[Auto- and alloreactivity of T lymphocytes in myelodysplastic syndrome]. / Auto- a aloreaktivita T lymfocytu u myelodysplastického syndromu.

BACKGROUND: Successful therapy with ATG and cyclosporine A in some myelodysplastic syndrome (MDS) patients led us to study the existence of T cells attacking autologous hemopoietic cells. In our study, we attempted to give the direct prove of autoreactive T cells in MDS (autoreactivity analysis). Simultaneously, we analysed the capacity of MDS patients to respond to allogeneic cells from unrelated individuals (alloreactivity analysis). METHODS AND RESULTS: Autoreactive lymphocytes directed against own bone marrow mononuclear cells were analysed using the modification of cell mediated cytotoxic reaction. With one exception we did not confirm the presence of autoreactive T cells among 10 patients examined. Analysis of alloreactivity was performed by means of standard cell mediated cytotoxic reaction and mixed lymphocyte reaction. Surprisingly, the cytotoxic response to allogeneic cells was negative in 11 MDS patients from 16 analysed. When comparing refractory anaemia (RA) and refractory anaemia with ring sideroblasts (RARS) patients, the proportion of negative results was higher in RA (78 %) than in RARS (40 %). In mixed lymphocyte reaction, the response of MDS cells to allogeneic cells of unrelated individual was positive in all tested patients. The preliminary testing of TNF and IFNgamma secretion examined in supernatants of effector cells showed impaired levels of both cytokines in RA and normal levels in RARS in accordance with the findings achieved in alloreactivity analysis. CONCLUSIONS: Autoreactive T cells were not found in MDS patients using our experimental arrangement. Analysis of alloreactivity showed the defect in effector--cytotoxic--phase of cell mediated cytotoxic reaction in the majority of MDS patients. The initial phase of this reaction represented in vitro by mixed lymphocyte reaction gave normal results. The possible reasons of disturbed alloreactivity and its relevance to immunity in MDS are commented in discussion.

The relationship between HA-1 compatibility and the activation of helper T lymphocyte precursors.

The relationship between the compatibility in minor histocompatibility HA-1 antigen and the activation of helper (IL-2 producing) T lymphocyte precursors in vitro was studied in the group of 17 HLA-A2 positive HLA identical siblings. Although the number of pairs studied is still small, no correlation has been found between HA-1 compatibility and helper T lymphocyte precursors activation. The results presented here could suggest the possibility that the HTLp assay does not have to be a relevant parameter for the detection of HA-1 mismatches in HLA identical siblings.

Transplantation of allogeneic peripheral blood progenitor cells--a single centre experience.

13 patients have been transplanted at Institute of Hematology and Blood Transfusion since 1995 using allogeneic PBPC either alone or with bone marrow as a source of progenitor cells. All donors were G-CSF mobilised HLA identical family members. PBPC harvests were performed on D 4,5, (6) of G-CSF administration. The medium content of TNC, CD34+, CD3+, CD4+and CD8+cells/kg b.w. of the recipients in the grafts were: 13,1x10(8)(TNC), 11,4x10(6)(CD34+), 393x10(6)(CD3+) 243x10(6)(CD4+), 125x10(6)(CD8+) The patients received either BuCy2 or CyTBI preparative regimen and Cyclosporin A + short course of Methotrexate for GVHD prophylaxis. Engraftment of ANC >500 was achieved by D+16 and PLT >20.000 by D+19. Three of ten evaluable patients developed acute and three of nine chronic GVHD. 8 patients survive with the longest follow up 776 days.

Effects of aflatoxin B1 on myelopoiesis in vitro.

The short-term cultures of mouse myeloid progenitor cells for granulocytes and monocytes, granulocyte-monocyte colony-forming units (CFU-GM) (CFU-GM assay) and mouse long-term bone marrow culture (LTBMC) were used to investigate the hemopoietic suppression caused by aflatoxin B1 (AFB1). A dose-related suppression of granulopoiesis in short-term bone marrow cultures was seen when the cultures were treated with 10, 5, 1, 0.5 and 0.1 micrograms of AFB1/ml. Two selected doses of AFB1 (5 and 0.5 micrograms/ml) considered to be highly and slightly suppressive in CFU-GM assay exerted a strong suppression of myelopoiesis in LTBMC when applied long-term. Short-term (2 h) exposure of LTBMC to 5 micrograms of AFB1/ml caused a small damage to the myelopoiesis detected in the non-adherent layer. Short-term exposure to 0.5 micrograms AFB1/ml was without any effect on myelopoiesis in LTBMC. The production of colony-stimulating activity (CSA) by an adherent layer of LTBMC was decreased on the second and fifth day after the short-term exposure to both doses of AFB1 and comparable with non-treated culture on the seventh day after the exposure. Presented results indicate that both short-term culture of CFU-GM and LTBMC can be used in the definition and the prediction of host toxicity of AFB1 to hemopoiesis. However, comparing these two in vitro systems, the LTBMC appears to be more sensitive and discriminatory in an evaluation of hemopoietic toxicity.

Alterations in the growth and cycling status of granulocyte-monocyte colony-forming units (CFU-GM) in rats injected single doses of aflatoxin B1.

To determine the toxic effect of aflatoxin B1 (AFB1) on granulopoiesis in vitro, cultures of myeloid progenitor cells for granulocytes and macrophages CFU-GM in semisolid agar medium were used to evaluate colony formation and tritiated thymidine suicide technique for analysis of cycling status of CFU-GM. Male Fischer rats, 5 to 6 weeks old, were given a single i.p. injection of 1 mg/kg or 0.1 mg/kg of AFB1 which were approximately 1/5 or 1/50, respectively, of LD50 dose of AFB1. The administration of either dose of AFB1 caused a significant reduction of the number of CFU-GM derived colonies on the first and the second day after injection. This reduction was followed by a strong enhancement in CFU-GM colony formation on the third day, and by restoration to the normal level on the fourth day. The increase in percentage of CFU-GM in S-phase preceded one day the peak in CFU-GM colony formation. The two different doses of AFB1 exerted similar effects on the growth and cycling status of CFU-GM. Our data outline an early suppressive effect exerted by AFB1 to the rat granulopoiesis in vivo.

Helper T lymphocyte precursor frequency analysis in alloreactivity detection.

The utility of IL-2 secreting helper T lymphocyte precursors (HTLp) frequency testing has been evaluated for detecting alloreactivity. The frequency of HTLp was approached by limiting dilution assay. High HTLp frequency was detected in 20 out of 30 HLA matched unrelated pairs (67%). The comparison of HTLp and CTLp (cytotoxic T lymphocyte precursors) frequencies in HLA matched unrelated pairs showed that the two examinations are not fully alternative in detecting alloreactivity. This could suggest the utility of combined testing of both HTLp and CTLp frequencies for alloreactivity assessment. In contrast, five positive HTLp values were only found among 28 HLA genotypic identical siblings (18%). Previous CTLp limiting dilution studies showed very low or undetectable CTLp frequency results in that group. For that, HTLp assay remains to be the only cellular in vitro technique detecting alloreactivity in these combinations.

Inhibitor of normal granulopoiesis produced by cells of MDS patients.

Low-density blood cells from patients with refractory anemia with excess of blasts (RAEB) and RAEB in transformation (RAEB-T) release a high molecular weight inhibitory substance that reduces the entry of normal progenitor cells of granulocytes and macrophages (CFU-GM) into the S-phase. Out of 20 patients with refractory anemia (RA and RAS) only 3 were positive. One patient with CMML was negative. Serial examination of 3 patients (two RA and one CMML) revealed that the production of the inhibitory activity preceded the development of the disease into RAEB, RAEB-T, or AML. With one exception, the inhibitory activity in positive cases was neutralized by antiserum against human placental ferritin.

Frequency analysis of cytotoxic T lymphocyte precursors in search for donors in bone marrow transplantation.

The usefulness of cytotoxic T lymphocytes precursors (CTLp) frequency analysis in the search for donors in bone marrow transplantation was studied. The frequency of anti-recipient CTLp was approached by limiting dilution assay in HLA matched unrelated, HLA partially matched related and HLA genotypically identical donors. The majority of patients examined were affected with different hematological malignancies. Alloreactive CTLp recognizing non-HLA gene products were not detected in pretransplant examination of two pairs of HLA identical siblings. However, an increased incidence of allospecific CTLp was identified in HLA matched MLC negative unrelated pairs. Thus, CTLp assay allowed to uncover the residual Class I incompatibilities that remained hidden in standard serotyping. In two matched unrelated pairs with high pretransplant CTLp frequency the severe acute graft-versus-host disease (GVHD) developed after bone marrow transplantation. Examination of other relatives in patients lacking an HLA identical sibling showed the importance of Class I incompatibility for CTLp generation as well. The lack of correlation between CTLp frequency and HLA-D disparity could suggest that Class II antigens do not participate in CTLp induction. With one exception we had good correlation between MLC and DNA analysis of Class II antigens demonstrating that MLC gives interpretable results even in unrelated pairs. Our results demonstrate the significance of CTLp frequency assay in detection of residual Class I incompatibilities in matched unrelated pairs and in assessment of Class I compatibility in related pairs. For that it should be used in the final selection of BMT donors.

Cytotoxic T lymphocyte precursor frequency analysis in the selection of HLA matched unrelated donors for hematopoietic stem cell transplantation: the correlation of CTLp frequency with HLA class I genotyping and aGVHD development.

The selection of human leukocyte antigen (HLA) compatible unrelated donors for hematopoietic stem cell transplantation (HSCT) is based on the direct genotyping of HLA class I and class II alleles (HLA-A, -B, -C, -DRB1, -DQB1 loci). The cellular test estimating the frequency of cytotoxic T lymphocyte precursors (CTLp) has been included into the selection procedure of unrelated donors to detect the class I alloreactivity and to predict acute graft versus host disease (aGVHD) occurrence and severity. The relationship between HLA-A, -B, -C high/medium resolution genotyping and CTLp activation was analysed in the cohort of 78 unrelated donor/patient pairs indicated for HSCT. The high frequency of CTLp (> 1:100,000) correlated significantly (p < or = 0.0002) with the incompatibilities in alleles of HLA-A, -B, -C loci. Nevertheless, the results of HLA-A, -B, -C genotyping and CTLp assay are not fully alternative, suggesting that the CTLp test gives its specific information. The high CTLp frequency (CTLpf) in 14/35 pairs fully matched by HLA class-I alleles genotyping could reflect the influence of another factors upon the CTLp activation. On the contrary, the low CTLp frequency values (< or = 1:100,000) found in 8/43 pairs with existing HLA class-I alleles incompatibilities could indicate the immunological permissivity of these particular mismatches. The clinical relevance of the CTLp test for aGVHD prediction has been also analysed. The relationship between CTLp activation in vitro and the incidence and severity of aGVHD was evaluated in 37 patients who underwent allogeneic HSCT. The severe form of aGVHD (grade III-IV) developed in 9 of 18 cases (50%) with the high pretransplant CTLpf value. The patients with the low CTLpf (n = 19) suffered from the severe form of aGVHD in 2 cases (10%) only, the remaining 17 patients from this group were without aGVHD symptoms or developed only the mild form of aGVHD (I-II). The relationship between CTLp results and the incidence and severity of aGVHD was found statistically significant (p < or = 0.01).

The role of gene products of non-H-2 loci and M locus in secondary MLC response.

The response in the secondary MLC after priming in vivo was studied in combinations of mouse strains H-2 identical and differing at genetic loci that segregate independently of the H-2 system (non H-2 loci including M locus). After priming with gene products of non-H-2 loci and M locus the response in secondary MLC in H-2-identical strains is influenced by the H-2 genotype (combinations compatible for H-2b and H-2k haplotypes were compared). With the H-2k compatibility, the type of secondary MLC response depends on the non-H-2 genotype of the responder strain. The same gene products of the M locus activate a cell suppressor mechanism, which suppresses the blastic response of lymphocytes, in some responder strains and an early proliferative response in others.

The MLC response of lymphocytes from mice neonatally primed and tolerating skin allografts.

Newborn B10.A mice were primed intravenously with semiallogeneic spleen cells differing in the whole H-2 complex or in individual regions of H-2. Neonatally treated mice were grafted with respective skin allografts and cells from tolerant animals were tested in mixed lymphocyte culture. These cells were found to be a reactive or hyporeactive to MLC-stimulating determinants. Using a panel of restimulating cells, MLC tolerance was found to be immunologically specific. Lymphocytes from tolerant mice did not react only to the alloantigens used for neonatal priming, whereas they showed similar reactions to third party antigens as did lymphocytes from normal untreated mice. The results indicate that neonatal priming leads to the tolerance which is manifested already in the recognitive phase of the allotransplantation reaction and that this MCL tolerance is specific, at least in B10.A mice.

The distribution of HLA-d (MLC) locus determinants in the Czech population.

The distribution of 8 HLA-D determinants in the group of 83 unrelated individuals of the Czech population was ascertained by means of homozygous typing cells using the mixed lymphocyte culture test. In the panel tested, the gene frequencies of HLA-D determinants, the distribution of HLA-D phenotypes and the magnitude of the linkage disequilibrium between the determinants of the HLA-B and HLA-D loci were determined.

HLA-D antigens in a Czech population and their association with HLA-DR antigens.

HLA-D specificities Dwl through 11 and HLA-DR antigens DRwl through WIA8 were studied on a panel of 101 unrelated individuals of the Prague HLA panel. THe HLA-D specificities were defined by means of the HLA-D homozygous typing cells in MLC test, the HLA-DR specificities were tested on B cells using VIIth Histocompatibility Workshop antisera. In the panel tested, the antigen and gene frequencies of eleven HLA-D determinants, the associations between the HLA-B and HLA-D antigens as well as between the HLA-D and HLA-DR antigens were determined. Significant associations between some of the HLA-D and HLA-DR antigens were found.

Frequencies of HLA-DRB1, -DQB1 and -DPB1 alleles in Czech population.

The frequencies of phenotypes, alleles and allelic subtypes of DRB1 and DQB1 HLA loci in 420 unrelated individuals from the Czech population were determined. The frequencies of DPB1 alleles of the HLA locus were determined in 92 individuals. The assays were performed using the polymerase chain reaction (PCR) method or the restriction fragment length polymorphism (RFLP) analysis. The most frequent DRB1 allele was *07, the most frequent DQB1 allele was *03 and the most frequent DPB1 allele detected was *04. These assays define the extent of polymorphism of the HLA system and are useful for determining the selection strategy of HLA-identical donor-recipient pair suitable for bone marrow transplantation.

Predictive value of HLA class II PCR typing for the outcome of mixed lymphocyte reaction.

The use of HLA-DRB1 and -DQB1 polymerase chain reaction-sequence-specific primer (PCR-SSP) typing at different levels of resolution for MLR prediction was assessed in 54 HLA-A and -B matched donor/recipient unrelated pairs and 89 HLA-A and -B identical siblings. Graft-versus-host (GvH) direction one-way MLR was evaluated unless stated otherwise. The typing of DRB1 alleles satisfactory for MLR prediction in HLA identical siblings (P = 0.0015) does not appear to be sufficient in matched unrelated pairs (P = 0.2407). Using more discriminatory PCR-SSP typing, the disparity in DRB1 allelic subtypes was predominantly found in the category of DRB1 allele compatible, MLR positive unrelated pairs. Besides, DRB1 allelic subtype mismatches were revealed in five of the forty-one DRB1 allele compatible, MLR negative unrelated pairs. More discriminatory typing made the correlation between DRB1 compatibility and MLR negativity extremely significant (P = 0.0001). As for these five exceptional cases, the reciprocal host-versus-graft (HvG) direction MLR was considered, too. This allowed HLA-D disparity to be disclosed in two of them. An uninterpretable result reflecting defective MLR reactivity occurred in one case. Negative reciprocal MLR in the last two DRB1 allelic subtype incompatible pairs is hardly to explain without postulation of MLR silent DRB1 allelic subtype mismatches. An analysis in unrelated pairs showed a role of some DQB1 gene products in the proliferative response too. GvH direction positive MLR was found in two HLA identical siblings among the 89 tested. The DPB1 incompatibility detected in one of them could be a potential cause of proliferative response but MLR reactivity in the other, DPB1 identical, pair cannot be interpreted easily.

HLA class II gene frequency in a Czech Population.

In a sample of 212 healthy, unrelated individuals of a Czech (Central Bohemian) population, the phenotype and allele frequencies of HLA-DRB1 and -DQB1 loci were determined. DNA typing technique used was the restriction fragment length polymorphism (RFLP) analysis. The restriction enzymes were TaqI and HindIII and specific DRB1 and DQB1 probes were applied. The most frequent DRB1 and DQB1 alleles found were DRB1*11 and DQB1*06, their respective frequencies being 0.1698 and 0.2594.

The search for HLA-compatible bone marrow donors. Procedure currently used in the Institute of Hematology and Blood Transfusion, Prague.

The search for compatible donors is based on the HLA types of donors and recipients. HLA-A, -B and -DRB1 antigens or alleles must be unequivocally typed in donors and recipients. The typing of HLA-DQB, -DPB and -C gene products is also useful to characterize the HLA phenotype, but it is not absolutely necessary in the donor selection. The standard serological methods using alloantisera and one-dimensional isoelectric focusing are used for the typing of HLA class I antigens. Recently, DNA analysis of class I alleles has been introduced. In HLA class II typing the serological analysis was generally replaced by DNA analysis. In addition to typing techniques that determine the individual HLA alleles or HLA gene products, the cellular matching techniques are used in the selection procedure (mixed lymphocyte culture, cytotoxic T lymphocyte precursor frequency assay, and IL-2-producing helper T lymphocyte precursor frequency assay). The cellular matching techniques determine the compatibility in the regions of HLA comprising several HLA loci; some of them may detect minor histocompatibility (non-HLA) gene disparities as well.

[Inhibitory factors in hematopoiesis]. / Inhibicní faktory v hematopoeze.

The author presents a short review of inhibitory factors which affect hemopoietic stem and progenitor cells. At present, the existence of naturally occurring inhibitory factors which take part in the control of hemopoiesis under pathological conditions in which they were detected, and probably even under physiological conditions, has been proved. Through their effects the inhibitory factors create a complex network of negative feed-back regulations. Their cooperation with stimulatory factors seems to be mechanism which ensures the balance in hemopoiesis and recovers it in the case of a disorder.

[Frequency of cytotoxic and helper T-lymphocyte precursors in donors with alloreactivity to recipient histocompatibility antigens. Possible use in predicting acute graft vs host disease in bone marrow transplantation]. / Frekvence prekurzoru cytotoxických a pomocných T lymfocytu dárce aloreaktivních vuci histokompatibilitním antigenum príjemce. Moznosti vyuzití pro predpoved aGVHD pri transplantaci kostní drene.

BACKGROUND: The utility of cytotoxic T lymphocyte precursor (CTLp) and helper T lymphocyte precursor (HTLp) frequencies estimation for detecting alloreactivity and for the prediction of acute graft versus host disease (aGVHD) has been evaluated. METHODS AND RESULTS: The limiting dilution assay and a maximum likelihood statistical programme were used for CTLp and HTLp frequency estimation. A high CTLp frequency suggesting the presence of hidden class I mismatches was detected in 41.2% of unrelated pairs. HLA-A and -B matched by serological typing and DRB1 and DQB1 matched by DNA analysis. Severe aGVHD (grade III-IV) occurred in all patients of this group who underwent bone marrow transplantation (BMT). In two patients of the three evaluated with low pretransplant CTLp frequency a mild form (grade I) or no aGVHD developed after unrelated BMT. Positive frequency of alloreactive HTLp was found in 50% of HLA matched unrelated pairs. The comparison of CTLp and HTLp values in the same individuals showed that these two methods are not fully alternative in detecting alloreactivity. In the group of HLA identical siblings, 18.7% of positive HTLp results were only found. Besides HLA-DP incompatibilities, the differences in non-HLA genes could cause this alloreactivity. CONCLUSIONS: CTLp assay has a potential for the prediction for aGVHD development following BMT from HLA matched unrelated donors. CTLp results suggest the necessity of more accurate class I typing in these cases. The comparison of CTLp and HTLp frequencies showed that the results can differ in some unrelated donor-recipient BMT pairs suggesting the convenience of simultaneous performing of both assays for the alloreactivity assessment. More cases have to be considered to determine the relationship between pretransplant HTLp frequency and posttransplant aGVHD development in HLA identical siblings.

[Allogenic transplantation of hematopoietic stem cells in the treatment of children with high-risk acute leukemia]. / Alogenní transplantace kmenových bunek krvetvorby v lécbe detí s vysoce rizikovou akutní leukémií.

BACKGROUND: Most children with acute lymphoblastic leukemia (ALL) and increasing number of children with acute myelogenous leukemia (AML) are currently cured with conventional chemotherapy. Despite of this success there is a subset of patients with high-risk features at diagnosis who are predisposed to a very high risk of relapse. Relapse of AML and early bone marrow relapse of ALL can not be cured by conventional chemotherapy. Allogeneic hematopoietic stem cell transplantation (HSCT) is therapeutic option in these children with very high-risk acute leukemia. METHODS AND RESULTS: Between XI/1989-XII/1996 33 children with acute leukemia (ALL: 22, AML: 11) underwent an allogeneic HSCT from HLA identical related donors (HLA-identical sibling: 30, twin: 1, other HLA-identical relative: 2) at the 2nd Dept. of Pediatrics, University Hospital Motol. Median age of our group was 9 years (1.5-19 y.), boys (n = 23) clearly dominated over the girls (n = 10). The resource of stem cells was bone marrow in 31 children, bone marrow and peripheral blood progenitor cells (PBPC) and PBPC in one child respectively. Myeloablative conditioning regimen varied, consisting of total body irradiation and chemotherapy in 21 children and chemotherapy in 12 children. HSCT was performed in first complete remission of acute leukemia in 9 children (AML: 7, ALL: 2), in second remission in 14 children (AML: 2, ALL: 12), in third remission in 4 children (ALL: 4). Six children underwent HSCT in first partial remission (n = 1) and in second (n = 4) or third (n = 1) chemoresistant relapse. Seven (21%) children died due to post-transplant complications. Nine (28%) children suffered from clinically significant acute graft-versus-host reaction (GVH) and 15% (4/27) children who survived 100 days post-transplant suffered from chronic GVH disease. Relapse of leukemia was diagnosed in 39% (12/31) children. Fourteen (42%) children are alive and well in continuous remission with median follow-up 42 months. CONCLUSIONS: Allogeneic HSCT can cure children with very high-risk acute leukemia in the situations where conventional chemotherapy fails. Relapse of leukemia and GVH reaction are most important causes of post-transplant morbidity and mortality.