Enhanced production of IL-2 from anti-CD3 antibody-stimulated mouse spleen cells by caffeic acid phenethyl ester, a major component of Chinese propolis.

OBJECTIVES: We evaluated the immune-modulatory effects of Chinese propolis (CP) and its major constituent, caffeic acid phenethyl ester (CAPE), on the cytokine production of anti-CD3 antibody-stimulated mouse spleen cells. METHODS: Mouse spleen cells stimulated by anti-CD3 monoclonal antibody were co-cultured with CP, CAPE, and HC030031, a specific antagonist of the TRPA1 Ca2+-permeable cation channel. Cytokine production was assayed by enzyme-linked immunosorbent assay. Interleukin (IL)-2 mRNA expression was examined by reverse transcription-quantitative polymerase chain reaction. RESULTS: In stimulated spleen cells treated with 1/16,000 CP diluent, IL-2 production was markedly enhanced, while IL-4 and IL-10 productions were not significantly affected. In contrast, interferon (IFN)-γ, IL-6, and IL-17 productions were markedly reduced. These effects of CP were reproduced by the CAPE treatment. A time-course observation demonstrated that, compared to control cells, IL-2 mRNA expression and production were significantly enhanced in the spleen cells stimulated by CAPE; however, IL-2 production was markedly delayed compared to that in the untreated control cells. The enhancement of IL-2 production by CAPE was scarcely alleviated by the addition of HC030031. These effects of CAPE upon IL-2 mRNA production were abolished in spleen cells without anti-CD3 antibody stimulation. CONCLUSIONS: CAPE is an important regulator of CP for cytokine regulation in anti-CD3 antibody-stimulated spleen cells. The agent specifically reduced IFN-γ, IL-6, and IL-17 and slightly enhanced Th2 cytokine production while significantly enhancing IL-2 production at the transcriptional level.

Enhanced production of IL-2 from anti-CD3 antibody-stimulated mouse spleen cells by artepillin C, a major component of Brazilian green propolis.

OBJECTIVES: In this report, we attempt to clarify the immune modulatory effects of Brazilian green propolis (BGP) and its major component, artepillin C, on the cytokine production of anti-CD3 antibody-stimulated mouse spleen cells. We also estimate the physiological mechanism affecting artepillin C's upon the cells. METHODS: Male C3H/HeN mouse spleen cells stimulated by antiCD3 monoclonal antibody were co-cultured with BGP, artepillin C, and HC030031, a transient receptor potential ankyrin 1 (TRPA1) Ca2+ channel antagonist. The synthesis of interferon (IFN)-γ, interleukin (IL)-6, IL-17, IL-4, IL-10, and IL-2 was assayed by enzyme-linked immunoassay. The expression of IL-2 mRNA and the protein product were examined by reverse transcription-quantitative polymerase chain reaction and Western blot analyses, respectively. RESULTS: The production of IL-2 was markedly enhanced, while that of IL-4 and IL-10 was not significantly affected; by contrast, the production of IFN-γ, IL-6, and IL-17 was significantly reduced in the antibody-stimulated spleen cells treated with BGP at a non-cytostatic concentration. These effects were reproduced in the cells treated with artepillin C. The expression of IL-2 mRNA was unaffected; however, that of the protein was significantly enhanced in the artepillin C-treated cells compared to untreated control cells. The enhancement of protein expression and the production of IL-2 by artepillin C was significantly alleviated by adding HC030031. CONCLUSIONS: Artepillin C is an important regulator of cytokine synthesis from activated spleen cells. The agent specifically augmented the expression of IL-2 via the Ca2+-permeable cation channel, TRPA1, at least in part, at the translational or secretion levels.

Enhanced cellular engraftment of adipose-derived mesenchymal stem cell spheroids by using nanosheets as scaffolds.

The short survival time of transplanted adipose-derived mesenchymal stem cells (ASCs) is a problem for skin wound healing. Transplantation after the formation of cellular spheroids has been investigated as a promising method for prolonging cellular survival. However, there have been technical restrictions for transplantation of spheroids in clinical practice. Here, we show an effective method for transplantation of ASC spheroids onto skin wounds in order to efficiently cure refractory ulcers. To assist anchoring of spheroids onto skin wounds, we used a 120-nm-thick free-standing film (nanosheet) that has a highly adhesive property. Bioluminescence imaging showed that ASC spheroids carried by the nanosheet survived for 14 days, which is about two-times longer than that previously reported. Wounds treated with a nanosheet carrying ASC spheroids were 4-times smaller than untreated wounds on day 14. This method for transplantation of spheroids could be applied to cell therapy for various refractory skin wounds.

Augmented secretion of IL-1α from mouse oral squamous cell carcinoma (OSCC) vcells caused by serum deprivation and hypoxia promotes immune suppressive activity of mesenchymal stromal cells.

OBJECTIVES: We have previously reported that mouse oral squamous carcinoma (OSCC), Sq-1979-1, produces interleukin (IL)-1α, which specifically enhances the immunosuppressive activity of co-cultured mesenchymal stromal 10T1/2 cells. This study assessed the conditions promoting the production of IL-1α in Sq-1979-1 cells, which could further enhance the immunosuppressive function of 10T1/2 cells, and evaluated its expression in OSCC tissues. METHODS: The expression of IL-1α was examined by RT-PCR, western blotting, and enzyme-linked immune sorbent assay (ELISA). The interferon (IFN)- γ-producing capability of anti-CD3 antibody-stimulated mouse spleen cells co-cultured with 10T1/2 cells and conditioned medium (CM) from Sq-1979-1 cells was examined by ELISA. The function of IL-1α was examined using an anti-IL1α antibody. Immunohistochemical analysis of the OSCC tissues was performed. RESULTS: The production of IL-1α from Sq-1979-1 cells was synergistically enhanced in lower serum (0.5% or 1.0% FBS) at the transcriptional level, and under hypoxia (1.0% oxygen) at the release level compared to that in the control medium supplemented with 10% FBS under normoxia. The IFN-γ-producing capability of stimulated spleen cells co-cultured with 10T1/2 cells was significantly reduced in the CMs prepared with the lower serum or under hypoxia. These functions of CMs were completely abolished by the anti-IL-1α antibody. The expression of IL-1α in OSCC tissues was prominent in the midst of a carcinomatous cellular lesion or a nearby necrotic lesion, where a supply deficiency could occur. CONCLUSION: s: IL-1α production by Sq-1979-1 cells was synergistically augmented under low serum and hypoxic conditions, which could promote the immunosuppressive activity of mesenchymal cells.

Highly reliable, targeted photothermal cancer therapy combined with thermal dosimetry using a near-infrared absorbent.

Photothermal therapy (PTT) using a photo-absorbent in the near-infrared (NIR) region is an effective methodology for local cancer treatment. Before PTT using a NIR absorbent is executed, the operator generally determines the two parameters of fluence rate and irradiation time. However, even if the irradiation parameters are unchanged, the therapeutic effect of PTT is often different for individual tumors. Hence, we examined the therapeutic effect of PTT using a NIR absorbent (ICG lactosome) while changing two parameters (fluence rate and irradiation time) in various combinations. As a result, there was no robust correlation between those parameters and the therapeutic effect. Compared to those parameters, we found that a more reliable determinant was maintenance of the tumor temperature above 43 °C during NIR irradiation. To reconfirm the significance of the determinant, we developed a new system that can regulate the temperature at the NIR irradiation site at a constant level. By using the new system, we verified the treatment outcomes for tumors in which the NIR absorbent had accumulated. All of the tumors that had been kept at 43 °C during NIR irradiation were cured, while none of the tumors that had been kept at a temperature below 41 °C were cured. In conclusion, PTT using a NIR absorbent with thermal dosimetry is a highly reliable treatment for cancer.

PIRs mediate innate myeloid cell memory to nonself MHC molecules.

Immunological memory specific to previously encountered antigens is a cardinal feature of adaptive lymphoid cells. However, it is unknown whether innate myeloid cells retain memory of prior antigenic stimulation and respond to it more vigorously on subsequent encounters. In this work, we show that murine monocytes and macrophages acquire memory specific to major histocompatibility complex I (MHC-I) antigens, and we identify A-type paired immunoglobulin-like receptors (PIR-As) as the MHC-I receptors necessary for the memory response. We demonstrate that deleting PIR-A in the recipient or blocking PIR-A binding to donor MHC-I molecules blocks memory and attenuates kidney and heart allograft rejection. Thus, innate myeloid cells acquire alloantigen-specific memory that can be targeted to improve transplant outcomes.

Immunomodulatory aspects in the progression and treatment of oral malignancy.

Inflammation substantially affects the risk of oral malignancy. Pro-inflammatory cytokine, interferon (IFN)-γ, confers anti-tumor activity using several different mechanisms. Conversely, higher expression of interleukin (IL)-17 is associated with worse prognosis. Monocyte chemotactic protein (MCP)-1 correlates positively with poor long-term survival of head and neck squamous cell carcinoma (HNSCC) patients. IL-1α affects cancer associated fibroblasts and macrophages, and promote several malignant phenotypes including immune suppression. Some anti-inflammatory cytokines, including IL-10 and transforming growth factor (TGF)-ß, relate to pro-tumoral activities. Among immune checkpoint modulators, programmed death (PD-)1 and PD-ligand (L)1 facilitate oral squamous cell carcinoma (OSCC) cell evasion from immune surveillance, and the expression status of these has a prognostic value. OSCCs contain tumor associated macrophages (TAMs) as major stromal cells of their tumor microenvironment. Among the two distinctive states, M2 macrophages support tumor invasion, metastasis and immune suppression. Crosstalk between TAMs and OSCC or cancer-associated fibroblasts (CAF) plays an important role in the progression of OSCC. Clinical trials with blocking antibodies against IL-1α or melanoma-associated antigens have been reported as therapeutic approaches against OSCCs. The most promising approach activating antitumor immunity is the blockade of PD-1/PD-L1 axis. Manipulating the polarization of pro-tumorigenic macrophages has been reported as a novel therapeutic approach.

The luminance ratio of autofluorescence in a xenograft mouse model is stable through tumor growth stages.

The aim of this research was to investigate the value of autofluorescence imaging of oral cancer across different stages of tumor growth, to assist in detecting tumors. A xenograft mouse model was created with human oral squamous cell carcinoma cell line HSC-3 being subcutaneously inoculated into nude mice. Tumor imaging was performed with an autofluorescence imaging method (Illumiscan®) using the luminance ratio, which was defined as the luminance of the tumor site over the luminance of normal skin tissue normalized to a value of 1.0. This luminance ratio was continuously observed postinoculation. Tumor and normal skin tissues were harvested, and differences in the concentrations of flavin adenine dinucleotide and nicotinamide adenine dinucleotide were examined. The luminance ratio of the tumor sites was 0.85 ± 0.05, and there was no significant change in the ratio over time, even if the tumor proliferated and expanded. Furthermore, flavin adenine dinucleotide and nicotinamide adenine dinucleotide were significantly lower in tumor tissue than in normal skin tissue. A luminance ratio under 0.90 indicates a high possibility of tumor, irrespective of the tumor growth stage. However, this cutoff value was determined using a xenograft mouse model and therefore requires further validation before being used in clinical diagnosis.

Suppression effect on IFN-γ of adipose tissue-derived mesenchymal stem cells isolated from ß2-microglobulin-deficient mice.

Administration of bone marrow-derived mesenchymal stem cells (MSCs) is a possible treatment for graft-versus-host disease (GVHD) following allogeneic hematopoietic stem cell transplantation and other inflammatory conditions. To address the mechanism of immunosuppression by MSCs, in particular those derived from adipose tissue (AMSCs), AMSCs were isolated from three different mouse strains, and the suppressive capacity of the AMSCs thus obtained to suppress interferon (IFN)-γ generation in mixed lymphocyte reaction cultures serving as an in vitro model of GVHD were assessed. It was revealed that the AMSCs had a potent capacity to suppress IFN-γ production regardless of their strain of origin and that such suppression was not associated with production of interleukin-10. In addition, the results demonstrated that ß2-microglobulin (ß2m)-deficient AMSCs from ß2m-/- mice were also potent suppressor cells, verifying the fact that the mechanism underlying the suppression by AMSCs is independent of major histocompatibility complex (MHC) class I expression or MHC compatibility. As AMSCs appear to have immunosuppressive properties, AMSCs may be a useful source of biological suppressor cells for the control of GVHD in humans.

Perspectives of Immune Suppression in the Tumor Microenvironment Promoting Oral Malignancy.

INTRODUCTION: In order to survive, cancers control immune systems and evade immune detection using mediators consisting of immune checkpoint molecules and cellular systems associated with immune suppression. METHODOLOGY: During the development of cancer and chronic infections, the immune checkpoints and cellular components including regulatory T cells, myeloid derived suppressor cells and cancer associated fibroblasts are often enhanced as a mechanism of immune subversion and have therefore become very important therapeutic targets. CONCLUSION: In this review, we will discuss the complexity of immune-suppressive mechanisms in the tumor milieu of cancers, including oral malignancy.

Cytokine Expression and Macrophage Localization in Xenograft and Allograft Tumor Models Stimulated with Lipopolysaccharide.

T cell-deficient mice such as nude mice are often used to generate tumor xenograft for the development of anticancer agents. However, the functionality of the other immune cells including macrophages, dendritic cells (DCs), and myeloid-derived suppressor cells (MDSCs) in the xenograft are largely unknown. Macrophages and dendritic cells (DCs) acquire functionally distinct properties in response to various environmental stimuli; the interaction of these cells with MDSCs in tumor microenvironments regulates cancer progression. Nude mice are less likely to reject human cancer cells because of major histocompatibility complex (MHC) mismatches. The tumor microenvironment in a xenograft, comprising human and mouse cells, exhibits more complex bidirectional signaling and function than that of allograft. Here, we evaluated the differences of myeloid cells between them. Plasma interferon-γ and interleukin-18 concentrations in the xenograft tumor model after lipopolysaccharide (LPS) administration were significantly higher than those in the allograft tumor model. MHC class I, II, and CD80 expression levels were increased in CD11b⁺ and MDSC populations after LPS administration in the spleen of a xenograft tumor model but not in that of an allograft tumor model. Additionally, the number of CD80- and mannose receptor C type 1 (MRC1)-expressing cells was decreased upon LPS administration in the tumor of the xenograft tumor. These results suggest that functions of macrophages and DCs are sustained in the xenograft, whereas their functions in response to LPS were suppressed in the allograft. The findings will encourage the consideration of the effects of myeloid cells in the xenograft for drug development.

Gene expression analyses associated with malignant phenotypes of metastatic sub-clones derived from a mouse oral squamous cell carcinoma Sq-1979 cell line.

To elucidate the genetic events that occur during the development of OSCC, the present study established a model of oral malignancy using a mouse oral squamous cell carcinoma (OSCC) Sq-1979 cell line. Sq-1979 cells were implanted into syngeneic C3H mice. Subsequently, 233 cells and metastatic sub-clones (L cells) from primary OSCC, as well as the metastasized lymph node tissues of Sq-1979-implanted mice were established. Compared with parental Sq-1979 and 233 cells, the majority of L cells exhibited a higher proliferation rate and transplantability, and conferred a lower survival rate on the implanted mice. To investigate the genetic background of L cells, preferentially expressed genes in L cells were identified by cDNA microarray and reverse transcription-polymerase chain reaction analyses. The expression of FYN-binding protein (Fyb), solute carrier family 16 member 13 (Slc16a13), keratin 7, transmembrane portion 173 and Slc44a3 mRNAs was significantly elevated in L cells compared with that in Sq1979 and 233 cells. The mRNA expression was also evaluated in human OSCC and leukoplakia (LP) tissues. Among the 5 aforementioned mRNAs, the expression of FYB and SLC16A13 was significantly higher in OSCC than in LP tissues. Furthermore, the expression of SLC16A13 mRNA was significantly elevated in highly invasive OSCCs, which were classified as grades 3 and 4 by the Yamamoto-Kohama (YK) classification of invasion, compared with those in lower grades (YK-1 and -2). The model proposed in the present study could thus describe essential marker genes for the diagnosis of oral malignancies.

Tumor growth limited to subcutaneous site vs tumor growth in pulmonary site exhibit differential effects on systemic immunities.

To evaluate systemic immunity associated with tumor growth limited to a subcutaneous site versus growth proceeding at multiple tumor sites, we established syngeneic mouse subcutaneous and pulmonary tumor models by local subcutaneous and intravenous injection of colon carcinoma CT26 cells. We found that splenic myeloid-derived suppressor cell (MDSC) levels were significantly increased in the subcutaneous tumor model but not in the pulmonary tumor model. Furthermore, both CD4+ and CD8+ T cells as well as CD4+ Foxp3+ T cells were significantly decreased in the subcutaneous tumor model and were largely unchanged in the pulmonary tumor model. In addition, the subcutaneous model, but not the pulmonary model, displayed a Th1 polarization bias. This bias was characterized by decreased IL-4, IL-9, and IL-10 production, whereas the pulmonary model displayed increased production of IL-10. These results suggest that the mode of tumor development has differential effects on systemic immunity that may, in turn, influence approaches to treatment of cancer patients.

Metastasized murine oral squamous cell carcinoma cells induce intratumoral polymorphonuclear myeloid derived suppressor cells.

Myeloid derived suppressor cells (MDSCs) localize to hematopoietic organs and peripheral blood during inflammation or tumor tissues and lymph nodes in the presence of a tumor. However, whether there are differences in MDSCs found in the primary tumor and metastases is unknown. In the present study, we established a cell line of metastasized tumor cells to a lymph node, L5-11, which were derived from the Sq-1979 mouse buccal mucosa squamous cell carcinoma cell line. We then analyzed tumor immunogenicity, especially with regard to MDSCs, to clarify the differences between the primary tumor and metastases, using an isogenic heterotopic tumor transplantation model. Our data showed that the population of intratumoral MDSCs, especially polymorphonuclear MDSCs in the lymph node metastasis model were significantly increased compared with syngeneic grafts from the primary cell line Sq-1979 after 21 days. Furthermore, we identified that the lymph node metastasis cell line had increased expression of genes that promote the expansion of MDSCs, tumor growth and metastasis. Hence, these data suggest that tumor immunosuppression can occur via activation of MDSCs. However, further examination is required to clarify whether all or a subset of these factors from the lymph node metastasis tumor cells are required to induce intratumoral MDSCs.

Promotion of Nickel (Ni) Allergy by Anamnestic Sensitization with a Bacterial Component, Lipopolysaccharide (LPS), in Mice.

BACKGROUND/OBJECTIVE: Lipopolysaccharides (LPS) promote allergic responses to nickel (Ni) both in the sensitization and elicitation steps. In this study, we examine the effect of pre-sensitization to LPS on the occurrence of Ni allergy using a mouse model. METHOD: A 100 mg of LPS was injected into C57BL/6J mice intraperitoneally (ip). Three weeks later, the mice were subsequently injected with 0.3 µ moles of nickel dichloride (NiCl2) and 100 µg of CpG-DNA, which acted as an adjuvant. The mice were repeatedly immunized with the 0.3 µg of nickel sulfate (NiSO4), along with 300 µl of the adjuvant, Inject Alum (Pierce, USA). Then we examined the producing capabilities of T helper type 1 (Th1) and 2 (Th2) cytokines (interferon-gamma- (IFN)-γ and interleukin (IL)-10, respectively) from anti CD3 antibody-stimulated spleen cells. RESULTS: Pre-treatment with LPS, followed by repeated challenges with Ni2+ and adjuvants significantly enhanced the IFN-γ-producing capability of spleen cells (n=5, p<0.01); however, that could not enhance the capability of spleen cells by a single challenge with Ni2+ and adjuvants (n=5). In contrast, without LPS treatment, single or even repeated challenges by Ni2+ could not enhance the IFN-γ-producing capability. On the other hand, the IL-10-producing capability of spleen cells was not enhanced even by LPS and repeated challenges with Ni2+ and adjuvants. CONCLUSION: The solitary pre-sensitization to LPS is essential for the onset of Ni allergy by shifting the Th1/Th2 immune balance toward a Th1 dominant.

Transient Tcf3 Gene Repression by TALE-Transcription Factor Targeting.

Transplantation of hematopoietic stem and progenitor cells (HSCs) i.e., self-renewing cells that retain multipotentiality, is now a widely performed therapy for many hematopoietic diseases. However, these cells are present in low number and are subject to replicative senescence after extraction; thus, the acquisition of sufficient numbers of cells for transplantation requires donors able to provide repetitive blood samples and/or methods of expanding cell numbers without disturbing cell multipotentiality. Previous studies have shown that HSCs maintain their multipotentiality and self-renewal activity if TCF3 transcription function is blocked under B cell differentiating conditions. Taking advantage of this finding to devise a new approach to HSC expansion in vitro, we constructed an episomal expression vector that specifically targets and transiently represses the TCF3 gene. This consisted of a vector encoding a transcription activator-like effector (TALE) fused to a Krüppel-associated box (KRAB) repressor. We showed that this TALE-KRAB vector repressed expression of an exogenous reporter gene in HEK293 and COS-7 cell lines and, more importantly, efficiently repressed endogenous TCF3 in a human B lymphoma cell line. These findings suggest that this vector can be used to maintain multipotentiality in HSC being subjected to a long-term expansion regimen prior to transplantation.

Producing Capabilities of Interferon-gamma and Interleukin-10 in Peripheral Blood from Oral Squamous Cell Carcinoma Patients.

In order to evaluate the Th1 and Th2 responses of Oral Squamous Cell Carcinoma (OSCC) patients, we investigated the cytokine producing capability of peripheral blood (PB), and compared it with clinicopathological appearances of OSCC patients. The production of a Th1-type cytokine, interferon (IFN)-γ, from lipopolysaccharide (LPS)-stimulated PB correlated positively with the frequency of lymph node metastasis. We also investigated the production of a Th2-type cytokine, IL-10, however, no significant correlation was observed with the clinicopathological appearances. Our results suggested that the IFN-γ producing capability was specifically regulated and dependent on the regional metastatic potencies of OSCCs.

Increase of peripheral blood CD57+ T-cells in patients with oral squamous cell carcinoma.

BACKGROUND/AIM: The subset of T-cells positive for expression of cluster of differentiation (CD) 57 has been associated with various cancer phenotypes. However, the presence of CD57(+) T-cells in patients with oral squamous cell carcinoma (OSCC) has yet to be confirmed. In the present study, we examined the diagnostic significance of the presence of CD57(+) T-cells in peripheral blood (PB) from patients with OSCC. MATERIALS AND METHODS: The subset of CD57(+) T-cells in PB was analyzed in 43 patients with OSCC by fluorescence-activated cell sorting (FACS) analysis. RESULTS: The proportion of CD57(+) T-cells, including both CD8(+) and CD4(+) subsets, significantly increased with clinical stage, especially in parallel with tumor size. CONCLUSION: Our results suggest that an increase in the population of CD57(+) T-cells is a potent prognostic marker and may also influence the systemic immunity of patients with OSCC.

Actively induced antigen-specific CD8+ T cells by epitope-bearing parasite pre-infection but not prime/boost virus vector vaccination could ameliorate the course of Plasmodium yoelii blood-stage infection.

The lack of MHC molecules on red blood cells (RBCs) has led to questions regarding the immunological function of CD8(+) T cells against malarial blood-stage (MBS). However, several recent reports contradicting with this concept have suggested that they play an important role in the course of MBS infection. The present study generated genetically engineered murine malaria, Plasmodium yoelii, which expresses a well-defined Trypanosoma cruzi-derived, H-2K(b)-restricted CD8(+) T cell epitope, ANYNFTLV. Prime/boost vaccination by the use of recombinant adenovirus and recombinant modified vaccinia virus Ankara (MVA), which induced an enhanced number of ANYNFTLV-specific CD8(+) T cells, failed to prevent a pathological outcome to occur upon ANYNFTLV-expressing murine MBS infection. This outcome did not change even with the combination of passive transfer of an appreciable number of in vitro-expanded ANYNFTLV-specific CD8(+) T cells. In contrast, the pre-infection of mice with T. cruzi, which intrinsically bears the same CD8(+) T cell epitope significantly improved the survival of ANYNFTLV-expressing malaria-infected mice but not that of control malaria-infected ones. This protective effect was abrogated by the use of a CD8(+) T cell-depleting monoclonal antibody. Although the protective effect was observed only in certain situations, the actively induced antigen-specific CD8(+) T cells could ameliorate the pathologies caused by the MBS. This is the first study to implicate that the active induction of antigen-specific CD8(+) T cells should be included in the development of a vaccine against MBS.

Simultaneous detection of T lymphocyte-related antigens (CD4/CD8, CD57, TCRß) with nuclei by fluorescence-based immunohistochemistry in paraffin-embedded human lymph node, liver cancer and stomach cancer.

OBJECTIVE: To develop a method for immunohistochemical staining of three different T lymphocyte antigens (CD4 or CD8, CD57 and TCRß) on the same tissue section and to determine whether tissues have been infiltrated with T lymphocytes expressing these markers. STUDY DESIGN: Commercially available antibodies were tested for immunohistochemical usefulness in a dye-based conventional single-immunostaining method after antigen retrieval on paraffin-embedded human lymph nodes. We searched for the combination of antibodies that could detect T lymphocyte antigens on the same section without any cross-reactivity and that have fluorescent signals robust enough to overcome paraffin autofluorescence. RESULTS: Application of the antigen retrieval technique and the Sudan black B quenching technique enabled staining of paraffin-embedded tissue sections with fluorescent-labeled secondary antibodies. The combination of primary and secondary antibodies that could simultaneously detect the T lymphocyte antigens CD4 or CD8, CD57 and TCRß in histochemical analysis of a paraffin-embedded human lymph node section was established, and was successfully applied to a human tissue section infiltrated with T lymphocytes that express these markers. CONCLUSION: The antibodies listed here would be helpful for histopathologists who wish to investigate T lymphocytes in the paraffin-embedded sections that have accumulated in pathology labs throughout the world.