Lesions of T-Cell-Mediated Kidney Allograft Rejection in Mice Do Not Require Perforin or Granzymes A and B

Organ allograft rejection is strongly associated with the presence of alloreactive cytotoxic T cells but the role of cytotoxicity in the pathologic lesions is unclear. Previous studies showed that the principal lesions of kidney rejection - interstitial infiltration, tubulitis, and endothelial arteritis - are T-cell-dependent and antibody-independent. We studied the role of cytotoxic granule components perforin and granzymes A and B in the evolution of the T-cell-mediated lesions of mouse kidney transplant rejection. By real-time RT-PCR, allografts rejecting in wild-type hosts at days 5, 7, 21, and 42 showed massively elevated and persistent expression of perforin and granzymes A and B, but evolution of tubulitis and arteritis did not correlate with increasing granzyme or perforin expression. Allografts transplanted into hosts with disrupted genes for perforin or granzymes A and B showed no change in tubulitis, arteritis, or MHC induction. Thus the development of the histologic lesions diagnostic of T-cell-mediated kidney transplant rejection are associated with but not mediated by perforin or granzyme A or B. Together with previous graft survival studies, these results indicate that the granule-associated cytotoxic mechanisms of T cells are not the effectors of T-cell-mediated allograft rejection.     

Granzyme B-positive primary gastric T-cell lymphoma: gastric T-cell lymphoma with the possibility of extrathymic T cell origin

A case of primary gastric T-cell lymphoma, which was positive for granzyme B, is reported. The patient was a 47-year-old Japanese female who complained of a dull upper abdominal pain. Radiographic and endoscopic examinations revealed an ulcerative infiltrative lesion in her stomach. Following the confirmation of a high-grade malignant lymphoma, a distal gastrectomy with regional lymph nodal dissection was performed. The histology of the gastric lesion revealed a malignant lymphoma of the diffuse pleomorphic type without lymph nodal involvement. Immunohistochemistry revealed that the tumor cells were positive for LCA, CD3, TIA-1 and granzyme B, but were negative for CD4, CD8, CD56, CD30, L-26, EMA, TCR alpha/beta and TCR gamma/delta. Because the tumor cells showed T cell nature with cytotoxic activity proved by TIA-1 and granzyme B, and without evidence of further maturation of T cell, a malignant lymphoma originating from extrathymic-derived T cells was suggested.     

Activated status of tumour-infiltrating lymphocytes and apoptosis in testicular seminoma.

Testicular seminoma is characterized by a prominent lymphoid infiltrate and an excellent prognosis. Cytotoxic T-lymphocytes (CTLs) infiltrating seminoma tumour nests constitute a major subset of the lymphoid infiltrate. The objective of this study was to determine whether CTLs express markers of cytotoxic potential and activity and whether the number of activated CTLs correlates with the extent of apoptosis in testicular seminomas, as opposed to non-seminomatous testicular germ cell tumours (NSTGCTs). Twenty cases of pure seminoma as well as 20 cases of NSTGCTs including 16 mixed germ cell tumours (MGCTs) were studied. Immunohistochemistry for the cytotoxic markers TIA-1 (cytotoxic potential) and granzyme B (cytotoxic activity) and the T-cell markers CD3 and CD8 was performed on formalin-fixed, paraffin-embedded sections. The apoptotic index (AI) was determined by the TUNEL method. The number of CD3(+), CD8(+), TIA-1(+), and granzyme B(+) cells in tumour cell nests was markedly increased in testicular seminomas, compared with NSTGCTs (p<0.01). Activated granzyme B(+) cells numbered 25.6+/-5.2 per high power field in seminomas and 8.9+/-3.2, 8.1+/-3.9, and 0.4+/-0.2 for embryonal carcinomas, yolk sac tumours, and immature teratomas, respectively. Double immunohistochemical staining for granzyme B and CD8 revealed that 82.6+/-8.5% of granzyme B-expressing cells were CD8(+). The tumour cell AI was significantly increased in embryonal carcinoma, compared with the seminoma, yolk sac tumour, and immature teratoma subgroups (6.7+/-1.3, 2.3+/-0.3, 3.0+/-1.1, and 2.3+/-1.1, respectively, p<0.001). TUNEL/CD3 double immunostaining revealed that a significant proportion of the apoptotic seminomatous tumour cells were in direct contact with one or more CD3(+) lymphocytes (47.2+/-6.2%). The number of activated granzyme B(+) CTLs showed a strong linear correlation with the AI in the seminoma group (r=0.71, p<0.0001) but not in other subgroups. TUNEL/granzyme B double immunolabelling revealed that a proportion of activated granzyme B(+) lymphocytes (20%) were often seen in close contact with apoptotic tumour cells. The presence of increased numbers of activated cytotoxic lymphocytes in testicular seminomas suggests that apoptotic tumour cell death in this neoplasm may be triggered by cytotoxic granule effectors. This phenomenon may be one of the key host immune mechanisms leading to the excellent prognosis in this tumour.     

外周血淋巴细胞穿孔素和颗粒酶B在诊断移植肾急性排斥中的作用

目的探讨外周血淋巴细胞（peripheral blood lymphocyte，PBL）穿孔素和颗粒酶B表达水平在肾移植诊断急性排斥反应（acute rejection，AR）和抗排斥疗效中的关系。方法采用定量逆转录PCR（RT-PCR）方法动态测定AR（n=7）、肾功能延迟恢复（n=8）、近期肾功能正常（n=27）、长期肾功能稳定（n=25）组共67例肾移植患者移植前后PBL穿孔素和颗粒酶B表达水平和AR的关系。结果肾移植术后患者PBL穿孔素和颗粒酶B表达强度依次为AR组、肾功能延迟恢复组、近期肾功能正常组、长期肾功能稳定组，AR组与其它3组有显著性差异（P〈0．01）；其升高时间比临床上出现AR的症状早3d左右，随着AR的逆转，其表达也逐渐降至原有基础水平。结论定量RT-PCR测定PBL穿孔素和颗粒酶B的表达可以是一种无创的、较敏感的早期诊断肾移植AR的发生。并可预测抗排斥反应治疗效果。     

Identification of CD56dim subpopulation marked with high expression of GZMB/PRF1/PI-9 in CD56+ interferon-α-induced dendritic cells

As the sentinels of innate and adaptive immune system, dendritic cells (DCs) have been considered to hold a great promise for medical application. Among the diverse types of DCs, monocyte-derived DCs (mo-DCs) generated in vitro have been most commonly employed. We have been improving the culture protocol and devised a protocol to produce mature interferon-α-induced DCs (IFN-DCs), hereinafter called (mat)IFN-DCs. While exploring the relationship between the expression of CD56 and the cytotoxic activity of (mat)IFN-DCs, we unexpectedly found that sorting of (mat)IFN-DCs with CD56 antibody-coated microbeads (MB) resulted in fractionating cells with tumoricidal activity into the flow-through (FT) but not MB-bound fraction. We uncovered that the FT fraction contains cells expressing low but substantial level of CD56. Moreover, those cells express granzyme B (GrB), perforin (PFN), and serpin B9 at high levels. By employing a specific inhibitor of PFN, we confirmed that direct tumoricidal activity relies on the GrB/PFN pathway. We designated subpopulation in FT fraction as CD56^dim and that in CD56 positively sorted fraction as CD56^bright, respectively. This is the first time, to our knowledge, to identify subpopulations of CD56-positive IFN-DCs with distinct tumoricidal activity which is ascribed to high expression of the components of GrB/PFN pathway.     

Comprehensive Profiling of an Aging Immune System Reveals Clonal GZMK+ CD8+ T Cells as Conserved Hallmark of Inflammaging

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外周血淋巴细胞穿孔素和颗粒酶B mRNA检测对肾移植急性排斥反应的早期诊断价值

探讨外周血淋巴细胞(peripheral blood lymphocyte,PBL)穿孔素和颗粒酶B mRNA检测对肾移植急性排斥反应(AR)的早期诊断价值。采用RT-PCR方法动态检测67例肾移植AR患者外周血淋巴细胞穿孔素和颗粒酶B mRNA的表达水平,同时检测所有患者肌酐(Cr)表达水平情况,比较各时间点穿孔素、颗粒酶B mRNA及Cr表达水平,并探讨穿孔素、颗粒酶B与AR的关系。移植后1d与移植前1d比较,PBL穿孔素、颗粒酶B mRNA表达水平比较无明显差异性(P>0.05),Cr表达水平明显下降(P<0.05);AR前3d较之前各时间点比较,PBL穿孔素、颗粒酶B mRNA表达水平明显上升(P<0.05),而Cr表达水平明显下降(P<0.05);AR后1d时PBL穿孔素、颗粒酶B mRNA表达水平达到最高峰,随后逐渐下降(P<0.05),而AR后5d时Cr表达水平达到最高峰,随后逐渐下降(P<0.05);PBL穿孔素、颗粒酶B mRNA表达水平升高时间比Cr早4d,AR患者经MP/OKT3冲击治疗后,PBL穿孔素和颗粒酶B mRNA表达水平逐渐降低至原有基础水平。PBL穿孔素和颗粒酶B mRNA在AR早期诊断和抗排斥反应疗效评估等方面具有一定的临床价值。     

CD1d-unrestricted NKT cells are endowed with a hybrid function far superior than that of iNKT cells

Invariant natural killer T (iNKT) cells to date represent the best example of cells known to have a hybrid function, representing both innate and adaptive immunity. Shared phenotypic similarities with NK cells together with a rapid response to a cytokine stimulus and a productive TCR engagement are the features that underline the hybrid nature of iNKT cells. Using these criteria, we provide molecular and functional evidence demonstrating that CD1d-independent (CD1d^ind) NKT cells, a population of CD1d-unrestricted NKT cells, are endowed with a hybrid function far superior to that of iNKT cells: (i) an extensive shared program with NK cells, (ii) a closer Euclidian distance with NK cells, and (iii) the ability to respond to innate stimuli (Poly:IC) with cytotoxic potential in the same manner as NK cells identify a hybrid feature in CD1d^indNKT cells that truly fulfills the dual function of an NK and a T cell. Our finding that CD1d^indNKT cells are programmed to act like NK cells in response to innate signals while being capable of adaptive responses is unprecedented, and thus might reemphasize CD1d-unrestricted NKT cells as a subset of lymphocytes that could affect biological processes of antimicrobial and tumor immunity in a unique way.Natural killer T (NKT) cells are increasingly regarded as cells endowed with a hybrid function between an NK cell and a T cell (1, 2). The current classification of NKT cells places them into three categories: type I, type II, and NKT-like cells (1). Type I comprises invariant NKT (iNKT) cells that recognize the glycolipid α-galactosylceramide (α-GalCer) loaded into the MHC class I molecule, CD1d, and contain an invariant TCR repertoire of Vα14-Jα18 (3–5). Type II NKT cells are also CD1d dependent but do not respond to α-GalCer in the same way as iNKT cells do (6, 7). NKT-like cells encompass all other NKT cells and are CD1d independent (CD1d^ind) (8); they are by far the most heterogeneous and the least characterized.Recent studies have increasingly shown a shared expression of NK cell-related receptors on other effector cells. CD8⁺ T cells are known to up-regulate NK markers, such as NK1.1, and can even respond quickly like NK cells (9). Other work has described NKT cells that express NKp46 (10), a marker selectively associated with conventional NK cells and NK22 cells in the gut (11). Moreover, γδ T cells have been shown to express NK markers and display an innate-like response (12). Collectively, these reports converge to raise the following key questions. What qualifies as an NKT cell? Do the cells need to express only NK1.1 and CD3 to be eligible for NKT nomenclature? With the continuous development of both NK and T-cell fields, the simplistic definition that NKT cells are subsets of T cells that express the NK1.1 marker is becoming increasingly misleading and even inaccurate. For instance, NK1.1 complex is expressed in the BALB/c strain but there are allelic divergences with the polymorphism leading to the PK136 antibody not reacting to the BALB/c NK.1.1 (NKrp1) complex (13). This definition is also limited in the C57BL/6 strain because of the discovery of NK1.1⁻CD1d⁺ NKT cells (14). Although phenotypic similarities can be misleading, the criteria that best describes an NKT cell is the ability to perform with a hybrid function between an NK cell and a T cell (2).Nonetheless, the concept of hybrid function is also an elusive notion allowing for a gradient of functions. A number of works refer to an NKT hybrid function as the ability of a T cell with phenotypic similarities to NK cells to perform with innate-like response. The best example of cells endowed with a hybrid NKT cell function are thought to be iNKT cells (2). In this study, we provide molecular and functional evidence demonstrating that CD1d^indNKT cells—a population of MHC-unrestricted T cells—are endowed with a hybrid function that associates them to the NK cell lineage in a manner far superior to the known link between NK and iNKT cells. An extensive shared program with NK cells, a similarity in the gene expression profile with NK cells, and their ability to respond (like NK cells) not only to cytokine signals (IL-12 plus IL-18) but also to innate stimuli [in vivo treatment with Poly:IC (Fisher)] with massive production of key effector players of the cytotoxic pathway collectively identify a hybrid feature in CD1d^indNKT cells that uniquely fulfills the function of an NK cell and a T cell.     

颗粒酶B在心血管疾病中的重要作用

细胞凋亡在心血管疾病如动脉粥样硬化,缺血性心脏病,充血性心力衰竭中起着重要的作用。以往的研究已经证明氧化应激、生理应激、肿瘤坏死因子和Fas配体参与了心血管系统的细胞凋亡。而最近的研究证明了另一个凋亡相关因子,即颗粒酶B/穿孔蛋白系统也参与了心血管系统的细胞凋亡。在这篇综述中,我们阐述了颗粒酶B在心血管疾病中的作用,并且发现颗粒酶B抑制剂的实验应用可以减轻心血管病的发生发展,从而使抑制颗粒酶B/穿孔蛋白系统治疗心血管疾病成为可能。     

Association between granzyme B and perforin I polymorphisms and allograft outcomes in Hispanic kidney transplant recipients

Granzyme B (GZMB) and perforin 1 gene (PRF1) are key effector molecules of cytotoxic T lymphocytes, in causing acute and chronic solid organ transplant rejection. In this study, we analyzed the impact of GZMB and PRF1 polymorphism on kidney allograft outcomes. In all, 527 de novo kidney Hispanic allograft recipients were genotyped for PRF1 (rs10999426, rs35947132) and GZMB (rs8192917, rs7144366). PRF1 (rs10999426, rs35947132) G alleles and GG genotypes were negatively associated with allograft rejection, demonstrating protection against allograft rejection (OR = 0.61, p = 0.005 for rs1099946; OR = 0.4, p = 0.01 for rs 35947132). On the other hand, the GA heterozygosity of PRF1 was found marginally associated with the rejection group (OR = 1.53, p = 0.05 for rs10999426; OR = 2.24, p = 0.07 for rs35947132). There was a significant increase in allograft survival in time period studied for the PRF1 (rs10999426) GG genotype, while the GA heterozygosity was associated with graft failure. We found no association for polymorphic markers in GZMB gene with allograft rejection. Survival was significantly improved for patients who were homozygous TT for the GZMB (rs8192917) (TT vs. CC/TT, p = 0.041). The result suggests that PRF1 and GZMB gene polymorphisms may determine the incidence of acute rejection or graft survival among Hispanic allograft recipients.