Surface markers of cloned human T cells with various cytolytic activities.

Human T cells stimulated in secondary allogeneic mixed lymphocyte culture (MLC) were cloned under limiting conditions in microculture systems using T cell growth factor and irradiated allogeneic cells. Clones with lytic activity against either phytohemagglutinin-induced blast cells bearing the stimulating alloantigen(s) (cytotoxic T lymphocyte [CTL] activity), L1210 mouse lymphoma cells coated with rabbit antibody (antibody-dependent cell-mediated cytotoxicity [ADCC]), or K562 human target cells were selected, expanded, and then analyzed for different surface markers, including rosette formation with sheep erythrocytes (E rosettes), receptors for the fc portion of IgG or IgM (Fc gamma R and Fc mu R), and a group of antigens recognized by monoclonal antibodies including Ia, 4F2, OKT8,a nd OKT4. All the cytotoxic cells were E rosette+, Ia+ and 4f2+. Expression of Fc gamma R was restricted to the clones active in ADCC. CTL clones were either OKT8+ or OKT8-. Furthermore, three of the OKT8- CTL clones were OKT4+. In addition, some cytolytic clones devoid of specific CTL activity were OKT8+. It thus appears that the claim that human CTL are OKT8+, OKT4-, and Ia- is not supported by the analysis of their phenotype at the clonal level.

Atrial fibrillation is associated with hypermethylation in human left atrium, and treatment with decitabine reduces atrial tachyarrhythmias in spontaneously hypertensive rats.

Atrial fibrillation (AF) is the most common cardiac arrhythmia. As the molecular mechanisms underlying the pathology are largely unknown, this cardiac arrhythmia remains difficult to treat. To identify specific molecular actors involved in AF, we have performed a transcriptomic analysis on left atrium (LA) from patients with valvular heart disease with or without AF. We showed that 1627 genes had altered basal expression level in LA tissue of AF patients compared with the control group. The significantly enriched gene ontology biological process "anatomical structure morphogenesis" contained the highest number of genes in line with changes in structure that occur when the human heart remodels following AF development (ie, LA dilatation and interstitial fibrosis). We then focused the study on Pitx2 (paired-like homeodomain 2), being the most altered transcription factor in LA from AF patients and from which compelling evidence have indicated that its reduced expression can be considered as a marker for the disease. In addition, its expression was inversely correlated with LA size. We demonstrated that AF is associated with Pitx2 promoter hypermethylation both in humans and arrhythmic aging spontaneously hypertensive rats. Chronic administration of a DNA methylation inhibitor (ie, 5-Aza-2'-deoxycitidine) improved ECG arrhythmic profiles and superoxide dismutase activities and reduced fibrosis in the left ventricle of spontaneously hypertensive rats. Taken together, these data support the notion that AF is associated with epigenetic changes in LA and provide a proof-of-concept that hypomethylating agents have to be considered in the treatment of atrial arrhythmias.

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.

Renal toxicity after allogeneic bone marrow transplantation: the combined effects of total-body irradiation and graft-versus-host disease.

PURPOSE: To evaluate retrospectively the cumulative risk probability and factors correlated with renal dysfunction after allogeneic bone marrow transplantation (BMT). PATIENTS AND METHODS: From October 1984 to July 1994, 84 patients with malignant hematopoietic diseases received allogeneic BMT after conditioning with high-dose chemotherapy and total-body irradiation (TBI). Seventy-nine patients with normal renal function before conditioning are included in this study. Conditioning included high-dose cyclophosphamide without (n = 46) or with (n = 33) other agents (daunorubicin, busulfan, cytarabine, and thiotepa) followed by TBI. The TBI dose prescribed to the center of the abdomen was 10 Gy for 24 patients, 12 Gy for 32, and 13.5 Gy for 23. In vitro T-cell depletion was undertaken in 48 cases. The post-BMT nephrotoxicity of aminoglycosides, vancomycin, amphotericin, and cyclosporine was assessed. Time to renal dysfunction was defined as the time to a persistent increase of serum creatinine (SCr) level greater than 110 mumol/L. The potential influence of sex, age, diagnosis, chimerism, and graft-versus-host disease (GvHD) on renal dysfunction was also assessed. RESULTS: The 18-month probability of renal dysfunction-free survival (RDFS) for the whole group was 77%. Only TBI dose and presence of GvHD were significantly correlated with renal dysfunction by multivariate analysis. The 18-month probabilities of RDFS were 95%, 74%, and 55% for the patients conditioned with 10, 12, and 13.5 Gy, respectively. The 18-month RDFS probabilities were 88% and 61% for patients without and with GvHD, respectively. Combining both variables, we have defined two risk categories: low-risk (ie, 10 Gy TBI with/without GvHD and 12 Gy TBI without GvHD) and high-risk (ie, 12 Gy TBI with GvHD and 13.5 Gy TBI with/without GvHD). The predicted 18-month RDFS rates were 93% and 52% for the low- and high-risk groups, respectively. CONCLUSION: Renal dysfunction after allogeneic BMT is strongly related to the delivered TBI dose (and dose per fraction) and to the presence of GvHD. Renal shielding should be recommended if a TBI dose greater than 12 Gy (fractionated twice daily over 3 days) is to be prescribed. Furthermore, in those cases with a high risk of developing GvHD (eg, unrelated allogeneic BMT, absence of T-cell depletion), these data suggest that kidney doses greater than 10 Gy should be avoided.

Stem cell transplantation from identical twins in patients with myelodysplastic syndromes.

In a multicentre retrospective EBMT database study, we analysed factors influencing outcome in 38 patients with MDS/sAML who were transplanted with stem cells from their syngeneic twin and compared those to 1444 patients who were transplanted from an HLA-identical sibling. The median time to leukocyte and platelet engraftment was faster in the twin group: 14 vs 17 (P=0.02) and 16 vs 26 days (P=0.09), respectively. The 5 years cumulative incidence of treatment-related mortality (TRM) was higher in the sibling than in the twin group (38 vs 27%; P=0.05). The 5 year cumulative incidence of relapse was 32% (95% CI: 29-35%) for the siblings and 39% (95% CI: 26-60%; P=0.6) for the twins. A trend for better 5-years disease-free and overall survival was observed in the twin group: 34% (95% CI: 14-54%) vs 28% (95% CI: 25-31%; P=0.2) and 36% (95% CI: 15-57%) vs 32% (95% CI: 29-35%; P=0.09), respectively. In a multivariate analysis, stem cell transplantation from identical twins had a lower TRM: HR: 0.4 (95% CI: 0.2-0.9; P=0.03). The relapse rate was similar for both groups with a HR of 1.2 (95% CI: 0.07-2.1; P=0.5), with a better survival for the twins: HR 0.6 (95% CI: 0.4-1.0; P=0.07). We conclude that twin transplantation in MDS/sAML is associated with a similar relapse risk, a lower TRM and a trend for better overall survival in comparison to transplantation from HLA-identical siblings.

Malignant histiocytosis in the leukaemic stage: a new entity (M5c-AML) in the FAB classification?

Malignant histiocytosis (MH) is a rare, rapidly fatal disorder, characterized by systemic proliferation of abnormal histiocytes. Most patients present with pancytopenia. We report the case of a patient with MH in the leukaemic stage, who presented extremely pronounced general symptoms and multisystemic involvement. Determination of serum cytokines showed high levels of tumor necrosis factor (TNF), interleukin 6 (IL-6) and interleukin 1 receptor antagonist (IL-1ra). Cytogenetic studies proved the monoclonality of the histiocytic proliferation. These findings strongly suggest that we are dealing with a proliferation of activated macrophages (= histiocytes). By analogy with the monoblastic (M5a) and monocytic (M5b) acute leukaemia of the French-American-British (FAB) classification, we propose a new entity, 'M5c', designating acute histiocytic leukaemia.

Optimized lentiviral transduction of erythroid precursors from healthy adults and patients with myelodysplastic syndromes.

Lentivectors, derived from human immunodeficiency virus-1 (HIV-1), represent a novel investigational and therapeutic tool for targeting hematopoietic progenitor cells. We describe a new protocol whereby we achieved a highly efficient lentiviral transduction of erythroid precursor cells originating from the bone marrow of healthy adults and patients with myelodysplastic syndromes (MDS). CD34(+) stem cells from healthy subjects were cultured with erythropoietin, IL-3 and stem cell factor, and thereby expanded approximately 300-fold. When these cultures were transduced with a lentiviral vector expressing GFP as a reporter gene, 70% glycophorin(+) cells were GFP(+). Although proliferation and levels of transduction were reduced in cultures of CD34(+) stem cells from patients with myelodysplastic syndromes, 50% of glycophorin(+) cells became GFP(+), amongst which 30% were sideroblastic erythroid precursors. This study demonstrates that lentiviral vectors are capable of efficiently transducing MDS precursors and offers new perspectives to investigate the influence of specific genes on normal erythroid differentiation. This may eventually help to correct defects in patients suffering from myelodysplastic syndromes.

Quantity and nature of residual bone marrow T cells after treatment of the marrow with Campath-1.

Precise characterization of T-cell depletion in marrows used for transplantation is necessary for the evaluation of their potential contribution to engraftment and to graft-versus-host disease. A limiting dilution culture method that allows the determination of small numbers of bone marrow T cells is described. It can detect less than one T cell in 10(4) cells. Bone marrow T-cell depletion with the rat monoclonal antibody Campath-1 and fresh human complement results in a median decrease of 1.7 log (range, 1.6-2.1 log) of marrow T cells. A 2.7 to 3.3-log reduction is obtained when peripheral blood T cells are treated. A second incubation with fresh complement removes additional T cells from peripheral blood and from marrow samples, indicating the importance of bystander marrow cells to complement activity. The assay system described also allows the phenotypic study of responding cells growing in culture. These studies demonstrate that, after treatment with Campath-1 plus complement, no T-subset imbalance occurs. Furthermore, the T cells in these cultures are derived from mature T cells contained in the samples.

CD34(+) cord blood cells expressing cutaneous lymphocyte-associated antigen are enriched in granulocyte-macrophage progenitors and support extensive amplification of dendritic cell progenitors.

OBJECTIVE: We evaluated the frequency of hematopoietic progenitor cells (HPC) in CD34(+)CLA(+) (cutaneous lymphocyte-associated antigen) and CD34(+)CLA(-) cord blood cells, and followed cellular growth and HPC production during cultures in Flt3 ligand, thrombopoietin, and stem cell factor (FTS). MATERIALS AND METHODS: Immunomagnetic bead-purified CD34(+) cells were sorted into CD34(+)CLA(+) or CD34(+)CLA(-) cells. HPC frequency was assessed by clonal assays in methylcellulose either ex vivo or after, 7, 14, or 21 days of culture with FTS. Dendritic cell (DC) progenitors were evaluated after induction of FTS-amplified cells into DC using secondary cultures containing granulocyte-macrophage colony-stimulating factor and interleukin-4. RESULTS: Ex vivo, granulocyte-macrophage progenitors were more frequent and erythroid progenitors were less frequent in the CLA(+) fraction. In FTS culture, CD34(+)CLA(+) cells produced greater absolute numbers of CD34(+) cells, granulocyte-macrophage-, erythroid-, and DC (including Langerhans cell-related) progenitors compared to CD34(+)CLA(-) cells. In CD34(+)CLA(+) cultures, CLA(+) cells steadily decreased with time, and CD34(+)CLA(-) cells appeared. In CD34(+)CLA(-) cultures, CLA(+) cells were generated, increased up to day 7, and decreased thereafter. CLA was expressed only on CD34(-) cells in these cultures. Ex vivo, CD34(+)CLA(+) cells could be subdivided further into CD38(low) and CD38(high) cells. Cord blood and growth factor-mobilized CD34(+) cells contained more CLA(+)CD38(low) cells than nonmobilized peripheral blood CD34(+) cells and proliferated more extensively with FTS than the latter cells. CONCLUSIONS: CD34(+)CLA(+) cells contain a rather immature progenitor capable of high proliferation and extensive amplification of HPC in vitro. This progenitor may be localized in the CD34(+)CLA(+)CD38(low) fraction. In addition, cultures of CD34(+)CLA(+) cells from cord blood produced CD34(+)CLA(-) cells, suggesting that these cells may derive directly from CD34(+)CLA(+) cells in vivo.

Invasive aspergillosis. Clinical features of 35 proven cases at a single institution.

Thirty-five patients with clinical features and histologically or microbiologically proven infection met predetermined stringent criteria for invasive aspergillosis over a 5-year period at our institution. Underlying conditions included hematologic malignancy, solid tumor, bone marrow and solid organ transplantation, and immunosuppressive therapy. The majority of patients (94%) presented with respiratory symptoms and abnormal pulmonary chest radiography; only 40% had neutropenia at time of infection. Invasive aspergillosis was suspected in only 21 cases (60%). Concomitant infections were present in 83% of patients. Half of patients had pathogenic or potentially pathogenic microorganisms other than Aspergillus spp. isolated from pulmonary specimens at time of aspergillosis. Aspergillus spp. were recovered from sputum in 75% of patients and from bronchoalveolar lavage in only 52%. Invasive aspergillosis is an unexpectedly unrecognized disease with poor outcome; overall mortality was 94% in our series. The lack of sensitivity of diagnostic procedures, together with the high frequency of concomitant infections, delays the time of diagnosis. Early diagnostic tests are needed, and presumptive antifungal therapy among high-risk patients is mandatory.

An efficient method for purification of human T-cell growth factor.

A 1000-fold purification of human T-cell growth factor (TCGF) was achieved starting from supernatants of human spleen cells stimulated with phytohaemagglutinin (PHA) in culture medium containing 0.5% serum. The purification scheme involved precipitation with ammonium sulphate, gel filtration and blue-Sepharose chromatography. The use of polyethylene glycol 6000 (PEG 6000) was critical during the chromatographic steps in order to obtain high final recoveries or activity (40-50%). Purified preparations of TCGF labelled with 125I by the chloramine T method revealed that the activity co-migrated with 2 molecular species of 14,000-17,000 daltons in SDS-PAGE under non-reducing conditions.

Total body irradiation before allogeneic bone marrow transplantation: is more dose better?

PURPOSE: This study was performed to retrospectively assess the potential influence of total-body irradiation (TBI) dose on overall survival in patients undergoing allogeneic bone-marrow transplants (BMT) for hematologic malignancies. METHODS AND MATERIALS: Between October 1984 and December 1996, 116 patients were conditioned with high-dose chemotherapy and fractionated TBI before allogeneic BMT. The median age was 34 years (range 3-60). The TBI dose was given in 6 fractions, twice-a-day, over 3 days before BMT. The total dose was 10 Gy in 24 patients, 12 Gy in 66 patients, and 13.5 Gy in 26 patients. RESULTS: TBI dose was inversely correlated with overall survival. Five-year survival was 62% for patients conditioned with 10 Gy, 55% for patients conditioned with 12 Gy, and 46% for patients conditioned with 13.5 Gy. Age at BMT was also independently correlated with survival, with the best outcome for patients < 40 years old. CONCLUSION: A TBI dose (fractionated) > 10 Gy may not necessarily be associated with a better outcome in patients undergoing allogeneic bone-marrow transplant for hematologic malignancies.

Recognition of major histoincompatibilities after transplantation with marrow from HLA closely matched donors.

BACKGROUND: To determine the extent to which major histoincompatibilities are recognized after bone marrow transplantation, we characterized the specificity of the cytotoxic T lymphocytes isolated during graft-versus-host disease. We studied three patients transplanted with marrow from donors who were histoincompatible for different types of HLA antigens. METHODS: Patient 1 was mismatched for one "ABDR-antigen" (HLA-A2 versus A3). Two patients were mismatched for antigens that would usually not be taken into account by standard selection procedures: patient 2 was mismatched for an "HLA-A subtype" (A*0213 versus A*0201), whereas patient 3 was mismatched for HLA-C (HLA-C*0501 versus HLA-C*0701). All three HLA class I mismatches were detected by a pretransplant cytotoxic precursor test. RESULTS: Analysis of the specificity of the cytotoxic T lymphocyte clones isolated after transplantation showed that the incompatibilities detected by the pretransplant cytotoxic precursor assay were the targets recognized during graft-versus-host disease. CONCLUSIONS: Independent of whether the incompatibility consisted of a "full" mismatch, a "subtype" mismatch, or an HLA-C mismatch, all clones recognized the incompatible HLA molecule. In addition, some of these clones had undergone antigen selection and were clearly of higher specificity than the ones established before transplantation, indicating that they had been participating directly in the antihost immune response.

HA-1 and the SMCY-derived peptide FIDSYICQV (H-Y) are immunodominant minor histocompatibility antigens after bone marrow transplantation.

BACKGROUND: Allogeneic bone marrow donors can be incompatible at different levels. Even HLA-identical pairs will be still incompatible for numerous minor histocompatibility antigens (mHag). Nevertheless, some incompatibilities are found to be associated with an increased risk of graft-versus-host disease (GVHD), which could be related to the way the immune system recognizes these antigens. METHODS: We determined the specificity of cytotoxic T-cell clones isolated during acute GVHD or during bone marrow graft rejection in patients (n=14) transplanted with marrow from donors who were histoincompatible for different minor and/or major histocompatibility antigens. RESULTS: We found a clear hierarchy among the different types of histoincompatibilities. In three combinations mismatched for a class I allele, all 27 clones isolated during GVHD were specific for the incompatible HLA molecule. In the 11 class I-identical combinations, 14 different mHags were recognized. The mHag HA-1, known to have a significant impact on the development of GVHD, was recognized in the two HA-1-incompatible combinations. In one of these combinations, which was sex mismatched, all 56 clones analyzed were directed against HA-1, demonstrating the dominance of this mHag. In the four HA-1-compatible, sex-mismatched combinations, the anti-H-Y response was directed against one immunodominant epitope rather than against multiple Y-chromosome-encoded epitopes. All male specific cytotoxic T lymphocytes (n=15) recognized the same high-performance liquid chromatography-purified peptide fraction presented by T2 cells. Moreover, all cytotoxic T lymphocytes tested (n=6) were specific for the SMCY-derived peptide FIDSYICQV, originally described as being the H-Y epitope recognized in the context of HLA-A*0201. CONCLUSIONS: Some histocompatibility antigens are recognized in an immunodominant fashion and will therefore be recognized in the majority of mismatched combinations. Only for such antigens, correlations between mismatches and the occurrence of GVHD or graft rejections will be found.

The frequency of pretransplant donor cytotoxic T cell precursors with anti-host specificity predicts survival of patients transplanted with bone marrow from donors other than HLA-identical siblings.

Transplantation with bone marrow from other than genotypically HLA-identical donors is associated with an increased incidence and severity of graft-versus-host disease (GvHD). The precise influence of HLA incompatibilities is not easy to analyze as even perfectly matched, HLA-identical unrelated donors might still express HLA differences that remain undetected by conventional typing. To measure T cell activity against serologically detectable and nondetectable HLA antigens, we analyzed the frequencies of CTL precursors (CTLp) between 11 unrelated HLA-matched and five related haploidentical donor/recipient pairs in graft-versus-host direction. Our results show that whenever HLA class I disparities could be identified by serology, high precursor frequencies (1/28,000-1/94,000) were measured. In contrast, in donor/recipient pairs that differed for class II only, no precursors were detected. CTLp were elevated in two out of eight fully matched donor/recipient combinations. These combinations displayed activities as high (1/21,000; 1/52,000) as the combinations that were serologically HLA class I disparate. The incompatibilities detected by the cellular assay were highly significant for the clinical results after transplantation. High CTLp frequencies before transplantation correlated with unfavorable clinical results independent of the incidence of detected HLA differences. Five out of the six patients with high (> 1/100,000) CTLp frequencies died within 120 days after transplantation. GvHD IV was the cause of death for all (3/5) patients who had received an unmanipulated bone marrow. In the group with intermediate or undetectable CTLp frequencies, eight out of 10 patients are alive, seven (CTLp frequency undetectable) without GvHD more severe than grade II, while one patient (CTLp frequency = 1/180,000) suffered from GvHD grade III. One patient rejected the graft and was rescued by an autologous BMT.

T-cell repertoire complexity after allogeneic bone marrow transplantation.

We analyzed the T-cell repertoire in patients transplanted with bone marrow from an HLA identical sibling by determining the TCR diversity through Vbeta-CDR3-size spectratyping with Vbeta/Cbeta- and Vbeta/Jbeta-specific primers. Using the Vbeta/Cbeta primers, we observed limited TCR diversity only in recipients of a T-cell-depleted graft, whereas the TCR diversity of patients transplanted with an unmanipulated graft seemed to be indistinguishable from the one of a normal individual. However, with Vbeta/Jbeta-specific primers, increase of the resolution by approximately 10-fold also allowed the detection of imbalances in the TCR repertoire of recipients of an unmanipulated graft. This demonstrates that when high numbers of T cells are cotransfused with marrow, the TCR repertoire is more complete but still not as complete as in normal individuals, thereby emphasizing the important role of coinfused mature T cells in the restoration of the T-cell compartment after bone marrow transplantation.

HLA-B35-subtype mismatches in ABDR serologically matched unrelated donor-recipient pairs.

We have characterized HLA incompatibilities in a group of 17 B35-positive patients who were ABDR matched (AB serology and oligotyping for DR1-14) with their 28 (unrelated) potential bone marrow donors. High-resolution oligotyping for DR subtypes disclosed that nine combinations were in fact DR mismatched. Cytotoxic T-lymphocyte (CTL) activity was detected in nine combinations (32%). In the group matched for DR subtypes, three (16%) of 19 combinations were CTL positive. Patient-specific cytotoxic activity appeared to be directed against HLA C (two cases) or against a subtype of B35. In the group of DR-subtype-mismatched combinations, CTL activity was found in six (67%) of nine pairs. In all four cases that were studied in detail, however, CTL reactivity appeared to be directed against a variant subtype of B35. We have studied the B35 incompatibilities recognized in five different combinations by specificity analysis of the B35-specific CTLs and by partially sequencing of relevant segments of B35 exon 3. Preliminary data show that, within this relatively small Caucasoid group, at least five B35-variant subtypes could be distinguished. This would make B35 an antigen that will be frequently subtype mismatched, in particular when DR matching is done with low resolution (DR1-14) only.

High-resolution histocompatibility testing of a group of sixteen B44-positive, ABDR serologically matched unrelated donor-recipient pairs. Analysis of serologically undisclosed incompatibilities by cellular techniques, isoelectrofocusing, and HLA oligotyping.

We have characterized HLA incompatibilities in a group of 16 B44-positive patients who were serologically ABDR matched with their 23 (unrelated) potential bone marrow donors. After analysis with a combination of cellular techniques, IEF for HLA-A/B and oligotyping for class II and HLA-B44, 44% of the patients revealed one or more HLA incompatibility with at least one of their potential donors. CTL activity was detected in 12 of the 22 combinations tested. CTL incompatibility occurred more frequently in DR subtype-mismatched combinations, but CTL reactivity was always directed against class I. To characterize these incompatibilities between matched unrelated individuals, we analyzed the specificity of T-cell clones from seven primary CTL cultures. In three combinations, CTL reactivity was directed against a subtype of B44. In two combinations, the CTL reactivity was directed against a non-B44 class I subtype. In two of seven combinations, the CTLs recognized an antigen that, though unconditionally associated with B4403, was expressed by 60% of the B4403+ cells only. Because all 12 of these B4403+ targets recognized could be typed for one HLA-C allele only (Cwl-Cw8), we believe that this alloreactivity might be directed against a serologically undefined Cw antigen.

Clinical consequences of sensitisation to minor histocompatibility antigens before allogeneic bone marrow transplantation.

To study sensitisation to minor histocompatibility antigens (mHag) before and after BMT, we measured antidonor CTL activity in five patients who had rejected their graft, and in a control group of 10 leukemic patients who engrafted without complications. All patients were transplanted with marrow from an HLA-identical sibling. Fourteen patients were conditioned with cyclophosphamide (120 mg/kg) and TBI (1350 cGy) and received a T cell-depleted graft, while one patient with aplastic anaemia received cyclophosphamide alone and unmanipulated marrow. Before transplantation, anti-donor CTL activity was detected in two of the 15 patients. These patients rejected their grafts at days 21 and 58, respectively. In the other three patients who rejected their grafts at days 41, 60 and 250, CTL activity was found only after transplantation. In contrast, no anti-donor CTLs could be detected at any time in the 10 patients who engrafted permanently. We have identified some of the mHags recognised during graft rejection by cloning and subsequent specificity analysis of the recipient CTLs. In the patient who rejected at day 41 without detectable immunisation before BMT, the response was directed against HA-1, a minor antigen known to play a role in GVHD. In the other combinations, a significant part of the CTL activity was directed against the male antigen H-Y. In the patient who rejected the marrow of her HLA-identical brother at day 250, two clones recognised H-Y, while five others recognised at least three distinct autosomal mHags. This patient had an HLA-identical sister who expressed only one autosomal mHag that had been recognised by one single T cell clone. After re-transplantation with the marrow of this second donor, the CTL activity could no longer be detected and the patient engrafted without further complications.