Lack of recognition of HLA class I mismatches outside α1/α2 domains by CD8+ alloreactive T lymphocytes: the HLA-B44 paradigm.

Transplantation with hematopoietic stem cells (HSC) from a donor with a single human leukocyte antigen (HLA) mismatch can be proposed to those patients lacking an HLA identical sibling donor or an unrelated donor matched for the HLA-A, -B, -C, DRB1, DQB1 loci. Incompatibilities at HLA classes I and II loci are associated with an increased risk of graft-versus-host disease (GVHD) and mortality, although no consensus exists yet on the relative importance of specific allele disparities on clinical outcome. Donor search algorithms are now complicated by the growing number of new HLA alleles, in particular those that differ outside the peptide-binding site of the HLA molecules. We report here an in vitro cellular assay to quantify CD8+CD137+ alloreactive cytotoxic T lymphocytes (CTLs) in a one-way mixed lymphocyte reaction. Two unique combinations with a single HLA mismatch in the HLA-B44 serotype differing by one amino acid in the α3 domain were investigated. We show that the B*44:27 versus B*44:02 mismatch was not recognized by CTLs in both directions. At days 10 and 20, the frequency of CD8+CD137+T cells was comparable to that measured in the autologous stimulation (0.3-3.9%). A B*44:02 versus B*44:03 mismatch was, however, well recognized at day 10 (7.2%) and day 20 (17.8%). This is the first demonstration that a single HLA-B mismatch involving a residue outside the peptide-binding site is not recognized in an in vitro functional assay and may probably be considered as a permissive incompatibility in vivo.

Basic characteristics and safety of donation in related and unrelated haematopoietic progenitor cell donors - first 10 years of prospective donor follow-up of Swiss donors.

Since July 2007 prospective life-long follow-up (FU) for unrelated (URD) and related donors (RD) is mandatory in Switzerland and data on every allogeneic haematopoietic progenitor cell (HPC) donation are collected prospectively. We report the real-world experience of HPC donation during a 10-year study period (01.07.2007-30.06.2017) with basic characteristics and FU data. 1105 donors underwent 1155 HPC donation procedures. Eighty percent of first donations performed by 802 (73%) RDs and 303 (27%) URDs were peripheral blood stem cells (PBSC), 20% bone marrow (BM). Male donors were over-represented as URD (60% male vs 40% female). Main differences between RDs and URDs concerned age and pre-existing health disorders. RDs were significantly older at first donation (median age 48 years) compared to URD (34 years, p < 0.0001) and had more pre-existing health problems: 25% vs 9% in URD (p < 0.0001). No fatal complications occurred, collection related severe adverse events (SAE) after first donation were not significantly different between groups (RD 1.2%, URD 0.99%), incidence rates for neoplastic and autoimmune diseases did not exceed the rates of the general population. RDs are a more heterogeneous and potentially more vulnerable group, but if donor evaluation is performed appropriately, HPC donation is still safe.

Changes of circulating antibody levels induced by ABO antibody adsorption for ABO-incompatible kidney transplantation.

ABO-incompatible kidney transplantation using immunoadsorption to remove anti-A/B antibodies has become a successful clinical practice. Since the data on the specificity of the ABO columns are controversial, the present study assessed the efficiency and specificity of the ABO immunoadsorption, the effect on total immunoglobulins and antibodies previously induced by vaccination. Anti-A/B antibodies were measured by agglutination and ABO flow cytometry, total IgG/IgM, carbohydrate- and protein-specific antibodies by nephelometry and ELISA. The first immunoadsorption not only efficiently reduced donor-specific anti-A/B IgM (81%) and IgG (56%) but also reduced compatible anti-A/B IgM (59%) and IgG (34%). The measurements of antidonor A/B antibodies by direct agglutination (IgM) or flow cytometry better represented the effective antibody levels than the indirect agglutination test (IgG). The median reduction of total IgM and total IgG levels after a single immunoadsorption was 34% and 18%, respectively. Antibodies against pneumococcus and haemophilus polysaccharide antigens were significantly reduced, whereas antitetanus and antidiphtheria protein antibodies were not affected. Intravenous immunoglobulin administration restored the protective anticarbohydrate antibody levels. In summary, immunoadsorption efficiently removed antidonor A/B antibodies, but was not specific for A/B antigens. Anti-A/B antibody levels as determined by ABO flow cytometry are useful to establish the minimal number of immunoadsorptions needed for successful ABO-incompatible transplantation.

EXPOSURE OF A SERVICE TECHNICIAN REPAIRING A BIPLANE X-RAY SYSTEM.

While repairing a biplane cardiovascular X-ray system in a hospital, a service technician accidentally activated the system's floor pedal. He continued his work under unnoticed exposure for about 5 min until the system alarm was automatically activated. About 2 h after the exposure, the technician developed an erythema on parts of his left face and neck. The next day, he reported his incident to the competent authorities and was hospitalised in a unit specialised in treating heavily irradiated patients. Frequent blood analysis did not show any signs for a significant exposure to radiation. The Federal Office of Public Health then conducted extensive dose estimations. It could be shown that the dismounted collimator was always in front of the lateral X-ray tube, shielding the technician from the direct beam. The dose estimations came to the following conservative results: an effective dose of 5 mSv, a skin dose of 200 mSv, an eye lens dose of 100 mSv and an extremity dose (arm) of 700 mSv. The cause of the erythema remains unclear since the estimated doses are thought to be too low to induce any visible effect on the skin.

The probability of identifying a 10/10 HLA allele-matched unrelated donor is highly predictable.

Identification of an unrelated HLA allele-matched hematopoietic stem cell (HSC) donor is a costly and time-consuming procedure. To improve search logistics, we have limited the search period to 6 months and have introduced a probability estimate of the chances of identifying a 10/10 HLA allele-matched donor. Probabilities were classified as high (>95%), intermediate (50%) and low (<5% chance) based on allele and haplotype frequencies. By analyzing 350 consecutive searches between 2002 and 2005 (1719 donors tested), the probability estimates turned out to be correct for 96% (high), 88% (low) and 56% (intermediate) patients. For searches with a high probability of success, at least one of the 10 most frequent haplotypes in Caucasoids was found in 69% of the patients, but in only 11% of the patients with a low-probability estimate (P<0.00001). Survival probability at 3 years was significantly higher for HSCT patients classified with a high-probability estimate when compared to patients in the intermediate/low-probability groups (74 vs 51 and 54% respectively, P=0.01). The same difference in survival probabilities was observed when only 10/10 matched unrelated HSCT patients were analyzed. In the intermediate-/low-probability groups, patients with alternative (haploidentical, autologous) or mismatched unrelated donors had similar survival estimates. Probability prediction is therefore feasible in the search process for unrelated donors and can guide the therapeutic strategy.

Favorable long-term outcome of nephrotic syndrome after allogeneic hematopoietic stem cell transplantation.

BACKGROUND: The development of nephrotic syndrome (NS) after allogeneic hematopoietic stem cell transplantation (HS-CT) is a rare complication with few long-term outcome data. PATIENTS: Clinical course and long-term outcome of three adult patients and one child with NS after HSCT (total number of transplants n = 533) are presented. RESULTS: The median age at onset of NS was 35 years (range 15 - 56), occurring at a median of 17 months (range 11 - 21) after HSCT. Discontinuation of cyclosporine A (CSA) prior to onset of NS was a consistent feature and occurred a median of 6 months (range 2 - 10 months) prior to the development of NS. The histopathological lesion was membranous nephropathy (n = 3) and membranoproliferative glomerulonephritis Type 1 (n = 1). History of acute or concomitant clinically apparent chronic graft versus host disease (GVHD) was present in all cases except the pediatric patient who had abundant DR-activated cytotoxic T cells without evidence of viral reactivation. Long-term immunosuppression for 11 - 36 months with steroids (n = 1), combined steroids and CSA (n = 2) or CSA alone in steroid-refractory NS (n = 1) resulted in sustained remission of the NS in all patients (12 months - 8 years off immunosuppression). CONCLUSION: NS after HSCT seems to be etiologically related to subclinical or overt chronic GVHD, which flares up after discontinuation of CSA. However, resumption of immunosuppression can reverse NS as well as GVHD and induce favorable sustained long-term remission.

Impact of high-resolution matching in allogeneic unrelated donor stem cell transplantation in Switzerland.

It is currently unknown what degree of human leukocyte antigen (HLA)-mismatching is acceptable in unrelated donor hematopoietic stem cell transplantation (UD-HSCT). Mismatches at some loci may be more permissive than others. We have analyzed the effect of high-resolution HLA-matching on outcome of all 214 consecutive recipients of UD-HSCT carried out in Switzerland. All typing was by the Swiss reference laboratory. Donor-recipient pairs were HLA-10/10 matched (n=130) or mismatched for either HLA-A/-B/-DRB1/multiple loci (n=33; (HLA-A/-B=10); (-DRB1=8); (multiple=15)); HLA-C (n=29) or HLA-DQ/-DRB3 (n=22; (DQ=16); (-DRB1=6)). The median follow-up was 32 months. Survival probabilities (+/-95% confidence interval) at 3 years were 57 (+/-10)% for recipients of HLA 10/10-matched transplants, 53 (+/-22)% for recipients of HLA-DQ/-DRB3-mismatched transplants, 44 (+/-20)% for recipients of HLA-C-mismatched transplants and 0% for recipients of transplants mismatched at HLA-A/-B/-DRB1/multiple loci (P<0.0001). In multivariate analyses, HLA compatibility was the variable most significantly associated with survival and treatment-related mortality. We found important differences in survival in recipients of UD-HSCT with best results for transplants from 10/10 matched donors. Single mismatches at HLA-DQ/-DRB3 were well tolerated, mismatches at HLA-C had intermediate results and mismatches at HLA-A/-B/-DRB1/multiple loci resulted in poor survival.

Haematopoietic stem cell transplantation in Switzerland. Report from the Swiss Transplant Working Group Blood and Marrow Transplantation (STABMT) Registry 1997-2003.

In 1997, the Swiss Transplant Working Group Blood and Marrow Transplantation (STABMT) initiated a mandatory national registry for all haematopoietic stem cell transplants (HSCT) in Switzerland. As of 2003, information was collected of 2010 patients with a first HSCT (577 allogeneic (29%) and 1433 autologous (71%) HSCT) and 616 additional re-transplants. This included 1167 male and 843 female patients with a median age of 42.4 years (range 0.2-76.6 years). Main indications were leukaemias (592; 29%) lymphoproliferative disorders (1,061; 53%), solid tumours (295; 15%) and non-malignant disorders (62; 3%). At the time of analysis 1,263 patients were alive (63%), 747 had died (37%). Probability of survival, transplant related mortality or relapse at 5 years was 52%, 21%, 36% for allogeneic and 54%, 5%, 60% for autologous HSCT. Outcome depended on indication, donor type, stem cell source and age of patient. HSCT is an established therapy in Switzerland. These data describe current practice and outcome.

Sensitization and desensitization of burn patients as potential candidates for vascularized composite allotransplantation.

Sensitization describes the acquired ability of the immune system to react to foreign human leukocyte antigens (HLA) by producing antibodies and developing memory cells. In the field of transplantation, recipient preformed HLA antibodies due to previous sensitization have been identified - beneath ABO incompatibility - as a major factor for acute graft rejection. Several reasons for sensitization have largely been studied, such as previous blood transfusions, pregnancies or former transplants. Recent studies indicate that the use of assist devices (e.g. ECMO) or cadaveric skin allotransplantation providing temporary coverage in burn patients may lead to additional sensitization. As vascularized composite allotransplantation (VCA) has become a rapidly advancing therapeutic option for reconstruction of complex tissue defects in burns, it seems even more important to become familiar with immunological principles and to be cautiously aware of both sources of sensitization and therapeutic concepts in burns avoiding sensitization. This may also include emergency VCAs in burn patients as potential strategy for early definitive reconstruction avoiding procedures triggering HLA antibody formation. We hereby provide an overview on current evidence in the field of pre- and peritransplant sensitization, followed by posttransplant strategies of desensitization and their potential impact on future treatments of burn patients.

Levels of anti-A/B antibodies after ABO-incompatible hematopoietic stem cell transplantation.

In contrast to solid organ transplantation, ABO incompatibility is generally not associated with survival differences in hematopoietic stem cell transplantation. Therefore, patients receiving ABO-incompatible stem cell transplantation can be analyzed to study the mechanism of tolerance induction after antigen-mismatched transplantation. The goal of this study was to analyze the levels of anti-A/B antibodies after ABO-incompatible transplantation. Host-derived antidonor antibodies disappeared rapidly after transplantation and did not reappear in the further posttransplant course. Donor-derived antihost antibodies did not significantly increase and compatible anti-A/B antibodies remained positive after hematopoietic stem cell transplantation. Thus, there is no evidence for stimulation of donor B lymphocytes to produce antirecipient antibodies suggesting a potential B cell tolerance.

High-resolution HLA matching in unrelated donor transplantation in Switzerland: differential impact of class I and class II mismatches may reflect selection of nonimmunogenic or weakly immunogenic DRB1/DQB1 disparities.

Unrelated donor searches in Switzerland require high-resolution HLA typing for HLA-A/B/C/DRB1/DRB3,4/DQB1 loci. We evaluated this strategy accepting donors with ⩾9/10 match. Of 802 unrelated donor transplants in 2000-2013, 570 were 10/10 matched, 31 were DRB3/4 mismatched, 261 were single-allele mismatched and 13 had 2 allele mismatches. Of the 261 single-allele disparities, 60 concerned HLA-A/-B, 55 HLA-C and 73 HLA-DRB1/-DQB1 loci. Transplants were reduced intensity conditioning (289, 36%), marrow (187, 23%), EBMT risk score was low in 39, intermediate I in 331, intermediate II in 333 and high in 99 patients. Five-year survival was 48±4%. HLA affected survival in the multivariate model adjusted for risk score. HLA-A/-B and HLA-C mismatches had twice the mortality risks, whereas HLA-DRB1/-DQB1 mismatches were similar to matched transplants. HLA-DRB3/4 mismatches were associated with a nonsignificant increased mortality risk. HLA-DRB3/4 mismatches had higher graft-versus-host disease and transplant-related mortality risks and lower relapse rates compared with matched transplants. We show significant effects of HLA class I, but not HLA class II, mismatches. The lack of impact of DRB1 disparities may be related to the lower immunogenicity of the DRB1*11:01/11:04 and DRB1*14:01/14:54 mismatches, representing 46% of DRB1 incompatibilities. These results support a matching algorithm that prioritizes mismatches considered as more permissive.

The relative radioresistance of interleukin-2 production by human peripheral blood lymphocytes: consequences for the development of a new limiting dilution assay for the enumeration of helper T lymphocyte precursor frequencies.

We describe a limiting dilution assay for the enumeration of alloreactive helper T lymphocyte precursor frequencies in human peripheral blood. The proliferation rate of the murine indicator cell line, cytotoxic T lymphoblastic line 2 (CTLL-2) induced by interleukin-2 (IL-2) culture supernatants was determined by staining with the fluorescent DNA dye propidium-iodide. Lymphocytes from healthy individuals as well as from patients with end stage kidney disease and no previous allosensitization exhibited a relative radioresistance of their IL-2 production up to gamma irradiation doses of 40-60 Gy. This differs from previous findings in the literature, showing a total inhibition of the IL-2 production in unsensitized individuals using a gamma irradiation dose of 20 Gy. The consequences of this relative radioresistance are that for a reliable stimulator cell inactivation in assays for the enumeration of helper T lymphocyte precursors gamma irradiation doses of at least 50 (-60) Gy are needed. Increasing the gamma irradiation dose for the inactivation of the stimulator cells can result in a decrease of the antigen presenting capacity of these cells.

Additional inhibitory effects of intravenous immunoglobulins in combination with cyclosporine A on human T lymphocyte alloproliferative response in vitro.

Intravenous immunoglobulins (IvIgG) are often used in patients receiving a basic immunosuppressive therapy with CsA either for prevention of infectious complications or as an additional prophylaxis of graft versus host disease in clinical bone marrow transplantation. As far as we know, the combined in vitro immunosuppressive effects of these 2 drugs have not been investigated yet. In this study, we compared the effect of CsA, IvIgG, and CsA combined with IvIgG on the proliferative capacity of peripheral blood mononuclear cells in a mixed lymphocyte culture system. The concentration-dependent inhibition of peripheral blood mononuclear cell proliferation in the mixed lymphocyte culture system by CsA is a well established phenomenon. By adding IvIgG to the cultures (n=20) containing CsA, we were able to show a significantly (P<0.0002) higher inhibition compared with the inhibitory capacity of CsA alone. Cyclosporine A was added to the cultures at concentrations ranging from 25 to 400 ng/ml, and IvIgG was added in 3 different fixed concentrations: 1.25, 2.5, and 5 mg/ml. These are all concentrations which one usually obtains in patients during therapy with these drugs. Even with a minimal concentration of CsA (25 ng/ml) plus IvIgG (1.25 mg/ml), we achieved a mean inhibition of 77.7 +/- 7.9%, which is in the range of the mean inhibition (84.3 +/- 4.7%) with the highest concentration of CsA (400 ng/ml tested. Our in vitro results could suggest that the additional therapy with IvIgG in patients receiving CsA might cause a CsA sparing effect. This might lead to a combined therapeutic regimen with a good immunosuppressive efficacy and minimal drug associated adverse effects.

Differential inhibition of primed alloreactive CTLs in vitro by clinically used concentrations of cyclosporine and FK506.

Previous studies in highly sensitized patients waiting for a renal transplant showed a lack of correlation between the formation of alloantibodies and the alloantigen specific cytotoxic T cell precursor frequency. The frequencies of CTLp against HLA class I antigens, toward which patients had formed antibodies (not acceptable mismatches, NAM), were similar to those against HLA antigens, toward which no antibodies were present (acceptable mismatches, AM). In more recent studies we tested whether the immunological triggering leading to antibody formation might have resulted in a different population of cytotoxic T cells. Limiting dilution assays performed in the absence or presence of antibodies against CD8 showed that CTL directed against NAM were significantly less inhibited by anti-CD8 compared with those directed against AM. Those "primed" CTL could also be distinguished from the more "naive" CTL on the basis of their resistance to cyclosporine. In contrast to CsA, therapeutic concentrations of FK506 were able to inhibit both "naive" and "primed" CTLs. The clinical relevance of these data is currently being investigated.

Cytotoxic T lymphocytes against HLA-B antigens are less naive than cytotoxic T lymphocytes against HLA-A antigens.

In healthy individuals, like in highly sensitized patients, CTL precursors (CTLp) frequencies against HLA-A antigens were found to be significantly lower than CTLp frequencies against HLA-B antigens. In the present study, we tested whether not only quantitative but also qualitative differences exist between CTLs against HLA-A antigens versus HLA-B antigens. Experiments performed in the presence of antibodies against CD8 showed that CTLp directed against HLA-B antigens were significantly less inhibited by anti-CD8 compared with CTLp against HLA-A. The low anti-CD8 inhibition of the CTLp directed against HLA-B antigens may be explained by a higher affinity of the CTLp for HLA-B antigens on the target cells. CTLp directed against HLA-B antigens were also found to be less sensitive to CsA, which suggests that their high affinity is due to previous priming. A clinical relevance of these findings is suggested by the observation that matching for HLA-B antigens is of greater importance for kidney graft survival than matching for HLA-A.

Rapid and automated processing of bone marrow grafts without Ficoll density gradient for transplantation of cryopreserved autologous or ABO-incompatible allogeneic bone marrow.

The growing number of BMTs has increased interest in safe and standardized in vitro bone marrow processing techniques. We describe our experience with a rapid automated method for the isolation of mononuclear cells (MNC) from large volumes of bone marrow using a Fenwal CS-3000 cell separator without employing density gradient materials. Forty bone marrow harvests with a mean volume of 1650 +/- 307 ml were processed. A mean of 75 +/- 34% (50 percentile range 54-94%) of the original MNCs were recovered in a volume of 200 ml with only 4 +/- 2% of the starting red blood cells (RBC). Removal of granulocytes, immature myeloid precursors and platelets proved to be sufficient to permit safe cryopreservation and successful autologous BMT (n = 25). Allogeneic BMT (n = 14, including three major ABO-incompatible) could be performed without additional manipulation. In both groups of patients timely and stable engraftment comparable to historical controls receiving Ficoll gradient processed autologous (n = 17) or unprocessed allogeneic BMT (n = 54) was observed. Moreover, 70 +/- 14% of the RBC could be recovered from the grafts. They were used for autologous RBC support of donors, rendering unnecessary autologous blood pre-donations.

Delaying treatment with granulocyte colony-stimulating factor after allogeneic bone marrow transplantation for hematological malignancies: a prospective randomized trial.

The use of granulocyte colony-stimulating factor (G-CSF) has been established to improve hematological recovery after allogeneic bone marrow transplantation (BMT). The optimal timing to start with G-CSF has not been determined. This study investigates whether delayed use of G-CSF starting on day 6 is as safe and efficient as starting treatment with G-CSF immediately after BMT. Thirty-eight patients undergoing allogeneic BMT were randomized to either receive post-transplant G-CSF treatment starting at day 1 or at day 6. The time to hematological recovery was monitored and the groups were compared with respect to peritransplant morbidity and mortality. Recovery of the neutrophil granulocyte counts (PMN) to >100/microl, >500/microl and >1000/microl was comparable in both groups. The nadir of the PMN counts after stopping G-CSF was also similar. There was no difference in the recovery of red blood cells and platelet counts and no difference between the two groups with respect to the number of febrile episodes, number of days with antibiotics or number of documented bacterial, fungal or viral infections. Delayed treatment with G-CSF resulted in a reduction of G-CSF treatment from 19 days to 14 days (P = 0.0017). Reducing the length of treatment by 5 days lowered G-CSF treatment costs by 26.3%. Therefore, postponing treatment with G-CSF has no influence on the hematological recovery after allogeneic BMT. There is an economical benefit of postponing G-CSF use without any clinical disadvantages.