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Peripheral T-cell lymphoma (PTCL) is rare in children. Expression of cytotoxic molecules (CM) in nodal PTCL has unique clinicopathologic features, including an Epstein-Barr virus (EBV) association. However, CM+, EBV-associated PTCL is extremely rare in the childhood, with only 1 study having been reported to date, including both pediatric and adult patients. We report a case of CM+ PTCL in a 20-month-old boy with left neck lymphadenopathy as well as multiple visceral lesions. A biopsied lymph node was diffusely infiltrated by atypical lymphoid cells with a CD4/CD8, granzyme B+, perforin+, and TIA-1+ phenotype, and EBV positivity by in situ hybridization. Rearrangements of the TCR γ-chain and β-chain genes were demonstrated by polymerase chain reaction. Ancillary genetic studies detected trisomy 2, trisomy 10, a structurally abnormal 6p, and additional copies of the IRF4 gene. Multiple bone marrow biopsies failed to show any evidence of tumor, histiocytic hyperplasia, or hemophagocytosis. This lesion was therefore diagnosed as "CM+, EBV-associated high-grade peripheral T-cell lymphoma." After 5 cycles of chemotherapy, the patient was in remission 8 months following initial diagnosis. To our knowledge, this represents the youngest child with this rare tumor in the published literature, and showing an unusually favorable initial response to therapy.

Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) is relatively common, and patients occasionally develop other neoplasms; however, patients who develop other types of lymphomas are rare. We encountered 3 patients with CLL/SLL (one 59-y-old man and 2 women aged 56 and 66 y) who developed T-cell lymphomas. Both women developed ALK anaplastic large cell lymphomas (ALCLs), whereas the man developed CD8 peripheral T-cell lymphoma, not otherwise specified. All 3 T-cell lymphomas expressed granzyme B and perforin, indicating a cytotoxic immunophenotype. In 1 case, the first presentation was a lymph nodal composite lymphoma. In the other 2 cases, the T-cell lymphomas arose <1 year after the diagnosis of CLL/SLL and were identified in a lymph node in one case and in the spleen in the other. The patient with a composite lymphoma (SLL/ALK ALCL) was treated and was free of disease at last follow-up, whereas the other 2 patients succumbed to their disease, 1 month and 7 months after the diagnosis of T-cell lymphoma. Peripheral T-cell lymphomas rarely occur in CLL/SLL patients. On the basis of our small series, those with a cytotoxic phenotype appear to be more common in this setting. The occurrence of ALK ALCL in 2 older patients was especially surprising and suggested that CLL/SLL may have played a role in the development of ALCL.

Enzyme-linked immune absorbent spot (Elispot) is a quantitative method for measuring relevant parameters of T cell activation. The sensitivity of Elispot allows the detection of low-frequency antigen-specific T cells that secrete cytokines and effector molecules, such as granzyme B and perforin. Cytotoxic T cell (CTL) studies have taken advantage with this high-throughput technology by providing insights into quantity and immune kinetics. Accuracy, sensitivity, reproducibility, and robustness of Elispot resulted in a wide range of applications in research as well as in diagnostic field. Actually, CTL monitoring by Elispot is a gold standard for the evaluation of antigen-specific T cell immunity in clinical trials and vaccine candidates where the ability to detect rare antigen-specific T cells is of relevance for immune diagnostic. The most utilized Elispot assay is the interferon-gamma (IFN-γ) test, a marker for CD8(+) CTL activation, but Elispot can also be used to distinguish different subsets of activated T cells by using other cytokines such as T-helper (Th) 1-type cells (characterized by the production of IFN-γ, IL-2, IL-6, IL-12, IL-21, and TNF-α), Th2 (producing cytokines like IL-4, IL-5, IL-10, and IL-13), and Th17 (IL-17) cells. The reliability of Elispot-generated data, by the evaluation of T cell frequency recognizing individual antigen/peptide, is the core of this method currently applied widely to investigate specific immune responses in cancer, infections, allergies, and autoimmune diseases. The Elispot assay is competing with other methods measuring single-cell cytokine production, e.g., intracellular cytokine by FACS or Miltenyi cytokine secretion assay. Other types of lymphocyte frequency and function assays include limiting dilution assay (LDA), cytotoxic T cell assay (CTL), and tetramer staining. Compared with respect to sensitivity the Elispot assay is outranking other methods to define frequency of antigen-specific lymphocytes. The method described herein would like to offer helpful and clear protocols for researchers that apply Elispot. IFN-γ and perforin Elispot assays are described.

Ziram is a carbamate pesticide, which is widely used throughout the world as a fungicide in agriculture and as an accelerating agent in latex production. In the present study, we investigated the effect of ziram at 0.031-4 μM in vitro on human natural killer (NK) and lymphokine-activated killer (LAK) and murine cytotoxic T lymphocyte (CTL) activity and found that it significantly inhibited all three activities in a concentration-dependent manner. To explore the mechanism of ziram-induced inhibition of NK activity, NK-92MI cells, a human NK cell line, were used. We previously confirmed that NK-92MI cells express CD56, perforin, granzyme (Gr) A, GrB, Gr3/K, and granulysin and are highly cytotoxic to K562 cells in the chromium release assay. NK-92MI cells were treated with ziram at 0.125-4 μM for 4 or 16 h at 37°C in vitro. Then, intracellular levels of perforin, GrA, GrB, Gr3/K, and granulysin were determined by flow cytometry. It was found that ziram significantly reduced Gr3/K, granulysin, perforin, GrA, and GrB levels. The extent of the decrease differed among the proteins, and the order was as follows: Gr3/K > granulysin > perforin, GrA, and GrB. Taken together, these findings suggest the ziram-induced inhibition of NK, LAK, and CTL activities to be at least partially mediated by decreases in the intracellular levels of Gr3/K, granulysin, perforin, GrA, and GrB.

Background: Anti-CD20 monoclonal antibodies are major therapeutic agents for patients with follicular lymphoma and work through complement-mediated cytotoxicity and antibody-dependent cellular cytotoxicity. Optimization of antibody-dependent cellular cytotoxicity, in particular by amplifying its effectors, could further increase the efficacy of anti-CD20 monoclonal antibodies., Design and Methods: We investigated the cytotoxic activity of Vγ9Vδ2 T cells against follicular lymphoma cells and whether this killing could be increased by promoting antibody-dependent cellular cytotoxicity with anti-CD20 monoclonal antibodies, in particular a type-II glycoengineered anti-CD20. Vγ9Vδ2 T cells were expanded in vitro in the presence of bromohydrin pyrophosphate (Phosphostim) and interleukin-2 and their ability to kill follicular lymphoma primary cells or cell lines was evaluated by flow cytometry cytotoxic T-lymphocyte assays in the presence or absence of three anti-CD20 monoclonal antibodies: the afucosylated GA101, the chimeric rituximab or the humanized ofatumumab. The ability of these cells to release perforin/granzyme and secrete interferon-γ when co-cultured with follicular lymphoma primary cells or cell lines in the presence or not of the three anti-CD20 monoclonal antibodies was also evaluated by CD107a staining and Elispot assays., Results: Phosphostim and interleukin-2 expanded Vγ9Vδ2 T cells were cytotoxic to primary follicular lymphoma cells and their cytotoxic potential was dramatically increased by GA101, a type II glycoengineered anti-CD20 monoclonal antibody, and to a lesser extent, by rituximab and ofatumumab. The increased cytotoxicity was associated with increased secretion of perforin/granzyme and interferon-γ., Conclusions: In-vitro expanded Vγ9Vδ2 T cells efficiently kill primary follicular lymphoma cells and express CD16; anti-CD20 monoclonal antibodies, in particular GA101, dramatically increase the cytotoxic activity of expanded Vγ9Vδ2 T cells. These preclinical results prompt the development of clinical trials using this antibody dependent cellular cytotoxicity property of Vγ9Vδ2 T cells and anti-CD20 monoclonal antibodies.

Perforin is one of the key effector molecules of cytotoxic T cells and natural killer cells. The influence of HLA-DRB1 alleles on peripheral blood perforin-positive CD4, CD8, CD16 and CD 56 cells was studied by flow cytometry. HLA-DRB1 typing was done in normal healthy subjects (NHS: n = 156) and patients with pulmonary tuberculosis (PTB: n = 102) by polymerase chain reaction-based sequence-specific oligonucleotide hybridization method. We observed a significantly decreased percentage of total perforin-positive cells (per(+)) (P = 0.0004); CD8(+)/Per(+) (P = 0.0005); CD16(+)/Per(+) (P = 0.05) and CD 56(+)/Per(+) cells (P = 0.001) in HLA-DR2-positive PTB patients compared to non-DR2 patients. Subtyping of HLA-DR2-positive subjects at the allelic level revealed that the percentage of CD8(+)/Per(+) cells did not differ among DRB1*1501 and DRB1*1502 patients while a trend towards a decreased percentage of CD16(+)/Per(+) and CD 56(+)/Per(+) cells was noticed in DRB1*1501 patients compared to DRB1*1502 patients. The present study suggests that HLA-DR2 may be associated with down-regulation of perforin-positive cytotoxic lymphocytes and natural killer cells in pulmonary tuberculosis.