing GL183 (2DL2/3, 2DS2) and with alloreactivity against C1 targets could be generated in vitro from 2DS2-positive, C2 homozygous donors. A majority of these rare GL183-positive clones did not demonstrate inhibitory function against the HLA class I deficient 721.221 transfected with Cw3 (C1-group), and GL183 cross-linking of the clones resulted in increased cytokine production. Thus, KIR2DS2 is an activating receptor in NK clones from C2 homozygous donors, but does not appear to recognize C1 ligand. We next investigated 2DS2 function in donors heterozygous for the C groups (i.e. C1/C2). Analysis of NK cell function in a 2DS2-positive, C1/C2 donor revealed a “missing HLA-KIR ligand” effect for the C2 group. Cytotoxicity by IL2-propagated, polyclonal NK cells and NK clones revealed allocytotoxicity against targets lacking the C2 group (p<0.001). In addition, a repertoire analysis on 138 NK clones generated from this donor revealed a marked increase in the number of EB6 (KIR2DL1/S1)-expressing NK clones (95%) compared to both the fresh (10%–50%) and the IL-2-expanded polyclonal NK repertoire (12%–60%). Additionally, all EB6-expressing clones from this donor were inhibited by the C2 ligand. Subsequent studies in freshly isolated NK cells following activating receptor cross-linking (NKp46, NKG2C, and CD16) or by alloantigen activation demonstrated that the functioning subset of NK cells in this donor predominantly expressed the EB6 receptor. Other inhibitory receptors (e.g. NKG2A, KIR3DL1, and KIR3DL2) did not contribute significantly to the functional subset of NK cells. Presence of 2DS2 in this donor was therefore associated with a “skewing” of the NK repertoire towards EB6 positivity, and dominated by functional NK cells that were inhibited by the “self” C2 ligand. Collectively, these studies provide the first evidence that activating KIR can influence the NK cell repertoire. Furthermore, our studies would indicate that presence of activating KIRs in HCT donors for recipients homozygous HLA-KIR ligands might induce post-transplantation graft versus host NK alloreactivity.

Hierarchy of the Human Natural Killer Cell Response Is Determined by Class and Quantity of Inhibitory Receptors for Self-HLA-B and HLA-C Ligands

The interaction of NK inhibitory killer Ig-like receptors (KIRs) with self-MHC class I molecules mediates NK tolerance to self while conferring functional competence. Through single-cell analysis of intracellular IFN-gamma production and NK clone cytotoxicity we evaluated the resting NK repertoire, analyzing the responsiveness of NK subgroups expressing discrete combinations of non-KIR and KIR class I-specific receptors. CD94:NKG2A and Ig-like transcript 2 (ILT2)-expressing cells have a modest response to class I-negative target cells, but NK cells expressing inhibitory KIRs to self-MHC class I ligands, both HLA-B and HLA-C ligands, achieve significantly higher effector capacity. There is a dose effect of KIR for self-MHC on effector capacity, but even in the most highly responsive NK cells expressing more than one inhibitory KIR for self-MHC the presentation of only one cognate MHC ligand is sufficient to abolish response. Among KIR(+) cells there is preferential expression for inhibitory KIR for self-MHC. The likelihood of KIR expression is influenced by whether other KIRs are already expressed on the same cell, supporting a model of serial acquisition of KIR expression. These findings define how inhibitory receptor and autologous HLA interactions impact single-cell function and demonstrate that the resting human NK repertoire is highly attuned but variegated in response. These findings have important implications for the resting NK response to viral pathogens and malignancy, for donor selection in allogeneic hemopoietic cell transplantation, and for models of NK tolerance.

KIR2DS1-Positive NK Cells Mediate Alloresponse against the C2 HLA-KIR Ligand Group In Vitro

The inhibitory 2DL1 and activating 2DS1 killer Ig-like receptors (KIR) both have shared ligand specificity for codon sequences in the C2 group HLA-Cw Ags. In this study, we have investigated NK cell activation by allogeneic target cells expressing different combinations of the HLA-KIR ligand groups C1, C2, and Bw4. We demonstrate that fresh NK cells as well as IL-2-propagated NK cells from 2DS1-positive donors that are homozygous for the C1 ligand group are activated in vitro by B lymphoblastoid cell lines expressing the C2 group. This response is, in part, due to the absence of C1 group recognition mediated by the inhibitory receptor 2DL2/3. This "missing self" alloresponse to C2, however, is rarely observed in NK cells from donors lacking 2DS1. Even in presence of 2DS1, the NK alloresponse is dramatically reduced in donors that have C2 group as "self." Analysis of selected NK clones that express 2DS1 mRNA and lack mRNA for 2DL1 demonstrates that activation by the C2 ligand and mAb cross-linking of 2DS1 in these clones induces IFN-gamma. Furthermore, this C2 group-induced activation is inhibited by Abs to both HLA class I and the receptor. Collectively, these studies demonstrate that NK cells from 2DS1-positive donors are activated by target cells that express the C2 group as an alloantigen. This leads to increased IFN-gamma-positive fresh NK cells and induces NK allocytotoxicity in IL2-propagated polyclonal NK cells and NK clones. This study also provides support for the concept that incompatibility for the HLA-KIR ligand groups C1, C2, and Bw4 dominates NK alloactivation in vitro.

The Human Cytomegalovirus MHC Class I Homolog UL18 Inhibits LIR-1+ but Activates LIR-1− NK Cells

The inhibitory leukocyte Ig-like receptor 1 (LIR-1, also known as ILT2, CD85j, or LILRB1) was identified by its high affinity for the human CMV (HCMV) MHC class I homolog gpUL18. The role of this LIR-1-gpUL18 interaction in modulating NK recognition during HCMV infection has previously not been clearly defined. In this study, LIR-1(+) NKL cell-mediated cytotoxicity was shown to be inhibited by transduction of targets with a replication-deficient adenovirus vector encoding UL18 (RAd-UL18). Fibroblasts infected with an HCMV UL18 mutant (DeltaUL18) also exhibited enhanced susceptibility to NKL killing relative to cells infected with the parental virus. In additional cytolysis assays, UL18-mediated protection was also evident in the context of adenovirus vector transduction and HCMV infection of autologous fibroblast targets using IFN-alpha-activated NK bulk cultures derived from a donor with a high frequency of LIR-1(+) NK cells. A single LIR-1(high) NK clone derived from this donor was inhibited by UL18, while 3 of 24 clones were activated. CD107 mobilization assays revealed that LIR-1(+) NK cells were consistently inhibited by UL18 in all tested donors, but this effect was often masked in the global response by UL18-mediated activation of a subset of LIR-1(-) NK cells. Although Ab-blocking experiments support UL18 inhibition being induced by a direct interaction with LIR-1, the UL18-mediated activation is LIR-1 independent.

Validation of Flow Cytometric Measurements of NK Cytotoxicity Against Chromium Release Assay for Use in Clinical Monitoring of Immune Function.

Chromium (51Cr) release assays have long been a standard technique for measuring the cytolytic activity of various immune effector cells. Although relatively easy to perform, these assays suffer from problems with sensitivity and require special radiation precautions limiting their routine use in clinical laboratories. Several flow cytometric techniques measuring cytotoxicity have been introduced and used as a measure of immune response. These assays are more rapid than the 51Cr based assay and have the added advantage of freeing the laboratory from the precautions required when working with radioactivity. Such testing has however largely been restricted to the research setting. In an attempt to extend these benefits to a clinical laboratory, we attempted to validate two commercially available flow cytometry kits (CyToxiLux Plus® & GranToxiLux®, OncoImmunin, Gathersburg, MD) measuring either caspase or granzyme B activity inside K562 target cells induced by interaction with NK cells. These assays were then compared with a standard NK cytotoxicity assay using 51Cr release technique. With K562 as a target, we parallel tested three CD56+CD3-KIR- NK clones; both the 51Cr and caspase based cytotoxicity values were highly positive. With WH autologous EBV cell line as target, we parallel tested five NK clones and five normal control donors as effectors. As expected, all cytotoxicity values for both assays were <10%. The caspase and granzyme assays were then compared and the degree of separation in the flow cytometric plots of the granzyme based assay were found to be marginally superior facilitating analysis of the data. The granzyme B assay therefore was chosen for investigation in the clinical samples. The granzyme and 51Cr assays were then compared both in 49 normal control donors and in 10 patients. There was a high level of concordance between the two assays. Comparing all results using linear correlation yielded a squared correlation (R2) value of 0.6878. However, given the wide range of NK activity in normal control donors the degree of positivity may not be clinically significant. Therefore, the data was examined in the context of a positive 51Cr response. Using a 2×2 table to exam the level of agreement, the kappa statistic was 0.63, indicating good agreement between the assays. When progressively fewer effector cells were added to a fixed number of target cells, the % cytotoxicity decreased from 39% to 0% with the flow cytometric assay and from 26% to 1% with the 51Cr assay. In the normal control donors the positive values ranged from 17% to 65% cytotoxicity using the flow assay compared with the previous range of 15% to 72% established with 51Cr. Patients having cytotoxicity values below 17% were deemed as having an unreactive or abnormal low result. One of the patients with few CD56+CD3- NK cells had consistently low cytotoxicity each time tested. For the patients who have either undergone allogeneic stem cell transplantation or have a known immunodeficiency, further clinical follow-up will serve to establish the clinical relevance of these measurements. These preliminary data suggest that the flow cytometric technique can be used in the clinical setting for immune monitoring.

Complete Remission of Immunocytoma without Graft Versus Host Disease Caused by Allo-HLA-DP Specific T Cells.

Mismatching for HLA-DPB1 in unrelated donor hematopoietic stem cell transplantation (URD-SCT) has been associated with a significant decreased risk of disease relapse, indicating that HLA-DP might be a target for a graft versus leukemia (GVL) effect in HLA-class II expressing hematological malignancies. To determine whether a specific GVL effect could be caused by allo-HLA-DP specific T cells, we analyzed the immune response in a patient with a refractory immunocytoma responding to donor lymphocyte infusion (DLI) after HLA-DP mismatched URD-SCT. Patient and donor were fully matched for HLA-A, -B, -C, -DR and -DQ, but differed for both HLA-DP alleles (donor HLA-DPB1*0402/0501; patient HLA-DPB*020102/0301). The patient received a T cell depleted URD-SCT after a non-myeloablative conditioning regimen, resulting in mixed chimerism (75% donor) without GVHD. Because of a hematological relapse, a single DLI was given 6 months after SCT, resulting in a profound anti-leukemic effect with only grade I GVHD, treated with topical corticosteroids. 6 weeks after DLI, malignant cells in peripheral blood (PB) had dropped from 72% to 47%. 7 weeks later, only 3% malignant cells were present, and after 4 months, complete remission and conversion to full donor chimerism in the absence of GVHD was observed. To determine whether allo-HLA-DP specific T cells were involved in the immune response, leukemia-reactive donor T cell clones were isolated from PB or bone marrow at different time points during the response to DLI. Patient derived T cells were overnight stimulated with irradiated leukemic cells harvested before transplantation, and clonal IFNγ producing T cells were sorted and expanded. 21 CD4+ T cell clones, 19 CD8+ T cell clones and 6 NK cell clones were tested for recognition of patient or donor derived cells as measured by IFNγ production and cytotoxic activity. The CD8+ or NK clones did not recognize patient leukemic cells. However, all 21 CD4+ clones produced INFγ in response to patient leukemic cells but not to donor cells. To determine whether these CD4+ T cell clones were capable of killing the leukemic cells, a CFSE based cytotoxicity assay was performed. 8 clones showed 30–90% lysis of the leukemic cell population. To further analyze the specificity of these CD4+ clones, blocking and panel studies were performed. Blocking with the HLA-DP specific mAb B7.21 abrogated IFNγ production by all clones, confirming HLA-DP restricted recognition. A panel study using 12 unrelated EBV-LCL expressing different HLA-DP alleles identified 18 clones specific for HLA-DPB1*0301, and 3 clones specific for HLA-DPB1*0201. To analyze the polyclonality of the immune response, the distribution of TCR Vβ chains was characterized by RT-PCR and sequence reactions. 7 different Vβs were found within the HLA-DPB1*0301 specific clones and 3 different Vβs within the HLA-DPB1*0201 specific clones. T cells using the same Vβ could be isolated at different time points during the clinical response, demonstrating the significance of this anti-HLA-DP response. In conclusion, we observed in a patient with an HLA-class II positive B cell malignancy a profound GVL effect without GVHD, caused by a polyclonal immune response comprising both T helper and cytotoxic CD4+ HLA-DP specific T cell clones directed against both HLA-DP alleles. These data indicate that in HLA-class II expressing hematological malignancies HLA-DP mismatched SCT may be preferable over a fully matched SCT making use of HLA-DP as a specific target for immunotherapy.

Alloreactivity of Natural Killer Cell Clones Against Childhood Precursor-B Lymphoblastic Leukemia Cells Is Determined by Differential KIR Expression.

Relapse after allogeneic stem cell transplantation of patients with acute lymphoblastic leukemia (ALL) remains a major problem. A beneficial impact of alloreactive NK cells has been reported for myeloid malignancies, but has been questionable for B-lineage-ALL. Here we show that the majority of NK cell clones from healthy donors could effectively lyse primary blasts from a representative childhood precurser-B-ALL and demonstrate the relevance of differential surface expression of KIRs (killer cell immunglobulin like receptors CD158a, CD158b and CD158e) for antileukemic alloreactivity. More than 79% of clones exerted specific lysis >40% against primary ALL blasts. Mismatch of effector/target HLA-C type (KIR-ligand incompatibility) did not correlate with antileukemic alloreactivity, although non-malignant lymphoblastoid cell lines were lysed according to KIR-ligand incompatibility. In contrast differential surface expression of the three major KIRs showed significant impact on the antileukemic activity against precursor-B-ALL blasts. NK clones with none of the three KIRs or a single KIR that recognized no ligand, were not inhibited by the targets and exerted higher lysis (p = <0.03) in comparison to NK clones with expression of one or more KIRs with a ligand on the ALL blasts. In conclusion the differential surface expression of KIRs is relevant to predict NK cell activity against childhood lymphoid leukemia.