In the U.S., approximately 1.7 million people suffer traumatic brain injury each year, with many enduring long-term consequences and significant medical and rehabilitation costs. The primary injury causes physical damage to neurons, glia, fiber tracts and microvasculature, which is then followed by secondary injury, consisting of pathophysiological mechanisms including an immune response, inflammation, edema, excitotoxicity, oxidative damage, and cell death. Most attempts at intervention focus on protection, repair or regeneration, with regenerative medicine becoming an intensively studied area over the past decade. The use of stem cells has been studied in many disease and injury models, using stem cells from a variety of sources and applications. In this study, human adipose-derived mesenchymal stromal cells (MSCs) were administered at early (3 days) and delayed (14 days) time points after controlled cortical impact (CCI) injury in rats. Animals were routinely assessed for neurological and vestibulomotor deficits, and at 32 days post-injury, brain tissue was processed by flow cytometry and immunohistochemistry to analyze neuroinflammation. Treatment with HB-adMSC at either 3d or 14d after injury resulted in significant improvements in neurocognitive outcome and a change in neuroinflammation one month after injury.

Most patients with acquired pure red cell aplasia (PRCA) and some with acquired aplastic anemia (AA) respond well to cyclosporine (CsA), but thereafter often show CsA dependency. The mechanism underlying this dependency remains unknown. We established a reliable method for measuring the regulatory T cell (Treg) count using FoxP3 and Helios expression as markers and determined the balance between Tregs and other helper T cell subsets in 16 PRCA and 29 AA patients. The ratios of interferon-γ-producing CD4 + (Th1) T cells to Tregs in untreated patients and CsAdependent patients were significantly higher (PRCA 5.77 ± 1.47 and 7.38 ± 2.58; AA 6.18 ± 2.35 and 8.94 ± 4.06) than in healthy volunteers (HVs; 3.33 ± 0.90) due to the profound decrease in the percentage of Tregs. In contrast, the ratios were comparable to HVs in convalescent CsA-treated AA patients (4.74 ± 2.10) and AA patients in remission after the cessation of CsA treatment (4.24 ± 1.67). Low-dose CsA (100 ng/ml) inhibited the proliferation of conventional T cells (Tconv) to a similar degree to the inhibition by Tregs in a co-culture with a 1:1 Treg/Tconv ratio. The data suggest that CsA may reverse the hematopoietic suppression in PRCA and AA patients by compensating for the inadequate immune regulatory function that occurs due to a profound decrease in the Treg count.

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The presence of leukocytes lacking one haplotype of the human leukocyte antigen (HLA) gene as a result of copy-neutral loss of heterozygosity of chromosome 6p (6pLOH) is compelling evidence that cytotoxic T cells (CTLs) have a key role in the development of acquired aplastic anemia (AA). Pathogenic auto-antigens are presumed to be presented by particular HLAs, such as HLA-B*40:02, HLA-A*31:01, HLA-A*02:01 and HLA-A*02:06, all of which are frequently lost due to 6pLOH (Katarigi, Blood 2011). However, this presumption may be incorrect, because the frequency of lacking haplotypes that contain KIR-ligands (KIR-Ls), such as HLA-A*24:02 and HLA-B*52:01, the most frequent alleles of HLA-A and HLA-B, may be underestimated as a result of the killing of 6pLOH(+) leukocytes by NK cells. Indeed, all four frequently missing HLA alleles are non-KIR-Ls. To address these issues, we conducted a mass screening of 6pLOH(+) leukocytes in newly diagnosed AA patients using an assay that allows us to detect 6pLOH(+) cells within one day, and reanalyzed haplotypes that are likely to be lost. The HLA-B and –C allele-specific duplex real time PCR (2qPCR) that we established compared the copy number of heterozygous HLA alleles in a single reaction mixture using two allele-specific TaqMan probes labeled with two different fluorochromes (VIC and FAM) and one primer pair complementary to consensus sequences, which allowed us to detect as small as 5% 6pLOH(+) leukocytes in the total leukocytes. The HLA haplotypes of 6pLOH(+) patients were determined based on the HLA-A antigen expression as demonstrated by flow cytometry and the HLA-B and -C allele data from the 2qPCR, in combination with the haplotype database of the Japanese population available in the HLA laboratory website. A total of 498 patients with AA were subjected to this analysis and 60 6pLOH(+) patients, including 39 6pLOH(+) patients that had been identified by our previous study were used in this analysis. The allelic loss frequencies of HLA-B*40:02 (25%, 33/132) and HLA-A*31:01 (19%, 14/75) were markedly higher than those of the other HLA-B (2.9%, 24/814, P = 7x10-16) and HLA-A (4.9%, 36/731, P = 0.002) alleles, while the frequencies of HLA-A*02:01 (11%, 11/99) and HLA-A*02:06 (11%, 11/99) were similar to those of the other HLA-A alleles. In 41 6pLOH(+) patients possessing either HLA-B*40:02 or HLA-A*31:01, these alleles were all contained in the missing haplotype. On the other hand, four of the 15 patients with HLA-A*02:01(+) haplotypes and two of the 13 patients with HLA-A*02:06(+) haplotypes had these alleles in the retained haplotype. The 6pLOH(+) patients could be divided into three groups according to the status of KIR-Ls (HLA-Bw4 and HLA-C1/C2) of their haplotype; A, the lack of the KIR-L does not occur when either of the two haplotypes is lost; B, the lack of KIR-L occurs when one specific haplotypes is lost; C, the lack of KIR-L occurs when either haplotype is lost. These groups comprised 42%, 53% and 5% of the total. The proportion of group C was much lower than that expected in the general population (16%) and only five (16%) of the 31 group B patients lacked KIR-Ls, thus suggesting that NK cells had an effect on the appearance of 6pLOH(+) leukocytes in AA patients. However, the very high frequency of HLA-loss in the HLA-B*40:02 and HLA-A*31:01 alleles could not be explained by the absence of KIR-Ls in the missing haplotype. Of particular note, the lack of KIR-Ls occurred in eight patients as a result of 6pLOH; six of the eight lost a haplotype containing HLA-B*40:02 and one lost an A*31:01-containing haplotype, suggesting that CTLs specific for autoantigens presented by these class I alleles more dominantly inhibit HSCs than NK cells. Together, these results indicate that HLA-B*40:02 and HLA-A*31:01 have particularly important roles in the presentation of autoantigens to T cells in AA. Studies of T-cell responses to autoantigens restricted by these class I alleles are thus warranted. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

Background: HLA-A allele-lacking leukocytes (HLA-LLs) derived from hematopoietic stem cells (HSCs) with copy-number neutral loss of heterozygosity (LOH) in chromosome 6p (6pLOH) are detected in 13% of patients with acquired aplastic anemia (AA). These defective leukocytes account for more than 90% of granulocytes in some AA patients in remission after immunosuppressive therapy (IST). However, it remains unclear how a few HSC clones with genetic abnormalities sustain hematopoiesis for a long time in AA patients. The fate of 6pLOH(+) HSCs can be easily monitored by revealing HLA-LLs using monoclonal antibodies (mAbs) specific for HLA-A alleles. Phenotypic analyses of hematopoietic progenitor cells (HPCs) in bone marrow (BM) and gene expression profiling of HLA-A allele-lacking HPCs may help to clarify the mechanisms underlying clonal hematopoiesis by 6pLOH(+) HSCs in patients with AA. Methods: The HLA-A allele expression of BM CD34+ cell subsets, including common myeloid progenitors (CMPs), megakaryocyte/erythroid progenitors (MEPs) and granulocytic/monocyte progenitors (GMPs) in three patients possessing HLA-LLs was determined using mAbs specific for CD34, CD38, CD45RA, CD123 and three different HLA-A alleles. The HLA-A allele-lacking and wild-type CMPs were sorted, and the gene expression levels were compared between the two subsets using an Agilent Whole Human Genome Oligo Microarray (4x44K). The CXCR4 expression by CMPs was compared among different types of BM failure patients. Results: The percentages of HLA-A allele-lacking cells in peripheral blood granulocytes, GMPs, MEPs and CMPs of the three patients were 54.1%/67.3%/63%/1.7%, 98.1%//97.2%/98.8%/4.5% and 97.2%/97.8%/96.9%/12.9%, indicating that the hematopoiesis of these patients was being supported by HLA-A-lacking cells at the CMP level, which accounted for only a small percentage of the total CMPs. To characterize the CMPs capable of supporting hematopoiesis, the gene expression profiles were compared between HLA-A-lacking CMPs that substantially contributed to hematopoiesis and HLA-A-retaining (wild type) CMPs that did not contribute to hematopoiesis. One of the striking differences in the gene expression profile between the two groups was a lower expression of CXCL12 in the HLA-lacking CMPs compared to the wild type CMPs (1 vs. 27.6). Because CD34+ cells are known to negatively regulate themselves by secreting CXCL12, the expression of its receptor, CXCR4, on CMPs was examined using flow cytometry. Surprisingly, all HLA-A-lacking cells were negative for CXCR4, while most of the wild-type cells expressed the CXCL12 receptor (Figure). Examination of the CD34+ cell subsets from 10 healthy individuals showed that the percentages of CXCR4-negative cells were 25.9±8.7% (mean±SD) in CMPs, 98.7±6.4 in MEPs, 98.7±2.1 in GMPs and 77.1±11.5 in CD34+ cells. When the CXCR4 expression levels of CMPs were compared among different types of BM failure, the mean fluorescence intensity (MFI) of CXCR4 in the CMPs from four patients with AA (550±76, mean±SD) was significantly lower than that in three patients with MDS (901±332) and 10 healthy individuals (1440±229). Conclusions: Hematopoiesis in patients with AA is supported by a limited number of CXCR4(-) cells at the CMP level. Decreased CXCR4 expression by CMPs of AA patients may represent immune pressure applied to HSCs that elicits the participation of dormant CMPs in hematopoiesis. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.