Regional Lymph Node Lymphocytes Research Articles

Dysbiosis Contributes to Arthritis Development via Activation of Autoreactive T Cells in the Intestine.

The intestinal microbiota is involved in the pathogenesis of arthritis. Altered microbiota composition has been demonstrated in patients with rheumatoid arthritis (RA). However, it remains unclear how dysbiosis contributes to the development of arthritis. The aim of this study was to investigate whether altered composition of human intestinal microbiota in RA patients contributes to the development of arthritis. We analyzed the fecal microbiota of patients with early RA and healthy controls, using 16S ribosomal RNA-based deep sequencing. We inoculated fecal samples from RA patients and healthy controls into germ-free arthritis-prone SKG mice and evaluated the immune responses. We also analyzed whether the lymphocytes of SKG mice harboring microbiota from RA patients react with the arthritis-related autoantigen 60S ribosomal protein L23a (RPL23A). A subpopulation of patients with early RA harbored intestinal microbiota dominated by Prevotella copri; SKG mice harboring microbiota from RA patients had an increased number of intestinal Th17 cells and developed severe arthritis when treated with zymosan. Lymphocytes in regional lymph nodes and the colon, but not the spleen, of these mice showed enhanced interleukin-17 (IL-17) responses to RPL23A. Naive SKG mouse T cells cocultured with P copri-stimulated dendritic cells produced IL-17 in response to RPL23A and rapidly induced arthritis. We demonstrated that dysbiosis increases sensitivity to arthritis via activation of autoreactive T cells in the intestine. Autoreactive SKG mouse T cells are activated by dysbiotic microbiota in the intestine, causing joint inflammation. Dysbiosis is an environmental factor that triggers arthritis development in genetically susceptible mice.

CD169 identifies an activated CD8+ T cell subset in regional lymph nodes that predicts favorable prognosis in colorectal cancer patients

ABSTRACTPurpose: CD169 was first identified on macrophages (Mφ) and linked to antigen presentation. Here, we showed CD169 expression on some CD8+ T lymphocytes in regional lymph nodes (LNs) and investigated the function and clinical relevance of CD169+CD8+ T cells in tumor-draining LNs of colorectal cancer (CRC) patients. Experimental design: Fresh tumor-draining LN tissues from 39 randomly enrolled patients were assessed by flow cytometry for activation and differentiation of CD169+CD8+ T cells and T cell-mediated killing of tumor cells. In total, 114 tumor-draining LN paraffin sections from CRC patients were analyzed by multiple-color immunofluorescence for CD169+CD8+ T cell distribution and clinical values. The prognostic significance of CD169+CD8+ T cells was evaluated by Kaplan–Meier analysis. Results: A fraction of CD8+ T cells in regional LNs, but not peripheral blood, tonsils, or tumors, expressed surface CD169. In situ detection of draining LNs revealed preferential localization of CD169+CD8+ T cells to subcapsular sinus and interfollicular regions, closely associated with CD169+ Mφ. CD169+CD8+ T cell ratios were significantly lower in peri-tumor LNs than distant-tumor LNs. CD169+CD8+ T cells predominantly expressed activation markers (CD69, HLA-DR, PD-1) with slightly lower CD45RA and CD62L levels. They produced high granzyme B, perforin, TNF-α, and IFNγ levels, and promoted tumor-killing efficiency ex vitro. Moreover, CD169+CD8+ T cells infiltrating tumor-draining LNs decreased with disease progression and were strongly associated with CRC patient survival. Conclusions: We identified novel activated/cytolytic CD169+CD8+ T cells selectively present in regional LNs, potentially serving as a powerful prognostic factor and indicator for selecting patients for immunotherapy.

Clinical significance of the frequency of regulatory T cells in regional lymph node lymphocytes as a prognostic factor for non-small-cell lung cancer

BackgroundRegulatory T cells (Tregs) are potent immunosuppressive cells that play a crucial role in tumor immune escape. The purpose of the present study was to evaluate the prognostic significance of the frequency of CD4+CD25+Foxp3+ Tregs in the regional lymph node lymphocytes (RLNL) and peripheral blood lymphocytes (PBL) in patients who underwent surgical resection of non-small cell lung cancer (NSCLC). MethodsThe RLNL and PBL in 158 NSCLC patients who underwent complete surgical resection were collected at the time of surgery. The proportions of CD4+CD25+Foxp3+ cells in the RLNL and PBL were determined by flow cytometry. ResultsThe average proportions of Tregs in the RLNL and PBL were 1.28% and 0.76%, respectively. The proportion of Tregs in the RLNL was significantly higher than that in the PBL (p<0.0001). The 5-year overall survival rates of the patients according to the proportion of Tregs in the RLNL were 84.4% and 63.5% in the lower and higher groups, respectively. A significant difference was observed in the survival rate between the higher and lower groups (p=0.0056). Among the patients with stage I disease, the 5-year survival rate (91.4%) was significantly higher in patients with the lower proportion of Tregs in RLNL that in the higher group (72.1%) (p=0.0147). ConclusionsThe higher proportion of Tregs in the RLNL was a significant unfavorable prognostic factor, even in patients with node-negative NSCLC. The information about the proportion of Tregs in the RLNL might improve the discriminatory power for assessing the risk of the recurrence of NSCLC.

Immunosuppressive effect of regulatory T lymphocytes in lung cancer, with special reference to their effects on the induction of autologous tumor-specific cytotoxic T lymphocytes

It is not easy to induce cytotoxic T lymphocytes (CTLs) against cancer in in vitro culture. Regulatory T cells (Tregs) are considered to play a pivotal role in tumor immune escape. In this study, we analyzed the distribution of Tregs among tumor-infiltrating lymphocytes (TILs), regional lymph node lymphocytes (RLNLs) and peripheral blood lymphocytes (PBLs) in patients with lung cancer, and analyzed the effect of Tregs on the induction of CTLs in vitro. A total of 84 patients with non-small cell lung cancer underwent surgery between January 2003 and December 2004. The TILs, RLNLs and PBLs from these patients were subjected to a comparison analysis. The proportion of CD4(+)CD25(+)Foxp3(+) cells in these lymphocytes was determined by flow cytometry. The effects of Tregs on the induction of CTLs was analyzed by the depletion of Tregs in mixed lymphocyte-tumor cell culture (MLTC). The average proportions of Tregs in the TILs, RLNLs and PBLs were 10.4±9.5, 4.4±2.4 and 2.8±2.1%, respectively. The proportion of Tregs in the RLNLs was significantly higher than that in the PBLs (P<0.001); furthermore, TILs contained a larger number of Tregs than RLNLs (P=0.034). These Tregs substantially suppressed the induction of CTLs against autologous tumor cells. The depletion of Tregs in the MLTC resulted in the successful induction of CTLs. Tregs were found at a higher frequency in the TILs and RLNLs than in the PBLs in lung cancer patients. Since Tregs inhibited the induction of CTLs, the depletion of Tregs may represent a new therapeutic strategy for lung cancer patients.

アレルギー性接触皮膚炎の感作相は少なくとも2つのステップより構成され，その完成は皮膚の抗原に対する反応性の向上に重要である

We examined whether 0.1% 2,4-Dinitorofluorobenzene (DNFB), which is a concentration insufficient to induce contact dermatitis with a single application, can induce dermatitis with repeated applications. 0.1% DNFB was painted on the shaved backs of C57BL/6 mice 2 consecutive days a week and 6 applications (3 weeks) induced allergic contact dermatitis. A hapten-specific lymphocyte proliferative response was observed in regional lymph node cells obtained 2 applictions (1 week) of 0.1% DNFB. Four applications (2 weeks) of 0.1% DNFB did not elicit dermatitis, despite the appearance of hapten-specific lymphocytes; therefore, immunological memory is not sufficient to complete the sensitization phase. Interferon (IFN)-γ was produced at 1 week and in significantly higher amounts at 2 weeks. The redox status of regional lymph node cells was examined, because reductive cells have been reported to produce IFN-γ. The number of reductive cells was also increased at 1 week and significantly higher at 2 weeks. Most of the increased reductive cells were considered to be antigen non-specific because even repeated immunization can not induce such a high number of antigen specific cells. If the higher number of antigen non-specific reductive cells play a crucial role in completing the sensitization phase, even irrelevant antigens could induce dermatitis after 4 applications (2 weeks) of DNFB. Although 2.5% or 3.5% oxazolone could not induce dermatitis with a single application, it could induce dermatitis, if applied following 4 applications (2 weeks) of 0.1% DNFB. These results demonstrate that the sensitization phase of allergic contact dermatitis consists of at least 2 steps: the first step, which is antigen specific, is the appearance of antigen specific lymphocytes in regional lymph nodes, and the second step, which is antigen non-specific, is increases in IFN-γ production and the number of reductive cells. Entry of an antigen to the skin plays a crucial role in the response to all antigens.

Effect of cocoa-enriched diets on lymphocytes involved in adjuvant arthritis in rats

Cocoa and its flavonoids have potential anti-inflammatory properties in vitro and in acute inflammation models in vivo. The aim of the present study was to ascertain the effects of two cocoa-enriched diets on adjuvant arthritis (AA) in rats, considering not only clinical and biochemical inflammatory indices, but also antibody response and lymphocyte composition. Female Wistar rats were fed with a 5 or 10 % cocoa-enriched diet beginning 2 weeks before arthritis induction and until the end of the study. AA was induced by an intradermal injection of heat-killed Mycobacterium butyricum suspension. The hind-paw swelling (plethysmometry), serum anti-mycobacterial antibody concentration (ELISA), blood and inguinal lymph node lymphocyte subset percentage (flow cytometry), and IL-2, interferon γ and PGE₂ released from splenocytes (ELISA) were assessed. Although the cocoa diets had no significant effect on hind-paw swelling, a tendency to reduce it was observed at the end of the study. Cocoa-enriched diets were able to decrease the serum anti-mycobacterial antibody concentration and the splenocyte PGE2 production, as well as the proportion of T-helper (Th) lymphocytes in blood and regional lymph nodes, which probably includes cells responsible for the arthritic process. The cocoa diets prevented a decrease in the proportion of regulatory T-cells in blood and a disequilibrium between inguinal lymph node natural killer (NK) CD8⁺ and NK CD8⁻ subsets. In conclusion, the cocoa-enriched diets during AA were not able to significantly decrease joint inflammation but modified Th-cell proportions and prevented specific antibody synthesis.

Distribution of Th17 cells and FoxP3(+) regulatory T cells in tumor‐infiltrating lymphocytes, tumor‐draining lymph nodes and peripheral blood lymphocytes in patients with gastric cancer

Although Th17 cells reportedly play critical roles in the development of autoimmunity and allergic reactions, information on Th17 cells in cancer-bearing hosts is still limited. In the present study, we investigated the distribution of Th17 cells in relation to regulatory T cells (Treg) in the tumor-infiltrating lymphocytes (TILs), regional lymph node lymphocytes, and peripheral blood lymphocytes of gastric cancer patients. Interleukin (IL)-17-producing CD4(+) cells as Th17 cells and CD4(+)CD25(+)FoxP3(+) cells as Treg were evaluated by flow cytometry and expressed as a percentage of the total CD4(+) cells, in addition to performing a Th1/Th2 balance assay. Moreover, immunohistochemical staining for IL-17 and FoxP3 were performed. In TILs from patients with early disease (n = 27), the frequency of Th17 cells was significantly higher than that in the normal gastric mucosa (23.7 ± 8.9 vs 4.5 ± 3.1%). In TILs from patients with advanced disease (n = 28), the frequency of Th17 cells was also significantly higher, but lower compared to early disease, than that in the normal gastric mucosa (15.1 ± 6.2 vs 4.0 ± 2.0%). This observation for Th17 cell-distribution was also confirmed by immunohistochemistry. When the ratio of Th17/Treg in TILs was evaluated in individual cases, it was more markedly increased in early than in advanced disease. In conclusion, the accumulation of Th17 cells as well as Treg in the tumor microenvironment of gastric cancer occurred in early disease and then the infiltration of Th17 cells gradually decreased according to the disease progression, in contrast to increased Treg.

Identification of ribosomal protein L19 as a novel tumor antigen recognized by autologous cytotoxic T lymphocytes in lung adenocarcinoma

The purpose of the present study was to identify a novel tumor-specific antigen capable of inducing a specific cellular immune response in lung cancer patients. The co-culture of regional lymph node lymphocytes and the CD80-transfected autologous lung adenocarcinoma cell line H1224L resulted in a successful induction of bulk cytotoxic T lymphocytes (CTL). CTL clone L7/8 was established by the limiting dilution method from these bulk CTLs and lysed H1224L but not autologous Epstein-Barr virus-transformed B cells or K562. The CTL clone also recognized allogeneic lung cancer cell lines in an HLA-A*31012-restricted manner. Using the CTL clone, an antigen-coding gene was identified using the cDNA expression cloning technique, which encodes ribosomal protein L19 (RPL19). Finally, a 9 mer antigenic peptide was identified by means of construction of mini-genes. RPL19 was overexpressed in the lung cancer tissue from patient H1224. All of the normal tissues examined expressed lower levels of RPL19 mRNA than that of the lung cancer tissue. RPL19 was also found to be overexpressed in 12 of 30 (40%) non-small-cell lung cancer tissues by immunohistochemical staining. The expression level of RPL19 in tumor cell lines correlated positively with the production of interferon (IFN)-gammaby CTL clone L7/8 in response to such cell lines. In addition, the suppression of RPL19 expression by transfection with small interfering RNA resulted in the suppression of cyclinD1, D3 synthesis, and the growth inhibition of lung cancer cell lines overexpressing RPL19. Therefore, this growth suppression could be ascribed to the inhibition of the cell cycle. These results may indicate that RPL19 is a novel overexpressed antigen which may therefore be a useful candidate as a target for specific immunotherapy.

The significance of Treg cells in defective tumor immunity

Regulatory T cells (Treg) enriched in FoxP3+, glucocorticoid-induced TNF receptor+, and cytotoxic T-lymphocyte-associated antigen-4+ exert a potential to suppress effector T cells in the periphery. These cells exist in markedly higher proportions within tumor-infiltrating lymphocytes, peripheral blood lymphocytes, and/or regional lymph node lymphocytes of patients with cancer and their frequencies are suggested to be strongly related to tumor progression and inversely correlated with the efficacy of treatment. Tumor-specific Treg cells require ligand-specific activation and cell-to-cell contact to exert their suppressive activity on tumor-specific effector cells (CD8+ cytotoxic T lymphocytes and CD4+ Th cells), which includes decreased cytotoxity, proliferation, and Th1 cytokine secrection. Depletion or blockade of Treg cells can enhance immune protection from tumor-associated antigens that are expressed as self antigens. Recent studies revealed that lymphoma T cells might adopt a Treg profile as well. Studies assessing the influence of chemotherapy on Treg cells have also been included in this review.

Detection of novel cancer‐testis antigen‐specific T‐cell responses in TIL, regional lymph nodes, and PBL in patients with esophageal squamous cell carcinoma

We recently identified three HLA-A2402-restricted epitope peptides derived from cancer-testis antigens (CTA), TTK protein kinase (TTK), lymphocyte antigen 6 complex locus K (LY6K), and insulin-like growth factor (IGF)-II mRNA binding protein 3 (IMP-3) for the development of immunotherapies against esophageal squamous cell carcinoma (ESCC). In order to evaluate their immunotherapeutic potential in ESCC patients, we estimated by ELISPOT assay the TTK-, LY6K-, or IMP-3-specific T-cell immune responses in tumor-infiltrating lymphocytes (TIL), regional lymph node lymphocytes (RLNL), and peripheral blood lymphocytes (PBL) expanded from 20HLA-A2402 (+) ESCC patients, and correlated their immune activity with the expression levels of TTK, LY6K, and IMP-3, and MHC class I in the tumors. Induction of TTK-antigen specific T-cell response in TIL to the peptide-pulsed target cells was detected in 14 out of 20 (70%) cases, while LY6K or IMP-3 specific T-cell activity was observed in 11 of 20 (55%) or in eight of 20 (40%) cases, respectively. Furthermore, T-cell activity in RLNL and PBL was detectable in the similar proportion of the 20 ESCC patients. Interestingly, CTA-specific T-cell immune response was found in 13 of 14 (93%) TIL obtained from ESCC tumors with strong MHC class I expression, while it could be observed only in two of six (33%) TIL from ESCC tumors with weak MHC class I expression. These results strongly suggest the pre-existence of specific T-cell responses to HLA-A24-restricted epitope peptides from TTK, LY6K, and IMP-3 in ESCC patients. Monitoring antigen-specific T-cell responses, as well as the expression levels of MHC class I and epitope CTA in tumors, should be a selection index for application of cancer vaccine therapies to the patients who are likely to show good immune response.

Lack and restoration of sensitivity of lung cancer cells to cellular attack with special reference to expression of human leukocyte antigen class I and/or major histocompatibility complex class I chain related molecules A/B

Both cytotoxic T lymphocytes (CTL) and natural killer (NK) cells may play major roles in the host defense against cancer. However, their relationship against the same tumor remains to be elucidated. Among 26 human lung cancer cell lines established in our laboratory, 10 (38%) exhibited human leukocyte antigen (HLA)-class I haplotype loss and three (12%) lost HLA-class I expression totally by flow cytometry analysis. The two cell lines (E522L and C831L) that lost their expression of HLA-class I in vitro and in vivo were applied for further evaluations. Genetic abnormalities of beta2-microglobulin gene were observed in both E522L (loss of mRNA) and C831L (point mutation). Transduction of the wild-type beta2-microglobulin gene rendered them positive for HLA-class I expression. The CTL were induced from autologous peripheral blood mononuclear cells or regional lymph node lymphocytes by stimulation with wild-type beta2-microglobulin transduced-E522L or -C831L, and they showed tumor-specific cytotoxicity against wild-type beta2-microglobulin-transductant, but not parental cells. In NK cell cytotoxicity, E522L showed high sensitivity to NK cells; however, C831L showed resistance despite loss of HLA-class I expression. E522L expressed MHC class I chain related molecules A/B, but C831L did not. The transduction of the MHC class I chain related molecule A gene from E522L rendered C831L positive for expression and sensitive to NK cell cytotoxicity. Reconstruction of HLA-class I and MHC class I chain related molecules A expression could abrogate evasion from cellular attack by CTL and NK cells, and it may lead to a breakthrough in the development of cancer immunotherapy.

Identification of HLA‐A24 restricted shared antigen recognized by autologous cytotoxic T lymphocytes from a patient with large cell carcinoma of the lung

The aim of the present study was to elucidate the tumor-specific cellular immunological responses occurring in a patient with large cell carcinoma of the lung who had no evidence of recurrence following surgical resections of both a primary lung lesion and a metastatic adrenal lesion. We analyzed an autologous tumor-specific cytotoxic T lymphocytes (CTL clone F2b), which were HLA-A*2402 restricted from regional lymph node lymphocytes. The F2b possessed T cell receptor (TCR) using the Valpha5 and Vbeta7 gene segment. The existence of precursor CTL (pCTL) against autologous tumor cells (A904L) was analyzed using CTL clone-specific PCR. Lymphocytes with the same TCR as F2b were detected in the primary tumor tissue, regional lymph node and the peripheral blood collected from the patient 3 years after the operation. Using the F2b, we identified a cDNA clone encoding the tumor antigen using cDNA expression cloning method. The gene was found to encode splicing variant of the Tara gene. Finally, we identified the 9-mer Ag peptide, using constructions of mini-genes. The F2b recognized 3 out of 7 HLA-A24 positive allogeneic tumor cell lines and in 1 out of 7 HLA-A24 negative allogeneic tumor cell lines when transfected with HLA-A24. This peptide is therefore considered to be potentially useful for performing specific immunotherapy in a significant proportion of lung cancer patients bearing HLA-A24.

Identification of a New Cancer/Germline Gene,KK-LC-1, Encoding an Antigen Recognized by Autologous CTL Induced on Human Lung Adenocarcinoma

The purpose of our present study is to identify a tumor-specific antigen capable of inducing a specific cellular immune response in lung cancer patients. We established a lung adenocarcinoma cell line, designated as F1121L, and induced tumor-specific CTL clone H1 from regional lymph node lymphocytes of patient F1121. CTL clone H1 lysed autologous tumor cells in an HLA-B*1507-restricted manner, but not autologous EBV-B, phytohemagglutinin-blast cells, and K562. The CTL clone also recognized allogeneic HLA-B*1501- or 1507-positive lung cancer cell lines in the HLA-restricted manner. Using the CTL clone, we identified an antigen-coding gene by cDNA expression cloning technique. The gene consisted of 556 bp, including an open reading frame consisted of 113 amino acids, designated as Kita-kyushu lung cancer antigen 1 (KK-LC-1). A 9-mer peptide (KK-LC-1(76-84); RQKRILVNL) was identified as an epitope peptide. The genomic DNA of this antigen was located in chromosome Xq22. A reverse transcription-PCR analysis revealed that the mRNA of this gene was only expressed in the testis among normal tissues. It was expressed in 9 of 18 (50%) allogeneic non-small-cell lung cancer cell lines and in 40 of 100 (40%) non-small-cell lung cancer tissues. We thus identified a new tumor antigen-coding gene categorized as a cancer/germline gene by an autologous lung cancer and CTL system. The new cancer/germline gene was located in Xq22, which is apparently different from the locations of previously reported cancer/germline genes.

Identification of the HLA-Cw*0702-Restricted Tumor-Associated Antigen Recognized by a CTL Clone from a Lung Cancer Patient

A large number of tumor-associated antigens have been used in vaccination trials for mainly melanomas. Our purpose of this study is to identify a novel tumor antigen useful for immunotherapy of lung cancer patients. Analysis of an autologous tumor-specific CTL clone F2a that was established from regional lymph node lymphocytes of a patient with lung cancer (A904) by a mixed lymphocyte-tumor cell culture. F2a recognized and killed autologous tumor cells (A904L), whereas it did not respond to autologous EBV-transformed B cells, phytohemagglutinin-blastoid T cells, and K562 cells. cDNA clone 31.2 was isolated by using cDNA expression cloning method as a gene encoding antigen. This gene was identical to the reported gene whose function was unknown. The antigen encoded by the cDNA was recognized by the CTL in a HLA-Cw*0702-restricted manner. Furthermore, a 9-mer peptide at positions 659 to 685 in cDNA clone 31.2 was identified as a novel epitope peptide. The CTL recognized some allogeneic cancer cell lines with HLA-Cw*0702 as well as some HLA-Cw*0702-negative cell lines when transfected with HLA-Cw*0702, thus indicating that the identified antigen was a cross-reactive antigen. Although exact mechanism to process the encoded protein and present the antigen in the context of HLA class I remains to be elucidated, the CTL recognized some of tumor cells in the context of HLA-Cw*0702 but did not recognize a variety of normal cells and also autologous EBV-transformed B cells. These results indicated that the antigen identified in this study may therefore be a possible target of tumor-specific immunotherapy for lung cancer patients.

609. Long Term Persistence of Canine Factor IX-Specific Th1 Lymphocytes in Regional Lymph Nodes after AAV1-Mediated Intramuscular Gene Transfer in a Hemophilia B Dog

Previous studies on AAV-mediated gene therapy for hemophilia B showed that underlying factor IX (F.IX) mutation, vector dose, serotype, and route of administration all play a role in the risk of anti-F.IX antibody formation. Intramuscular (IM) administration of an AAV2 vector in normal and hemophilia B (HB) mice invariably activates F.IX-specific Th2 lymphocytes with production of mainly IgG1 and IgG2b antibody subclasses. Similarly, HB dogs with a missense mutation usually do not develop an immune response against canineF.IX when injected IM with low doses of an AAV2 vector. At high vector doses, a neutralizing antibody response has been observed, characterized by the production of both IgG1 and IgG2 subclasses, with levels of IgG2>IgG1. Although previous observations ruled out the involvement of CD8+ T cells, a recent study on the ovalbumin mouse model of AAV-mediated muscle-directed gene transfer showed a CTL response against the transgene protein at high vector doses (Wang et al., Blood, in press). An HB dog from the Chapel Hill colony (E57) received by IM injection an AAV1 vector expressing canine F.IX under the control of the CMV promoter at a dose of 2.4 |[times]| 10^11vg/kg, vector was co-administered with immunosuppression (IS) (Arruda et al., Blood 2004). One week after vector administration the whole blood clotting time (WBCT) fell to the normal range (18min) and circulating F.IX levels reached 104 ng/ml. However, after discontinuing IS a long-lasting neutralizing antibody response was observed (6 BU >3 years after injection) with production of both anti-cF.IX IgG1 and IgG2 and levels of IgG1>IgG2. In order to study in detail T cell responses against the cF.IX, a peptide library was produced spanning the F.IX protein sequence. PBMCs were isolated from whole blood of E57 and, from two HB dogs that received an AAV2-cF.IX vector delivered to the muscle. Lymphocytes were tested by ELISpot for secretion of IL-4, IL-10 and IFN-|[gamma]| and, interestingly, dogs injected with the AAV2 vector showed a positive IL-10 response while the AAV1 injected dog did not. Next, E57 and a normal dog were sacrificed and lymphocytes from spleen, draining lymph nodes (LN) and non-draining LN were isolated. Lymphocytes from E57 draining LN showed positive results on ELISpot for IFN-|[gamma]| for 2 cF.IX peptide pools. No response was detected for the other tissues and for the control dog. IF staining for CD8+ cells and H&E staining of muscle sections from E57 were negative. These results suggest a long-lived immune response detectable locally in the draining LN. Cytokine and antibody subclass profiles are typical of a Th1 response vs. the Th2 response seen in AAV2 injected dogs. Whether the shift from a Th2 to a Th1 response is driven by higher levels of antigen expression of by the switch in serotypes is currently under study. We conclude that in AAV-mediated, muscle-directed gene transfer, there may be long-term persistence of F.IX-specific Th1 lymphocytes in the regional LN.

Tumor specific expression of survivin-2B in lung cancer as a novel target of immunotherapy

Survivin-2B is reported to be specifically expressed in numerous malignant tissues. Furthermore, survivin-2B includes the epitope peptide (survivin-2B(80-88)) which is capable of binding to HLA-A24. In this study, we evaluated whether survivin-2B could be a novel vaccine target against lung cancer. (1) The differences in the survivin-2B expression between 15 sets of lung cancer tissues and normal lung tissues were investigated using RT-PCR. (2) The expression of survivin-2B was further examined in 42 lung cancer tissues, and the relationship between the expression and clinicopathologic factors was analyzed. (3) To compare the frequency of precursor CTL between survivin-2B positive and negative lung cancer patients, surivivin-2B(80-88) peptide-specific CTL were induced from regional lymph node lymphocytes (LNL) of four HLA-A24 (+) lung cancer patients, in whom two showed a positive survivin-2B expression of lung cancer while another two were negative, after stimulation with surivivin-2B(80-88). Survivin-2B was specifically expressed in lung cancer tissues, and was expressed in 17 of 42 lung cancer tissues (42.9%). Histologically, it was significantly more frequently expressed in squamous cell carcinoma than in adenocarcinoma (p=0.014). The frequency of precursor CTL in LNL was approximately one in 2.0 x 10(7) in patients with survivin-2B expression (-) lung cancer, however, it was one in 5.0 x 10(6) to 6.0 x 10(6) in those with survivin-2B expression (+) lung cancer. Survivin-2B was specifically expressed in lung cancer tissue, and found to specifically elicit a cellular immune response in lung cancer patients and therefore it may be a novel candidate for peptide vaccination.

A different pattern of cytotoxic T lymphocyte recognition against primary and metastatic tumor cells in a patient with nonsmall cell lung carcinoma

Lung carcinoma represents the most frequent cause of cancer death worldwide because of tumor metastases. The objective of the current study was to analyze the immunologic response during the progress of lung carcinoma metastasis. The authors established two tumor cell lines that were derived from primary and metastatic lesions in a patient with lung carcinoma (Patient G603). One cell line (G603L) was established from the primary lesion, and the other cell line (G603AD) was established from a metastatic lesion in the right adrenal gland 7 months after the patient underwent surgery for the primary lesion. Autologous regional lymph node lymphocytes were stimulated with CD80-transfected G603L cells, then cytotoxic T lymphocytes (CTLs) were induced against both lung carcinoma cell lines. Both G603L cells and G603AD cells expressed Class I human leukocyte antigen, intracellular cell adhesion molecule 1, and lymphocyte-associated antigen type 3 (LFA-3), but not Fas or Fas ligand on their surfaces. By stimulation with CD80-transfected G603L cells, 2 CTL clones (H2/17 and H2/36) were established from the bulk CTLs. CTL clone H2/17 lysed G603L cells but not G603AD cells, suggesting that the antigen recognized by CTL clone H2/17 was abrogated during the process of metastasis. In contrast, CTL clone H2/36 lysed both G603L cells and G603AD cells, indicating that the antigen recognized by CTL clone H2/36 was maintained in the tumor cells throughout tumor progression. The results demonstrated the possibility that some tumor-associated antigens may be abrogated during the process of metastasis, although others are maintained. The identification of these antigens will lead to a better understanding of their immunologic role during disease progression in patients with lung carcinoma.

Intranasal HIV-1-gp160-DNA/gp41 Peptide Prime-Boost Immunization Regimen in Mice Results in Long-Term HIV-1 Neutralizing Humoral Mucosal and Systemic Immunity

An intranasal DNA vaccine prime followed by a gp41 peptide booster immunization was compared with gp41 peptide and control immunizations. Serum HIV-1-specific IgG and IgA as well as IgA in feces and vaginal and lung secretions were detected after immunizations. Long-term humoral immunity was studied for up to 12 mo after the booster immunization by testing the presence of HIV-1 gp41- and CCR5-specific Abs and IgG/IgA-secreting B lymphocytes in spleen and regional lymph nodes in immunized mice. A long-term IgA-specific response in the intestines, vagina, and lungs was obtained in addition to a systemic immune response. Mice immunized only with gp41 peptides and L3 adjuvant developed a long-term gp41-specific serum IgG response systemically, although over a shorter period (1-9 mo), and long-term mucosal gp41-specific IgA immunity. HIV-1-neutralizing serum Abs were induced that were still present 12 mo after booster immunization. HIV-1 SF2-neutralizing fecal and lung IgA was detectable only in the DNA-primed mouse groups. Intranasal DNA prime followed by one peptide/L3 adjuvant booster immunization, but not a peptide prime followed by a DNA booster, was able to induce B cell memory and HIV-1-neutralizing Abs for at least half of a mouse's life span.

Epidermal Langerhans cell migration and sensitisation to chemical allergens

Epidermal Langerhans cells (LC) form part of the wider family of dendritic cells (DC; professional antigen-processing and antigen-presenting cells). LC are considered to serve in the skin as sentinels of the adaptive immune system, surveying the local environment and transporting foreign antigen for presentation to responsive T lymphocytes in regional lymph nodes. As such, LC play pivotal roles in the initiation of cutaneous immune responses, including immune responses to chemical allergens encountered at skin surfaces. Here we explore two aspects of LC function in the context of sensitisation to chemical allergens. The first is consideration of the cytokine and chemokine signals that regulate and counter-regulate the mobilisation and migration of LC from the epidermis to skin-draining lymph nodes following topical sensitisation. The second is examination of the ways in which LC may influence the polarity of induced T lymphocytes, and thereby the quality of immune responses.

Prevention of adjuvant arthritis by the W3/25 anti-CD4 monoclonal antibody is associated with a decrease of blood CD4(+)CD45RC(high) T cells.

Imbalance between Th1 and Th2 functions is considered to play a key role in the induction and development of several autoimmune diseases, and the correction of that imbalance has led to effective therapies of some experimental pathologies. To examine whether CD4(+)CD45RC(high) (Th1-like) and CD4(+)CD45RC(low) (Th2-like) lymphocytes play a role in the pathogenesis of adjuvant arthritis (AA) and in its prevention by anti-CD4 antibody, CD45RC expression on CD4(+) T cells was determined in arthritic rats and in animals treated with an anti-CD4 MoAb (W3/25) during the latency period of AA. The phenotype of regional lymph node lymphocytes from arthritic rats in the active phase of the disease was determined by flow cytometry. Peripheral blood lymphocytes from rats treated with W3/25 MoAb were also analysed for 2 weeks after immunotherapy finished. IgG2a and IgG1 isotypes of sera antibodies against the AA-inducing mycobacteria, considered to be associated with Th1 and Th2 responses, respectively, were also determined by ELISA techniques. Fourteen days after arthritis induction, regional lymph nodes presented an increase in CD4+CD45RC(high) T cell proportion. Preventive immunotherapy with W3/25 MoAb inhibited the external signs of arthritis and produced a specific decrease in blood CD4(+)CD45RC(high) T cells and a diminution of antibodies against mycobacteria, more marked for IgG2a than for IgG1 isotype. These results indicate a possible role of CD4(+)CD45RC(high) T lymphocytes in the pathogenesis of AA, and suggest that the success of anti-CD4 treatment is due to a specific effect on CD4(+)CD45RC(high) T subset that could be associated with a decrease in Th1 activity.