| Adverse drug reaction (ADRs) is one of the most common clinical diseases, accounting for 3%-6% of all the cause of hospitalization. The clinical manifestations of ADRs are diverse, the skin of patients is frequently affected, and the degree of harmfulness is different. Among them, drug-induced hypersensitivity syndrome (DIHS) is a type of serious skin adverse drug reaction syndrome which is life-threatening. DIHS is also called drug reaction with eosinophilia and systemic symptoms(DRESS), typically develops during 2 to 6 weeks after the application of causative drugs, the patients show fever, rash (facial and periorbital edema, trunk limbs symmetrically lesions like measles or exfoliative dermatitis damage), lymph node swelling and multiple organs (liver, lung, kidney and heart) are affected with hematologic abnormalities (eosinophils, atypical lymphocytes and/or monocytes are increased). The sensitive drugs of DIHS contain aromatic anticonvulsants, allopurinol, minocycline, sulfonamides and antiviral drugs (abacavir, nevirapine). According to the epidemiological survey, in the population of patients treated with the anticonvulsant phenytoin, the incidence of DIHS is about 1/10000-1/1000, and the mortality rate is up to 20%-30%, if multiple organs are affected, the risk of patients’death is significantly increased. In addition, the relapse rate of DIHS is high, even though culprit drug are discontinued for weeks or months, the patients often show relapse. At present, the exact pathogenesis of DIHS remains unknown and need to be clarified for preventing its occurrence and guiding clinical treatment.The pathogenesis remains to be determined but three components are considered: drug pharmacokinetics and metabolism abnormalities, HLA genetic susceptibility and sequential reactivation of herpesvirus family. Aromatic anticonvulsants such as carbamazepine, phenytoin and phenobarbital are metabolized by the hepatic cytochrome P450 (CYP) enzymes and undergo oxidation by aromatic hydroxylation, with the formation of toxic arene oxides. These reactive intermediaries are normally enzymatically converted to nontoxic metabolites by epoxide hydroxylase or glutathione transferase. A deficiency of epoxide hydroxylase can lead to the accumulation of arene oxides, which may offer abnormal immune responses or cytotoxic reaction. Due to eosinophils and macrophages are stimulated by toxic metabolites, and abnormal activation of T-lymphocytes, cytokines especially interleukin-5 (IL-5) secretion is induced, which then cause hypersensitivity reaction. At present, there are two interpretions on drug hypersensitivity hypothesis: hapten/pre-hapten theory and pharmacology theory (pharmacologic interaction of durg with immune receptor, P-I concept). The former show that drugs are small molecules and no immunogenicity. However, while drugs are linked with large molecular proteins, they can become semi-antigen with immunogenicity. Therefore, the drugs-proteins compounds can be processed and presented to the major histocompatibility complex (MHC) by antigen-presenting cells (APC), and hapten peptide-MHC complex can be formed and then recognized by T cell receptor (TCR), T cells are subsequently activated and eventually drug hypersensitivity is caused. Nevertheless, P-I theory show that some drugs with MHC molecule on the surface of APC can be directly recognized by TCR and then T cells are activated, and some drugs show strong relevance with TCR. For example, the metabolite of allopurinol can bind with auto-immune antigen peptides and HLA-B*5801 gene of MHC-I molecules and then was recognized by CD8+ effector T cells. The activated cytotoxic reaction of CD8+ effector T cells damage the mucosa of skin, suggesting that DIHS is genetic susceptibility. In addition, the occurrence and aggravation of DIHS are associated with herpes virus sequential reactivation. Recently, studies have found that HHV-6 infection is closely related to DIHS. In addition to HHV-6, Epstein-Barr virus (EBV) and cytomegalovirus (CMV) reaction in the pathogenesis of DIHS can be also detected. Picard et al detected the peripheral blood mononuclear cells and serum of 40 DIHS patients by using polymerase chain reaction (PCR), and found that the DNA of EBV was detected in all patients and the EBV reaction positive ratio was 42%(16/38) and HHV-6 reaction positive ratio was 45% (17/38), but EBV and HHV-6 infection was not detected in the control group.The latest study showed that DIHS was the type of sensitized immune response to special drugs. Due to the drug itself had the ability to activate T cells, T cells could specially proliferate after drugs were recognized by T cells. Meanwhile, viral genome was integrated into the nucleus of T cells, which induced cellular immune response to the HHV. And ultimately caused multiple organ damage associated DIHS. In addition, at the early stage of the disease, the level of IgG, IgA and IgM and the counts of B cells were all decreased, accompanied with T cells proliferation induced by the cross-interaction of virus and causative drugs. Even though drugs withdrawal, immune response could be not stopped owing to virus antigen. Thus, the complication of DIHS with repeatedly fever and relapse after drug withdrawal is associated with HHV infection. Another study showed that CD8+ T cells are the major effector cells involved in the pathogenesis of DIHS by secreting perforin, granzyme, IL-5 and IFN-y. Above all, CD8+ T cells play an important role in the pathogenesis of DIHS, and TCR is the key point of CD8+ T-cell recognition and activation.The TCR-mediated cross-reactivation between virus-activated T-cell and some sensitizing drugs, which reflected by valid TCR gene segment rearrangement, was a critical driven force in the pathogenesis of DIHS. Recently, richly expensive progresses were archived in research of TCR gene segment rearrangement, such as anti-virus infection, GVHD, cancer and autoimmune disease. TCR gene rearrangement is completely random, and displays the signatures of multi-family and multi-clonality. Complementarity determining region 3 (CDR3) was the direct contact site with antigens in T-cell, and, an amino acid sequence of CDR3 represented a unique T-cell clones. In physiological circumstance, the spectrum of CDR3 length distribution exhibited the Gaussian distribution pattern with 8-10 peaks. However, single or less than 8 of peaks in CDR3 length distribution, commonly referred as TCR repertoire shifting, which will be observed in pathological conditions if one or several sub-family T-cells were mono or oligo-clonal amplified by certain antigens. The analysis and sequencing of TCR CDR3 region, and the characterization of disease-specific CDR3 pattern, has an important significance to understand the severity of disease, personalized treatment and the prevention of DIHS. However, the pathogenic role and the clinic relevance of TCR repertoire shifting in DIHS is still not clear.According to the pathogenesis of DIHS, we selected 8 cases of DIHS patients with different stages of disease, collected peripheral blood and sorted CD4+/CD8+ T-cells in PBMC and subjected to high-throughput repertoire sequencing to characterize the spectrum of TCR CDR3 region, establish the TCR features of common motifs, and analysis of its relationship with the occurrence, development and severity of DIHS.Main results:1. Signatures and dynamic modification of CD4+/CD8+T-cell repertoire in DIHS1.1. Principal component analysis indicated that the overall usage of the TRBV/J segment was highly dependent on the individual, without overt general bias in either DIHS patients or healthy donors. These data suggested that an aberrant TRBV/J usage pattern is not a valid biomarker for diagnosis of DIHS.1.2. We compared the proportion of highly expanded clones (HECs, defined as clonotypes having an abundance beyond a cutoff value in their repertoires) between patients and condition-matched healthy donors. To determine the "highly expanded" threshold, a gradient cutoff (ranging from 0.1% to 10%, at intervals of 0.1%) was introduced to avoid artificial bias. The proportion of expanded CDR3 AA clonotypes (defined as the T cell population in which the amino acid sequence in the CDR3 region were identical) tended to show a clear increase, in DIHS patients, in contrast to those in healthy donors. These results suggested that the dominant clonotypes are substantially elevated in the patients’repertoire relative to that in healthy donors, which strongly implicated the occurrence of selective proliferation events in DIHS.1.3. To quantify repertoire dynamics over time, we used a novel analysis approach, employing the MHSI, which is a statistical measure of clonal similarity of two repertoires. The pattern for the CD8+ T cell repertoire tended toward a polarized distribution, being either persistently stable or markedly unstable. In contrast, CD4+ T cells showed a uniform distribution without any potential clustering across the whole range of analysis. Furthermore, the average MHSIs of CD4+ and CD8+ T cell subsets in all intervals for each patient were clustered based on the expectation maximization algorithm under Gaussian mixture models.1.4. Dichotomous aspect of the CD8+ T cell repertoire hierarchy mainly reflects the disparate abilities to maintain the stability of dominant clones in the repertoires. Repertoires harboring overlapping dominant clones would be clustered closely in a binary tree, the "flat" group of 4 patients exhibited unique signatures throughout the course of the disease. Conversely, the "fluctuant" group of patients did not exhibit such well-organized distributions, but rather were arranged loosely in the dendrogram. These findings supported our previous similarity analysis and suggested that the loss-of-reproducibility of dominant clones may make the main contribution to reformation in the CD8+ repertoire hierarchy.2. Clinic relevance of CD8+ T-cell repertoire and potential pathogenic mechanism2.1. To assess the clinical relevance of the fluctuation in the CD8+ T cell repertoire hierarchy, the association between the MHSI and clinical severity was established. The MHSI of each CD8+ T cell-sampling interval correlated positively with KPS. Less strongly positive correlation, was observed for the CD4+ compartment. RegiSCAR scoring system confirmed that, disease severity was reproducibly associated with the level of fluctuation in the CD8+ repertoire.2.2. Assessment of anti-viral antibodies in the "flat" and "fluctuant" groups demonstrated a significant elevation of IgM anti-HHV-6 antibodies in the "fluctuant" group, compared to the "flat" group, at the time of diagnosis. Furthermore, we found that the IgG antibodies to EBV-EBNA and HHV-6, and IgM antibodies to HHV-6, from the samples taken over the course of the DIHS syndrome were also significantly elevated in the "fluctuant" group compared to the "flat" group. These data strongly implicated much higher B cell responses to a virus-challenge in patients in the "fluctuant" group than in the "flat" group.2.3. The proportion of CD4+ homologous EBV-specific CDR3 AA clones was almost independent of the reformation of repertoires (R= 0.06, p= 0.8121), indicating that the CD4+ repertoire may not the driving force in repertoire modification. In contrast, we found that these homologous EBV-specific CDR3 AA clones in the CD8+ repertoire correlated well with repertoire fluctuation (R=-0.58, p= 0.0049), providing strong evidence that expansion of EBV-specific CD8+ T cells may contribute to the marked repertoire fluctuation during DIHS progression.2.4. When comparing the percentage of homologous EBV-specific CDR3 AA clones in the "flat" and "fluctuant" groups, a 2-fold increase in the percentage of CD8+ homologous clones was identified in the "fluctuant" group (p= 0.0077). Consistent with previous findings, we failed to observe any significant difference in homologous clones for the case of CD4+ counterpart. Taken together, this substantial difference reinforced our observation that persistent arising of CD8+ homologous EBV-specific clones may be a driving force in repertoire fluctuation.Conclusion:1. Dominant clonotypes are substantially elevated in the patients’repertoire relative to that in healthy donors, which strongly implicated the occurrence of selective proliferation events in DIHS,2. Dichotomous aspect of the CD8+ T cell repertoire hierarchy mainly reflects the disparate abilities to maintain the stability of dominant clones in the repertoires.3. Disease severity was reproducibly associated with the level of fluctuation in the CD8+ repertoire, but not the CD4+ repertoires.4. Proportion of homologous EBV-specific CDR3 AA clones in the CD8+ repertoire correlated well with repertoire fluctuation, and evelated in "fluctuation" patients, providing strong evidence that expansion of EBV-specific CD8+ T cells may contribute to the marked repertoire fluctuation during DIHS progression. |
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