• Journal of Microbiology and Biotechnology
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• v.17 no.12
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• pp.1955-1964
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• 2007

A recent report showed that analysis of CD154 expression in the presence of the secretion inhibitor Brefeldin A (Bref A) could be used to assess the entire repertoire of antigen-specific $CD4^+\;T$ helper cells. However, the capacity of intracellular CD154 expression to identify antigen-specific $CD8^+\;T$ cells has yet to be investigated. In this study, we compared the ability of intracellular CD154 expression to assess antigen-specific $CD8^+\;T$ cells with that of accepted standard assays, namely intracellular cytokine IFN-${\gamma}$ staining (ICS) and MHC class I tetramer staining. The detection of intracellular CD154 molecules in the presence of Bref A reflected the kinetic trend of antigen-specific $CD8^+\;T$ cell number, but unfortunately showed less sensitivity than ICS and tetramer staining. However, ICS levels peaked and saturated 8 h after antigenic stimulation in the presence of Bref A and then declined, whereas intracellular CD154 expression peaked by 8 h and maintained the saturated level up to 24 h post-stimulation. Moreover, intracellular CD154 expression in antigen-specific $CD8^+\;T$ cells developed in the absence of $CD4^+\;T$ cells changed little, whereas the number of IFN-${\gamma}$-producing $CD8^+\;T$ cells decreased abruptly. These results suggest that intracellular CD154 could aid the assessment of antigen-specific $CD8^+\;T$ cells, but does not have as much ability to identify heterogeneous $CD4^+\;T$ helper cells. Therefore, the combined analytical techniques of ICS and tetramer staining together with intracellular CD154 assays may be able to provide useful information on the accurate phenotype and functionality of antigen-specific $CD8^+\;T$ cells.

• IMMUNE NETWORK
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• v.7 no.4
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• pp.173-178
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• 2007

Background: We studied the role for expression of CD40 and CD40L by CD4 and CD8 T cells in the generation of CD8 T cell response to minor histocompatibility antigen, H60. H60 is a cellular antigen to which CD8 responses require CD4 T cell help. Methods: CD40- or CD40L-deficient mice were adoptively transferred with normal CD4 or CD8 T cells or with memory CD4 or CD8 T cells, and were immunized with male H60 congenic splenocytes to induce CD8 T cell response to H60. Peripheral blood CD8 T cell from the immunized mice were stained with the H60 tetramer. Results: CD8 T cell response to H60 was not induced in both CD40- and CD40L-deficient mice. Adoptive transfer of $CD40^{+/+}$ CD8 T cells into CD40-deficient mice did not compensate the defect in inducing CD8 T cell response to H60, while the H60-specific CD8 T cells were activated in the CD40-deficient mice that were adoptively transferred with $CD40^{+/+}$ CD4 T cells. Adoptive transfer of $CD40L^{+/+}$ CD4 T cells into CD40L-deficient mice induced primary CD8 T cell response for H60 and the presence of $CD40L^{+/+}$ CD4 T cells was required even for memory CD8 T cells response to H60. Conclusion: Our results suggest that the CD40-CD40L interaction mediates the delivery of CD4 T cell help to naive and memory H60-specific CD8 T cells. While the expression of CD40L by CD4 T cells is essential, signaling through CD40 on CD8 T cells is not required for the induction of CD8 T cell response to H60.

• IMMUNE NETWORK
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• v.6 no.4
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• pp.179-184
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• 2006

Background: Identification of antigen-specific T cells has yielded valuable information on pathologic process and the disease state. Assays for quantification of inflammatory cytokines or lytic-granule molecules have been generally used to evaluate antigen specific T cell response, however their applicability have been hampered due to the limited source of autologous antigen-presenting target cells (APC). Methods: K562, a leukemic cell line deficient of human leukocyte antigen (HLA), was transfected with a gene encoding HLA-A*02 (K562/ A*02) and its function as stimulator cells in inducing activation of HLA-matched T cells was evaluated by IFN-${\gamma}$ enzyme linked immunospot (ELISPOT) assay. Results: The stable transfectant K562/ A*02 pulsed with HLA- A*02 restricted peptide could specifically induce IFN-${\gamma}$ secretion by CD8+ T cells compared to no detectable secretion by CD4+ T cells. However, CD56+ NK cells secreted IFN-${\gamma}$ in both K562/ A*02 with peptide and without peptide. The number of IFN-${\gamma}$ secreted CD8+ T cells was increased according to the ratio of T cells to K562 and peptide concentration. Formalin-fixed K562/ A*02 showed similar antigen presenting function to live K562/ A*02. Moreover, K562/ A*02 could present antigenicpeptide to not only A*0201 restricted CD8+ T cells but also CD8+ T cells from A*0206 donor. Conclusion: These results suggest that K562/ A*02 could be generally used as target having specificity and negligible background for measuring CD8+ T cell responses and selective use of K562 with responsder matched HLA molecules on its surface as APC may circumvent the limitation of providing HLA-matched autologous target cells.

• IMMUNE NETWORK
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• v.12 no.3
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• pp.118-125
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• 2012

CD40-CD40L-mediated help from CD4 T cells is essential to induce primary CD8 T cell responses specific to the non-inflammatory cell-based antigen H60. In this study, using H60 as a model antigen, we generated recombinant vaccinia viruses (rVVs) expressing the H60 CD8 epitope and investigated whether CD4 help was required to activate the CD8 T cell response specific to the virally expressed H60. The immune response after infection with rVVs expressing H60 was similar to that after immunization with H60 congenic splenocytes, with a peak frequency of H60-specific CD8 T cells detected in the blood on day 10 post-infection. A CD8 T cell response specific for virally derived H60 was not induced in CD4-depleted mice, but was in CD40-deficient mice. These results provide insights into the characterization of the CD8 T cell response specifically for antigens originating from cellular sources compared to viral sources.

• Korean Journal of Microbiology
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• v.47 no.4
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• pp.342-347
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• 2011

T cells play a key role in viral infection. However, in patients with chronic hepatitis C virus (HCV) infection, HCV-specific T cells are dysfunctional and impaired in the liver, which is the primary site for HCV replication. There are multiple potential mechanisms for HCV-specific T cell dysfunction including induction of immune inhibitory pathways (program death-1; PD-1, cytotoxic t lymphocyte associated antigen-4; CTLA-4) and immune tolerance induced specific for the liver. However, the interaction between hepatocytes and HCV-specific CD8 T cells has not clearly established. In this study, we confirmed huh (human hepatoma) 7.5 cells expressing HLA (human leukocyte antigen) A2 presented antigen to activate HCV-specific CD8 T cells in HLA A2-restricted manner and expression of PD-L (program death ligand) 1 on huh7.5 cells reduced HCV-specific CD8 T cell activation, suggesting an immune modulatory activity. Loss of HCV-specific tetramer responses following antigenic stimulation correlated with increased caspase-3 activity. In addition, PD-L1 on huh7.5 cells rescued HCV-specific CD8 T cells from apoptosis. Our results suggest that the interaction between PD-L1 and PD-1 can recover the function of HCV-specific CD8 T cells in the liver, which could be applied in therapy of HCV chronic infection.

• IMMUNE NETWORK
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• v.7 no.2
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• pp.80-86
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• 2007

Background: CD40-activated B (CD40-B) cells might be an attractive source of autologous antigen-presenting cells (APCs) for immunotherapy due to the convenience to obtain from peripheral blood and expand in vitro. Moreover, CD40-B cells were found to be comparable with DCs in their capacity to raise antigen-specific CD8+ T cells. Here, we have established K562 cells expressing CD40L to expand CD40-activated B cells used for APCs. Methods: After activation of B cell by K562/CD40L, CD40-B cells were examined by counting B cell numbers. Surface expression of CD54, CD80, CD86 and HLA class II was measured by flow cytometry. The CD40-B cells were tested for its function as APC by mixed lymphocyte reactions (MLR) and by induction of T cell responses specific for pp65 peptide in vitro. Results: The expansion of B cells by K562/CD40L increased about 6-folds compared with anti-CD40 or K562. Furthermore, the expression of CD54, CD80, CD86 and HLA class II was up-regulated by K562/CD40L. B cells by K562/CD40L showed comparable antigen presentation activity with mature DCs as shown in MLR, INF-${\gamma}$ ELISPOT assay. Conclusion: These results suggest that K562/CD40L could be used to generate activated B cells as potent APCs which could be useful for cellular vaccination and adoptive immunotherapy.

• Molecules and Cells
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• v.44 no.5
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• pp.328-334
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• 2021

The advent of the major histocompatibility complex (MHC) multimer technology has led to a breakthrough in the quantification and analysis of antigen-specific T cells. In particular, this technology has dramatically advanced the measurement and analysis of CD8 T cells and is being applied more widely. In addition, the scope of application of MHC multimer technology is gradually expanding to other T cells such as CD4 T cells, natural killer T cells, and mucosal-associated invariant T cells. MHC multimer technology acts by complementing the T-cell receptor-MHC/peptide complex affinity, which is relatively low compared to antigen-antibody affinity, through a multivalent interaction. The application of MHC multimer technology has expanded to include various functions such as quantification and analysis of antigen-specific T cells, cell sorting, depletion, stimulation to replace antigen-presenting cells, and single-cell classification through DNA barcodes. This review aims to provide the latest knowledge of MHC multimer technology, which is constantly evolving, broaden understanding of this technology, and promote its widespread use.

• Molecules and Cells
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• v.42 no.4
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• pp.313-320
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• 2019

Intraepithelial lymphocytes (IELs) develop through the continuous interaction with intestinal antigens such as commensal microbiome and diet. However, their respective roles and mutual interactions in the development of IELs are largely unknown. Here, we showed that dietary antigens regulate the development of the majority of $CD8{\alpha}{\beta}$ IELs in the small intestine and the absence of commensal microbiota particularly during the weaning period, delay the development of IELs. When we tested specific dietary components, such as wheat or combined corn, soybean and yeast, they were dependent on commensal bacteria for the timely development of diet-induced $CD8{\alpha}{\beta}$ IELs. In addition, supplementation of intestinal antigens later in life was inefficient for the full induction of $CD8{\alpha}{\beta}$ IELs. Overall, our findings suggest that early exposure to commensal bacteria is important for the proper development of dietary antigen-dependent immune repertoire in the gut.

• BMB Reports
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• v.55 no.2
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• pp.57-64
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• 2022

Autoimmune disease is known to be caused by unregulated self-antigen-specific T cells, causing tissue damage. Although antigen specificity is an important mechanism of the adaptive immune system, antigen non-related T cells have been found in the inflamed tissues in various conditions. Bystander T cell activation refers to the activation of T cells without antigen recognition. During an immune response to a pathogen, bystander activation of self-reactive T cells via inflammatory mediators such as cytokines can trigger autoimmune diseases. Other antigen-specific T cells can also be bystander-activated to induce innate immune response resulting in autoimmune disease pathogenesis along with self-antigen-specific T cells. In this review, we summarize previous studies investigating bystander activation of various T cell types (NKT, γδ T cells, MAIT cells, conventional CD4⁺, and CD8⁺ T cells) and discuss the role of innate-like T cell response in autoimmune diseases. In addition, we also review previous findings of bystander T cell function in infection and cancer. A better understanding of bystander-activated T cells versus antigen-stimulated T cells provides a novel insight to control autoimmune disease pathogenesis.

• BMB Reports
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• v.30 no.6
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• pp.415-420
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• 1997

Although $CD4^+$ T cell responses to protein-derived antigen have well been understood, the epitopes recognized by hapten-specific $CD4^+$ T cells have not been fully defined. In this study, we characterized the response of a T cell hybridoma (5Di0.1B8) which is specific for a hapten. N-hydroxysuccinimidyl-4-azidobenzoate (HSAB) restricted by MHC class II $I-A^d$. Using three different antigen presenting cells (APCs) expressing $I-A^d$, the role of class II MHC proteins in haptenic antigen presentation and subsequent activation of 5D10.1B8 has been examined. Activation of 5D10.1B8 T cells by HSAB analogs was also performed. Our results show that each APC activated T cells differentially and that interleukin-2 (IL-2) augmented antigen-presenting ability of all the APCs, suggesting that increased expression of class II MHC protein by IL-2 played an important role in HSAB presentation and T cell activation. Finally, early T cell receptor-dependent signals induced by HSAB or its analogs were examined by phosphotyrosine immunoblot analysis, and showed that tyrosine phosphorylation level of a 18-20 kD protein increased upon stimulation.