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. 2014 Jul;142(3):506-16.
doi: 10.1111/imm.12282.

Lack of transglutaminase 2 diminished T-cell responses in mice

Jin-Hee Kim  1 , Jun Man Hong, Eui Man Jeong, Wang Jae Lee, Hang-Rae Kim, Jae Seung Kang, In-Gyu Kim, Young-Il Hwang
Affiliations

Lack of transglutaminase 2 diminished T-cell responses in mice

Jin-Hee Kim et al. Immunology. 2014 Jul.

Abstract

Transglutaminase 2 (TG2) has been reported to play a role in dendritic cell activation and B-cell differentiation after immunization. Its presence and role in T cells, however, has not been explored. In the present study, we determined the expression of TG2 on mouse T cells, and evaluated its role by comparing the behaviours of wild-type and TG2(-/-) T cells after activation. In our results, naive T cells minimally expressed TG2, expression of which was increased after activation. T-cell proliferation, expression of activation markers such as CD69 and CD25, and secretions of interleukin-2 and interferon-γ were suppressed in the absence of TG2, presumably due, in part, to diminished nuclear factor-κB activation. These effects on T cells seemed to be reflected in the in vivo immune response, the contact hypersensitivity reaction elicited by 2,4-dinitro-1-fluorobenzene, with lowered peak responses in the TG2(-/-) mice. When splenic T cells from mice immunized with tumour lysate-loaded wild-type dendritic cells were re-challenged ex vivo with the same antigen, the profile of surface markers including CD44, CD62L, and CD127 strongly indicated lesser generation of memory CD8(+) T cells in TG2(-/-) mice. In the TG2(-/-) CD8(+) T cells, moreover, Eomes expression was markedly decreased. These results indicate possible roles of TG2 in CD8(+) T-cell activation and CD8(+) memory T-cell generation.

Keywords: CD8+ T cells; Eomes; T-cell activation; transglutaminase 2.

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Figures

Figure 1
Figure 1
Expression of transglutaminase 2 (TG2) in naive and activated T cells. Wild-type (WT) mouse splenic T cells were isolated and stimulated with anti-CD3 and anti-CD28 antibodies. After 24 or 48 hr, they were subjected to (a) RT-PCR and (b) Western blot analysis to determine the presence of TG2. For each result, a densitometric analysis was performed, the results of which are shown on the right side of each panel. (c) Immunofluorescence-staining for TG2 was minimal in the naive T cells (upper panels), but strongly positive in the 48 hr-stimulated T cells, both in the cytoplasm and on the cell surface (lower panels). (d) Using the same antibodies, naive and 48 hr-activated T cells were stained without permeabilization, and then subjected to flow cytometric analysis. (e) Intracellular TG2 enzyme activity was determined as described in the Materials and methods. HeLa cells were used as a positive control. All of the experiments were repeated more than three times; the results depicted are representative. DAPI, 4′,6-diamidino-2-phenylindole; DIC, differential interference contrast.
Figure 2
Figure 2
In vitro proliferation of wild-type (WT) and transglutaminase 2 deficient (TG2−/−) T cells with various stimulants. (a) Mouse splenic T cells were isolated and stimulated for 48 hr, [3H]thymidine was added to the culture medium for an additional 18 hr, and the counts per minute (CPM) were measured. (b) T cells were loaded with CFSE and activated with anti-CD3 and anti-CD28 antibodies for 72 hr, and then stained with anti-CD4-allophycocyanin and anti-CD8-peridinin chlorophyll protein (PerCP) antibodies. They were gated based on CD4 or CD8 staining and analysed for their CFSE profiles, respectively. (c) T cells were co-cultured with dendritic cells (DCs) from BALB/c mice for 48 hr, and subjected to a [3H]thymidine incorporation assay. (d) T cells were co-cultured with syngeneic DCs pre-stimulated with lipopolysaccharide. All of the experiments were repeated more than three times; the results depicted are representative. *P < 0·05; WT, wild-type; DC, dendritic cells; PMA/I, phorbol myristate acetate/ionomycin; CFSE, carboxyfluorescein succinimidyl ester.
Figure 3
Figure 3
Expression of activation markers upon in vitro stimulation. (a) Splenic T cells from wild-type (WT) and transglutaminase 2-deficient (TG2−/−) mice were isolated, stimulated with anti-CD3 and anti-CD28 antibodies for 3 days, stained for CD4, CD8, CD69 and CD25, and subjected to flow cytometric analysis. (b) In addition, T cells were co-cultured with allogeneic bone marrow-derived dendritic cells for 72 hr, stained, and processed in the same way. (c) The concentrations of interleukin-2 (IL-2) and interferon-γ (IFN-γ) in the experiment A supernatants were determined by ELISA. (d) For the evaluation of nuclear factor (NF) -κB activation, splenic T cells were stimulated with antibodies for 6 hr, harvested, and their relative amounts of nuclear NF-κB were estimated using commercial kits. All of the experiments were repeated more than three times; the results depicted are representative. *P < 0·05; **P < 0·01; WT, wild-type; DC, dendritic cells.
Figure 4
Figure 4
Contact hypersensitivity reaction. Mice were sensitized on days 0 and 1 with 2,4-dinitro-1-fluorobenzene (DNFB). (a) Forty days later, the mice were challenged with DNFB on the surface of the left pinna. The pinna thickness was measured every 24 hr until the swelling subsided. The percentage increase of pinna thickness was calculated and depicted in the graph. (b) Twenty-one days after challenge, splenic T cells were isolated and re-stimulated with dinitrobenzene sulphonic acid-loaded DCs for 24 hr, and a [3H]thymidine uptake assay was performed. (c) The re-stimulated T cells were stained for CD8 and intracellular interferon-γ (IFN-γ) and then subjected to flow cytometric analysis. (d) The concentrations of IFN-γ in the culture supernatants were determined by ELISA. All of the experiments were repeated twice and the representative one is shown. *P < 0·05; **P < 0·01; WT, wild-type.
Figure 5
Figure 5
T-cell responses after dendritic cell (DC) vaccination. Mice were immunized with wild-type ( WT) bone marrow-derived dendritic cells (DCs) loaded with tumour lysate. (a) Thirty days later, splenic T cells were isolated and re-stimulated with lysate-loaded DCs for 72 hr, and a [3H]thymidine uptake assay was performed. T cells from (b) the un-immunized mice and (c) the mice immunized 30 days earlier were isolated and stimulated with antigen-loaded DCs for 72 hr. Then, the cells were stained for CD8, CD44, CD62L, and CD127, gated on the basis of CD8, and were subjected to flow cytometric analysis to determine the cell fractions of CD62Llow CD44high and CD62Llow CD127+ cells. All of the experiments were repeated more than three times; the results depicted are representative. WT, wild-type, *P < 0·05.
Figure 6
Figure 6
Expression of molecules related to T-cell differentiation. Splenic CD8+ T cells were isolated, stimulated with anti-CD3 and anti-CD28 antibodies for 48 hr, re-plated at a density of 5 × 105 cells/ml of culture medium, and maintained for 8 days with every day recombinant murine, interleukin 2 (IL-2) supplementation. (a) The cells were harvested on the days indicated, total RNA was extracted, and gene amplification was performed using appropriate primer sets. (b) The results are depicted as densitometric values. This experiment was repeated twice; the representative one is shown. D, day; WT, wild-type.
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