Risk associations between HLA-DPB1 T-cell epitope matching and outcome of unrelated hematopoietic cell transplantation are independent of HLA-DPA1.

HLA-DP antigens are beta-alpha heterodimers encoded by polymorphic HLA-DPB1 and -DPA1 alleles, respectively, in strong linkage disequilibrium (LD) with each other. Non-permissive unrelated donor (UD)-recipient HLA-DPB1 mismatches across three different T-cell epitope (TCE) groups are associated with increased mortality after hematopoietic SCT (HCT), but the role of HLA-DPA1 is unclear. We studied 1281 onco-hematologic patients after 10/10 HLA-matched UD-HCT facilitated by the National Marrow Donor Program. Non-permissive mismatches defined solely by HLA-DPB1 TCE groups were associated with significantly higher risks of TRM compared to permissive mismatches (hazard ratio (HR) 1.30, confidence interval (CI) 1.06-1.53; P=0.009) or allele matches. Moreover, non-permissive HLA-DPB1 TCE group mismatches in the graft versus host (GvH) direction significantly decreased the risk of relapse compared to permissive mismatches (HR 0.55, CI 0.37-0.80; P=0.002) or allele matches. Splitting each group into HLA-DPA1*02:01 positive or negative, in frequent LD with HLA-DPB1 alleles from two of the three TCE groups, or into HLA-DPA1 matched or mismatched, did not significantly alter the observed risk associations. Our findings suggest that the effects of clinically non-permissive HLA-DPB1 TCE group mismatches are independent of HLA-DPA1, and that selection of donors with non-permissive DPB1 TCE mismatches in GvH direction might provide some protection from disease recurrence.

A phase II study of sirolimus, tacrolimus and rabbit anti-thymocyte globulin as GVHD prophylaxis after unrelated-donor PBSC transplant.

We report on a prospective phase II trial of 32 patients who underwent unrelated-donor hematopoietic cell transplantation, with a tacrolimus, sirolimus and rabbit anti-thymoctye globulin GVHD prophylactic regimen. The primary study endpoint was incidence of grades II-IV acute (aGVHD), with 80% power to detect a 30% decrease compared with institutional historical controls. Median age at transplant was 60 (19-71). In total, 23 patients (72%) received reduced-intensity conditioning, whereas the remainder received full-intensity regimens. Median follow-up for surviving patients was 35 months (range: 21-49). The cumulative incidence of aGVHD was 37.3%, and the 2-year cumulative incidence of chronic GVHD was 63%. We observed thrombotic microangiopathy in seven patients (21.8%), one of whom also developed sinusoidal obstructive syndrome (SOS). Four of the 32 patients (12.5%) failed to engraft, and 3 of these 4 died. As a result, enrollment to this trial was closed before the targeted accrual of 60 patients. Two-year OS was 65.5% and EFS was 61.3%. Two-year cumulative incidence of relapse was 12.5% and non-relapse mortality (NRM) was 15.6%. NRM and aGVHD rates were lower than historical rates. However, the unexpectedly high incidence of graft failure requires caution in the design of future studies with this regimen.

Incidence of humoral sensitization in HLA partially mismatched hematopoietic stem cell transplantation.

As part of the 15th International Histocompatibility and Immunogenetics Workshop (IHIWS), seven centers participated in a collaborative project to determine whether any significant humoral sensitization occurred post-transplant among recipients of HLA partially mismatched hematopoietic cell transplants (HCTs). A total of 140 donor/recipient pairs were enrolled with a total of 367 pre-and post-transplant sera analyzed. The majority of the samples (69.1%) were obtained within 30-90 days post-HCT. HLA-specific antibodies were defined using single antigen bead assays on a Luminex platform with a positive cutoff value of 1000 normalized median fluorescence intensity (MFI). There was an overall incidence of post-HCT sensitization toward donor HLA mismatches of 5.7%; however, all cases were among recipients of one HLA haplotype-mismatched grafts under nonmyeloablative, pre-transplant conditioning. Among the one haplotype-mismatched recipients, 15.7% (8/51) developed donor HLA-specific antibodies and 29.4% also had antibodies directed toward third party HLA antigens. Among the donor-specific antibodies, 9.8% were directed toward HLA class I antigens; 7.8% were against class II antigens; and 2.0% had both class I and II specificity. The relative strength of post-transplant antibodies was low with no significant difference in the mean maximum MFI values between third party and donor-specific antibodies. Because only a small number (10.2%) of the post-transplant samples were obtained 180 days or more post-HCT, longer term study is needed to evaluate any clinical relevance of these low-to-moderate levels of donor-specific antibody in one haplotype-mismatched recipients, as well as to determine whether any other antibodies occur at later times.

Feasibility of conditioning with thymoglobulin and reduced intensity TBI to reduce acute GVHD in recipients of allogeneic SCT.

Murine studies using anti-T-cell antibodies for conditioning in allogeneic SCT demonstrate engraftment with low rates of GVHD. On the basis of this preclinical model, we conditioned 30 patients with advanced hematologic malignancies with rabbit antithymocyte globulin (ATG) and TBI, to reduce rates of fatal acute GVHD. Patients were enrolled in two sequential groups: cohort 1 received ATG 10 mg/kg in divided doses (days -4 to -1)+200 cGy TBI (n=16), and cohort 2 received ATG (days -10 to -7)+450 cGy TBI (n=14). Median donor blood chimerism for the combined group was 94, 93 and 93% in the first, second and third months after transplant. Only three developed grade II acute GVHD despite 43% of patients receiving unrelated donor transplants. One-year survival was 71+/-11 and 54+/-14%, respectively, in recipients of related and unrelated donor SCT. Donor lymphocyte infusions were needed in 12 patients for the management of relapse and for mixed donor-recipient chimerism in 4 patients. We conclude that 10 mg/kg ATG and TBI allows engraftment with a low risk of acute GVHD; however, further dose optimization of ATG is required to achieve a balance between GVHD and disease relapse.

Killer Ig-like receptor (KIR) compatibility plays a role in the prevalence of acute GVHD in unrelated hematopoietic cell transplants for AML.

Killer Ig-like receptor (KIR) is a major cluster of the natural killer cell receptors and may play a role in the outcome of hematopoietic cell transplants. A total of 65 AML cases transplanted with T-replete hematopoietic cells from unrelated donors were retrospectively KIR-genotyped by a multiplex PCR method of our own design. The KIR gene frequency and genotype patterns in these 130 samples were consistent with the data in the literature. Based upon overall inhibitory and activating KIR genes in both donors and patients, we developed an algorithm to calculate a compatibility score for each transplant case as plus, zero or minus. Significantly higher incidence (18/20, 90%) of acute (a) GVHD (grade II-IV) was found in the transplant cases with plus scores than that (25/45, 56%) in the cases with zero or minus scores (P < 0.01). When the scores are sorted in the opposite way, fewer cases (13/26, 50%) of aGVHD were found in the transplants with minus scores than that (30/39, 77%) in the transplants with zero or plus scores (P < 0.05). The difference of aGVHD prevalence between the plus score and minus score groups is highly significant (P < 0.01). KIR genotype compatibility calculated by this algorithm may predict aGVHD incidence and be helpful in choosing donors.

Development of a multiplex PCR-SSP method for Killer-cell immunoglobulin-like receptor genotyping.

Killer-cell immunoglobulin-like receptors (KIRs) on natural killer (NK) cells recognize groups of HLA class I alleles. Recent work suggests that KIR genotype may affect the outcome of hematopoietic stem-cell transplants and that prospective KIR typing maybe of benefit in future matching of donors and recipients. A simple and informative KIR genotyping method was developed using a multiplex polymerase chain reaction-sequence-specific primer strategy. This method contains four multiplex reactions for detecting all functional KIR genes, including some 2DS4 variants that harbor a common deletion. Primer pairs were designed to provide short amplicons (108-565 bp) that can be analyzed by agarose gel electrophoreses or by automated electrophoretic systems. This method was evaluated in a blinded survey with the NK/KIR Phase II QC Panel (a total of 16 cell lines) from the 14th International Histocompatibility Workshop (IHWS), and the results are 100% concordant with the consensus genotype. Results in further KIR genotyping of 20 reference cell lines from the 10th IHWS were consistent with previously published genotypes, matching those of one study in instances where different genotypes have been previously reported. The genotypes obtained in this study may be helpful to other labs developing KIR genotyping methods in resolving typing discrepancies and in detecting common deletion variants of 2DS4. This method can save labor and reagent costs. It provides good results from partially degraded template DNA due to short amplicons in this method. It is convenient to use in both clinical and research laboratories.

Cyclosporine and mycophenolate mofetil prophylaxis with fludarabine and melphalan conditioning for unrelated donor transplantation: a prospective study of 22 patients with hematologic malignancies.

In an attempt to decrease toxicity in high-risk patients undergoing unrelated donor hematopoietic stem cell transplantation (URD HSCT), we tested a combination of cyclosporine (CSP) and mycophenolate mofetil (MMF) as graft-versus-host disease (GVHD) prophylaxis with the reduced-intensity conditioning regimen fludarabine/melphalan (Flu/Mel). A total of 22 adult patients with advanced myeloid (n=15) and lymphoid (n=7) malignancies were treated. All patients received Flu 25 mg/m2 for 5 days and Mel 140 mg/m2, with CSP 3 mg/kg daily and MMF 15 mg/kg three times a day. The median age was 49 years (range 18-66). Durable engraftment was seen in all but one patient with myelofibrosis. The 1-year nonrelapse mortality was 32%, 27% from GVHD. The cumulative incidence of acute GVHD grade 2-4 and 3-4 was 63 and 41%, respectively. With a median follow-up of 18 months, the disease-free survival (DFS) and overall survival (OS) are 55 and 59%, respectively. For patients with AML and MDS (n=14), the DFS and OS is 71%. For patients undergoing a second transplant (n=14), the DFS and OS is 57%. In conclusion, this regimen is associated with acceptable toxicity but high rates of GVHD in high-risk patients undergoing URD HSCT. Encouraging disease control for patients with advanced myeloid malignancies was observed.

Reduced-intensity allogeneic stem cell transplantation for patients whose prior autologous stem cell transplantation for hematologic malignancy failed.

Autologous hematopoietic stem cell transplantation (autoSCT) is an effective treatment for patients with various hematologic malignancies. Despite the significant improvement in the overall outcome, disease progression after transplantation remains the major cause of treatment failure. With longer follow-up, therapy-related myelodysplasia/acute myelogenous leukemia is becoming an important cause of treatment failure. The prognosis for these 2 groups of patients is very poor. Allogeneic hematopoietic stem cell transplantation (alloSCT) is a potential curative treatment for these patients. However, the outcome with conventional myeloablative alloSCT after failed autoSCT is typically poor because of high transplant-related mortality. In an attempt to reduce the treatment-related toxicity, we studied a reduced-intensity conditioning regimen followed by alloSCT for patients with progressive disease or therapy-related myelodysplasia/acute myelogenous leukemia after autoSCT. This report describes the outcomes of 28 patients with hematologic malignancies who received a reduced-intensity alloSCT after having treatment failure with a conventional autoSCT. Fourteen patients received a hematopoietic stem cell transplant from a related donor and 14 from an unrelated donor. The conditioning regimen consisted of low-dose (2 Gy) total body irradiation with or without fludarabine in 4 patients and the combination of melphalan (140 mg/m(2)) and fludarabine in 24. Cyclosporine and mycophenolate mofetil were used for posttransplantation immunosuppressive therapy, as well as graft-versus-host disease (GVHD) prophylaxis, in all patients. All patients engrafted and had >90% donor chimerism on day 100 after SCT. Currently, 13 patients (46%) are alive and disease free, 7 patients (25%) developed disease progression after alloSCT, and 8 (32%) died of nonrelapse causes. Day 100 mortality and nonrelapse mortality were 25% and 21%, respectively. With a median follow-up of 24 months for surviving patients, the 2-year probabilities of overall survival, event-free survival, and relapse rates were 56.5%, 41%, and 41.9%, respectively. Six patients (21%) developed grade III to IV acute GVHD. Among 21 evaluable patients, 15 (67%) developed chronic GVHD. We conclude that (1) reduced-intensity alloSCT is feasible and has an acceptable toxicity profile in patients who have previously received autoSCT and that (2) although follow-up was short, a durable remission may be achieved in some patients who would otherwise be expected to have a poor outcome.

Soluble interleukin-2 receptor levels in lupus nephritis.

Soluble interleukin-2 receptor (IL-2R) levels were measured and correlated prospectively with clinical, histologic and serologic findings over a 9-month period in 62 lupus patients. Initially, 39 patients had clinical nephritis and 23 patients did not have nephritis. The 62 lupus patients has significantly higher IL-2R than 15 normal controls, most of this difference attributable to patients with nephritis. During lupus nephritis flare 9 of 10 patients showed significant elevations of IL-2R while only 6 of the 10 patients showed either elevation of anti-DNA antibody or decrease in CH50. During disease remission or stable clinical activity changes in IL-2R levels paralleled changes in anti-DNA antibody and CH50. Nephritis patients with cellular proliferative histology had significantly higher IL-2R levels than those with membranous or mesangial nephropathy. IL-2R correlated strongly with histologic activity and chronicity indices, IgG and C3 deposition whereas anti-DNA antibody and CH50 levels did not. IL-2R levels did not correlate with serum creatinine suggesting that elevations of IL-2R were not simply due to decreased clearance. These observations suggest that serum IL-2R level is a useful marker of disease activity in lupus nephritis and may serve as a helpful adjunct in management of this disorder.

HLA associations in heroin-associated nephropathy.

Heroin-associated nephropathy (HAN) occurs almost exclusively in black heroin abusers, suggesting a genetic link to the disease. To further study this possibility, the frequencies of HLA-A, B, C, and DR antigens were determined in a group of 47 black patients with HAN. Included in the analyses is a subgroup of 16 patients with biopsy-proven focal glomerulosclerosis. Patient frequencies were compared with three separate control populations, the first a normal black population from New York City, the second from a national registry, and the third a group of blacks with idiopathic focal glomerulosclerosis. Only the frequency of HLA-BW53 was consistently increased significantly in the patients as compared with the control groups. This finding supports the notion that a genetic predisposition may exist in the addicted population for the development of renal disease.

Recurrent membranoproliferative glomerulonephritis type 1 in successive renal transplants.

We report a man who developed renal failure due to membranoproliferative glomerulonephritis (MPGN) type 1 which recurred in two cadaveric kidney transplants. This is the third such case in the literature. Nephrotic syndrome developed within 1 month following transplantation and histologic evidence of disease recurrence was documented in both kidneys 2 months after transplantation. Both grafts failed within 18 months. Factors which determine disease recurrence remain obscure.

Ethylnitrosourea-induced transplacental carcinogenesis in the mouse: tumor response, DNA binding, and adduct formation.

We have confirmed previous results which suggest that transplacental exposure of fetal mice to carcinogens does not cause an increase in tumor incidence as they mature unless treatment occurs after midorganogenesis. In C3HeB/FeJ mice we found a negligible increase in tumor incidence and multiplicity following transplacental exposure to the direct-acting carcinogen ethylnitrosourea (ENU) on gestation day 10, but significant increases in lung and liver tumor incidence following exposure on days 13 or 15 or in adults. To explore the possibility that this observed difference is due to differences in the biodistribution of the carcinogen or its interaction with cellular macromolecules, the level of covalent binding between ENU and fetal and maternal DNA following an i.p. injection of a dose of 50 mg/kg of tritium-labeled ENU was measured 30 min after its injection into pregnant females on days 10, 13, and 15 of gestation. The DNA from fetal and maternal lung, liver, and brain was isolated and the amount of covalent binding estimated from the dpm/mg DNA recovered. Samples of DNA were hydrolyzed and chromatographed to determine that the bound tritium was associated with ENU-DNA adducts and not as a product of DNA synthesis. The level of binding of ENU to fetal DNA was equivalent at all gestation days studied but was significantly less than maternal tissues. Binding to the DNA of maternal liver was 4-fold greater than to fetal DNA while maternal lung and brain DNA were bound at intermediate levels. We conclude that the lack of carcinogenic response to ENU documented here, in fetal mice exposed early in gestation (day 10), is not due to differences in ENU binding to fetal DNA during development.

Infectious antecedent of immunoblastic lymphoma. Progressive immunosuppression in a patient with lymphogranuloma venereum.

Angioimmunoblastic lymphadenopathy is a nonmalignant disease of unknown etiology often progressing to immunoblastic lymphoma. Immunologic deficiency is evident in these patients as well as in those with various infections found in association with the acquired immune deficiency syndrome (AIDS). This report describes a previously healthy young woman in whom angioimmunoblastic lymphadenopathy developed in association with lymphogranuloma venereum, with progressive loss of immunologic competence. This deterioration paralleled the evolution of angioimmunoblastic lymphadenopathy into a rapidly fatal immunoblastic lymphoma.

Protein A-binding immunosuppressive mouse serum factors.

In this study protein A of Staphylococcus aureus has been used to isolate an immunosuppressive component present in mouse serum. The suppressive effect of mouse serum on lymphocyte activation was partially abrogated by prior adsorption on protein A, and also by ammonium sulfate precipitation or specific immune precipitation with anti-IgG but not with anti-IgM. Protein A-binding material was isolated after chromatography on protein A-Sepharose and studied in spleen cell cultures. Protein A eluates from normal or NZB/NZW mice were found to suppress concanavalin A (Con A)-activated normal mouse spleen cells, and suppression was more potent with NZB/NZW serum isolates. Suppressive activity was dependent upon the dose of eluate added to cell cultures. The suppressive effect of NZB/NZW protein A-binding material was apparent in both Con A- and lipopolysaccharide (LPS)-stimulated normal mouse spleen cells, and required early addition to the cell cultures or preincubation with target lymphocytes. The suppressive activity was not detectably cytotoxic during a suppressive preincubation period. The possible relevance of these observations to experimental strategies in tumor immunotherapy is discussed.

Effect of mitogenic stimulation of murine splenocytes on PEG-induced cell fusion.

A cell fusion assay system was devised as a means of measuring cell activation. Polyethylene glycol (PEG) was used to induce fusion between spleen cells of various mouse strains and the BW5147 thymoma cell line. A fusion index (FI) was calculated by determining the ratio of the number of nuclei in fused cells to the number of nuclei in all cells and multiplying by 100 (the FI could range from 0 to 100). Spleen cells from BALB/c mice were compared in PEG-induced fusion assays. BALB/c spleen cells stimulated with phytohemagglutinin, leucoagglutinin, concanavalin A, pokeweed mitogen and LPS showed FI two- to three-fold higher than those found in unstimulated cultures, indicating that stimulated cells fuse at much higher rates. This response is mitogen dose-dependent and parallels DNA synthesis as measured by 3H-TdR incorporation. Treatment of spleen cells with cycloheximide 12 h prior to fusion had no effect on FI. In vivo and in vitro stimulation of BALB/c, C57BL/6 and NZW mice with LPS resulted in enhanced FI. This was not the case in low-responder DBA/2 and nonresponder C3H/HeJ animals.

Purification and characterization of mammary myoepithelial and secretory cells from the lactating rat.

Myoepithelial and secretory cells from the mammary gland of the lactating rat have been isolated, purified, and characterized. Mammary tissue was dissociated with collagenase into basket-like networks of myoepithelial cells and single secretory cells. Because of their larger size, the myoepithelial cell networks could be separated from other mammary and blood cells by differential centrifugation. Isolated secretory cells were purified by isopycnic centrifugation in 25% bovine serum albumin. The purified myoepithelial and secretory cells were viable, as shown by the incorporation of 32P into distinct macromolecules that were separable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both myoepithelial and secretory cells retained their characteristic morphology after isolation and purification, as shown by light, transmission, and scanning electron microscopies. The isolated myoepithelial cells were unique and, thus, distinguishable from other mammary cells in a number of respects; they 1) contracted in response to the addition of oxytocin, 2) bound [3H]oxytocin specifically, 3) accounted for the content of alkaline phosphatase and [Na+ + K+]ATPase in mammary tissue, and 4) reacted specifically with antiserum prepared against purified myoepithelial cells. The purified secretory cells were unique in possessing glucose-6-phosphate dehydrogenase activity. The different cell markers not only gave independent estimates of the purity of the cell fractions, but they also may be helpful in identifying mammary cells in stages of differentiation and neoplastic transformation.