Kidney-transplanted Adolescents-Nonadherence and Graft Outcomes During the Transition Phase: A Nationwide Analysis, 2000-2020.

BACKGROUND: The transition from pediatric to young adult care is a vulnerable period for the renal transplant patient. We aimed to identify medication nonadherence (noninitiation, nonimplementation, and nonpersistence) and graft loss rates among adolescents and young adults and elucidate the influence of the transition phase on transplant outcomes. METHODS: A retrospective nationwide cohort analysis of all renal transplantations in Norway from 2000 to 2020 was performed. Data were retrieved from the Norwegian Renal Registry, and adherence data from hospital charts. Patients transplanted aged <50 y, with functioning graft at 6 mo, were included. Recipients transplanted aged <26 y were compared with recipients transplanted aged 26-50 y. Graft loss, acute rejection, and development of de novo donor-specific antibodies were assessed in relation to the transition phase, defined as 14-26 y. RESULTS: Data from 1830 kidney recipients were included: 371 (20%) transplanted <26 y (64% male, 68% living donor) versus 1459 transplanted 26-50 y (63% male, 44% living donor). There were 298 graft losses, 78 (21%) in the <26-y group versus 220 (15%) in the 26- to 50-y group. During the transition phase, 36 grafts were lost, 29 (81%) after transfer to the adult service. Medication nonadherence was the reason for 58% (21 of 36) of the losses during the transition phase, versus 12% (27 of 220) in the 26- to 50-y group ( P < 0.001). The 5-y graft survival rate was 89% (95% confidence interval, 85%-92%) and 94% (92%-95%), respectively ( P = 0.01). CONCLUSIONS: Nonadherence was verified as the main cause of kidney graft loss in the transition phase.

Screening for antibodies to HLA class I in apheresis donors following Covid-19 or SARS-CoV-2 vaccination.

Transfusion of HLA-specific antibodies may play a role in induction of TRALI, the transfusion complication responsible for most transfusion-related deaths. In Oslo, we screen our apheresis donors and defer HLA-immunized donors from donation of plasma-rich blood components. During the second year of the Covid-19 pandemic and following the first months of SARS-CoV-2 vaccination, both the virus itself and the vaccines were suspected of inducing de novo production of antibodies to HLA class I in patients. For the blood center, the possibility of finding HLA-antibodies in an increased number of blood donors has serious implications. We therefore conducted a study to map the extent of de novo HLA-specific antibodies in representative donor groups. 106 apheresis donors were screened for antibodies to HLA class I/II following Covid-19 or vaccination with either mRNA or adenovirus-vector vaccines, and the findings were compared to pre-Covid blood samples from the same donors. In addition, we analyzed pre-Covid samples from 11 HLA-antibody-positive donors of Covid convalescence plasma. Only three established thrombapheresis donors were deferred due to vaccine-induced HLA-antibodies. In short, our findings did not support the hypothesis that SARS-CoV-2 virus or vaccination cause de novo HLA immunization in healthy blood donors. However, some donors with pre-existing antibodies showed increased antibody expression, confirming a general boost of the immune response following infection or vaccination.

Scandiatransplant acceptable mismatch program-10 years with an effective strategy for transplanting highly sensitized patients.

In March 2009, the Scandiatransplant acceptable mismatch program (STAMP) was introduced as a strategy toward improving kidney allocation to highly sensitized patients. Patients with a transplantability score ≤ 2% are potential candidates for the program. Samples are analyzed and acceptable antigens (HLA-A, B, C, DRB1, DRB3/4/5, DQB1, DQA1, DPB1, DPA1) are defined by the local tissue typing laboratory and finally evaluated by a steering committee. In the matching algorithm, patients have the highest priority when the donor's antigens are all among the recipient's own or acceptable HLA antigens. In the period from 2009 to 2020, we have transplanted 278 highly sensitized kidney patients through the program. The graft survival of the STAMP patients was compared with 9002 deceased donor kidney-transplanted patients, transplanted in the same time period. The 10-year graft survival was 73.4% (95% CI: 60.3-90.0) for STAMP and 82.9% (95% CI: 81.6-84.3) for the reference group. (p = .2). In conclusion, the 10-year allograft survival demonstrates that the STAMP allocation algorithm is immunological safe. The program is continuously monitored and evaluated, and the introduction of matching for all HLA loci is a huge improvement to the program and demonstrate technical adaptability as well as clinical flexibility in a de-centralized organization.

HLA class I depletion by citric acid, and irradiation of apheresis platelets for transfusion of refractory patients.

BACKGROUND: Patients can form antibodies to foreign human leukocyte antigen (HLA) Class I antigens after exposure to allogeneic cells. These anti-HLA class I antibodies can bind transfused platelets (PLTs) and mediate their destruction, thus leading to PLT refractoriness. Patients with PLT refractoriness need HLA-matched PLTs, which require expensive HLA typing of donors, antibody analyses of patient sera and/or crossmatching. An alternative approach is to reduce PLT HLA Class I expression using a brief incubation in citric acid on ice at low pH. METHODS AND MATERIALS: Apheresis PLT concentrates were depleted of HLA Class I complexes by 5 minutes incubation in ice-cold citric acid, at pH 3.0. Surface expression of HLA Class I complexes, CD62P, CD63, phosphatidylserine, and complement factor C3c was analyzed by flow cytometry. PLT functionality was tested by thromboelastography (TEG). RESULTS: Acid treatment reduced the expression of HLA Class I complexes by 71% and potential for C3c binding by 11.5-fold compared to untreated PLTs. Acid-treated PLTs were significantly more activated than untreated PLTs, but irrespective of this increase in steady-state activation, CD62P and CD63 were strongly upregulated on both acid-treated and untreated PLTs after stimulation with thrombin receptor agonist peptide. Acid treatment did not induce apoptosis over time. X-ray irradiation did not significantly influence the expression of HLA Class I complexes, CD62P, CD63, and TEG variables on acid treated PLTs. CONCLUSION: The relatively simple acid stripping method can be used with irradiated apheresis PLTs and may prevent transfusion-associated HLA sensitization and overcome PLT refractoriness.

Five decades with grandparent donors: The Norwegian strategy and experience.

Living donors (LDs) are preferred over DDs for renal transplantation in children due to superior GS. Oslo University Hospital has never restricted living donation by upper age. The aim of this study was to investigate long-term outcomes using grandparents (GPLD) compared to PLD. Retrospective nationwide review in the period 1970-2017. First renal graft recipients using a GPLD were compared to PLD kidney recipients for long-term renal function and GS. 278 children (≤18 years) received a first renal transplant: 27/251 recipients with a GPLD/PLD. GPLD (median 59 (42-74) years) were significantly older than PLD (median 41 (23-65) years, (P < .001). Median DRAD was 52 (38-70) vs 28 (17-48) years, respectively. GS from GPLD and PLD had a 1-, 5-, and 10-year survival of 100%, 100%, and 90% vs 93%, 82%, and 72%, respectively (P = .6). In a multivariate Cox regression analysis adjusted for gender, donor age, recipient age, and year of transplant, this finding was similar (HR 0.98; 95% CI 0.34-2.84, P = .97). Five-year eGFR was 47.3 and 59.5 mL/min/1.73 m2 in the GPLD and PLD groups (P = .028), respectively. In this nationwide retrospective analysis, GS for pediatric renal recipients using GPLD was comparable to PLD. Renal function assessed as eGFR was lower in the GPLD group. The GPLD group was significantly older than the PLD group, but overall this did not impact transplant outcome. Based on these findings, older age alone should not exclude grandparent donations.

Identification of MHC Class Ib Ligands for Stimulatory and Inhibitory Ly49 Receptors and Induction of Potent NK Cell Alloresponses in Rats.

Early studies indicate that rats may have a repertoire of MHC class Ib-reactive Ly49 stimulatory receptors capable of mounting memory-like NK cell alloresponses. In this article, we provide molecular and functional evidence for this assumption. Pairs of Ly49 receptors with sequence similarities in the lectin-like domains, but with opposing signaling functions, showed specificity for ligands with class Ia-like structural features encoded from the first telomeric MHC class Ib gene cluster, RT1-CE, which is syntenic with the H2-D/H2-L/H2-Q cluster in mice. The activating Ly49s4 receptor and its inhibitory counterparts, Ly49i4 and Ly49i3, reacted with all allelic variants of RT1-U, whereas Ly49s5 and Ly49i5 were specific for RT1-Eu NK cell cytolytic responses were predictably activated and inhibited, and potent in vivo NK alloresponses were induced by repeated MHC class Ib alloimmunizations. Additional Ly49-class Ib interactions, including RT1-Cl with the Ly49s4/Ly49i4/Ly49i3 group of receptors, were characterized using overexpressed receptor/ligand pairs, in vitro functional assays, and limited mutational analyses. Obvious, as well as subtle, Ly49-class Ib interactions led to ligand-induced receptor calibration and NK subset expansions in vivo. Together, these studies suggest that in vivo NK alloresponses are controlled by pleomorphic Ly49-class Ib interactions, some of which may not be easily detectable in vitro.

Portal vein reconstruction using primary anastomosis or venous interposition allograft in pancreatic surgery.

OBJECTIVE: Superior mesenteric vein/portal vein (SMV/PV) resection and reconstruction during pancreatic surgery are increasingly common. Several reconstruction techniques exist. The aim of this study was to evaluate characteristics of patients and clinical outcomes for SMV/PV reconstruction using interposed cold-stored cadaveric venous allograft (AG+) or primary end-to-end anastomosis (AG-) after segmental vein resections during pancreatic surgery. METHODS: All patients undergoing pancreatic surgery with SMV/PV resection and reconstruction from 2006 to 2015 were identified. Clinical and histopathologic outcomes as well as preoperative and postoperative radiologic findings were assessed. RESULTS: A total of 171 patients were identified. The study included 42 and 71 patients reconstructed with AG+ and AG-, respectively. Patients in the AG+ group had longer mean operative time (506 minutes [standard deviation, 83 minutes] for AG+ vs 420 minutes [standard deviation, 91 minutes] for AG-; P < .01) and more intraoperative bleeding (median, 1000 mL [interquartile range (IQR), 650-2200 mL] for AG+ vs 600 mL [IQR, 300-1000 mL] for AG-; P < .01). Neoadjuvant therapy was administered more frequently for patients in the AG+ group (23.8% vs 8.5%; P = .02). Patients with AG+ had a longer length of tumor-vein involvement (median, 2.4 cm [IQR, 1.6-3.0 cm] for AG+ vs 1.8 cm [IQR, 1.2-2.4 cm] for AG-; P = .01), and a higher number of patients had a tumor-vein interface >180 degrees (35.7% for AG+ vs 21.1% for AG-; P = .02). There was no difference in number of patients with major complications (42.9% for AG+ vs 36.6% for AG-; P = .51) or early failure at the reconstruction site (9.5% for AG+ vs 8.5% for AG-; P = 1). A subgroup analysis of 10 patients in the AG+ group revealed the presence of donor-specific antibodies in all patients. CONCLUSIONS: The short-term outcome of SMV/PV reconstruction with interposed cold-stored cadaveric venous allografts is comparable to that of reconstruction with primary end-to-end anastomosis. Graft rejection could be a contributing factor to severe stenosis in patients reconstructed with allograft.

Rat acute GvHD is Th1 driven and characterized by predominant donor CD4+ T-cell infiltration of skin and gut.

Acute graft-versus-host disease (aGvHD) remains a significant hurdle to successful treatment of many hematological disorders. The disease is caused by infiltration of alloactivated donor T cells primarily into the gastrointestinal tract and skin. Although cytotoxic T cells mediate direct cellular damage, T helper (Th) cells differentially secrete immunoregulatory cytokines. aGvHD is thought to be initiated primarily by Th1 cells but a consensus is still lacking regarding the role of Th2 and Th17 cells. The aim of this study was to determine the contribution of distinct T-cell subsets to aGvHD in the rat. aGvHD was induced by transplanting irradiated rats with T-cell-depleted major histocompatibility complex-mismatched bone marrow, followed 2 weeks later by donor lymphocyte infusion. Near complete donor T-cell chimerism was achieved in the blood and lymphatic tissues, in contrast to mixed chimerism in the skin and gut. Skin and gut donor T cells were predominantly CD4+, in contrast to T cells in the blood and lymphatic tissues. Genes associated with Th1 cells were upregulated in gut, liver, lung, and skin tissues affected by aGvHD. Increased serum levels of CXCL10 and IL-18 preceded symptoms of aGvHD, accompanied by increased responsiveness to CXCL10 by blood CD4+ T cells. No changes in the expression of Th2- or Th17-associated genes were observed, indicating that aGvHD in this rat model is mainly Th1 driven. The rat model of aGvHD could be instrumental for further investigations of donor T-cell subsets in the skin and gut and for exploring therapeutic options to ameliorate symptoms of aGvHD.

Liver transplantation with deceased ABO-incompatible donors is life-saving but associated with increased risk of rejection and post-transplant complications.

ABO-incompatible (ABOi) liver transplantation (LT) with deceased donor organs is performed occasionally when no ABO-compatible (ABOc) graft is available. From 1996 to 2011, 61 ABOi LTs were performed in Oslo and Gothenburg. Median patient age was 51 years (range 13-75); 33 patients were transplanted on urgent indications, 13 had malignancy-related indications, and eight received ABOi grafts for urgent retransplantations. Median donor age was 55 years (range 10-86). Forty-four patients received standard triple immunosuppression with steroids, tacrolimus, and mycophenolate mofetil, and forty-four patients received induction with IL-2 antagonist or anti-CD20 antibody. Median follow-up time was 29 months (range 0-200). The 1-, 3-, 5-, and 10-year Kaplan-Meier estimates of patient survival (PS) and graft survival (GS) were 85/71%, 79/57%, 75/55%, and 59/51%, respectively, compared to 90/87%, 84/79%, 79/73%, and 65/60% for all other LT recipients in the same period. The 1-, 3-, 5-, and 10-year GS for A2 grafts were 81%, 67%, 62%, and 57%, respectively. In conclusion, ABOi LT performed with non-A2 grafts is associated with inferior graft survival and increased risk of rejection, vascular and biliary complications. ABOi LT with A2 grafts is associated with acceptable graft survival and can be used safely in urgent cases.

Presensitization revisited: pitfalls of vascular allografts in transplant candidates.

Vascular allografts in end-stage renal disease (ESRD) patients represent a particular immunological challenge. A broad HLA immunization led us to study in depth the history of two patients with vascular allografts. In Case 1 the allograft was added to a Gore-Tex graft used for haemodialysis access and no immunosuppression was administered. In Case 2 the allograft was used to prolong a renal artery from living donor and immunosuppression was suboptimal. In vascular surgery, immunosuppression is mainly used to improve graft patency. ESRD patients are potential organ recipients and immunosuppression should therefore be tailored to reduce HLA immunization.

Early versus late acute antibody-mediated rejection in renal transplant recipients.

BACKGROUND: Over the last decade, the diagnostic precision for acute antibody-mediated rejection (aABMR) in kidney transplant recipients has improved significantly. The phenotypes of early and late aABMR may differ. We assessed the characteristics and outcomes of early versus late aABMR. METHODS: Between January 1, 2005 and December 31, 2010, aABMR was diagnosed in 67 grafts in 65 kidney recipients, with a median follow-up of 3.6 years (range, 61 days-7.3 years). Recipients were stratified by early aABMR (<3 months after transplantation; n=40) and late aABMR (>3 months after transplantation; n=27). The main outcome was kidney allograft loss. Outcome of aABMR was compared with recipients with acute early (n=276) or late (n=100) non-ABMR during the same period. RESULTS: Recipients with late aABMR had significantly reduced graft survival compared with recipients with early aABMR (P<0.001, log-rank test; 40% vs. 75% at 4 years; hazard ratio, 3.72; 95% confidence interval, 1.65-8.42). Graft survival in late aABMR was also inferior to late non-ABMR acute rejections (P=0.008). At transplantation, more patients were presensitized to human leukocyte antigens (22 [55%] vs. 4 [15%] in the early vs. late aABMR group). The late aABMR group was characterized by younger recipient age (37.9 ± 12.9 vs. 50.9 ± 11.6 years; P<0.001), increased occurrence of de novo donor-specific antibodies (52% vs. 13%; P=0.001), and nonadherence/suboptimal immunosuppression (56% vs. 0%; P<0.001). CONCLUSION: Compared with early aABMR, late aABMR had inferior graft survival and was characterized by young age, frequent nonadherence, or suboptimal immunosuppression and de novo donor-specific antibodies.

Identification of an MHC class I ligand for the single member of a killer cell lectin-like receptor family, KLRH1.

Natural killer cells are able to recognize and kill target cells according to differences in MHC class I expression. In rodents, the Ly49 receptors are primarily responsible for this MHC differentiation. We previously described the cloning of a novel C-type lectin-like receptor, KLRH1, encoded in the NK complex adjacent to the Ly49 genes and expressed by subsets of NK and NKT cells. MHC influence on selection of KLRH1(+) NK cells in congenic strains suggested that KLRH1 may have an MHC ligand, although we were unable to identify any such ligand. In this study, we have used a sensitive reporter system and Fc fusion protein to demonstrate that KLRH1 binds specifically to the classical MHC class I molecule RT1-A2 of the RT1(n) haplotype. Cytolytic activity of KLRH1-transfected RNK-16 cells was also inhibited by target cells expressing RT1-A2(n). Thus, KLRH1 represents a novel family of MHC allele-specific inhibitory receptors expressed by NK cells.

The role of natural killer cells in the defense against Listeria monocytogenes lessons from a rat model.

Ly49 receptors in rodents, like killer cell immunoglobulin-like receptors in humans, regulate natural killer (NK) cell activity. Although inhibitory Ly49 receptors clearly recognize classical major histocompatibility complex class I (MHC-I) molecules, the role for the activating Ly49 receptors has been less well understood. Here, we discuss recent data from a rat model for listeriosis. Rats depleted of NK cells, or more specifically the Ly49 receptor-bearing cells, showed increased bacterial loads in their spleen. Athymic nude rats with no functional T cells but increased numbers of Ly49-expressing NK cells were more resistant to infection, indicating a central role of NK cells in early immune defense against Listeria in this species. Listeria infection of macrophages or enteric epithelial cells led to upregulation of MHC-I, including nonclassical (Ib) molecules not regularly recognized by T cells. We have shown that activating Ly49 receptors are more efficiently stimulated when binding to upregulated class Ib antigens on infected cells. From this we postulate that activating Ly49 receptors may have a sentinel function in the early immune response against Listeria in detecting diseased cells 'flagged' by increased MHC-Ib expression.

The rat NK cell receptors Ly49s4 and Ly49i4 recognize nonclassical MHC-I molecules on Listeria monocytogenes-infected macrophages.

Ly49 receptors in rodents, like KIRs in humans, regulate NK cell activity. Although inhibitory Ly49 receptors clearly recognize MHC-I molecules, ligands for the activating Ly49 receptors are less well defined. Here, we show that the activating Ly49s4 and the inhibitory Ly49i4 receptors recognize nonclassical MHC-I molecules on the rat macrophage cell line R2 (RT1(d)). Listeria infection of R2 macrophages led to increased expression of classical and nonclassical MHC-I molecules. Coincubation of these infected cells with reporter cells expressing Ly49i4 or Ly49s4 increased the reporter cell responses. These responses were blocked by mAb OX18 (anti-MHC-I) and AAS1 (anti-nonclassical MHC-I). IFN-γ treatment of normal R2 cells also increased the MHC-I expression and enhanced the reporter cell responses. These results suggest that activating and inhibitory Ly49 receptors monitor MHC-I expression on Listeria-infected cells.

Partial NK cell tolerance induced by radioresistant host cells in rats transplanted with MHC-mismatched bone marrow.

We have studied the effect of radioresistant host cells in inducing tolerance and adaptation of the MHC recognition repertoire of donor-derived NK cells in stem cell allotransplanted (allo-SCT) rats. Sub-lethally irradiated PVG.1AV1 rats (RT1(av1)) were transplanted with bone marrow from fully MHC-mismatched allotype-marked PVG.7B (RT1(c)) rats; MHC-identical PVG (RT1(c)) controls were transplanted in parallel. In the PVG.7B → PVG.1AV1 allogeneic chimeras, NK cells were donor derived and showed partial tolerance toward host cells. Allogeneic chimeras failed to efficiently reject PVG.1AV1 cells by an NK-mediated mechanism in vivo (allogeneic lymphocyte cytotoxicity), and IL-2-cultured NK cells derived from these chimeras showed diminished cytolytic activity against PVG.1AV1 cells in vitro. There were corresponding changes in the phenotype and function of the highly alloreactive Ly49i2(+) NK cells, which are specifically inhibited by a donor MHC class I ligand, RT1-A1(c). The ligand-negative host MHC haplotype apparently induced expression of a second uncharacterized inhibitory MHC receptor responsible for the partial tolerance toward host-derived cells, along with a modest increase in Ly49i2 receptor levels. The host MHC haplotype did not induce a general hyporesponsiveness in Ly49i2(+) NK cells, which showed normal activation responses in a panel of MHC congenic strains. The data suggest that the MHC constitution of radiation-resistant host cells can have permanent, albeit not fully tolerogenic, effects on the development of a functional NK repertoire following allo-SCT.

Listeria monocytogenes infection affects a subset of Ly49-expressing NK cells in the rat.

NK cells are protective against certain bacterial and viral infections, and their production of IFN-γ is important for the early innate immune defence against L. monocytogenes. We have previously shown that depletion of NK cells in rats leads to increased bacterial burden upon L. monocytogenes infection, and that a subset of NK cells encompassing the majority of Ly49 receptors (Ly49s3+ NK cells) contributed to this effect. In this study, we have further investigated how the Ly49s3+ NK cell subset is affected by L. monocytogenes infection. We observed an increased percentage of Ly49s3+ NK cells in the spleen and a reduction in the bone marrow within the first 48 hrs of L. monocytogenes infection. Concomitantly, we observed increased expression levels of the inflammatory chemokine receptors CCR5 and CXCR3 by Ly49s3+ bone marrow NK cells, as compared to Ly49s3- NK cells, suggesting involvement of Ly49s3+ NK cells in the early phase of infection. However, NK cell production of IFN-γ was independent of Ly49 receptor expression. Furthermore, we observed increased expression levels of MHC class I molecules on both macrophages and NK cells during the first 48 hrs of infection, paralleled by a reduction in the surface expression of Ly49s3 on NK cells. In conclusion, L. monocytogenes infection modulates the tissue distribution of Ly49s3+ NK cells, and induces increased MHC class I expression and hence reduced surface expression of Ly49 receptors on NK cells. These changes indicate that L. monocytogenes infection may have multiple effects on NK cells in vivo, and suggests the involvement of Ly49-expressing NK cells in the immune responses towards L. monocytogenes.

The activating rat Ly49s5 receptor responds to increased levels of MHC class Ib molecules on Listeria monocytogenes-infected enteric epithelial cells.

We have investigated whether rat Ly49 receptors can monitor Listeria-infected intestinal epithelial cells through altered expression of MHC class I molecules. The rat colon carcinoma epithelial cell line CC531 infected with Listeria expressed higher levels of both classical and nonclassical MHC-I molecules. Reporter cells expressing the activating Ly49s5 receptor displayed increased stimulatory responses when incubated with Listeria-infected CC531 cells in vitro, which could be blocked with mAb 8G10 specific for nonclassical MHC-I molecules of the RT1(u) haplotype, but not with mAb OX18 reacting with classical MHC-I molecules in this haplotype. Similar responses were observed against IFN-γ-treated cells that also upregulated their expression of MHC-I molecules. Thus, the Ly49s5 receptor can respond to increased levels of nonclassical MHC-I molecules induced on target cells by either bacterial infection or cytokine stimulation. We furthermore found that splenic NK and NKT cells produced IFN-γ in response to Listeria-infected CC531 cells, and that this was not limited to Ly49-expressing cells, since similar levels of IFN-γ production were observed in Ly49(+) and Ly49(-) NK cell subsets. Therefore, NK cells may recognize Listeria-infected cells through both MHC-I-dependent and -independent innate immune receptor systems.

Two complementary rat NK cell subsets, Ly49s3+ and NKR-P1B+, differ in phenotypic characteristics and responsiveness to cytokines.

Two major subsets of rat NK cells can be distinguished based on their expression of the Ly49s3 or the NKR-P1B lectin-like receptor. Ly49s3(+) NK cells, but not NKR-P1B(+) NK cells, express a wide range of Ly49 receptors. Here, we have examined differences between these two subsets in their expression of certain NK cell-associated molecules as well as their responses to cytokines. A microarray analysis suggested several differentially expressed genes, including preferential expression of NKG2A/C receptors by NKR-P1B(+) NK cells. This was confirmed by staining with tetramers of RT.BM1, the putative ligand of CD94/NKG2, indicating that Ly49 and CD94/NKG2 receptors separate into distinct NK cell compartments. Further, expression of CD25 by Ly49s3(+) NK cells was associated with more rapid proliferation in response to IL-2 as compared with NKR-P1B(+) NK cells. Thus, certain inflammatory situations may preferentially expand the Ly49s3(+) NK cells. Moreover, freshly isolated Ly49s3(+) and NKR-P1B(+) NK cells produce similar amounts of cytokines, and a minor Ly49s3(-)NKR-P1B(-) double-negative NK subset appears to be hyporesponsive based on its significantly lower IFN-gamma production. Collectively, our data demonstrate divergent profiles of NKR-P1B(+) and Ly49s3(+) NK cells, indicating distinct tasks in vivo.

Two major groups of rat NKR-P1 receptors can be distinguished based on chromosomal localization, phylogenetic analysis and Clr ligand binding.

A major subset of non-alloreactive NK cells in PVG strain rats is generally low in Ly49 receptors, but expresses the rat NKR-P1B(PVG) receptor (previously termed NKR-P1C). The NKR-P1B(+) NK subset is inhibited by a non-polymorphic target cell ligand, which we have shown here to be a C-type lectin-related molecule (Clr). Clr11 ligates two divergent NKR-P1B alleles as judged by an NFAT-driven reporter assay, and inhibits NK-cell cytotoxicity of NKR-P1B(+) NK cells. Clr11 also interacts with the prototypic NKR-P1A receptor and exerts a stimulatory influence on NK lysis. NKR-P1A and B are encoded by adjacent genes in the proximal part of the NK gene complex and show close sequence homology in their extracellular region. They diverge from another pair, NKR-P1F and -G, which is encoded by a second, distal Nkrp1 gene cluster. NKR-P1F and -G bind an overlapping panel of Clr ligands, but not Clr11. Rat Clr molecules appear to be constitutively expressed by hematopoietic cells; expression in tumor cell lines is more variable. The data show the existence of two phylogenetic groups of NKR-P1 molecules, which demonstrate conservation of ligand-binding properties independent of signaling function.

Impact of MHC mismatch and freezing on bone graft incorporation: an experimental study in rats.

Cortical bone graft failure develops for poorly defined reasons, and the effects of the immune responses on the incorporation of an allograft are less clear. In a rat model of tibial allotransplantation, we have studied biometric and histological changes of the graft and the humoral immune response against it. We have also compared fresh with prefrozen grafts to study putative effects of freezing on the healing of the graft and the immune response against it. Fresh and frozen cortical bone grafts matched or mismatched for major histocompatibility complex antigens (syngeneic and allogeneic grafts) were implanted in an 8-mm segmental defect in the tibia. The construct was stabilized with intramedullary nailing. Incorporation of the graft was assessed with use of conventional radiography, micro computed tomography (CT(, biomechanical testing and histological examination. The immune response was evaluated by monitoring distribution of leukocytes in the blood and by measuring antibodies in a tailor-made fluorescence activating cell scanning (FACS( analysis. We found that the fresh syngeneic grafts were well integrated after 8 weeks with intact bone cells. In the fresh allogeneic grafts, all cells were dead with radiological signs of resorption, and mechanical testing indicated failure of incorporation. The frozen grafts showed poorer overall reconstruction than the fresh syngeneic grafts, but the incorporation was better than the fresh allogeneic grafts. A measurable alloantibody response was only detected after fresh allografting. The combined results suggest that freezing of bone allograft impedes the antibody response against major histocompatibility complex (MHC( antigens and improves incorporation, but frozen allografts still perform poorer than do frozen syngeneic grafts.