Specific HLA Antigens Research Articles

Individual HLAs affect survival after allogeneic stem cell transplantation in adult T-cell leukaemia/lymphoma.

Allogeneic haematopoietic stem cell transplantation (allo-HSCT) is the only curative therapy for adult T-cell leukaemia/lymphoma (ATL). Specific HLAs are associated with outcomes of immunotherapy and allo-HSCT. We hypothesised that individual HLAs would affect the clinical outcomes of ATL patients after allo-HSCT. Using data from a Japanese registry, we retrospectively analysed 829 patients with ATL who received transplants from HLA-identical sibling donors or HLA-A, -B, -C or -DRB1 allele-matched unrelated donors between 1996 and 2015. We evaluated the overall mortality risk of HLA-A, -B and -DR antigens with frequencies exceeding 3%. Outcomes were compared between transplants with or without specific HLA antigens. Of the 25 HLAs, two candidates were identified but showed no statistically significant differences by multiple comparison. HLA-B62 was associated with a lower risk of mortality (hazard ratio [HR], 0.68; 95% confidence interval [CI]: 0.51-0.90; p = 0.008), whereas HLA-B60 was associated with a higher risk of mortality (HR, 1.64; 95% CI: 1.19-2.27; p = 0.003). In addition, HLA-B62 was associated with a lower risk of transplant-related mortality (TRM) (HR, 0.52; 95% CI: 0.32-0.85, p = 0.009), whereas HLA-B60 was associated with a higher risk of grades III-IV acute graft-versus-host disease (HR, 2.63; 95% CI: 1.62-4.27; p < 0.001). Neither HLA influenced relapse. The higher risk of acute GVHD in HLA-B60-positive patients and the lower risk of TRM in HLA-B62-positive patients were consistent with previously obtained results from patients with other haematological malignancies. Consideration of HLA in ATL patients may help to predict risk and outcomes after allo-HSCT.

Homozygosity in any HLA locus is a risk factor for specific antibody production: the taboo concept 2.0.

In a cooperative study of the University Hospital Leipzig, University of Leipzig, and the Charité Berlin on kidney transplant patients, we analysed the occurrence of HLA-specific antibodies with respect to the HLA setup of the patients. We aimed at the definition of specific HLA antigens towards which the patients produced these antibodies. Patients were typed for the relevant HLA determinants using mainly the next-generation technology. Antibody screening was performed by the state-of-the-art multiplex-based technology using microspheres coupled with the respective HLA alleles of HLA class I and II determinants. Patients homozygous for HLA-A*02, HLA-A*03, HLA-A*24, HLA-B*07, HLA-B*18, HLA-B*35, HLA-B*44, HLA-C*03, HLA-C*04, and HLA-C*07 in the class I group and HLA-DRB1*01, HLA-DRB1*03, HLA-DRB1*07, HLA-DRB1*15, HLA-DQA1*01, HLA-DQA1*05, HLA-DQB1*02, HLA-DQB1*03(7), HLA-DQB1*06, HLA-DPA1*01, and HLA-DPB1*04 in the class II group were found to have a significant higher antibody production compared to the heterozygous ones. In general, all HLA determinants are affected. Remarkably, HLA-A*24 homozygous patients can produce antibodies towards all HLA-A determinants, while HLA-B*18 homozygous ones make antibodies towards all HLA-B and selected HLA-A and C antigens, and are associated with an elevation of HLA-DRB1, parts of DQB1 and DPB1 alleles. Homozygosity for the HLA class II HLA-DRB1*01, and HLA-DRB1*15 seems to increase the risk for antibody responses against most of the HLA class I antigens (HLA-A, HLA-B, and HLA-C) in contrast to HLA-DQB1*03(7) where a lower risk towards few HLA-A and HLA-B alleles is found. The widely observed differential antibody response is therefore to be accounted to the patient's HLA type. Homozygous patients are at risk of producing HLA-specific antibodies hampering the outcome of transplantation. Including this information on the allocation procedure might reduce antibody-mediated immune reactivity and prevent graft loss in a patient at risk, increasing the life span of the transplanted organ.

Development of electrochemical biosensor: Voltammetric analysis of lymphocytes and indication activation of complement system

This work reports the development of an electrochemical biosensor after immobilization of the lymphocytes to detect the reaction between antibodies and specific HLA antigens present in the serum samples. A clean homemade gold electrode with voltammetric polycrystalline characteristics was used. Lymphocytes were immobilized and tested with positive and negative human serum and complements on the gold electrode. The experiments were carried out in a cell with three electrodes: working - gold, reference - Ag/AgCl/sat. KCl and auxiliary - platinum. The cyclic voltammetric analyses of immobilized lymphocytes on the gold surface presented anodic current equal to 1.78 μA at c.a. 0.50 V vs. Ag/AgCl/sat. KCl. The electrochemical responses of the serum (positive and negative) and complement do not show signs of oxidation or reduction in the potential range used. The electrodes with cells and positive serum showed the amplified current signal in the oxidation potential of the cells. The electrode was developed to verify the antigen antibody reaction, present lymphocyte cell and human serum samples. The electrode was qualitatively efficient when compared to the methods of flow cytometric analysis and complement dependent cytotoxicity, being able to be used with operational and economic advantages.

HLA expression levels of unstimulated and cytokine stimulated human umbilical vein endothelial cells.

Transplant rejection occurs following recipient recognition of mismatched HLA on donor tissue, but active rejection is dependent not only upon the severity of the T cell or alloantibody response, but also upon the cell surface expression of target HLA molecules. To investigate the variation in HLA expression using a model of endothelium, human umbilical vein endothelial cell (HUVEC) cultures were generated from 48 umbilical cords donated consecutively following planned caesarean section. HUVECs were stimulated using the cytokines tumour necrosis factor alpha and interferon gamma and HLA expression of unstimulated and stimulated cells determined using flow cytometry. HLA-A2, HLA-A3 and HLA-C antigens all showed a modest increase in expression for 12 hours post cell activation, followed by a more pronounced response over the next 24 to 36 hours. Each of these antigens increased by up to 40 times over unstimulated levels and in addition cells homozygous for specific HLA antigens on average had twice the amount of antigen expressed compared with cells heterozygous for that antigen, both when unstimulated and following cytokine stimulation. Cell activation is an important consideration in the assessment of transplant risk and may help progress towards understanding why rejection does not always occur in the presence of significant donor specific antibody. This data also confirms guidelines for transplantation, which recommend doubling the specific antibody level when considering immunological risk for homozygous donors.

P045 Kidney transplant with historic but no current DSA – A case study

Presence of donor-specific HLA antibodies (DSA) at the time of transplant is associated with poor outcome. DSA may be present in historic serum samples but not at the time of a kidney offer. Going forward with the transplant holds the risk of a memory B-cell response upon re-exposure to a specific HLA antigen or epitope. A patient with X-linked Alport syndrome was investigated for a kidney transplant. Anti-HLA class II antibodies were observed following pregnancy, which were specific to her husband’s mismatched DR and DQ antigens. A year later, she received a blood transfusion and had an episode of aseptic peritonitis while on dialysis. Class I HLA antibodies appeared but were observed over a period of 7 months. Thereafter, anti-HLA antibody screening using FlowPRA beads and identification using Luminex single antigen beads showed disappearance of class I antibodies but persistence of a single anti-HLA-DR antibody developed after prior pregnancy. Three years later, a kidney from a DCD donor was offered and transplanted despite presence of historic DSA against 2 HLA-B antigens. A CDC crossmatch proved negative using a current serum whereas a flow cytometric crossmatch using 3 DSA-containing historic sera gave positive but inconsistent results (T + B+, T-B+, T-B-). One week after transplant, historic DSA reappeared (3000 MFIs). Despite absence of biopsy-proven rejection, short-term treatment comprising plasmapheresis, rituximab and low-dose IVIg was initiated. DSA quickly disappeared from circulation with only an HLA-DR-specific reactivity from the previous pregnancy. Without any further desensitization treatment, the patient has not demonstrated re-appearance of DSA nor experienced antibody-mediated rejection (AMR) as demonstrated upon for-cause and protocol biopsies, up to 8 months posttransplant at the time of abstract submission. This case demonstrates that, although DSA may reappear following re-exposure to a sensitizing HLA antigen/epitope, acceptable transplant outcome can be achieved without AMR. Time between DSA development and transplant could be a factor in identifying acceptable HLA mismatches.

HLA Donor Specific Antibody and HLA Antigen Bias are Independent Risk Factors for Chronic Lung Allograft Dysfunction

Immune responses to both HLA donor specific antibody (DSA) as well as lung associated self antigens have been implicated in chronic lung allograft dysfunction (CLAD). Furthermore, several studies have suggested that a bias in the HLA-DR antigens may promote a T-cell driven response to self-antigens. The goal of this study was two-fold; firstly we investigated the association of de novo HLA DSA with bronchiolitis obliterans syndrome (BOS) as well as restrictive allograft syndrome (RAS). Secondly, we investigated the HLA genotypes of both recipients and donors to determine if there was any antigen bias associated with either form of CLAD.

Current Perspectives on Stevens-Johnson Syndrome and Toxic Epidermal Necrolysis.

Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are considered a delayed-type hypersensitivity reaction to drugs. They represent true medical emergencies and an early recognition and appropriate management is decisive for the survival. SJS/TEN manifest with an "influenza-like" prodromal phase (malaise, fever), followed by painful cutaneous and mucous membrane (ocular, oral, and genital) lesions, and other systemic symptoms. The difference between SJS, SJS/TEN overlap, and TEN is defined by the degree of skin detachment: SJS is defined as skin involvement of <10%, TEN is defined as skin involvement of >30%, and SJS/TEN overlap as 10-30% skin involvement. The diagnosis of different degrees of epidermal necrolysis is based on the clinical assessment in conjunction with the corresponding histopathology. The mortality rates for SJS and TEN have decreased in the last decades. Today, the severity-of-illness score for toxic epidermal necrolysis (SCORTEN) is available for SJS/TEN severity assessment. Drugs with a high risk of causing SJS/TEN are anti-infective sulfonamides, anti-epileptic drugs, non-steroidal anti-inflammatory drugs of the oxicam type, allopurinol, nevirapine, and chlormezanone. Besides conventional drugs, herbal remedies and new biologicals should be considered as causative agents. The increased risk of hypersensitivity reactions to certain drugs may be linked to specific HLA antigens. Our understanding of the pathogenesis of SJS/TEN has improved: drug-specific T cell-mediated cytotoxicity, genetic linkage with HLA- and non-HLA-genes, TCR restriction, and cytotoxicity mechanisms were clarified. However, many factors contributing to epidermal necrolysis still have to be identified, especially in virus-induced and autoimmune forms of epidermal necrolysis not related to drugs. In SJS/TEN, the most common complications are ocular, cutaneous, or renal. Nasopharyngeal, esophageal, and genital mucosal involvement with blisters, erosions as well as secondary development of strictures also play a role. However, in the acute phase, septicemia is a leading cause of morbidity and fatality. Pulmonary and hepatic involvement is frequent. The acute management of SJS/TEN requires a multidisciplinary approach. Immediate withdrawal of potentially causative drugs is mandatory. Prompt referral to an appropriate medical center for specific supportive treatment is of utmost importance. The most frequently used treatments for SJS/TEN are systemic corticosteroids, immunoglobulins, and cyclosporine A.

P084 De novo HLA-DQ donor specific antibodies are associated with worse long term allograft survival after lung transplantation

Aim The long term graft survival of lung transplantation remains significantly worse than any other solid organ transplants. De novo HLA donor specific antibodies (dnDSA) have been implicated with antibody-mediated rejection (AMR), a recognized cause of allograft dysfunction in lung transplant recipients. It remains unclear what dnDSA against specific HLA antigens play in long term graft function and patient survival. We have observed that HLA class II dnDSA are common in lung transplant recipients. In this study, we further characterized that HLA dnDSA implicated allograft function and recipient survival. Methods We retrospectively investigated lung transplant recipients received transplantation between Jan, 2004 and December, 2015 in a single center. HLA DSA were tested using Luminex LabSceeen single antigen bead assays per recipient’s clinical indications. Graft function and patient survival were followed up until December 31, 2016. Data were analyzed using cox hazards regression model and logistic regression. Results A total of 187 patients were followed up for a median of 2.67 years (1–12 years). Among them, 54% were female and 46%, male and 78% were Caucasians. Fifty-eight (31%) patients developed dnDSA, i.e., 8 (4.2%) class I, 42 (22.4%) class II and 8 (4.2%) class I and class II DSA, respectively. Among these DSA positive patients, 36 (62%) patients developed HLA-DQ DSA. Both Class II dnDSA and/or DQ DSA were significantly associated with worse allograft survival and patient survival (P Conclusions HLA-DQ dnDSA are dominate in lung transplant recipients after transplantation which are associated with worse long term allograft and recipient survival. To prevent HLA-DQ dnDSA by better HLA-DQ match or to properly treat patients with HLA-DQ dnDSA may improve long term outcomes for lung transplant recipients.

De novo DQ donor-specific antibodies are associated with worse outcomes compared to non-DQ de novo donor-specific antibodies following heart transplantation.

Antibody-mediated rejection (AMR) resulting from de novo donor-specific antibodies (dnDSA) leads to adverse outcomes following heart transplantation (HTx). It remains unclear what role dnDSA to specific HLA antigens play in adverse outcomes. This study compares outcomes in patients developing dnDSA to DQ antigens with those developing non-DQ dnDSA and those free from dnDSA. The present study was a single-center, retrospective analysis of 122 consecutive HTx recipients. The primary outcome was a composite of death or graft dysfunction. After 3.3years of follow-up, 31 (28%) patients developed dnDSA. Mean time to dnDSA was 539days. Of 31 patients, 19 developed DQ antibodies and 12 developed non-DQ antibodies. Compared to non-DQ dnDSA, DQ antibodies presented with higher MFI values (P=.001) were more likely persistent (P=.001) and appeared later post-HTx (654 vs 359days, P=.035). In a multivariable analysis, DQ dnDSA was associated with increased risk of the primary endpoint (HR 6.15, 95% CI 2.57-14.75, P=.001), whereas no increased risk was seen with non-DQ dnDSA (P=.749). dnDSA to DQ antigens following HTx are associated with increased risk of death and graft dysfunction.

Research Highlights

Melanoma Risk and Survival Among Organ Transplant Recipients. Robbins HA, Clarke CA, and Arron ST, et al. J Invest Dermatol. 13 August 2015 The risk of melanoma in solid organ transplant recipients is significantly increased compared to nonimmunosuppressed patients.1 The authors assessed the epidemiology of melanoma in close to 140 000 white patients based on who received a transplant in the United States. Invasive melanoma was diagnosed in 519 transplanted patients and in situ melanoma in 190 patients. The risk of invasive melanoma was elevated greater than 2-fold compared with the general population; most melanomas were detected on an “in situ” level with the most prominent location on head and neck. Tumors were classified as local only, regionally spread, or distantly spread. The risk for regional melanoma increased markedly within 4 years after the transplantation and remained consistently increased (1.5- to 2-fold) long term for the occurrence of in situ melanoma. A multivariate analysis for regional/distant stage melanoma, male, age, and an induction treatment with polyclonal antibodies represented particular risks. Survival was monitored in greater than 130 000 patients of which 27% died subsequent to melanoma compared to 12% in the general population. Melanoma-specific mortality was elevated 3-fold in transplant recipients, suggesting that tumor progression may be more aggressive in immunosuppressed patients. Moreover, the incidence of localized tumors was increased in patients on azathioprine potentially linked to an accelerated UVR-induced DNA damage in this patient group.2 However, the authors only documented immunosuppression at baseline. For regional- and distant-stage melanoma, the authors report a high risk shortly after transplantation that peaked at 4 years and was furthermore augmented when T-cell–depleting polyclonal antibodies were used. Of interest, melanoma incidence declined subsequent to graft failure in kidney recipients when immunosuppression had been discontinued or significantly reduced. The authors postulate that patients might have melanocytic precursors or early-stage melanomas undiagnosed at the time of transplantation that progressed rapidly with intense immunosuppression. Of note, melanomas express a range of neoantigens that can serve as a target for T cells. This article highlights the importance of screening patients on the waitlist thoroughly before transplantation to remove small melanomas and precursor lesions in time. REFERENCES Dahlke E, Murray CA, Kitchen J, et al. Systematic review of melanoma incidence and prognosis in solid organ transplant recipients. Transplant Res. 2014;3:10. Clarke LE, Warf BM, Flake DD, et al. Clinical validation of a gene expression signature that differentiates benign nevi from malignant melanoma. J Cutan Pathol. 2015;42:244–252. Preformed Circulating HLA-Specific Memory B Cells Predict High Risk of Humoral Rejection in Kidney Transplantation. Lucia M, Luque S, and Crespo E, et al. Kidney Int. 15 July 2015 Traditionally, clinicians use donor-specific antibodies (DSA) to assess the risk for antibody-mediated rejection in transplant candidates. It has been recognized that HLA-specific antibodies appear and disappear from time to time, and that it is possible to get histological evidence of humeral rejection in the absence of circulating DSAs. The authors investigated the significance of the memory B-cell pool in patients with antibody-mediated rejection in addition to highly sensitized patients on the waitlist; nonsensitized patients or transplanted patients who did not experience humoral rejections served as controls. The authors developed a B-cell ELIspot assay to detect circulating HLA-specific memory B Cells (mBC) capable of producing anti-class I/II HLA antibodies subsequent to polyclonal stimulation. Sensitized patients on the waitlist and patients who experienced an episode of antibody-mediated rejection demonstrated a detectable broad range of HLA-sp mBC even before transplantation. Moreover, the authors reported on a significant correlation of HLA sp mBC responses and acute vascular lesions by histology. Donor-specific mBC responses were detectable in 10/16 patients who developed antibody-mediated rejection, even before transplantation. Monitoring HLA-sp mBC seems to be more sensitive and specific than monitoring DSA in transplanted patients. HLA-specific mBC responses were able to predict HLA-specific antibody responses and humoral rejections early and accurately. Moreover, the presence of HLA-specific mBC predicted a rapid differentiation into antibody-producing cells when in contact with specific HLA antigens in presensitized patients.

Multicolor Flowcytometric Measurement of Alloantigen-Specific Antibody Secreting Cells in Highly Sensitized Patients in Renal Transplantation.

Not all pretransplantation donor-specific antibodies (DSA) cause worse graft outcome. The increase of DSAs post-transplantation can be more important marker than the level of DSA in pretransplant sera. The assessment of sensitized cells which can secrete DSA is needed. Our aim was to develop an assay for antibody secreting cells (ASC) by multicolor flowcytometry in short-term culture of peripheral blood. Five highly-sensitized patients who were awaiting or received renal transplantaion and 3 non-sensitized patients were included. PRA screening and identification were performed with LIFECODES Life-Screen Deluxe and Class I and Class II ID kits (Gen-probe). Peripheral blood mononuclear cells were seeded with CpG 2006: 5'-TCGTCGTTTTGTCGTTTTGTCGTT-3'), (Bioneer, Korea), 500 ng/ml human CD40 Ligand (Sigma), and 50 ng/ml IL-21 (PeproTech). Lymphocyte lysates of HLA types (corresponding to specificities detected in the serum of the patient to be tested) were added to each test cells. After 72 hrs, ELISPOT analysis with Human IgG ELISpotPlus kit (Mabtech, Sweden) and flowcytometric analyses with Anti-CD19-PE, CD27-PerCP-CY5.5, CD38-APC, IgG-FITC antibodies (BD Pharmingen) were performed. The proportion of ASC was significantly increased when stimulated with lymphocyte lysate of donor specific HLA antigen than controls in highly sensitized renal transplantation patients. In non-sensitized patients, there was no difference between culture with lymphocyte lysate and controls. The absolute number of ASC was also significantly increased when stimulated with lymphocyte lysate of donor specific HLA than controls in highly sensitized patients. The number of total recovered cells and the number of ASC in ELISPOT analysis stimulated with lymphocyte lysate of donor specific HLA showed tendency of increase but didn't reach a statistical significance compared to controls in highly sensitized patients. Three day-culture condition using peripheral blood in highly sensitized patients can differentiate ASC with flowcytometric analysis. It can be applied for future study to see the increase of ASC can predicts renal tranplantation outcome.

Impact of Pre Transplant Donor Specific Antibodies in the Setting of a Negative Cell Based Flow Cytometry Cross Match On Graft Outcomes.

Background:Presence of antibodies to donor specific HLA antigens (DSA) detected by single antigen bead (SAB) analysis prior to kidney transplant has been associated with inferior graft outcomes. However, it is unclear if low-level DSA (e.g. in a negative flow cytometry crossmatch (FCXM)) leads to a higher risk of rejection or a later graft dysfunction. Most studies,limited by small sample size, have not considered flow cytometry cross match results in the analysis. Methods: 547 consecutive recipients of a first kidney or kidney/pancreas transplant with a negative pre-transplant FCXM between September 2007 and August 2012 at our center were analyzed. All patients underwent cell-based FCXM and SAB analysis (LABScreen beads, One Lambda Inc.) on current and historic sera prior to transplantation. Patients were placed on triple immunosuppression. Graft outcomes were compared for patients with (DSA+) and without (DSA-) pre-transplant DSA. Results: Of 547 patients with negative pre-transplant FCXM, 196 had DSA (MFI ≥ 500) detected prior to transplant by SAB analysis. Acute rejection (AR) rates at one year were similar in DSA+ vs. DSA- (15% vs. 12% respectively, p=0.22) although AR occurred earlier in the DSA+ group. With median follow up period of 2.3 years, mean GFR and mean proteinuria (0.27 g/day vs 0.35 g/day for DSA+ vs. DSA- groups) was similar for both groups. Three year graft survival by Kaplan-Meier estimate was similar in DSA+ and DSA-patients (p= 0.81) and remained similar when defining DSA as MFI ≥ 1000.Figure: No Caption available.Conclusion: To date, this is the largest series characterizing post-transplant outcomes of patients with pre-transplant +DSA/-FCXM without desensitization therapies. Presence of pre-transplant DSA in the setting of negative FCXM was neither associated with higher AR rates nor with longer term graft outcomes. These data suggest that DSA detected by SAB alone may confer minimal immunologic risk and do not require preemptive B-cell/antibody-targeted therapies.

HLA typing in systemic sclerosis

OBJECTIVE: The aim of the study was to investigate the relationship between Systemic Sclerosis (SSc) and HLA antigens, and to correlate these antigens with the clinical manifestations of the disease. MATERIALS AND METHODS: 55 patients were stratified according a) to the cutaneous involvement b) to the positivity of Scl-70 and anticentromere antibody and c) to the internal organ involvement, in particular we used HRCT to demonstrate lung fibrosis, echocardiography for the diagnosis of pulmonary hypertension, blood creatinine, urinalysis and arterial hypertension to demonstrate renal failure, and esophagus double-contrast barium swallow for the diagnosis of esophagopathy. The control group consisting of 2000 healthy Caucasian subjects was recruited from the same population. RESULTS: The frequency of the antigens A23 (p=0.003, RR=3.69), B18 (p<0.0001, RR=3.57), and DR11 (p<0.0001, RR=6.18) was statistically increased in the patients population compared with the healthy controls. Although there is no any significant correlation between HLA antigens and different clinical subsets of scleroderma, antigens B18 and DR11 could be associated with more severe clinical features. CONCLUSIONS: The presence of a significant association between SSc and specific HLA antigens (A23, B18, and DR11) could link the HLA system with SSc.

A Retrospective Study on Standard Regimen for Vaccination in Celiac Children

Background: HLA system plays an essential role in the human immune system activity and the expression of some spe-cific HLA antigens could modify the immune response to vaccinations. Celiac Disease is included among the diseases associated to specific HLA profiles, principally characterized by the expression of the HLA DQ2 antigen. Object: Our study was a retrospective study, leaded on a group of celiac children, with the object to evaluate their immunological response to both obligatory and recommended vaccinations in childhood. Materials and methods: It was a retrospec-tive study, including 66 patients affected by celiac disease, between 3 and 15 years of age, and a control group of 50 children of the same age. All patients performed both obligatory and recommended vaccinations as indicated in the Italian standard regimen for vaccinations. The immunologic response to each vaccine was analysed and compared in the two groups. Moreover, authors also studied the immunologic response to vaccines in celiac children comparing patients whose diagnosis was made before 18 months of age with those whose disease was diagnosed after 18 months of age. Results: Our results showed that in celiac patients the immunological response to vaccine is similar to that one found in general population, except for HBV vaccine. Conclusions: the valuation of immunological response to HBV vaccine should be regularly effectuated in celiac children and revaccination should be recommended.

HLA antigen frequencies and Wegener's granulomatosis

Previous reports of an association between HLA tissue type and Wegener's granulomatosis are contradictory. By using for the first time a highly sensitive restriction fragment-length polymorphism (RFLP) analysis in addition to standard microcytotoxicity assays, the largest series yet investigated (41 patients) was tissue typed. No association was found between any specific HLA antigen and Wegener's granulomatosis. Although the condition appears to be immunologically mediated, this study indicates that the HLA antigens do not have a major role.

Infantile Epileptic Encephalopathy with Hypsarrhythmia (Infantile Spasms/West Syndrome) and Immunity

West syndrome is a severe epilepsy, occurring in infancy, that comprises epileptic seizures known as spasms, in clusters, and a unique EEG pattern, hypsarrhythmia, with psychomotor regression. Maturation of the brain is a crucial component. The onset is within the first year of life, before 12 months of age. Patients are classified as cryptogenic (10 to 20%), when there are no known or diagnosed previous cerebral insults, and symptomatic (80 to 90%), when associated with pre-existing cerebral damages. The time interval from a brain insult to infantile spasms onset ranged from 6 weeks to 11 months. West syndrome has a time-limited natural evolutive course, usually disappearing by 3 or 4 years of age. In 62% of patients, there are transitions to another age-related epileptic encephalopathies, the Lennox-Gastaut Syndrome and severe epilepsy with multiple independent foci. Spontaneous remission and remission after viral infections may occur. Therapy with ACTH and corticosteroids are the most effective. Reports about intravenous immunoglobulins action deserve attention. There is also immune dysfunction, characterized mainly by anergy, impaired cell-mediated immunity, presence of immature thymocytes in peripheral blood, functional impairment of T lymphocytes induced by plasma inhibitory factors, and altered levels of immunoglobulins. Changes in B lymphocytes frequencies and increased levels of activated B cells have been reported. Sensitized lymphocytes to brain extract were also described. Infectious diseases are frequent and may, sometimes, cause fatal outcomes. Increase of pro-inflamatory cytokines in serum and cerebrospinal fluid of epileptic patients were reported. Association with specific HLA antigens was described by several authors (HLA-DR7, HLA-A7, HLA-DRw52, and HLA-DR5). Auto-antibodies to brain antigens, of several natures (N-methyl-d-aspartate glutamate receptor, gangliosides, brain tissue extract, synaptic membrane, and others), were described in epileptic patients and in epileptic syndromes. Experimental epilepsy studies with anti-brain antibodies demonstrated that epileptiform discharges can be obtained, producing hyperexcitability leading to epilepsy. We speculate that in genetically prone individuals, previous cerebral lesions may sensitize immune system and trigger an autoimmune disease. Antibody to brain antigens may be responsible for impairment of T cell function, due to plasma inhibitory effect and also cause epilepsy in immature brains. Keywords: West syndrome, Lennox-Gastaut syndrome, multifocal independent spikes, autoimmunity, cell-mediated immunity, humoral immunity

Two Novel Assays of Alloantibody-Secreting Cells Demonstrating Resistance to Desensitization With IVIG and rATG

Donor-specific alloantibody presents a major barrier to the successful transplantation of kidneys and hearts. However, the study of alloantibody production has been hampered by both an inadequate source of antibody-secreting cells (ASCs) and a paucity of assays to determine their function. We describe two new assays that allow for the determination of the frequency and specificities of allo-ASCs in humans using purified HLA as targets. These assays demonstrated allo-ASCs in the CD138(+) fraction of the bone marrow, but not in peripheral blood. Alloantibody specificities in these assays correlated well with those detected in the serum suggesting that bone marrow-derived ASCs are indeed a major source of alloantibody in vivo. However, ASCs for a specific HLA antigen were rare with an estimated frequency of only 1/2 x 10(6) marrow cells. Pretransplant treatment in vivo with multiple plasmaphereses and low-dose IVIG alone or in combination with rATG had no effect on ASC number or alloantibody production. These techniques allow for the study of allospecific ASCs and provide a method to test the potential efficacy of agents on alloantibody production in vivo.

Correlation of molecular HLA typing and outcome in high-risk melanoma patients receiving adjuvant interferon

8029 Background: Serological typing for both HLA class I and class II antigen expression, has previously shown association of specific HLA antigen expression with clinical response and survival in patients with metastatic melanoma treated with IL-2 (e.g. HLA-DQ1). Purpose: To evaluate the impact of HLA class I (low-resolution) and class II (high-resolution) expression, on the outcome of high-risk melanoma patients receiving adjuvant high-dose interferon. Methods: 181 stage IIB, IIC and III melanoma patients (88 female and 93 male), median age 52.1 years and 246 healthy controls were included in this study. DNA was used for the determination of HLA-A, HLA-B, HLA-Cw, HLA-DRB1 and HLA-DQB1 genotypes. Results: With a median follow-up of 37 months, 59 (group 1) patients have remained with no evidence of recurrence and 122 have recurred (group 2). Statistical significant differences between the two groups, were found in the following genotypes: HLA-A*02 (42% vs. 57.3%, p=0.08), HLA-A*33 (15.2% vs. 6.5%, p=0.05), HLA-B*51 (15.2% vs. 34.4%, p=0.01), HLA-B*57 (11.8% vs. 2.4%, p=0.02). Statistical significant differences between group 1 and healthy controls, were found in the following genotypes: HLA-A*33 (15.2% vs. 6.5%, p=0.05), HLA-B*51 (15.2% vs. 28.5%, p=0.05), HLA-B*57 (11.8% vs. 4.5%, p=0.05), HLA-Cw*03 (23.7% vs. 11%, p=0.01), HLA-Cw*06 (27.1% vs. 16.1%, p=0.06), HLA-DRB1*0701 (27.1% vs. 13.4%, p=0.01), HLA-DRB1*1601 (35.6% vs. 22.3%, p=0.01), HLA-DQB1*0202 (23.8% vs. 10.1%, p=0.09). Conclusions: Statistical significant differences were seen in HLA-A and HLA-B alleles between the patients with high-risk melanoma free of recurrence and those who recurred after treatment with adjuvant interferon. Additionally, differences were seen between healthy controls and melanoma patients free of recurrence. No significant financial relationships to disclose.

The Two-Event Model of Transfusion-Related Acute Lung Injury: Antibodies to HNA-3a Cause PMN Cytotoxicity.

Transfusion related acute lung injury (TRALI) is a serious complication of blood administration. TRALI is neutrophil (PMN)-mediated and the pathogenesis is due to the infusion of antibodies directed against specific HLA (class I or II) or granulocyte antigens and may also be the result of two events: 1) a predisposing clinical condition of the patient resulting in PMN pulmonary sequestration followed by 2) the infusion of biologic response modifiers (BRMs), e.g. lipids from stored blood. These BRMs prime PMNs in vitro and cause PMN cytotoxicity in vivo resulting in endothelial damage, capillary leak and TRALI. We hypothesize that antibodies directed against specific PMN antigens cause rapid PMN priming and cytotoxicity in a two-event model, specific to the antigen they recognize. PMNs were isolated from healthy donors and 5b− (HNA-3a−) donors by standard techniques and were incubated for 3 min with 10% fresh plasma (FP) from healthy donors, plasma from three donors who had antibodies against HNA-3a and were implicated in TRALI, or 10% immune complexes (ICs) made from sera (+ control). Selected wells had Fab′2 fragments against the Fc receptors (CD16, CD32 & CD64). The maximal rate of O2− production was measured as cytochrome c reduction at 550 nm. Priming is defined as augmentation of the fMLP-activated respiratory burst (Table 1). We used an in vitro model of TRALI (Wyman AJP Cell 283: C1592, 2002) in which Human plumonary microvascular endothelial cells (HMVECs) were grown to 90% confluence and incubated with buffer or endotoxin (LPS) [200 ng/ml] for 6 hours. HNA-3a+ PMNs were added ± Fab′2 fragments, allowed to settle, and incubated for 30 min with FP or HNA-3a+ plasma. The plates were forcibly decanted and the viable HMVECs per mm2 were counted (Table 2). HNA-3a+ plasma significantly primed the PMN oxidase as compared to FP-treated controls (Table 1). Pre-treatment of PMNs with Fab′2 inhibited IC priming but did not affect other groups, and HNA-3a+ plasma did not prime HNA-3a− PMNs (data not shown). HNA-3a+ plasma ± LPS without PMNs did not affect HMVEC viability (data not shown), and HMVECs activated with LPS caused widespread PMN adherence. HNA-3a+ plasma plus HNA-3a+ PMNs caused destruction of LPS-activated HMVECs that was partially inhibited by Fab′2 fragments (Table 2). We conclude that HNA-3a+ plasma rapidly, effectively, and specifically primes HNA3+ PMNs. HNA-3a+ plasma can also serve as the second event in a two-event model of PMN-mediated HMVEC damage. Thus, antibodies directed against specific granulocyte antigens and lipids from stored blood may cause TRALI through a common final pathway of PMN activation.HNA-3a+ (5b+) plasma priming of HNA-3a+ PMNsPre-tx/GroupControlICsFFPDonor 1Donor 2Donor 3Buffer2.7±0.58.8±0.7†2.5±0.45.7±1.8†6.5±†5.0±1.0†Fab′22.1±0.83.2±0.4*3.6±0.93.8±0.74.6±1.54.0±1.0Units=nmol O2−/min; †=P<0.05 versus FFP controls; *=p<0.05 versus ICs.HNA-3a+ (5b+) plasma causes PMN cytotoxicityPre-tx/GroupControlLPS/PMNsLPS/PMNs/FFPLPS/PMNs/5b plasmaBuffer245±1234±8229±9151±7†Fab′2no datano data240±9190±*Units=viable HMVECs/mm2; †=p<0.05 versus Controls and LPS/PMNs/FFP; *=p<0.05 versus LPS/PMN/5b+ plasma.

The Use of Algorithms with a Novel Score Standardization Method for the Selection of Candidate Immunogenic HLA-Specific Peptides Led to the Identification of New Immunogenic HLA-A*0201 Cytomegalovirus Epitopes.

The identification of HLA class I restricted epitopes within immunogenic proteins that are capable of stimulating memory T lymphocytes is an important step for both clinical vaccine and adoptive immunotherapy. Despite the strong immunogenicity demonstrated by specific HLA restricted peptides, the peptide's affinity to a specific HLA antigen may vary among individuals. Therefore, it is important to identify more than one immune determinant for each HLA antigen of interest. Two computer algorithms, one by Parker and another by Rammensee, are widely used in immunogenetic studies in order to predict which peptides bind best to specific HLA molecules. The use of these algorithms represents the first step in screening proteins. Parker's algorithm ranks potential peptide according to the predictive half-time disassociation of peptide/MHC complexes. Rammensee's algorithm ranks the peptides based on the presence of primary and secondary HLA-binding anchor residues. Starting with the sequence of Cytomegalovirus (CMV) phosphorylated matrix protein 65 (pp65; SwissProt ID: P06725), we derived from each algorithm a list of 250 nonamer (9-mer) peptides predicted to bind to the HLA-A*0201 allele. Standardizing the two groups of scores, corresponding to the endpoints of the stated interval, by the following formula: [(peptide score - mean of scores)/ standard deviation of scores], we obtained pure values that are independent of the unit of measurement, therefore homogeneous and thus comparable. This allowed us to link each peptide to a pair of values, one for each algorithm. The values derived from standardized Parker scores (X axis) were plotted against the values derived from standardized Rammensee scores (Y axis). We represented final results by a graph. All peptides having positive values with both algorithms and included in the first quadrant of the dot-scatter graph (double positive quadrant (+/+)) were likely to be highly immunogenic epitopes. Those peptides were included in a list of potential peptides to screen. In the presence of homoschedasticity, we selected only peptides showing mean values greater than 1 (among those having both the mean value greater than 0 and the standard deviation value greater than 1). Nine out of the 250 peptides were thus chosen within the CMV pp65 and their immunogenicity analyzed by ex vivo stimulation of PBMC and measured at the transcriptional level using the production of IFN-γ mRNA. Four out of nine peptides stimulated PBMCs from all HLA-A*0201 restricted CMV-seropositive subjects tested (p values: peptide #5, 0.011032; peptide #7, 0.015225; peptide #8, 0.025221; and peptide #9, 0.007302). The intracellular IFN-γ protein production by ex vivo peptide stimulation ranged from 0.14 to 0.62% compared to 0.03% for non stimulated cells. Moreover, each of the four selected peptide, either alone or in pool, was able to reactivate in vitro a strong immune T cell memory response, as measured by cytokine protein release and target cell lysis. The identification of multiple HLA class I restricted epitopes will allow the simultaneous administration of peptides with the ability to produce CMV-specific CTLs in patients bearing the same HLA types. This represents an extraordinary advantage for CMV adoptive immune therapy or vaccination.