Immunogenetic Analysis Reveals the Association of INF-γ (+874 A/T) Hypersecretor Genotype in AA and a Low Frequency of KIR-2DL3/C1 Mismatch in Responders to Immunosuppression.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1700-1700
Author(s):  
Bianca Serio ◽  
Giridharan Ramsingh ◽  
Ramon Tiu ◽  
Antonio M. Risitano ◽  
Mikkael A. Sekeres ◽  
...  
Keyword(s):  
Cytotoxic T Cells ◽  
Bone Marrow Failure ◽  
Receptor Gene ◽  
Low Frequency ◽  
Hematopoietic Stem ◽  
Hla Ligand ◽  
Bone Marrow Failure Syndromes ◽  
Immune Mediated ◽  
Self Tolerance ◽  
Tnf Α

Abstract Clinical and laboratory evidence support an immune pathogenesis in most cases of idiopathic aplastic anemia (AA) and closely related disorders such as paroxysmal nocturnal hemoglobinuria (PNH). While external triggers are likely necessary, a complex constellation of immunogenetic factors may determine disease susceptibility. Many immunogenetic factors can influence the quality of immune response and affect the propensity to immune-mediated attack on hematopoietic stem cells in AA. Here we investigated whether KIR and KIR-L (HLA-A) genotype and cytokine/receptor gene variants are over-represented in AA and PNH. We studied a cohort of 77 patients with AA (23 AA, 20 AA/PNH and 34 PNH), 10 with hypocellular MDS and 175 healthy controls. The following SNPs in immunoregulatory genes were analyzed: IL-1α (−889 T/C), IL-2 (−330 T/G +166 G/T), IL-4 (−1098 T/G −590 T/C −33 T/C), IL-1R (−1970 C/T), IL-1Rα (mspa111100 T/C), IL-4RA (+ 190 G/A), IL-1β (−511 C/T, +3962 T/C), IL-6 (−174 C/G, nt565 G/A), IL-10 (−1082 G/A, −819 C/T, −592 C/A), IL-12 (−1188 C/A), TGF-β (+10 C/T, +25 G/C), INF-γ (+874 A/T), TNF-α (−308 G/A, −238 G/A) and immunomodulatory receptor genes including CTLA-4 exon 6 (+49 G/A), FcRIIIa (158 F/V) and CD45-exons 6 (+138 A/G), and 4 (+54 A/G, +77 C/G). As binding of KIR to the appropriate HLA ligand (KIR-L) can modulate activation of NK and cytotoxic T cells, we examined the combined impact of KIR/KIR-L genotypes on the risk of AA and PNH syndrome. In AA we found a decreased frequency of inhibitory KIR-2DL3 genes (68% vs. 89%, p=.0002); analysis of the KIR genotype in correlation with the corresponding KIR-L profile, revealed a decreased frequency of stimulatory 2DS1/C2 mismatch resulting in a potentially enhanced cytotoxic activity (14% vs.44%, p=.003). No association was found for most of the SNPs tested. However, when we examined the frequency TGF-β genotypes, increased frequency of GG variant in codon 25 (61% vs. 35% in controls, p=.03), associated with the “high secretor” phenotype, was found in AA. This relationship was also present in hypocellular MDS (82% vs. 32%, p=.007). Additionally, we found a lower incidence of TT genotypes for the IL-1Rα gene (33% vs. 62% p=.02). We confirm that the hypersecretor genotype T/T of INF-γ was over-represented in AA (28% vs. 10% in controls, p=.02). Subgroup analysis revealed that the T/T genotype of IFN-γ (35% vs. 14% p=.01) correlated with presence of a PNH clone. Previously, we have shown the association of HLA-DR15 with responsiveness to immunosuppression. When AA patients were subgrouped according to response to ATG/CsA, therapy refractoriness correlated with the presence of the C2/C2 haplotype (30% vs. 0% p=.02) and inhibitory KIR-2DL3/C1 mismatch (70% vs. 0%, p=.01) which may result in a greater propensity to breach of self-tolerance. In comparison, in the total AA group, C2/C2 haplotype and KIR-2DL3/C1 mismatch were present in 17% vs. 24% and 8% vs. 16% of controls, respectively. An increase in the frequency of 2DL3 and a decrease in 2DS1 mismatch may result in imbalance between cytotoxicity and KIR inhibition. In sum, our findings demonstrate that complex inherited traits involving immunogenetic factors may genetically determine propensity to bone marrow failure syndromes.

scholarly journals Genetic Associations in Acquired Immune-Mediated Bone Marrow Failure Syndromes: Insights in Aplastic Anemia and Chronic Idiopathic Neutropenia

2012 ◽  
Vol 2012 ◽  
pp. 1-7 ◽  
Author(s):  
Irene Mavroudi ◽  
Helen A. Papadaki
Keyword(s):  
Bone Marrow ◽  
Aplastic Anemia ◽  
Transforming Growth Factor ◽  
Bone Marrow Failure ◽  
Genetic Associations ◽  
Hematopoietic Stem ◽  
Bone Marrow Failure Syndromes ◽  
Immune Mediated ◽  
Chronic Idiopathic Neutropenia ◽  
Activated T Lymphocytes

Increasing interest on the field of autoimmune diseases has unveiled a plethora of genetic factors that predispose to these diseases. However, in immune-mediated bone marrow failure syndromes, such as acquired aplastic anemia and chronic idiopathic neutropenia, in which the pathophysiology results from a myelosuppressive bone marrow microenvironment mainly due to the presence of activated T lymphocytes, leading to the accelerated apoptotic death of the hematopoietic stem and progenitor cells, such genetic associations have been very limited. Various alleles and haplotypes of human leucocyte antigen (HLA) molecules have been implicated in the predisposition of developing the above diseases, as well as polymorphisms of inhibitory cytokines such as interferon-γ, tumor necrosis factor-α, and transforming growth factor-β1 along with polymorphisms on molecules of the immune system including the T-bet transcription factor and signal transducers and activators of transcription. In some cases, specific polymorphisms have been implicated in the outcome of treatment on those patients.

TNF-α/Fas-RIP-1–induced cell death signaling separates murine hematopoietic stem cells/progenitors into 2 distinct populations

Blood ◽  
2011 ◽  
Vol 118 (23) ◽  
pp. 6057-6067 ◽  
Author(s):  
Yechen Xiao ◽  
Hongling Li ◽  
Jun Zhang ◽  
Andrew Volk ◽  
Shubin Zhang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Death ◽  
Bone Marrow Failure ◽  
Caspase 8 ◽  
Apoptotic Pathway ◽  
Interacting Protein ◽  
Hematopoietic Stem ◽  
Complete Inactivation ◽  
Bone Marrow Failure Syndromes ◽  
Tnf Α

AbstractWe studied the effects of TNF-α and Fas-induced death signaling in hematopoietic stem and progenitor cells (HSPCs) by examining their contributions to the development of bone marrow failure syndromes in Tak1-knockout mice (Tak1−/−). We found that complete inactivation of TNF-α signaling by deleting both of its receptors, 1 and 2 (Tnfr1−/−r2−/−), can prevent the death of 30% to 40% of Tak1−/− HSPCs and partially repress the bone marrow failure phenotype of Tak1−/− mice. Fas deletion can prevent the death of 5% to 10% of Tak1−/− HSPCs but fails to further improve the survival of Tak1−/−Tnfr1−/−r2−/− HSPCs, suggesting that Fas might induce death within a subset of TNF-α-sensitive HSPCs. This TNF-α/Fas-induced cell death is a type of receptor-interacting protein-1 (RIP-1)–dependent programmed necrosis called necroptosis, which can be prevented by necrostatin-1, a specific RIP-1 inhibitor. In addition, we found that the remaining Tak1−/− HSPCs died of apoptosis mediated by the caspase-8–dependent extrinsic apoptotic pathway. This apoptosis can be converted into necroptosis by the inhibition of caspase-8 and prevented by inhibiting both caspase-8 and RIP-1 activities. We concluded that HSPCs are heterogeneous populations in response to death signaling stimulation. Tak1 mediates a critical survival signal, which protects against both TNF-α/Fas-RIP-1–dependent necroptosis and TNF-α/Fas-independent apoptosis in HSPCs.

Tumor Necrosis Factor-Alpha and Interleukin-6 Promoter Gene Polymorphisms in Acquired Bone Marrow Failure Syndromes.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3707-3707 ◽  
Author(s):  
Vinod K. Gidvani ◽  
Shakti H. Ramkissoon ◽  
Elaine W. Wong ◽  
Lori Mainwaring ◽  
Elaine M. Sloand ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Failure ◽  
Cytokine Gene ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Bone Marrow Failure Syndromes ◽  
Immune Mediated ◽  
Tnf Α ◽  
Historical Controls ◽  
Pro Inflammatory Cytokines

Abstract Some acquired aplastic anemia (AA) results from immune mediated destruction of the hematopoetic stem cells. Immunosuppressive therapy is successful in majority of AA patients and substantial laboratory data are consistent with an immune pathophysiology. Substantial research has implicated differences in cytokine gene expression profiles and polymorphisms in the genes controlling cytokine expression in other autoimmune diseases such as lupus erythematosus and rheumatoid arthritis. Interlukin-6 (IL-6) and tumor necrosis alpha (TNF-α) are two potent pro-inflammatory cytokines that have implicated in a variety of immune-mediated conditions. TNF-α results in Fas expression and apoptosis of in progenitor cells and the TNF-alpha −308 allele was significantly associated with SLE in Caucasians. Levels both IL-6 and TNF-α have been reported elevated in AA patients. In the promoter region of the IL-6 gene, at position −174, exists a single nucleotide polymorphism (G/C) in close proximity to a glucocorticoid-responsive element; patients homozygous for the G allele have circulating IL-6 concentrations close to twice as high as those homozygous for the C allele. The TNF-α gene, located in the class III region of the major histocompatibility complex (MHC), has a polymorphism at position −308, TNF2, where the presence of adenine instead of guanine is associated with higher cytokine production. In our study, we characterized the IL-6/−174 and the TNF-α/−308 polymorphisms in patients with acquired bone marrow failure syndromes to assess if the higher production genotypes were more prevalent that in established controls. We identified seventy-three patients (age range 3–84) treated at our institution for AA. Following an established protocol for the identification of single nucleotide polymorphisms, genomic DNA was amplified with primers designed for the promoter regions of the IL-6 and TNF-α genes where intentional mismatches were inserted at 1–3 nucleotide positions to incorporate a restriction site for endonucleases. The amplicons were digested with four restriction endonucleases (BlsI, BsaBI, EcoNI, RsaI) then analyzed by electrophoresis in 3% agarose gels. The resulting fragments allowed for the identification and confirmation of the specific nucleotide polymorphism at the 174 and 308 position of the IL-6 and TNF-α promoter, respectively. The frequency of the high cytokine producing genotypes in the cohort was compared to established controls and the statistical significance determined by the two-tailed Fishers exact test. The GG genotype of the IL-6/−174 polymorphism was present in 32 of 73 (44%) of affected patients versus 80 of 250 (32%) historical controls of the control population (p =0.0698) while the AA genotype of the TNF-α/−308 polymorphism was found in 8 of 73 AA patients (11%) and in only 9 of 354 historical controls (2.5%) (p= 0.0034). Three of 73 AA patients had both gene polymophisms p<0.0001. Two patients’ BM was cultured and ELISA performed for TNF-α as part of a larger study, which included 20 normal controls and 30 patients with marrow failure; both of these patients demonstrated significant elevations in TNF-α. In conclusion, we showed that some patients with acquired bone marrow failure have cytokine gene polymophisms which are linked to high production of pro-inflammatory cytokines, particularly TNF-α.

scholarly journals Implications of hematopoietic stem cells heterogeneity for gene therapies

Gene Therapy ◽  
2021 ◽  
Author(s):  
Jeremy Epah ◽  
Richard Schäfer
Keyword(s):  
Immune Cell ◽  
Ex Vivo ◽  
Bone Marrow Failure ◽  
Cell Types ◽  
Blood Disorders ◽  
Hematopoietic Stem ◽  
Therapeutic Concept ◽  
Gene Therapies ◽  
Bone Marrow Failure Syndromes ◽  
Immunodeficiency Syndrome

AbstractHematopoietic stem cell transplantation (HSCT) is the therapeutic concept to cure the blood/immune system of patients suffering from malignancies, immunodeficiencies, red blood cell disorders, and inherited bone marrow failure syndromes. Yet, allogeneic HSCT bear considerable risks for the patient such as non-engraftment, or graft-versus host disease. Transplanting gene modified autologous HSCs is a promising approach not only for inherited blood/immune cell diseases, but also for the acquired immunodeficiency syndrome. However, there is emerging evidence for substantial heterogeneity of HSCs in situ as well as ex vivo that is also observed after HSCT. Thus, HSC gene modification concepts are suggested to consider that different blood disorders affect specific hematopoietic cell types. We will discuss the relevance of HSC heterogeneity for the development and manufacture of gene therapies and in exemplary diseases with a specific emphasis on the key target HSC types myeloid-biased, lymphoid-biased, and balanced HSCs.

Accelerated telomere shortening in glycosylphosphatidylinositol (GPI)–negative compared with GPI-positive granulocytes from patients with paroxysmal nocturnal hemoglobinuria (PNH) detected by proaerolysin flow-FISH

Blood ◽  
2005 ◽  
Vol 106 (2) ◽  
pp. 531-533 ◽  
Author(s):  
Fabian Beier ◽  
Stefan Balabanov ◽  
Tom Buckley ◽  
Klaus Dietz ◽  
Ulrike Hartmann ◽  
...  
Keyword(s):  
Telomere Length ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Bone Marrow Failure ◽  
Disease Stage ◽  
Healthy Individuals ◽  
Hematopoietic Stem ◽  
Replicative Stress ◽  
Selective Growth Advantage ◽  
Bone Marrow Failure Syndromes ◽  
The Impact

Abstract Telomere length has been linked to disease stage and degree of (pan-)cytopenia in patients with bone marrow failure syndromes. The aim of the current study was to analyze the impact of replicative stress on telomere length in residual glycosylphosphatidylinositol-positive (GPI+) versus GPI– hematopoiesis in patients with paroxysmal nocturnal hemoglobinuria (PNH). Peripheral blood granulocytes from 16 patients and 22 healthy individuals were analyzed. For this purpose, we developed proaerolysin flow-FISH, a novel methodology that combines proaerolysin staining (for GPI expression) with flow-FISH (for telomere length measurement). We found significantly shortened telomeres in GPI– granulocytes (mean ± SE: 6.26 ± 0.27 telomere fluorescence units [TFU]), both compared with their GPI+ counterparts (6.88 ± 0.38 TFU; P = .03) as well as with age-matched healthy individuals (7.73 ± 0.23 TFU; P &lt; .001). Our findings are in support of a selective growth advantage model of PNH assuming that damage to the GPI+ hematopoietic stem-cell (HSC) compartment leads to compensatory hyperproliferation of residual GPI–HSCs.

scholarly journals The Effectiveness of Rapamycin Combined with Eltrombopag in Murine Models of Immune-Mediated Bone Marrow Failure

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 10-11
Author(s):  
Rong Fu ◽  
Shaoxue Ding ◽  
Xiaowei Liang ◽  
Tian Zhang ◽  
Zonghong Shao
Keyword(s):  
Bone Marrow ◽  
Nk Cells ◽  
Standard Dose ◽  
Bone Marrow Failure ◽  
Statistical Significance ◽  
Murine Models ◽  
Immune Mediated ◽  
Tnf Α ◽  
Significant Difference ◽  
Ifn Γ

Recent research has found that Rapamycin (Rapa) was an effective therapy in mouse models of immune-mediated bone marrow failure. However, it has not achieved satisfactory effect in clinical application. At present, many studies have confirmed that Eltrombopag (ELT) combined with IST can improve the curative effect of AA patients. Then whether Rapa combined with Elt in the treatment of AA will be better than single drug application. In this study, we tested efficacy of Rapa combined with Elt as a new treatment in mouse models of immune-mediated bone marrow failure. Compared with AA group, the whole blood cell count of Rapa+Elt group increased significantly (Figure 1A) (P&lt;0.05). Survival of mice of Rapa+Elt group was significantly higher than that in the Rapa group (p &lt;0.01)(Figure 1B).There was no obvious difference in the numbers of NK cells and their subsets were noted in Rapa group,CsA group and Rapa+Elt group.The expression of NKG2D on peripheral functional NK cells was up-regulated in CsA group, Rapa group and Rapa+Elt group compared with AA group (P&lt;0.05). But there was no significant difference between effect of Rapa and CsA on the function of NK cells (Figure 1C).When Rapa combined with Elt, the expression of CD80 and CD86 are down-regulated more compared to Rapa group, but there is no statistical significance. Although these results suggested that Rapa+Elt had no statistical significance effect on numbers of mDC and expression of its functional molecule CD80 and CD86, the combined therapy still indicated that there is a potential synergy with immunosuppressant on AA mice to improve its outcome (Figure 1D).The results showed that CD4+/CD8+ ratio in CsA group, Rapa group, Rapa + Elt group had an obvious elevation than AA group (all P&lt;0.05). But there were no significant difference among the three groups on the CD4+/CD8+ ratio (Figure 1E,1F). As for INF-gamma, Rapa can reduce the secretion of IFN-γ from CD8+T cells with efficacy similar to that of the standard dose of CsA, and had a better outcome when combined with Elt in bone marrow failure mice (Figure 1E,1G).CsA group, Elt group, Rapa group, Rapa + Elt group showed notable increased ratio of Tregs compared with AA group, among which there were only Rapa group, Rapa + Elt group showed statistical significance(P&lt;0.05). for IL-10/Tregs ratio, Rapa group and Rapa +Elt group were superior to than CsA group(P&lt;0.05) (Figure 1H,1I).Rapa+Elt group and Rapa showed more lower level of IFN-γ compared with CsA group, and there was significant difference in Rapa+Elt group(P&lt;0.05). As for IL-10, IL-12p70, IL-2, IL-6, KC/GRO and TNF-α, the Rapa+Elt group showed more significant effect than Rapa or Elt alone(Figure1J). Thus, Rapa+Elt significantly down-regulated cytokines related to Th1 immune responses, such as IFN-γ, and upregulated cytokines related to Th2 immune responses, such as IL-10. To some extent, Rapa combined with Elt has a synergistic effect with CsA and Rapa alone in AA treatment. Conclusions In this study, Although Rapa combined with Elt had no significant improvement effect on the number and function of NK cells and their subsets, mDCs, and CD4+/CD8+ ratio in AA mice compared with Rapa alone, the Rapa+Elt can increase the secretion of IL-10 of Tregs and the number of Tregs, but has no significant effect on the number of Treg cells compared to with Rapa alone. Compared with AA group, the level of plasma IFN-γ, IL-2 and TNF-α decreased significantly (P&lt;0.05), but IL-10, IL-4, IL-5 and IL-1β increased significantly in Rapa group(P&lt;0.05). As for IL-10, IL-12p70, IL-2, IL-6, KC/GRO and TNF-α, the Rapa+Elt group showed more significant effect than Rapa alone. intervention treatment with Rapa in combination Elt in the AA mouse model more obviously ameliorated pancytopenia, improved bone marrow cellularity, and extended animal survival in a manner comparable to the standard dose of CsA and Rapa alone. Combination therapy support potential clinical utility in aplastic anemia treatment, which may further improve the efficacy of AA patients. Keywords: Rapamycin, Eltrombopag, murine models, bone marrow failure Figure 1 Disclosures No relevant conflicts of interest to declare.

scholarly journals Secondary CNL after SAA reveals insights in leukemic transformation of bone marrow failure syndromes

2020 ◽  
Vol 4 (21) ◽  
pp. 5540-5546
Author(s):  
Laurent Schmied ◽  
Patricia A. Olofsen ◽  
Pontus Lundberg ◽  
Alexandar Tzankov ◽  
Martina Kleber ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Aplastic Anemia ◽  
Myeloid Leukemia ◽  
Bone Marrow Failure ◽  
Driver Mutations ◽  
Leukemic Transformation ◽  
Hematopoietic Stem ◽  
Bone Marrow Failure Syndromes ◽  
Proinflammatory Responses ◽  
Stem And Progenitor Cells

Abstract Acquired aplastic anemia and severe congenital neutropenia (SCN) are bone marrow (BM) failure syndromes of different origin, however, they share a common risk for secondary leukemic transformation. Here, we present a patient with severe aplastic anemia (SAA) evolving to secondary chronic neutrophilic leukemia (CNL; SAA-CNL). We show that SAA-CNL shares multiple somatic driver mutations in CSF3R, RUNX1, and EZH2/SUZ12 with cases of SCN that transformed to myelodysplastic syndrome or acute myeloid leukemia (AML). This molecular connection between SAA-CNL and SCN progressing to AML (SCN-AML) prompted us to perform a comparative transcriptome analysis on nonleukemic CD34high hematopoietic stem and progenitor cells, which showed transcriptional profiles that resemble indicative of interferon-driven proinflammatory responses. These findings provide further insights in the mechanisms underlying leukemic transformation in BM failure syndromes.

scholarly journals Treatment of immune-mediated cytopenias in patients with primary immunodeficiencies and immune regulatory disorders (PIRDs)

Hematology ◽  
2020 ◽  
Vol 2020 (1) ◽  
pp. 673-679
Author(s):  
Markus G. Seidel
Keyword(s):  
Lupus Erythematosus ◽  
Bone Marrow Failure ◽  
Primary Immunodeficiencies ◽  
Definitive Treatment ◽  
Inborn Errors ◽  
Hematopoietic Stem ◽  
Rheumatologic Diseases ◽  
Inborn Errors Of Immunity ◽  
Immune Mediated ◽  
Regulatory Disorders

Abstract Severe immune cytopenias (SICs) are rare acquired conditions characterized by immune-mediated blood cell destruction. They may necessitate emergency medical management and long-term immunosuppressive therapy, strongly compromising the quality of life. The initial diagnostic workup involves excluding malignancies, congenital cytopenias, bone marrow failure syndromes, infections, and rheumatologic diseases such as systemic lupus erythematosus. Causal factors for SIC such as primary immunodeficiencies or immune regulatory disorders, which are referred to as inborn errors of immunity (IEIs), should be diagnosed as early as possible to allow the initiation of a targeted therapy and avoid multiple lines of ineffective treatment. Ideally, this therapy is directed against an overexpressed or overactive gene product or substitutes a defective protein, restoring the impaired pathway; it can also act indirectly, enhancing a countermechanism against the disease-causing defect. Ultimately, the diagnosis of an underling IEI in patients with refractory SIC may lead to evaluation for hematopoietic stem cell transplantation or gene therapy as a definitive treatment. Interdisciplinary care is highly recommended in this complex patient cohort. This case-based educational review supports decision making for patients with immune-mediated cytopenias and suspected inborn errors of immunity.

Investigation of TCR-Vβ CDR3 Spectrotypes in CD4 and CD8 Lymphocytes from Paroxysmal Nocturnal Hemoglobinuria (PNH) Patients.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2827-2827
Author(s):  
Akiko Nakamura ◽  
Tsutomu Shichishima ◽  
Hideyoshi Noji ◽  
Kazuhiko Ikeda ◽  
Yukio Maruyama
Keyword(s):  
Bone Marrow ◽  
Healthy Volunteer ◽  
Bone Marrow Failure ◽  
Interferon Γ ◽  
Relative Increase ◽  
Hematopoietic Stem ◽  
Bone Marrow Failure Syndromes ◽  
Abnormal Cells ◽  
Ifn Γ ◽  
Cd4 Lymphocytes

Abstract PNH is one disorder of bone marrow failure syndromes, including aplastic anemia and myelodysplastic syndrome. It is considered that immunologic mechanisms by cytotoxic T lymphocytes (CTLs) and interferon-γ (IFN-γ) contribute to hypoplastic bone marrow of these disorders. In addition, PNH is an acquired clonal disorder of the hematopoietic stem cell. Recently, it has been reported that analysis of T cell-antigen receptor (TCR)-Vβ repertoires, especially TCR-Vβ CDR3 (complementarity- determining region 3) spectrotypes, is an effective tool to study immunologic mechanisms by CTLs in pathophysiology of PNH (Karadimitris et al, Blood, 2000; Kook et al, Blood, 2002; Risitano et al, Blood, 2002). In the present study, we investigated 21 kinds of TCR-Vβ repertoires by flow cytometry in CD4 and CD8 lymphocytes from 5 PNH patients and a healthy volunteer and the TCR-Vβ CDR3 spectrotypes using polymerase chain reaction assay in CD4 and CD8 lymphocytes from 3 of 5 PNH patients and the control. We also quantitated intracellular IFN-γ in CD4 and CD8 lymphocytes from 5 PNH patients and the control according to the method by Sloand et al (Blood, 2002). We found no specific TCR-Vβ repertoires in CD4 and CD8 lymphocytes from PNH patients compared with the control. The TCR-Vβ repertoires with relative increase of CD4 or CD8 lymphocytes (over 10 of ratio of the proportion of each TCR-Vβ repertoire in a PNH patient/the proportion of the same TCR-Vβ repertoire in a healthy volunteer) were 13.6 or 4 and 22 in Case 1, 3 and 11 or 1 in Case 2, 3 and 13.6 or 3 in Case 3, 5.3 and 7.2 or 2, 3, 7, and 18 in Case 4, and 4, 5.2, 13.6, 16, and 23 or 1 and 14 in Case 5, respectively. TCR-Vβ CDR3 spectrotyping showed that in CD4 lymphocytes most CDR3 patterns were chiefly polyclonal, except for one oligoclonal (Case 1) and one monoclonal (Case 3) patterns of TCR-Vβ25; in CD8 lymphocytes most CDR3 consisted of polyclonal, oligoclonal, and/or monoclonal patterns, suggesting the possibility that CD8 lymphocytes recognize much more antigens of abnormal cells, probably including PNH clones, than CD4 lymphocytes. Unfortunately, we found the same patterns as described above in CD8 lymphocytes from the control, although CD4 lymphocytes from the control presented only polyclonal pattern of CDR3. Quantitative analyses of IFN-γ showed that index values of IFN-γ in CD4 and CD8 lymphocytes from PNH patients were higher than those from the control. However, we did not find any significant correlations between the spectrotypes of TCR-Vβ CDR3 and the index values of IFN-γ in PNH patients, suggesting that TCR-Vβ repertoires with monoclonal and oligoclonal CDR3 patterns do not necessarily produce much IFN-γ. In conclusion, our findings suggest that TCR-Vβ CDR3 spectrotyping is more effective tool to resolve some immune mechanisms of pathophysiology in PNH, especially by auto-reactive CTLs.

Sign in / Sign up

Export Citation Format

Share Document