Diversity of the T cell receptor β chain complementarity-determining region 3 in peripheral blood of neonates with sepsis: an analysis based on immune repertoire sequencing / 中国当代儿科杂志

OBJECTIVES@#To investigate the diversity of peripheral blood T cell receptor (TCR) β chain complementarity-determining region 3 (CDR3) based on immune repertoire sequencing in neonates with sepsis and the possible pathogenesis of neonatal sepsis.@*METHODS@#A total of 12 neonates with sepsis were enrolled as the case group, and 9 healthy full-term infants, matched for gestational age, birth weight, and age, were enrolled as the control group. Omega nucleic acid purification kit (SQ blood DNA Kit II) was used to extract DNA from peripheral blood samples, TCR β chain CDR3 was amplified by multiplex PCR, and then high-throughput sequencing was performed for the products to analyze the diversity of TCR β chain CDR3 and the difference in expression.@*RESULTS@#The length and type of TCR β chain CDR3 were similar between the case and control groups, and Gaussian distribution was observed in both groups. With D50 and Shannon-Wiener index as the evaluation indices for diversity, the case group had a significantly lower diversity of TCR β chain CDR3 than the control group (@*CONCLUSIONS@#There is a significant change in the diversity of TCR β chain CDR3 in the peripheral blood of neonates with sepsis, suggesting that it might be associated with the immune pathogenesis of neonatal sepsis.

Detection of Putative T cell Clones Using T cell Receptor beta Chain Gene Clonality Assay in Korean Patients with Aplastic Anemia / 대한진단검사의학회지

BACKGROUND: We analyzed T cell receptor beta chain (TCRB) gene to investigate the presence of putative T cell clones and its clinicopathologic implications in Korean patients with aplastic anemia (AA). METHODS: Twenty-nine bone marrow specimens were collected from 20 AA patients, 19 specimens from initial diagnosis and 10 from follow-up. T cell clonality assay was performed using IdentiClone(TM) TCRB Gene Clonality Assay kit (InVivoScirbe Technology, USA) and automatic genetic analyzer. Patients' clinical information and laboratory parameters were also analyzed. RESULTS: Five patients had definitive underlying factors related with aplastic anemia, such as hepatitis B virus (4 cases) and benzene exposure (1 case). Putative T cell clones were detected in bone marrow specimens of 11 (58%) out of 19 patients at diagnosis. The location of putative T cell clones of TCRB gene (diversity region, Dbeta; joining region, Jbeta; variable region, Vbeta) was distributed in Dbeta2+Jbeta2 (6 cases), Dbeta1+Jbeta1 (3 cases), Vbeta+Jbeta1 (2 cases), and Dbeta1+Jbeta2 (2 cases). Interestingly, among seven patients who underwent stem cell transplantation, five patients with no T cell clones detected at diagnosis developed new T cell clones during the follow-up. CONCLUSIONS: Putative pathogenetic T cell clones were detected in most of AA patients in the current study. T cell clonality assay would be useful for investigating the pathophysiology of acquired AA.

The presence of CD8+ invariant NKT cells in mice

Invariant natural killer T (iNKT) cells develop in the thymus upon recognition of CD1d expressed on developing thymocytes. Although CD4 and CD8 coreceptors are not directly involved in the interaction between CD1d and the T cell receptors (TCRs) of iNKT cells, a conspicuous lack of CD8+ iNKT cells in mice raised the question of whether CD8+ iNKT cells are excluded due to negative selection during their thymic development, or if there is no lineage commitment for the development of murine CD8+ iNKT cells. To address this question, we analyzed iNKT cell-specific TCR Valpha14+ transgenic mice, where the Valpha14 transgene forces the generation of iNKT cells. This allows detailed study of the iNKT cell repertoire. We were able to identify CD8+ iNKT cells which respond to the NKT cell-specific glycolipid ligand alpha-galactosylceramide. Unlike conventional iNKT cells, CD8+ iNKT cells produce predominantly IFN-gamma but not IL-4 upon antigen stimulation. We also confirmed the presence of CD8+ iNKT cells in wild type mice. Our results suggest that CD8+ NKT cells do exist in mice, although their population size is quite small. Their Th1-skewed phenotype might explain why the population size of this subtype needs to be controlled tightly.

Experimental autoimmune encephalomyelitis in cynomolgus monkeys

Experimental autoimmune encephalomyelitis was induced in macaques. T cell clones infiltrated into the brain lesion area were compared with those in blood. Intradermal immunization of macaques with brain white matter derived from healthy macaque in combination with pertussis toxin, induced neurological symptoms in two macaques. One died on day 25 after immunization, whereas the other survived. Gross examination of the brain from the dead macaque, showed clear hemorrhagic lesions in the white matter. Hematological analysis showed that drastic T cell response was induced in macaques immunized with white matter, but not in control macaques. Flow cytometric analysis of blood cells from the affected macaques demonstrated an increase of CD4 and CD8 T cell populations expressing the CD69 early activation marker. Single strand conformation polymorphism (SSCP) analysis of T cell receptor beta chain showed T cell clones infiltrated into the brain lesion, which were different from those found in the peripheral blood of the same monkey. The present paper shows that SSCP analysis of TCR is useful in studying clonality of T cells infiltrating into the brain tissue of macaque with EAE.

Endoplasmic reticulum retention and degradation of T cell antigen receptor beta chain

The T cell antigen receptor-CD3 (TCR/CD3) complex is assembled in the endoplasmic reticulum (ER) of T cells after synthesis of individual chains, and is transported to the cell surface for immune recognition and regulation. Partially assembled or unassembled TCR chains are retained and rapidly degraded in the ER. These processes are strictly regulated in the ER at post-translational level for the maintenance of cellular homeostasis. In order to identify the region responsible for the ER retention and rapid degradation of the TCR beta chain, number of mutants were engineered and their fates, after synthesis in the ER of the HeLa cells, were investigated. Extensive mutagenic analysis of TCR beta chain, including changing the charged amino acid residues and two tyrosine residues of the transmembrane region into hydrophobic amino acid residues, did not alter the ER retention and rapid degradation. Soluble TCR beta chain and cytoplasmic tail truncation mutant were also rapidly degraded in the ER. However, N-glycosylation rate of soluble TCR beta chain in the ER was significantly increased possibly due to the increased exposure of the N-glycosylation site. These results suggest that the ER retention of TCR beta chain is mediated through its extracellular and transmembrane-cytoplasmic regions and that the rapid ER degradation can be caused by an exposure of unassembled subregion of TCR beta chain, either extracellular domain or hydrophobic transmembrane region to the hydrophilic environment (lumen of the ER) rather than by presence of a specific degradation signal.

High frequency of specific T cell receptor rearrangements in rheumatoid arthritis: analysis of synovial fluid T lymphocytes

1. We have searched for rearrangements in the beta chain T cell receptor genes to identify clonal T lymphocyte populations in the synovial fluid of 10 patients with well established rheumatoid arthritis, using a T cell population unbiased by preselection. 2. Analysis of the restriction fragments with the beta chain constant region probe C beta 2 disclosed a rearranged band in 50 of cases (5/10). No significant differences in age, duration of the disease, treatment employed and presence of articular deformities or erosion upon X-ray examination were observed when patients with or without rearrangements were compared. 3. The rearranged band observed after BamH I digestion was of the same size in the 5 patients (14 kb). In addition, two patients presented a 10-kb rearranged band upon restriction with Hind III. 3. These data indicate that a significant number of rheumatoid arthritis patients probably present oligoclonal T cell proliferation of their synovial fluid lymphocytes.