Alloreactive Natural Killer Research Articles

Lack of Predictive Value of KIR-HLA Genotypes for Autologous Transplant Outcome for Multiple Myeloma.

In allogeneic hematopoietic cell transplantation (HCT), donor alloreactive natural killer (NK) cells can prevent leukemia relapse and prolong survival. Donor NK alloreactivity has been shown to be influenced by the killer Ig-like receptor (KIR) repertoire of the donor and the HLA class I KIR ligand phenotype of the recipient. In T-cell depleted HLA-identical sibling HCT, it has been shown that lack of recipient class I ligand for donor inhibitory KIR is associated with improved outcome in acute myelogenous leukemia. In the normal population, more than 60% of individuals lack class I ligand for one or more of their inhibitory KIR. Because autoreactive NK cells are known to occur in normal individuals with low frequency, we sought to determine if these same NK clones driven by autologous KIR-HLA interactions might expand and play a role following treatment involving autologous stem cell infusion for hematologic malignancy. 233 patients with multiple myeloma who underwent high-dose chemotherapy with autologous stem cell infusion were evaluated with HLA and KIR genotyping. KIR-HLA genotype combinations were then correlated with event-free survival (EFS) and overall survival (OS) following autologous HCT. Cox regression models were fit to examine the association of KIR-HLA combinations with the hazards of mortality and relapse. We found that there was no association between lack of KIR ligand for inhibitory KIR and improved EFS (p=0.69) or OS (p=0.36), nor was there any association between presence of specific activating KIRs and transplant outcome. Furthermore, the cumulative effect of more than one activating KIR did not affect outcome, including the specific combination of KIR2DS1 and KIR2DS2 (EFS p=0.86 and OS p=0.67), which have been previously reported to be associated with lower leukemia relapse in allogeneic HLA-identical HCT. The combination of activating KIR with their known or putative HLA ligands was also not associated with improvement in transplant outcome. Patients were also evaluated for KIR haplotype (AA versus AB and BB). There was no association between KIR haplotype and DFS (p=0.59) or OS (p=0.96). These data suggest that KIR genotypes and KIR-HLA genotype combinations are not associated with improved outcome in multiple myeloma patients undergoing autologous HCT. This implies that NK cells in autologous HCT do not significantly impact outcome, either because they are not expanded in the post-HCT period or because they have no activity against autologous multiple myeloma cells.

Mature T Cells in the Graft Improve NK Cells Reconstitution after Haploidentical Hematopoietic Stem Cell Transplantation.

We previously demonstrated that natural killer (NK) cells generated after haploidentical stem-cell transplantation (SCT) are blocked at an immature state characterized by phenotypic features and impaired functioning, a blockage that may affect transplantation outcome (Nguyen et al. Blood 2005). Hypothesizing that the absence of mature donor T cells in the graft may affect NK cell differentiation and function, we examined NK cells from 21 patients who received haploidentical SCT from relatives for advanced malignant hematopoietic disease and underwent either partial (pTCD) (CD3+ in the graft >1x105/Kg, mean: 6.9x105/Kg; n=11) or extensive (e-TCD) (CD3+ in the graft<1x105/Kg; mean: 0.35x105/Kg; n=10) T cell depletion and compared them with NK cells from their healthy donors. As previously described, compared with donor cells, recipient NK cells expressed lower levels of inhibitory KIR (in particular KIR2DL1 and KIR2DL2) and higher levels of CD94/NKG2A receptors after transplant (mean expression of CD94/NKG2A on recipient NK cell at 3 months post-transplant: 93.4%±7.2% versus 49.6%±10.9% on donor NK cells, p<0.0001), but these levels did not differ significantly between the pTCD and eTCD groups. However, the frequency of the immunoregulatory CD3−CD56bright NK subset was sharply lower in the pTCD than eTCD groups after transplantation (25.0%±9.6% versus 53.3%±18.0 at 3 months; p<0.001). The level of NKp30 receptors on NK cells was also higher after pTCD than eTCD transplantation (70.3%±7.1% versus 58.0%±6.5%, p=0.013) and that of pTCD patients resembled the donor NK repertoire. NK cytotoxicity against primary haplomismatched AML blasts was significantly more pronounced after pTCD than eTCD transplants (29.0%±8.9% specific lysis versus 6.7%±4.1% at a ratio Effector/Target (E/T):20/1, p=0.002), although still lower than in donor NK cells (mean specific lysis of donor NK cells from both groups against AML blasts: 43.5%±13% at a ratio E/T: 20/1). This more mature phenotypic and functional profile of NK cells after pTCD transplant was clinically associated with a lower rate of relapse and superior survival (1/11 relapse, 3/11 patients alive in complete remission at 11, 10 and 3 years) than in eTCD group (8/10 relapse; no patient alive at 1 year). These results support a model in which mature donor T cells in the graft may play a key role, in vivo, in NK cell differentiation by improving NK cell maturation and cytolytic function against leukemic blasts. They point to the dilemma of haploidentical hematopoietic SCT in leukemic patients: on the one hand, extensive T-cell depletion is associated with a risk of fatal leukemia relapse due to the loss of the GvL effect T cells, which can not be replaced by immature NK cells; on the other hand, partial T-cell depletion might increase the risk of GvHD but also improves the GvL effect mediated by NK cells. New treatments infusing mature haploidentical NK cells in leukemic patients should be used to test the efficiency of NK alloreactivity.

The Activating KIR Genes 2DS1 and 2DS2 Regulate NK Alloreactivity In Vitro.

The role of Natural Killer (NK) cells in host protection against viral infection and malignant transformation has been well described. NK cells may also lead to a reduction in post-transplant relapse and improved survival in hematopoietic stem cell transplantation (HSCT) for acute myelogenous leukemia (AML). It has been hypothesized that the genotype for the inhibiting killer immunoglobulin-like receptor (KIR) of the hematopoietic stem cell donor in combination with the HLA class I genotype of the recipient could control NK alloreactivity leading to a reduction in post-transplant complications. The KIR gene family encodes however both activating and inhibiting receptors. Here we test the hypothesis that activating KIRs with ligand specificity for HLA class I may contribute to alloreactivity, and potentially could be a genetic factor of significance in allogeneic HSCT. We tested this hypothesis in studies of two pairs of inhibiting and activating KIRs with highly homologous codon sequences in the extracellular domain, namely KIR2DL2/3-KIR2DS2 and KIR2DL1-KIR2DS1. Both the inhibitory 2DL1 and activating 2DS1 have ligand specificity for HLA-Cw group 2, and 2DL2 and 2DL3, have ligand specificity for HLA-Cw group 1, while the activating 2DS2 does not bind in vitro to C1 group. Using an EBV-transformed B-lymphoblastoid cell line (EBV-BLCL) target cell panel homozygous for HLA Class I alleles, we found that NK cells from donors with KIR haplotypes lacking KIR2DS1 or 2DS2 were not cytotoxic to allogeneic EBV-BLCL, independent of the target HLA class I genotype. Polyclonal NK cells obtained from KIR2DS1 positive and C1 group positive donors mediated NK cytotoxicity against C2 positive targets. In contrast, NK cells from KIR2DS1 positive, C2 group homozygous donors displayed minimal cytotoxicity against the C2 group targets (p<0.01). NK clones generated from 2DS1 positive, C2-group negative individuals were cytotoxic to C2-group target cells, while such NK clones could not be obtained from individuals positive for 2DS1 and cognate ligands. Similar findings were made for the relationship between 2DS2, 2DL2/3 and cognate ligand C1 group. Both polyclonal IL-2 propagated NK cells and NK clones from individuals positive for 2DS2 and homozygous for C2 group displayed specific cytotoxicity against C1 positive target cells. The cytotoxicity of 2DS2 positive, C1 group positive NK cells against the C1 positive BLCLs was minimal (p<0.01). These studies demonstrate that 2DS1 and 2DS2 are activating receptors that can induce an alloantigen response. We also present a model for combinations of KIR and HLA genotypes in which the allogeneic function of KIR2DS1 and 2DS2 is consistently seen in donor NK cells. Activating KIR may therefore play a role in allogeneic HSCT, and could contribute to the balance between activating and inhibiting signals for NK cells in HLA-Cw incompatible donor-recipient combinations. Activating KIR interactions with cognate ligand could potentially also play a role in the innate immune response. In the normal host, the increased affinity of the inhibiting KIR isoforms for HLA class I may prevent auto-reactivity, while the activating isoforms may only function in an HLA restricted pattern in context of specific pathogens or transformed cells. It is possible that the low affinity activating KIR may require additional co-stimulating signals that are up-regulated during cellular stress.

Strain-dependent expression of four structurally related rat Ly49 receptors; correlation with NK gene complex haplotype and NK alloreactivity

Natural killer (NK) cells from certain rat strains promptly kill MHC allogeneic lymphocytes in vivo, a rejection phenomenon termed allogeneic lymphocyte cytotoxicity (ALC). ALC can be reproduced in vitro, and is preferentially mediated by a subset of NK cells expressing the Ly49 stimulatory receptor 3 (Ly49s3) in PVG strain rats. Functional studies have suggested that Ly49s3 triggers NK cell alloreactivity, but its importance relative to other Ly49 receptors has not been investigated. In this study, we have characterized three rat Ly49 receptors with close sequence similarity to Ly49s3 in the extracellular region, i.e., Ly49s4, Ly49 inhibitory receptor 3 (Ly49i3), and Ly49i4. Similar to Ly49s3, Ly49s4 mediated cellular activation while Ly49i4 inhibited NK cytolytic function. Ly49s4, -i3, and -i4 all reacted with a previously described anti-Ly49s3 monoclonal antibody (mAb) (DAR13), but not a novel mAb (STOK6), which was shown to be specific for Ly49s3. Expression of these Ly49 receptors varied markedly between inbred strains, in patterns related to their NK gene complex (NKC) haplotype, and ability to mediate ALC. Three major groups of NKC haplotypes could be discerned by restriction fragment length polymorphism analysis. Ly49s3 was present in strains from one of the groups, which corresponded with the "high" ALC responders. Ly49s3 surface expression was also markedly reduced in the presence of its putative MHC class Ib ligand(s) in MHC congenic strains. These data support the notion that Ly49s3 functions as a triggering MHC receptor both in vitro and in vivo. MHC ligands for the other three Ly49 receptors remain to be determined.

Killer cell Ig‐like receptors ligand‐mismatched, alloreactive natural killer cells lyse primary solid tumors

Donor alloreactive natural killer (NK) cells have a potent antileukemic effect in haploidentical stem cell transplantation. Whether alloreactive NK cells are able to specifically kill fresh tumor cells from primary solid tumors was analyzed. NK cells were purified from healthy donors for the expression of inhibitory killer cell immunoglobulin (Ig)-like receptors (KIRs), ex vivo expanded, and used as effector cells. Their cytotoxic effect on tumor cells freshly obtained from surgical specimens was assessed by means of a single-cell cytotoxic assay (SCCA). Tumor cells from 1 ovarian, 1 gastric, 3 colon, and 4 renal cell cancers were analyzed and found susceptible to alloreactive NK cell killing (>20% lysis at an effector cell to target cell [E:T] ratio of 10:1 for tumor cells not expressing at least 1 human lymphocyte antigen [HLA] class I KIR-ligand group). Remarkably, NK cells that recognized specific HLA-C group mismatches were able to kill HLA-C KIR ligand-mismatched tumor cells, whereas no lysis of target cells occurred with KIR ligand-matched tumor targets. Alloreactive NK-cell mediated antitumor effects might provide useful insights for designing new cell therapy approaches against solid tumors.

NK Cells Mediate Costimulation Blockade-Resistant Rejection of Allogeneic Stem Cells During Nonmyeloablative Transplantation

Although T-cell CD28/CD40 costimulation blockade represents a powerful mechanism to promote immune tolerance during murine allotransplantation, it has not yet been successfully translated to clinical transplantation. We determined the impact of natural killer (NK) cells on costimulation blockade-resistant rejection of donor bone marrow. We found that NK cells represent a potent barrier to engraftment: host NK depletion led to increased donor stem cell survival, increased mixed hematopoietic chimerism and to engraftment of low doses of donor marrow (1 x 10(8)/kg) that were otherwise rejected. To understand the mechanisms of NK alloreactivity, we employed an in vivo NK-specific cytotoxicity assay. We found that an increased proportion of target cells were killed between days 2 and 8 after cell transfer, and that NK killing of parental targets was inducible: NK cells preprimed with allotargets were more efficient at their elimination upon reexposure. Finally, both transplant and in vivo NK-killing models were used to determine the contribution of LFA-1 to NK alloreactivity. Blockade of LFA-1 led to decreased NK-mediated killing, and increased alloengraftment. These results identify NK alloreactivity as an integral component to costimulation blockade-resistant rejection, and suggest that its inhibition may represent an important target in the clinical translation of tolerance-induction transplantation.

Killer Cell Immunoglobulin-like Receptor (KIR) Analysis in Adult Korean Patients with Acute Myeloid Leukemia

Background: The prevalent natural killer (NK) cells induce alloreaction against leukemic cells during post-transplant. NK cell alloreactivity depends on the compatibility of killer cell immunoglobulin-like receptors (KIR) epitopes for graft-versus-host disease. Genotypic expressions of inhibitory or activating KIR in patients with acute myelogenous leukemia (AML) and their HLA-matched sibling donors, as a model for Korean KIR haplotype diversity and NK alloreactivity, were investigated. Methods: Ninety-two patients in complete remission and their 76 HLA-matched sibling donors were enrolled in this study. All the patients were scheduled to receive allogeneic hematopoietic stem cell transplantations (HSCT). KIR PCR-SSP typing was performed for 19 different kinds of KIR genes and pseudogenes. The PCR data representing the KIR genotypes from both the patients and donors were compared. Results: We found 43 Korean KIR haplotypes. Thirty-three variable haplotypes for the AML patients, in addition to 25 haplotypes for the normal HSCT donors, were demonstrated. Of note, the expressions of specific genes such as 2DL2 (P=0.026), 2DS2 (P=0.042), and 2DS4 (P=0.037) revealed remarkable differences between the patients and the normal donors. Korean HLA-identical sibling pairs showed 38% KIR matches in terms of the gene content and allelic polymorphism. Although the KIR gene content was the same between the patients and the donors, 40% of those matched pairs of patients and donors showed allelic polymorphism, specifically in the context of 2DL5 and 2DS4 genes. Conclusion: These results indicate that the expressions of donor inhibitory and activating repertoire of KIR genotypes, even in the HLA-matched sibling setting, are unique parameters to be considered when we perform allogeneic sibling HSCT.

Negative Impact of KIR-Ligand Mismatch on Transplant-Related Mortality (TRM) in Umbilical Cord Blood Transplant (UCBT) Recipients.

Umbilical cord blood (UCB) is frequently considered as a suitable alternate source of hematopoietic stem cells (HSC) for both pediatric and adult patients who require HSC transplant for treatment of high-risk or relapsed hematologic malignancy. As mismatched killer Ig-like receptor ligands (KIR-L MM) has been associated with anti-host alloreactive natural killer (NK) cell activity and reduced risk of acute myeloid leukemia relapse in recipients of T-cell depleted haploidentical HSC, we hypothesized that GVL after UCBT may also be mediated by NK alloreactivity. We therefore assessed the effect of KIR-L MM in 243 recipients of UCB transplanted at the University of Minnesota between 1998 and 2004 for whom HLA-A, -B, -C and DRB1 typing was available for both patient and UCB unit(s). Median age, weight, and follow-up were 27 yrs (range, 0.2–69), 64.6 kg (range, 3.8–120.2), and 1.1 yr (range, 0.5–6.6), respectively. For recipients of double UCBT (n =106), we analyzed the KIR-L assignment of the engrafting unit only. KIR-L MM in the GVH direction was established using the algorithm of Ruggeri and Velardi and was found in 70 (29%) donor-recipient pairs. Probability of 2-year survival was 54% (95%CI: 42–66) vs. 47% (95%CI: 38–56) (p=0.88) in KIR-L MM vs. matched pairs, respectively. Intensity of the conditioning regimen was the only independent predictor of survival, with a RR of death 1.54 (95%CI: 1.07–2.22, p=0.02) for those who received a non-myeloablative conditioning. For the whole group, incidence of relapse at 1-yr was 17% (95%CI: 8–26) vs. 31% (95%CI: 24–38) (p=0.12); for myeloid malignancy patients, incidence of relapse at 1 year was 13% (95%CI:2–24) vs. 34% (95%CI:22–46)(p=0.10). Intensity of the conditioning regimen was the only independent predictor of relapse, with a RR 2.09 (95%CI: 1.31–3.35, p<0.01) for those who received a non-myeloablative conditioning. Incidence of graft failure and grade II–IV acute GVHD was 11% (95%CI: 3–18) vs. 10% (95%CI: 6–15) (p= 0.97) and 50% (95%CI: 37–63) vs. 50% (95%CI: 42–58) (p=0.67), respectively. Notably, the incidence of TRM was higher among recipients of KIR-L MM grafts [26% (95%CI: 15–26) vs. 13% (95%CI: 8–18), p=0.01]. KIR-L MM did not impact on outcome within the subgroups of patients based on conditioning, number UCB units (single vs. double), and those who had a myeloid malignancy (AML, CML or MDS). In multivariate analysis, a nucleated cell dose < 3.5 X 10E7/kg [RR 3.86 (95%CI: 1.84–8.12, p<0.01)] and KIR-L MM [RR 2.15 (95%CI: 1.15–4.01, p<0.01)] were independent predictors of increased TRM. In summary, in the setting of UCBT, KIR-L MM is associated with increased TRM with no obvious beneficial effect on engraftment, relapse risk or survival as previously demonstrated in recipients of TCD haploidentical HSC. Differences in NK cells reconstitution, presence of T-cells and/or use of immunosuppression may interfere with any potential beneficial effect alloreactive NK cells in the setting of UCBT. While additional studies are still needed, results to date fail to support a specific search for UCB units with a KIR-L MM.

Receptor Repertoire Reconstitution Suggests Acquisition of Patient-Specific Tolerance by Natural Killer Cells Arising from Hematopoietic Progenitor Stem Cells after Haploidentical Transplantation.

Donor-recipient incompatibility for human leukocyte antigen (HLA) ligands of killer cell immunoglobulin-like receptors (KIRs) in haploidentical hematopoietic stem cell transplantation (HSCT), has been associated with a selective graft versus leukemia (GvL) effect mediated by donor-derived alloreactive natural killer (NK) cells expressing KIRs whose ligands are missing in the recipient. In this study, we show that NK cells arising from hematopoietic stem cell progenitors after transplantation into haploidentical recipients, acquire a receptor repertoire that is compatible with patient-specific tolerance due to engagement of patient HLA ligands by inhibitory NK receptors. Using four-color immunofluorescence with monoclonal antibodies (mAbs) specific for the receptors CD94/NKG2A, KIR2DL1/2DS1, KIR2DL2/2DL3/2DS2 and KIR3DL1, we have analyzed NK receptor reconstitution kinetics in eleven adult patients affected by acute myeloid (n=9) or lymphoblastic (n=2) leukemia, who underwent HSCT from a KIR ligand matched (n=5) or mismatched (n=6) haploidentical family donor, using high doses (median 12.5x106/kg) of purified CD34+ progenitors. Nine patients achieved long-term (>150 days) complete remission of disease, independently from disease status at time of transplantation, and, importantly, from the presence (n=5) or absence (n=4) of donor NK alloreactivity. Within the first two months after transplantation, the vast majority (96% at 30 days, 86% at 60 days; SD 2% and 11%, respectively) of NK cells arising in the patients expressed the inhibitory receptor CD94/NKG2A, whose ligand HLA-E is ubiquitously expressed by cells positive for classical HLA class I molecules including leukemic blasts. As shown by mAb inhibition studies, lysis of patient-derived phytohemagglutinin-activated T cell blasts by these early arising NK cells was specifically inhibited by engagement of CD94/NKG2A. KIR expression was restored with variable kinetics in the later post-transplantation phase (3–9 months). Interestingly, however, during this period, NK cells devoid of CD94/NKG2A were found to express at least one KIR specific for an HLA ligand present in the patient, suggesting functional silencing of NK cells arising in the later phases after transplantation by acquisition of specific KIRs. Taken together, these data challenge current broad view on putative antileukemic effect of alloreactive NK cells reconstituting from haploidentical donor CD34+ cells and suggest that optimal exploitation of NK alloreactivity for GvL requires the presence of NK cells matured in the context of the donor's rather than the recipient's HLA repertoire. Ultimately, these findings provide a rationale for emerging clinical evidence in favor of efficacy of NK-based immunotherapy with mature donor NK cells.

In Vitro and In Vivo Immunomodulatory Effects of Mesenchymal Stem Cells from Adipose Tissue.

Previous studies have shown the immunomodulatory properties of bone marrow mesenchymal stem cells (BM-MSCs), opening the possibility of using these cells for the treatment of graft-versus-host disease (GVHD) in patients transplanted with allogeneic hematopoietic grafts. Additionally, Phase I studies in patients with Crohn's disease suggested the efficacy of adipose tissue-derived mesenchymal stem cells (Ad-MSCs) for the healing of Crohn's fistulas. In the present study we have investigated in vitro and in vivo, the immunomodulatory effects of Ad-MSCs, compared to BM-MSCs. We observed that both BM-MSCs and Ad-MSCs were negative for CD34, CD45, CD14, CD31 and MHC class I expression, while positive for CD29, CD44, CD90 and CD105. When studying the immunomodulatory effects of these cells in vitro, we found that - as happened with BM-MSCs - Ad-MSCs did not induce proliferation of allogeneic lymphocytes and were not lysed by cytotoxic T cells or alloreactive natural killer cells, indicating that Ad-MSCs are non-immunogenic. Additionally, the presence of Ad-MSCs inhibited in a dose-dependent fashion, both the mixed lymphocyte reaction (MLR) and the T cell proliferation induced by mitogens. To determine whether cell-to-cell contact between Ad-MSCs and PBMNCs was required for immunosuppression, transwell experiments were conducted. Phytohaemagglutinin (PHA)-stimulated lymphocytes were cultured in the upper chamber of a transwell, while irradiated Ad-MSCs remained in the lower chamber. As observed with BM-MSCs, Ad-MSCs were also capable of suppressing the lymphocytes proliferation in this transwell assay. When conditioned medium from Ad-MSCs was added to the MLR, the immuno-suppressive effect persisted, although at a lower level than that observed in a cell-to-cell contact system. Next we studied whether our in vitro findings were of significance in an in vivo mouse model of haploidentical transplantation. In these experiments irradiated F1(C57Bl/Balbc) recipient mice received 1x107 bone marrow cells from C57Bl mice, together with 2x107 splenocytes from the donor, to induce GVHD. One cohort of recipient mice received additional i.v. infusions of 5x105 mouse Ad-MSCs, administered at periodic intervals for up to 28 days post-transplant. When compared to the control group, the severity of the GVHD was significantly reduced in mice receiving Ad-MSCs. Our results suggest that Ad-MSCs obtained from adipose tissue may constitute a new and readily available source of immunomodulatory cells for the prophylaxis and/or treatment of GVHD in patients transplanted with allogeneic grafts.

Reduced-Intensity Conditioning (RIC) in Haploidentical Transplantation.

With the introduction of the concept of alloreactive Natural Killer (NK) cells, haploidentical transplantations are gaining increasing interest in the treatment of malignant diseases. While it has been shown in patients that alloreactive NK cells can exert an effective Graft-versus-Leukemia (GvL) effect, data in murine haploidentical transplantation models suggest that alloreactive NK cells also facilitate engraftment and reduce the risk of Graft-versus-Host Disease (GvHD). From these findings, we hypothesized that haploidentcial transplantation using Reduced Intensity Conditioning (RIC) regimens should be feasible in a clinical setting if the graft contains not only stem cells, but also large numbers of NK cells. Therefore, we have established a clinical method which allows the large scale depletion of CD3+ T-lymphocytes from mobilized peripheral haploidentical stem cells (PBSC's). We have initiated a clinical protocol using this approach in a patient population known to be at high risk for transplant-related toxicity and mortality (TRM). All patients suffered from chemorefractory leukemia/lymphoma or had a relapse after a previous myeloablative allogeneic transplant.As of June 2004, 18 pediatric patients have been transplanted. Seven patients had a chemorefractory disease and 11 patients had a relapse after a previous myeloablative allogeneic transplant. For all patients, haploidentical donors were mobilized using a combination of GM-CSF/G-CSF. Depletion of CD3+ T-cells was performed using the Clinimacs device. The RIC regimen consisted of Fludarabine (200 mg/m2), Thiotepa (10 mg/kg) and Melphalan (60 mg/m2 or 120 mg/m2, depending on the patient's clinical pretransplant condition). As an additional rejection prophylaxis, anti-CD3 antibody MuronomabR was given. A short course of posttransplant CellceptR was given for GvHD prophylaxis. For in vivo B-cell depletion, patients received one dose of RituxanR at day 0. The number of transplanted CD34+ stem cells was 19.3 x 106/kg (range 2.2 – 37.5) and the number of coinfused CD56+ NK cells was 103 x 106/kg (8.4 – 288). The number of residual CD3+ T-cells in the graft was 1.38 x 105/kg (0.07 – 4.5). Engraftment was rapid in 17 patients with 10.2 days (range 7–12) to reach an ANC>500. One patient did not engraft and one patient rejected his graft at day +12. Seventeen patients were available for chimerism analysis and in all patient, a 100% donor chimerism was seen after 14.5 days (range 6 – 21). Nine out of 15 evaluable patients had GvHD grade I–II and GvHD grade III or IV was not observed. One out of 8 evaluable patients had a limited chronic GvHD. One heavily pretreated patient who already had engrafted died due to a TRM (heart failure at day +10). No lethal infection was seen. One patient developed a slowly improving encephalopathy and one patient has an improving neuropathy. Of the 18 patients, 5 patients relapsed at a median of 73 days (range 35–126) after transplant. Twelve patients are alive without evidence of disease with a short median follow-up of 125 days (range 35 – 370). In 10 of these 12 patients, a 100% donor chimerism is maintained, whereas a decrease in donor chimerism was seen in 2 patients. The feasibility, low toxicity and the fast and sustained engraftment in this study is promising and argues for a wider use of RIC in haploidentical transplantation. In order to augment the antileukemic effect, this approach could be a platform for further posttransplant immune and cell therapy.

Mycophenolate mofetil does not suppress the graft-versus-leukemia effect or the activity of lymphokine-activated killer (LAK) cells in a murine model.

Graft-versus-leukemia (GVL) effect is an essential component in the course of allogeneic stem cell transplantation (SCT). However, both prevention and treatment of established graft-versus-host disease (GVHD), including with drugs such as cyclosporine, can suppress GVL effects. Mycophenolate mofetil (MMF) is becoming a standard of care in SCT recipients for better prevention of GVHD as well as for promoting stem cell engraftment. To evaluate the effect of MMF, an immunosuppressive drug increasingly used for prevention of GVHD, on disease recurrence following SCT in a preclinical animal model. Since GVL effects may be also induced by alloreactive natural killer (NK) cells, the goal was to investigate the effects of MMF on the activity of lymphokine-activated killer (LAK) cells. MMF was administered by daily intraperitoneal injection starting at day 1 post-SCT. Cytotoxic LAK activity was measured by 5-h 35S-release assay, and GVL was tested by the appearance of BCL1 leukemia in a semi-mismatched (C57BL/6 donors to [BALB/c x C57BL/6] F1 recipients) murine model. A dosage regimen of 28-200 mg/kg per day MMF had no negative effect on either cytotoxic LAK activity or GVL (as measured by finding of leukemic cells in recipient spleen by PCR or the appearance of clinical leukemia with adoptive transfer). These results suggest that MMF does not impair GVL effects or reduce LAK cell activity in mice.

Increased infection-related mortality in KIR-ligand-mismatched unrelated allogeneic hematopoietic stem-cell transplantation.

Recently, attention has been focused on the role of killer cell immunoglobulin-like receptor (KIR)-ligand incompatibility between donor and recipient in allogeneic hematopoietic stem-cell transplantation (ASCT). Although KIR-ligand mismatch is clearly associated with improved survival in haploidentical ASCT for acute myeloid leukemia, its role in unrelated human leukocyte antigen-mismatched ASCT is more controversial. Here we present a retrospective analysis of KIR-ligand-matched (n=167) and mismatched (n=23) unrelated ASCTs for hematologic malignancies performed at a single center. We observed that KIR-ligand mismatch was associated with increased transplantation-related mortality (P=0.02), leading to decreased overall survival (P=0.01). The increased transplantation-related mortality was a consequence of a higher rate of infections (P=0.01), whereas incidence of graft-versus-host disease and leukemic relapse did not differ significantly between the two groups. These results suggest that the presence of donor-derived, alloreactive natural killer cells may interfere with immunity to infection in the early posttransplantation period.

Phenotype and natural killer cell sensitivity of a radiation-induced acute T-cell leukaemia (Roser leukaemia) in PVG rats.

A radiation-induced T-cell leukaemia [Roser leukaemia (RL)] in the rat was conditioned for growth in vitro by repeated in vivo-in vitro passages. This in vitro cell line, termed RL-T, maintained its leukaemia-inducing property when transferred to syngeneic PVG rats. It expresses several T-cell markers and the T-cell alpha/beta receptor-CD3 complex. RL-T, furthermore, expresses major histocompatibility complex (MHC) I antigens, both classical (RT1.A) and nonclassical (RT1.C), which makes it susceptible to killing by alloreactive natural killer cells in vitro.

The beneficial role of inhibitory KIR genes of HLA class I NK epitopes in haploidentically mismatched stem cell allografts may be masked by residual donor-alloreactive T cells causing GVHD.

HLA allele mismatches will provoke T-cell alloreactivity after allogeneic stem cell transplantation. As donors and recipients are usually HLA matched, the public HLA epitopes that are recognized by natural killer (NK) cells (NK epitopes) are rarely mismatched, and therefore there is rarely potential for NK alloreactivity arising from the absence of ligands for inhibitory killer immunoglobulin-like receptors (KIR). Transplants using related donors sharing only one haplotype (haploidentical donors) represent a setting in which NK epitopes are often mismatched, thus resulting in the potential for NK alloreactivity. We have analyzed engraftment, acute graft vs host disease (GVHD), leukemia relapse, and survival in 62 haploidentical transplants in relationship with potential NK alloreactivity, inhibitory, and activating KIR genes of class I HLA NK epitopes. Potential NK alloreactivity in the rejection direction was not associated with any outcome variable. Potential NK alloreactivity in the GVHD direction was associated with an increased incidence of severe GVHD and poorer patient survival but not with non-engraftment nor leukemia relapse. A higher number of activating KIR receptors in the genome of the donor was associated with a higher prevalence of GVHD. These results suggest that lack of extensive T-cell depletion in haploidentical transplantation is associated with high GVHD rates and diminishes the benefits of NK-cell alloreactivity.

Immunotherapy with alloreactive natural killer cells in haploidentical haematopoietic transplantation.

Immunotherapy with alloreactive natural killer cells in haploidentical haematopoietic transplantation.

Relevance of KIR gene polymorphisms in bone marrow transplantation outcome

Natural Killer (NK) cells may be involved both in allogeneic bone marrow transplantation (BMT) rejection and graft-versus-host disease (GVHD). The physiologic functions of NK cells appear to be regulated by diverse non-inhibitory and inhibitory receptors including the killer cell immunoglobulin-like receptors (KIR). Although human leukocyte antigen (HLA) epitope mismatches are well-known causes of NK alloreactivity, the role of KIR genes in transplantation remains to be further investigated. In this study, we have evaluated whether KIR genotype differences between donors and recipients of HLA identical (related and unrelated) compared with HLA non-identical unrelated BMT, had an impact on transplantation outcome. Our results show that 5 of 15 KIR genes were always identical in donors and recipients and most variations were observed in the number and specificity of noninhibitory KIR genes. Based on the presence or absence of particular KIR genes, 70 different genotypes were obtained from all individuals. According to the donor or recipient KIR genotype, different combination patterns were described. Interestingly, when the recipient KIR genotype was “included” in the donor KIR genotype, 100% (11/11 pairs) of unrelated BMT developed GVHD compared with 60% (18/30) in all other combinations (p = 0.012). In contrast, no GVHD was observed in related BMT when the recipient KIR genotype was “included” in the donor KIR genotype (p = 0.0001). In conclusion, our results reveal a great diversity for KIR genotypes in donors and recipients of BMT and that the risk of GVHD was maximum in unrelated BMT when the recipient KIR genotype was “included” in the donor KIR genotype.

The athymic nude rat: an animal experimental model to reveal novel aspects of innate immune responses?

Athymic nude rats resemble nude mice in their lack of a normal thymus and functionally mature T cells. They have been useful in the study of mechanisms of tumor growth or graft rejection in immunocompromised hosts since they can accept major histocompatibility complex (MHC) mismatched organ allografts or xenografts for several months and because a number of tumor cell lines of human and rodent origin grow well in these rats. Injection of a few helper T (Th) cells from euthymic littermate rats partly restores the pool of mature T cells as well as full immunocompetence to reject organ allografts and has helped to reveal some of the cell interactions necessary for rejection to occur In contrast, immunologically naive athymic nude rats of certain strains, acutely reject allografts consisting of lymphocytes or bone marrow cells, which is due to the presence of alloreactive natural killer cells. These cells can recognize and kill MHC incompatible hematopoietic cells through the recognition of both mismatches within the classical (RT1.A) and nonclassical (RT1.C/E) MHC class I regions with a repertoire of inhibitory and activating killer lectin-like receptors (KLR) for MHC-I molecules, encoded by the Ly-49 portion of the rat natural killer cell gene complex (NKC). Some of these receptors have been identified and molecularly cloned and show similarities with NK receptors identified in the mouse. Other leukocytes in nude rats, such as dendritic cells, may also contribute to specific innate immune responses in the absence of mature T cells. Nude rats develop T-like cells expressing CD3 and T-cell receptor (TCR) with increasing age. Though their phenotype in peripheral lymphatic tissues resembles that of normal T cells, consisting mainly of CD4+ or CD8+ cells, they lack alloreactivity in vivo and their TCR repertoire is more of an oligoclonal nature. Their contribution to allograft rejection in T-cell-reconstituted rats is therefore questionable, and their role in innate immune response in these rats still enigmatic.

Long-term donor chimerism after MHC (RT1) mismatched bone marrow transplantation in the rat: the role of host alloreactive NK cells.

We have investigated the role of major histocompatibility complex (MHC) (RT1) disparities in the engraftment of bone marrow (BM) cells after whole body irradiation of rats. Mononuclear BM cells from PVG.RT7.2 (RT1c) rats were injected i.v. into sublethally (10Gy) whole body irradiated PVG (RT1c) rats and RT1 congenic and recombinant PVG rats. Repopulation of the BM, spleen, and blood with donor cells was assessed by FACS analysis of cells labelled with the fluorescein isothiocyanate (FITC)-labelled HIS41 monoclonal antibody (MoAb) against the RT7.2 marker. In RT1 matched (PVG.RT7.2 --> PVG) and RT1-mismatched combinations (PVG.RT7.2 --> PVG.1AV1), where radioresistant host natural killer (NK) cells could not recognize the BM inoculum as foreign, a donor chimerism close to 100% was observed after 6-8 weeks. However, in rat strain combinations where host NK cells could recognize an RT1 mismatch, almost no donor cells survived, and the rats were repopulated with leukocytes of host origin. In intra-MHC recombinant rat strains the element determining rejection or acceptance of the allograft mapped to the RT1-B/D-C/E/M region in PVG.R8 and PVG.R23 rats, in accordance with the patterns of NK alloreactivity in these strain combinations. NK cells may therefore be a primary obstacle to successful allogeneic BM engraftment in this model.

Activation of alloreactive natural killer cells is resistant to cyclosporine.

We have previously shown that cytotoxic T lymphocytes (CTL) with alloreactivity were induced when Wistar Furth (WF; RT1u) rats were immunized with allogeneic Brown Norway (BN; RT1n) cells. In contrast, when BN rats were immunized with WF cells, the allospecific response was confined to alloreactive natural killer (NK) cells, and no CTL activity was observed. In this study, the effect of cyclosporine (CsA) on the activation of alloreactive NK cells in vivo was analyzed. Distinct peritoneal effector cells from rats immunized with allogenic cells with or without concomitant CsA and/or interleukin (IL) 2 treatment were tested for specific cytolytic activity. Furthermore, the presumptive role of NK cells in rejection immunity was addressed in a cardiac graft model. The results showed that doses of CsA that completely inhibited the activation of alloreactive CTL, only marginally affected the activation of alloreactive NK cells. We also showed that CsA treatment failed to prolong graft survival in BN recipients of WF hearts. Treatment of BN rats with CsA/IL-2 during immunization with allogeneic WF cells resulted in concomitant induction of alloreactive NK cells and alloreactive CTL. We have demonstrated that CsA failed to suppress the activation of alloreactive NK cells. Consequently, the cardiac graft survival in the donor-recipient combination known to activate alloreactive NK cells was not significantly prolonged by CsA treatment, emphasizing the involvement of NK cells as effectors in organ rejection. Furthermore, the parallel emergence of alloreactive NK cells and CTL only in the presence of CsA/IL-2 indicated that CsA interfered with alloreactive NK cell-associated suppression of CTL activated by allogeneic tissue.