ALBERT

Description: How receptor acquisition correlates with the functional maturation of natural killer (NK) cells is poorly understood. We used quantitative real-time polymerase chain reaction (PCR) assays to compare NKG2 and killer immunoglobulin-like receptor (KIR) gene expression in NK cells from allogeneic transplant recipients and their donors. Marked differences were observed in the NK subsets of recipients who had 8-fold more CD56bright cells, diminished KIR expression (except 2DL4), and increased NKG2A. In normal blood not all CD56dim cells express KIR, and a novel subpopulation of cells committed to the NK-cell lineage was defined. These cells, which comprise 19.4% ± 2.8% of the CD56dim NK population in healthy donors, express the activating NKG2D and NKG2E receptors but no KIR or NKG2A. Although the CD56dim NKG2A− KIR− NK cells lack “at least one” inhibitory receptor for autologous MHC class I, they are not fully responsive, but rather functionally immature cells with poor cytotoxicity and IFN-γ production. Upon culture with IL-15 and a stromal cell line, CD56dim and CD56bright KIR− NK cells proliferate, express KIR, and develop cytotoxicity and cytokine-producing potential. These findings have implications for the function of NK cells reconstituting after transplantation and support a model for in vivo development in which CD56bright cells precede CD56dim cells.

Description: Organ transplantation is a life-saving procedure for patients. However, acute T-cell mediated graft rejection occurs in a significant quantity of recipients, which is a potentially life-threatening condition. Current diagnostic criteria for transplant rejection rely on invasive tissue biopsies and relatively non-specific clinical features. We hypothesized that nanoparticles could be developed that specifically bind to T-cells. Furthermore, we postulated that these nanoparticles could be detected non-invasively by a Superconducting Quantum Interference Device (SQUID). Nanoparticles were conjugated through either amino or carboxy termini to either anti-CD2 or anti-CD3 antibodies. All three cell types were found to bind exclusively to specifically targeted antibody-tagged nanoparticles as identified through light microscopy and SQUID detection. Using receptor site densities determined by flow cytometry and the magnetic moment per nanoparticle results, SQUID is capable of determining the number of labeled cells in a sample. Our results indicate that T-cells bind approximately 1×104 nanoparticles per cell. A SQUID sensor array also allows for in vitro localization of discrete sources of labeled T-cells. Using a phantom as a surrogate for organ or soft tissue, we also demonstrate that discrete signals of 105 T- cells can be detected at 10 cm from the SQUID detector. These results demonstrate the potential utility of antibody-tagged nanoparticles as a contrast agent and SQUID as a noninvasive and sensitive detection device in patients who may be undergoing T-cell mediated graft rejection. In addition, the extreme sensitivity of this technique may provide not only a means of early, non-invasive detection of impending T-cell mediated rejection, but also an approach to titrating chemotherapeutics for treating rejection.

Description: The importance of killer immunoglobulin-like receptors (KIR) in determining clinical outcome after hematopoietic cell transplantation (HCT) remains controversial. We analyzed 428 unrelated, T-cell replete transplants for acute myeloid leukemia (AML) facilitated by the National Marrow Donor Program between 1988 and 2003. Fifty seven KIR-ligand mismatched (thus HLA mismatched; KIR-Lmm/HLAmm) transplants were selected as cases. By matching on 15 clinical and demographic variables, KIR ligand matched, HLA mismatched (KIR-Lm/HLAmm; n = 162) and 10/10 HLA matched (HLAm; n = 209) comparison groups were selected. Using a validated single nucleotide polymorphism (SNP)-based MALDI-TOF assay, DNA samples were typed for 16 individual KIR genes which were used to estimate KIR haplotypes. About 1/3 of donors and recipients were A/A (lacking any activating KIR except 2DS4) and 2/3 possessed at least one B haplotype (B/X). As expected, 3 year survival was better in HLAm vs. HLAmm KIR-Lm or KIR-Lmm HCT (32%; p = .07 and 0.0004 respectively). However, survival was similar in both HLAmm groups (KIR-Lm 27% vs. KIR-Lmm 17%; p = .11). Importantly, transplants from donors with an activating B KIR haplotype had significantly better survival. Using a Cox regression that stratifies the baseline hazard according to sets of patients matched on important clinical variables, transplants using B KIR haplotype donors had a 42% reduced risk of mortality [B/X (vs. A/A) KIR haplotype donors (RR .58, 95% CI .44–.78; p = .0002]. Similarly, for disease-free survival (DFS), the risk of relapse and/or death decreased 43% [B/X (vs. A/A) RR .57, 95% CI .43–.76; p = .0001]. No differences were seen in risks of acute GVHD. The significant improvements in overall survival and DFS conferred by donor B KIR haplotype were seen in both the HLAm and KIR-Lm/HLAmm groups, but not in the KIR-Lmm/HLAmm group. The recipient KIR haplotype had no effect on outcomes, and the benefit of donor B KIR haplotype was equal regardless of recipient KIR haplotype (figure). Individual KIR genes were also analyzed and in Cox regression only donor 2DL2 and 2DS2 were favorable. As these genes are in strong linkage disequilibrium, the effects were similar (donor 2DL2 RR of death .70, 95% CI .54–.91; p = .008 and 2DS2 RR .66, 95% CI .50–.86; p = .002). 2DS2 defines a B KIR haplotype, confounding interpretation of these single gene effects. In summary, data from this analysis of the effect of KIR genes in unrelated T-replete HCT for AML suggest that using B KIR haplotype donors improves survival and DFS. Therefore, KIR genotyping could be incorporated into unrelated donor selection algorithms to identify B haplotype donors for patients with AML. Figure Figure

Description: In individuals with the plasma cell malignancy multiple myeloma, there is evidence that bone formation rates are reduced and that increased ostoeclastic bone resorption is associated with impaired osteoblast function. We investigated the osteogenic differentiation capacity of myeloma bone marrow mesenchymal progenitor cells (MPCs). We also examined the expression levels and activity of Runx2, the transcription factor required for osteogenesis. Bone marrow MPCs were cultured in osteogenic induction medium and assessed for bone alkaline phosphatase (bALP) expression (Sigma kit), induction of osteoblast specific genes like osteocalcin (RT-PCR) and mineralization by von Kossa staining. Immunoblot analysis and electrophoretic mobility shift assays (EMSA) were used to determine Runx2 expression and DNA-binding activity respectively. RT-PCR was used to sequence Runx2 and detect any mutations or deletions present within its domains. Transactivation ability of Runx2 was measured by its ability to activate osteocalcin promoter in transient transfection assays. Myeloma -derived MPCs showed reduced levels of bALP and osteocalcin transcript and a lower degree of mineralization, in osteogenic induction medium, as compared to that of the healthy donors. Immunoblot analysis and EMSA indicated equivalent Runx2 expression and DNA-binding capacity, respectively, in both healthy donor and myeloma-derived MPCs. Sequence analysis of Runx2 indentified a splice variant of Runx2 lacking exon 8 (Runx2D8) in myeloma patients with reduced transactivation ability. Co-transfection of the splice variant led to reduced transcriptional activity of the full-length Runx2. The reduced transactivation ability of spliced Runx2, as well as its suppressive action on the transactivation function of full-length Runx2, likely contributes to the defective osteogenesis clinically observed in myeloma pateints.

Description: The effector function of human NK cells is down-regulated via ligation of inhibitory receptors (killer immunoglobulin-like receptors [KIR] and NKG2A) that recognize self MHC. Although most NK cells express “at least one” self MHC receptor, the mechanism producing self-tolerance is not yet understood. In order to precisely evaluate receptor expression to better understand this mechanism we developed and validated quantitative, real time RT-PCR (Q-RT-PCR) assays to measure mRNA levels from individual NKG2 and KIR genes. Comparison of subpopulations of NK cells sorted from blood showed a high expression of NKG2A on CD56+bright cells but virtually no KIR expression except for KIR2DL4. To further define KIR expression patterns on more mature cells we sorted the CD56+dim NK cells into KIR+ and KIR− subpopulations using an antibody cocktail that recognizes 6 KIR genes. Comparing KIR gene expression by Q-RT-PCR in these subsets showed much higher transcript levels for the KIR genes included in the sorting cocktail in the KIR+ subpopulation than in the KIR− cells (median expression ratio of 221.3; n=43; P=

Description: NK cell KIR interactions are among the variables known to affect clinical outcomes including relapse, graft versus host disease (GVHD) and survival after HCT. We hypothesized that T cells in graft sources available for HCT may affect KIR recovery and the therapeutic potential of KIR alloreactivity. We studied KIR reconstitution (the percentage of KIR+ NK cells measured by flow cytometry) in blood collected from recipients at day +100 after T cell deplete (TCD-BMT) and unmanipulated (U-BMT) unrelated BM transplants. We found that KIR reconstitution was suppressed compared to the healthy donors, significantly more so after U-BMT transplants (donor: 48.42 ± 2.35% KIR+ NK cells versus recipient: 26.74 ± 1.94, n = 36; P 〈 .001) than after TCD-BMT transplants (donor: 53.34 ± 3.25% versus recipient: 42.68 ± 3.32%, n = 38; P = .017), with P = .001 between the recipient groups. Additionally, multivariate Cox proportional hazards models showed that improved KIR recovery independently correlated with improved survival and that higher NK cell IFN-γ production independently correlated with more frequent acute GVHD in that patient cohort. These data suggested that T cell number in the graft affects KIR reconstitution and transplant outcome. We next examined other sources of hematopoietic cells in which T cell function may be suppressed either by growth factor mobilization (sibling donor unmanipulated peripheral blood: SibU-PB) or the innate naivety of the T cells (umbilical cord blood: UCB). KIR+ NK reconstitution on recovering cells at day +100 after all HCT graft types was significantly less than that on normal donor cells (normals 55.33 ± 1.73%, n = 124; all P 〈 .0006). U-BMT recipients had significantly lower KIR+ NK recovery (27.31 ± 2.06%, n = 36 vs. SibU-PB: 37.58 ± 3.29%, n = 29; TCD-BMT: 42.68 ± 3.32%, n = 38; or UCB, 37.99 ± 2.54%, n = 49) when compared to all other transplant types. The highest absolute T cell inoculum, found in SibU-PB, showed KIR reconstitution similar to that of TCD-BMT, which had the lowest T cell content (p=0.29), perhaps due to the lower alloreactivity of the Sib grafts and to the G-CSF-priming which preferentially mobilizes T cells with a suppressive phenotype. Similarly, KIR reconstitution was better after UCB compared to U-BMT (P = .0027), possibly due to the more permissive interactions with naive T cells. These results suggest that reduced T cell number after T cell depletion, suppressed T cells found after growth factor mobilization, or naive T cells present in UCB grafts enhance in vivo KIR reconstitution after allogeneic HCT when compared to unmanipulated marrow grafts. Such enhanced KIR reconstitution may have clinical consequences. Graft T cells may directly compete for cytokines and growth factors, or may be a surrogate marker for other transplant factors such as the development of GVHD and the requirement for intensive post-transplant immunosuppression. Understanding these interactions will allow judicious selection of hematopoietic cell source to select for enhanced KIR recovery. For example, among unrelated unmanipulated donor grafts, KIR+ NK recovery was significantly better using UCB than adult donors and further investigation may show that this is advantageous to improve clinical outcomes.

Description: Osteoporosis is common in adults after hematopoietic cell transplantation (HCT). The data on bone mineral density (BMD) in children after HCT are limited. The goals of this prospective study were to determine the incidence, timing, magnitude and possible predictors of bone loss in children following HCT. The study population included 49 patients (age 5–18 years) who were eligible to receive HCT at the University of Minnesota. The patients were evaluated at baseline, 100 days, 6 months, and 1 year after HCT. Lumbar BMD (BMDL) was assessed by dual-energy x-ray absorptiometry. The number of patients with osteopenia increased from 18% at baseline to 33% one year after HCT, and with osteoporosis from 16% to 19%. Mean areal BMDL z-score decreased from −0.56 to −1.1 by 6 months and at 1 year was −0.94, which was significant compared to standard normal distribution (p=0.004 and p=0.022, respectively). The absolute loss of bone mineral corresponded to 5.3% reduction in areal BMDL and 4.8% reduction in volumetric BMDL. The level of bone-specific alkaline phosphatase decreased by 30% by day 100 (p=0.009), followed by recovery toward baseline by 6 months. The level of osteocalcin 〉6.5 ng/mL at day 100 predicted recovery from the initial bone loss by 1 year. A reduction in BMDL at 6 months correlated with a cumulative dose of glucocorticoids. In conclusion, this study demonstrates that bone loss is common in children after HCT and is primarily due to suppression of bone formation. Further studies are necessary to validate osteocalcin as a predictive biomarker.

Description: Survival for patients with acute myeloid leukemia (AML) is limited by treatment-related mortality (TRM) and relapse after unrelated donor (URD) hematopoietic cell transplantation (HCT). Natural killer (NK)–cell alloreactivity, determined by donor killer-cell immunoglobulin-like receptors (KIRs) and recipient HLA, correlates with successful HCT for AML. Hypothesizing that donor KIR genotype (A/A: 2 A KIR haplotypes; B/x: at least 1 B haplotype) would affect outcomes, we genotyped donors and recipients from 209 HLA-matched and 239 mismatched T-replete URD transplantations for AML. Three-year overall survival was significantly higher after transplantation from a KIR B/x donor (31% [95% CI: 26-36] vs 20% [95% CI: 13-27]; P = .007). Multivariate analysis demonstrated a 30% improvement in the relative risk of relapse-free survival with B/x donors compared with A/A donors (RR: 0.70 [95% CI: 0.55-0.88]; P = .002). B/x donors were associated with a higher incidence of chronic graft-versus-host disease (GVHD; RR: 1.51 [95% CI: 1.01-2.18]; P = .03), but not of acute GVHD, relapse, or TRM. This analysis demonstrates that unrelated donors with KIR B haplotypes confer significant survival benefit to patients undergoing T-replete HCT for AML. KIR genotyping of prospective donors, in addition to HLA typing, should be performed to identify HLA-matched donors with B KIR haplotypes.

Description: Although unrelated hematopoietic cell transplantation (HCT) is curative for many hematologic malignancies, complications and relapse remain challenging obstacles. Natural killer (NK) cells, which recover quickly after transplantation, produce cytokines and express killer immunoglobulin-like receptors (KIRs) that regulate their cytotoxicity. Some clinical trials based on a KIR ligand mismatch strategy are associated with less relapse and increased survival, but results are mixed. We hypothesized that T cells in the graft may affect NK cell function and KIR expression after unrelated transplantation and that these differences correlate with clinical outcomes. NK cell function was evaluated using 77 paired samples from the National Marrow Donor Program Research Repository. Recipient NK cells at 100 days after both unmanipulated bone marrow (UBM) and T-cell depleted (TCD) transplants were compared with NK cells from their healthy donors. NK cells expressed fewer KIRs and produced more interferon γ (IFN-γ) after UBM compared to TCD transplants. Multivariate models showed that increased NK cell IFN-γ production correlated with more acute graft-versus-host disease (GVHD), and decreased KIR expression correlated with inferior survival. These results support the notion that T cells in the graft affect NK cell reconstitution in vivo. Understanding these mechanisms may result in strategies to improve clinical outcomes from unrelated HCT.