Ferritin Light Chain: A Candidate Autoantigen in Immuno-Related Pancytopenia.

The characteristic feature of immune-related pancytopenia (IRP) is autoantibody-mediated bone marrow (BM) damage and peripheral blood cytopenia. We found that the potential antigen of IRP was Ferritin light chain (FTL) by SEREX (serological analysis of recombinant cDNA expression libraries) in the previous study. In this study, we tried to explore the antigenic epitopes of FTL and verify its antigenicity in IRP. We found the possible FTL epitope: VNLYLQASYTYLSLG by phage random peptide library. Through ELISPOT, it was found that peptide VNLYLQASYTYLSLG can significantly stimulate the production of interleukin-4 and cannot stimulate the production of interferon-γ, which suggested that the peptide can obviously activate Th2 cells. Peptide-major histocompatibility complex tetramer elicited antigen-specific T cell responses. The expression levels of FTL were significantly increased in the patients with untreated IRP (P < 0.05). In conclusion, we found that FTL is the target antigen for some patients with IRP. The peptide of VNLYLQASYTYLSLG is an epitope of the target antigen. The target antigen is abnormally overexpressed on the membrane of BM cells, especially on the surface of CD34+ BM cells of patients with IRP. In addition, it is related to the severity of disease. These results provide a possible new target for the treatment of IRP in the future.

Antibodies specific to ferritin light chain polypeptide are frequently detected in patients with immune­related pancytopenia.

Immuno-related pancytopenia (IRP) is characterized by pancytopenia resulting from bone marrow suppression or destruction mediated by auto­antibodies. In our previous study, a K562 cDNA library was established, which was used to screen for seven possible auto­antigens produced by hematopoietic cells in patients with IRP, including ferritin light chain (FTL). In the present study, FTL was expressed and purified, and the levels of the auto­antibodies specific to FTL were measured. Through ELISA, it was shown that the titer of anti­FTL antibodies was higher in patients with IRP without treatment compared with those who had recovered from IRP, those with severe aplastic anemia (SAA), those with myelodysplastic syndrome (MDS) and the healthy controls. Furthermore, the expression levels of FTL­mRNA were upregulated in patients with IRP without treatment compared with those who had recovered from IRP, those with MDS and the normal controls. The results suggest that FTL antibody expression is upregulated in patients with IRP. Detecting FTL antibodies may therefore have certain clinical value in differentiating between IRP, SAA and MDS. Furthermore, in specific patients with IRP, FTL as an auto­antigen may induce immune attack on hematopoietic stem cells.

Roles of immune responses in the pathogenesis of immunorelated pancytopenia.

Some patients with pancytopenia do not conform to any diagnostic criteria of known haematological or non-haematological diseases; however, they respond well to corticosteroid, high-dose intravenous immunoglobulin and rituximab treatment. This abnormality is termed immunorelated pancytopenia (IRP). Later studies indicated that IRP might be a kind of autoimmune disease in which T helper (Th) type 2 cell function is enhanced, resulting in the hyperfunction of B lymphocytes, which then produce excess autoantibodies that attack the bone marrow (BM) and cause cytopenia. Hypofunction of regulatory T (Treg) cells and enhanced Th17 cell function, an elevated percentage of plasmacytoid dendritic cells (pDCs) and a decreased percentage of natural killer (NK) cells help to promote the process. Moreover, increased expression of a synergistic stimulator of B lymphocytes, CD70 and the reactive overexpression of the BCR inhibitory coreceptor CD22 also support this claim. Candidate autoantigens targeted by autoantibodies on haematopoietic cell membranes have also been reported in IRP. This review is focused on studies that demonstrate the role of immune responses in the pathogenesis of IRP. Current diagnostic criteria and treatments for IRP are also referenced to provide a thorough understanding. Distinguishing IRP from idiopathic cytopenias of undetermined significance (ICUS) and other haematological disorders, for example myelodysplastic syndrome (MDS), aplastic anaemia (AA), paroxysmal nocturnal hemoglobinuria (PNH) and Evans syndrome, may help patients with pancytopenia benefit from proper treatment. Further studies are required to achieve new insight into the pathophysiology of IRP with regard to the immune system, which will be instrumental for the development of novel therapies for inhibiting disease initiation and/or progression.

Abnormal numbers of CD4+ T lymphocytes and abnormal expression of CD4+ T lymphocyte­secreted cytokines in patients with immune­related haemocytopenia.

In the past decade, a group of cases with persisting haemocytopenia were separated from those with idiopathic cytopenia of undetermined significance due to the optimal response of these patients to immunosuppression therapy and due to the detection of autoantibodies in the bone marrow of haemopoietic cells. This condition was termed immune­related haemocytopenia (IRH). However, the quantity of T lymphocytes remained unknown. In the present study, the percentage of CD4+ T­cell subsets and related cytokines was measured using flow cytometry and an enzyme­linked immunosorbent assay. An abnormal number of CD4+ T cell subsets was found, including increased percentages of T helper (Th)2, Th9 and Th17 cells and a decreased number of regulatory T (Treg) cells. In addition, the results showed downregulation in the levels of interleukin (IL)­2, transforming growth factor­ß and IL­35, and upregulation in the levels of IL­4, IL­6, IL­17, IL­23 and interferon­Î³ in patients who did not receive therapy (untreated patients). These levels were significantly associated with the number of peripheral blood cells and were recovered following treatment. In conclusion, an abnormal number of CD4+ T cell subsets and corresponding abnormal levels of regulatory cytokines resulted in the stimulation of B1 lymphocytes to produce autoantibodies in IRH, which may be considered as markers to evaluate disease prognosis and treatment strategies.

Lower level of IL­35 and its reduced inhibition in Th17 cells in patients with bone marrow mononuclear cells Coombs test­positive hemocytopenia.

Interleukin (IL)-35 is the latest member of IL-12 family, which plays an important role in other autoimmune diseases. Bone marrow mononuclear cells Coombs test­positive hemocytopenia, also termed immunorelated hemocytopenia (IRH) is a type of autoimmune-associated diseases. The present study investigated the relationship of IL­35 in patients with IRH. A total of 43 patients with IRH and 19 normal controls were enrolled in the current study. Serum levels of IL­35 and IL­17 in peripheral blood were evaluated by ELISA. Regulatory T cells (Tregs) level was detected by flow cytometry and IL­35 subunits mRNA in Treg was determined using reverse transcription­quantitative polymerase chain reaction: Epstein­Barr virus induced 3 (EBI3) and IL­12α chain p35. Effect of IL­35 on T helper 17 cells (Th17) cells was determined by mix­culture of IL­35 with CD4+ T lymphocytes. Serum level of IL­35 was decreased in untreated patients with IRH compared with remission patients (P<0.01) and was significantly associated with clinical indexes. Frequency of IL­35 produced Tregs was lower and IL­35 subunits mRNA in CD4+CD25+ Tregs were decreased in patients with IRH compared with health controls (P<0.01). Serum level of IL­17 was increased in patients with IRH (P<0.01) and there was a negative correlation between IL­35 and IL­17 (r=­0.553; P<0.01). The production of Th17 cells and IL­17A mRNA expression were reduced (P<0.05) after mix­culture of CD4+ T lymphocytes with IL­35 compared with mix­culture of CD4+ T lymphocytes without IL­35. In conclusion, the present study revealed that IL­35 may be a monitoring indicator of IRH occurrence and progression. IL­35 level was lower and the inhibition on Th17 cells was reduced in the patients with IRH.

Screening novel autoantigens targeted by serum IgG autoantibodies in immunorelated pancytopenia by SEREX.

Immunorelated pancytopenia (IRP) is characterized by pancytopenia caused by autoantibody-mediated destruction or suppression of bone marrow. However, the autoantigens targeted by autoantibodies in IRP remain unclear. In the present study, we screened novel autoantigens in IRP by serological analysis of recombinant cDNA expression libraries and compared anti-UQCR10 antibody levels between IRP and normal controls detected by immunoblotting. Our results indicate that we successfully constructed the K562 cDNA library, which we used to screen seven candidate autoantigens expressed in haematopoietic cells of IRP: ferritin, light polypeptide, ubiquinol-cytochrome c reductase, complex III subunit X (UQCR10), multifunctional methyl-transferase subunit TRM112-like protein isoform 1 (TRMT112), hemoglobin gamma-G, stathmin 1 (STMN1), transcript variant 3, phosphoglycerate kinase 1 (PGK1), and trafficking protein particle complex subunit 4 (TRAPPC4). Six of 17 (35.29%) IRP patients exhibited positive reactivity to UQCR10 antigen, while only one of 10 (10%) of normal controls reacted to UQCR10 antigen. The IRP patients with positive reactivity to UQCR10 antigen exhibited significantly improved total efficiency (6/6) compared with those with negative reactivity (5/11). Thus, UQCR10 may be implicated as one of the autoantigens involved in development of IRP.

HOTAIR is upregulated in acute myeloid leukemia and that indicates a poor prognosis.

Hox transcript antisense intergenic RNA (HOTAIR) is a long non-coding RNA, its overexpression has been documented in various human solid tumor and it can be considered as a potential cancer biomarker. However, little is known about the role of HOTAIR in acute myeloid leukemia (AML). In this study, We evaluated HOTAIR expression in bone marrow of de novo AML patients, AML-CR patients and normal controls by real-time quantitative reverse transcription-PCR (qRT-PCR), then we inhibited hotair expression of two cell lines by siRNA and evaluated their proliferation by CCK, finally we analyzed its relationship with the clinicopathological parameters of AML. We found that HOTAIR is significantly upregulated in de novo AML patients compared with those of AML-CR patients and normal controls; the reduction of HOTAIR by small interfering RNA (siRNA) repressed the proliferation of HL-60 and K562; the higher expression level of HOTAIR in AML patients was significantly correlated with NCCN high risk group. In conclusion, our study indicated that hotair is highly expressed in AML patients, and hotair expression significantly correlates with clinicopathological prognostic stratification in AML.