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アセチルコリン（ACh）は、最も古くから知られている神経伝達物質である。しかしながら、AChはリンパ系細胞でも産生され、種々のサイトカイン産生やナィーブT細胞の分化に影響を及ぼして免疫機能調節に関与していることが明らかになっている。ナマコ（sea cucumber）由来の抽出成分IJ-337には、サイトカイン産生抑制作用、神経性疼痛抑制作用や糖尿病性知覚異常改善作用などがあることが報告されている。本研究では、IJ-337のTリンパ球機能およびTリンパ球コリン作動系活性に及ぼす影響について検討した。T細胞のモデルとしてヒトT細胞系白血病細胞株MOLT-3を用いた。IJ-337は、MOLT-3細胞における転写調節因子c-fos遺伝子発現を抑制した。さらに、IJ-337は、インターロイキン-2 (IL-2) タンパク質産生量を減少させた。しかしながら、IJ-337はTリンパ球コリン作動系活性には影響を及ぼさなかった。IJ-337はMOLT-3細胞の増殖を抑制した。以上の結果より、IJ-337はコリン作動系には影響を与えることなく，T細胞機能を抑制して免疫活性を抑制する可能性が示唆された。Acetylcholine (ACh) is the oldest known neurotransmitter. However, ACh is also present in lymphoid cells, and it has been clarified that it is involved in the regulation of immune function through regulation of various cytokine expression and differentiation of naive T cells. It has been reported that extractive component, IJ - 337 derived from sea cucumber has an inhibitory effect on cytokine expression, anti-diabetic action and anti-inflammatory action. In this study, we investigated the effect of IJ-337 on T lymphocyte function and T lymphocyte cholinergic system activity. A human T cell lineage leukemia cell line MOLT-3 was used as a model of T cells. IJ-337 suppressed transcription regulatory factor c-fos gene expression in MOLT-3 cells. In addition, IJ-337 decreased interleukin-2 (IL-2) protein production. However, IJ-337 did not affect T lymphocyte cholinergic system activity. IJ-337 suppressed proliferation of MOLT-3 cells. Theseresults suggested that IJ-337 could suppress immune activity by suppressing T cell function, regardless of T lymphocyte cholinergic system., 論文

application/pdf, ２カ月前からの皮膚結節性病変発現の病歴を有する３歳５カ月齢のホルスタイン種乳牛が，起立不能を主訴として受診した．症例の頭部，頸部，肩部および背部には脱毛斑と結節性皮膚腫瘤が認められた．また，全身の体表リンパ節は腫大しており，直腸検査により骨盤腔内に腫瘤が触知された．皮膚結節のパンチ生検病理組織学的所見および腸骨下リンパ節の細針吸引細胞診検査において，異型性の高い大型独立円形細胞が観察された．リンパ球増多症はみられなかったが，LDH活性，LDHアイソザイム各分画およびチミジンキナーゼ活性の著しい高値が認められた．牛白血病ウイルス抗体は陰性であった．以上の所見より皮膚型牛白血病が疑われた．第４病日に行われた病理学的検索，および皮膚結節と腫大リンパ節の組織学的検索によりT細胞性皮膚型牛白血病と確定診断された．, A 41-month-old Holstein cow was presented with a chief complaint of astasia. It also had a history of nodular cutaneous lesions of 2 months' duration. Several alopecic spots and nodular cutaneous masses were observed on the head, neck, shoulder and back. Physical examination revealed enlargement of most of the surface lymph nodes. Masses were also palpated in the pelvic cavity by rectal palpation. Histopathological examination of punch biopsy samples from the skin lesions and fineneedle aspiration smear cytology of the iliac lymph node showed large, atypical round cells. Although lymphocytosis was not noted by hematology, activities of serum total lactate dehydrogenase（LDH）, LDH isozymes and thymidine kinase were elevated. Serum antibody against bovine leukemia virus was negative. The cutaneous form of bovine leukosis was strongly suspected from these findings. Necropsy and histopathological findings confirmed the diagnosis of cutaneous T-cell form bovine leukosis.

Toll-like receptors （TLRs） are highly expressed on cells of the innate immune system, such as macrophages and dendritic cells, and have been studied most extensively in them. TLRs-mediated signals have been found to enhance antigen presentation by upregulating co-stimulatory molecule expression and promoting the production of pro-inflammatory cytokines. Thus, TLRs are also involved in induction of the acquired immunity in which T cells plays a central role. It has been demonstrated that T cells themselves express TLRs and TLRs also control T cell responses directly by affecting clonal expansion of antigen-specific T cells and enhancing proliferation and survival of T cells and cytokine production by them. However, details on roles of TLRs in T cell responses still remain unknown. Therefore, this study was designed to investigate the roles of TLRs in the T-cell receptor （TCR）-mediated activation of T cells. First of all, the effects of the TLR2 ligand FSL-1 on the TCR-mediated activation of CD4+ T cells derived from C57BL/6 mice induced by anti-CD3ε antibody were investigated. It was found that FSL-1 exhibited synergic effects on the activation. In order to clarify the mechanism on the synergic effects, a crosstalk between the Src family kinase Lck, which plays important roles in the TCR-mediated activation, and TLR2 was investigated. Unexpectedly, overexpression of Lck in HEK293 cells expressing TLR2 （HEK293/TLR2 cells） significantly reduced the TLR2-mediated NF-κB activation induced by stimulation with the TLR2 ligand FSL-1. In addition, deletion mutants of SH3 and SH2 domains of Lck as well as the constitutively active mutant （LckY505F） attenuated the NF-κB activation more strongly than the wild-type Lck （Lckwt）, whereas deletion mutants of the kinase domain and the kinase-inactive mutants （LckK273R and LckY394F） significantly reversed the attenuation by the Lckwt. These results suggest that the kinase activity of Lck partially is involved in the attenuation of TLR2 signal by Lck. Furthermore, we found that TLR2 was associated with Lckwt through its kinase domain irrespective of the FSL-1 stimulation. Thus, this study is the first to report that the Src family kinase Lck suppresses the TLR2-mediated signal, although it was not able to explain the mechanism by which TLR2 signal synergistically enhances the TCR-mediated activation of T cells., TLRは自然免疫系で病原体の侵入を感知するだけでなく，T細胞が中心的な役割を果たす獲得免疫の誘導においても重要な役割を果たしている．これまで，T細胞自身もTLRを発現していることが明らかになっているが，T細胞の機能におけるTLRの役割はいまだ不明な点が多く残されている．本研究では，T細胞受容体を介したT細胞の活性化で重要な役割を果たしているSrcファミリーキナーゼのひとつであるLckに注目し，TLR2とLckのクロストークを検討した． C57BL/6マウスのCD4陽性T細胞を抗CD3ε抗体およびTLR2のリガンドであるリポペプチドFSL-1で刺激すると，抗CD3ε抗体またはFSL-1で単独刺激したものと比べ，共刺激したものではCD4陽性T細胞の増殖が相乗的に増加した．ヒト胎児腎細胞由来のHEK293細胞にTLR2遺伝子とLck野生型遺伝子をそれぞれ単独，あるいは同時に導入し，NF-κBの活性化を調べた．その結果，予想に反して，TLR2遺伝子を単独導入したものと比較し，TLR2遺伝子とLck野生型遺伝子を同時に導入したものでは，FSL-1刺激によるNF-κBの活性化が有意に抑制されることがわかった．さらに，Lckの各種欠失変異体（キナーゼ不活型点変異体K273R，Y394F，恒常的活性化型点変異体Y505F）を作製し，これらの遺伝子をHEK293細胞に導入し，FSL-1刺激によるNF-κBの活性化を調べたところ，キナーゼ活性がLckによるTLR2シグナルの抑制に部分的に関与するという結果を得た．また，TLR2とLckとの会合が免疫沈降法で確認された． 以上の結果から，TCRを介したT細胞の活性化に関与するLckがTLR2と会合し，下流のシグナルを負に制御することが示唆された．

Cryptosporidium parvumは生後間もない子牛に感染し下痢を引き起こす消化管内寄生原虫である．本研究ではC. parvumの感染制御において，あらかじめ大腸菌ワクチンを接種した母牛の初乳を介した新生子牛への免疫賦与が効果を持つか否かを検討した．まず，C. parvumが常在する農家の妊娠牛に，免疫を賦活する目的で大腸菌ワクチンを投与したところ，末梢血中のCD4+T細胞およびCD8+T細胞の増加がみられ，さらに分娩後の初乳ではCD4+T細胞，CD8+T細胞あるいはγδT細胞の割合が増加した．その初乳を給与された子牛では末梢血中のそれらのT細胞サブセットが増加する傾向がみられたが，これらの新生子牛の糞便からは対照群と同様にC. parvumオーシストが検出され，その排出数や下痢の程度，下痢の発現時期にもほとんど差異がみとめられなかった．牛におけるC. parvum感染防御においてCD4+T細胞およびCD8+T細胞がいずれも重要であるとされているが，今回の研究で大腸菌ワクチン投与によって誘導される各リンパ球サブセットをはじめとする種々の免疫賦活物質を初乳を介して新生子牛に賦与してもC. parvum感染防御効果は得られなかった．今後さらにC. parvum特異的免疫の移入を含むその他の免疫賦活法を検討していく必要がある．, Cryptosporidium parvum is an enteric protozoan that infects neonatal calves and causes severe diarrhea. We examined whether colostrum from E. coli vaccinated dams would provide better protection against C. parvum in neonatal calves. Pregnant Holstein cows kept at a C. parvumpositive farm were vaccinated against E. coli during the dry period. The number of CD4+ cells and CD8+ cells in the peripheral blood was higher in the vaccinated cows after parturition compared to those in non-vaccinated cows. The colostrum from these vaccinated cows contained higher percentages of CD4+, CD8+, and γδT cells compared to that from non-vaccinated cows. The calves fed the colostrum from vaccinated dams showed increased numbers of T cell subsets in the peripheral blood compared to calves fed the colostrum from non-vaccinated dams. However, the number of C. parvum oocyst detected in the feces, the severity of diarrhea and the timing of onset of diarrhea were similar for both groups of calves. Although CD4+ and CD8+ T cells are very important immune cells to protect calves from C. parvum infection, the colostrum from the E. coli vaccinated dams containing higher numbers of these T cells did not improve protection against C. parvum in calves. Further studies, including those focusing on C. parvum-specific immune protection, are needed to provide better immunity to calves.

It is known that nutritional state crucially affects immune function. In recent years, the contributionof the diet to the optimal working of the immune system has become wildly appreciatedand the influence of different dietary components on specific aspects of immune function has beenextensively studied. Soy isoflavone genistein suppresses antigen-specific immune response inBALB/c mice and suppresses development of atopic-like dermatitis in NC/Nga mice. In other soyisoflavone equol enhances antigen-specific IgE production via IL-13-mediated pathway. Further,equol exacerbates colitis of dextran sulfate sodium-treated mice. Therefore, immune stimulatoryeffects are strikingly different among soy isoflavones. In this reviews, in addition of the isoflavone,we overview the effects of dietary nucleic acid and protein-deficiency on antigen-specific immuneresponses. Furthermore, we present hot topics researches in the field of obesity and T cellfunction.

Article, 信州医学雑誌 57(4): 127-136(2009)

金沢大学医学部医学科

IgA nephropathy (IgAN) is characterized by the presence of IgA deposits, predominantly in the glomerular mesangium, and by mesangial proliferative glomerulonephritis (GN). Concerning its pathogenesis, several investigators have suggested that the deposited IgA is an antibody (Ab) to viral, bacterial or dietary antigens. Thus, the Ab is probably produced as part of the specific host immune response to various environmental antigens. Such reports strengthen the possibility of a relationship between mucosal immunity and the pathogenesis of IgAN. Nevertheless, attempts to isolate a specific IgA circulating immune complex associated antigen in patients with IgAN have been unsuccessful. We have shown that mucosal infection such as pharyngitis are often associated with the acute onset of IgAN. IgAN is, then, an immune complex disease that is caused by a poor mucosal immune response to environmental antigens to which the patient has been chronically exposed. We have observed previously that Haemophilus parainfluenzae (HP) is more commonly isolated from the pharynx of patients with IgAN than from those with other diseases.We have also identified the glomerular deposition of outer membranes of HP antigens (OMHP) and an increased serum concentration of IgA-Ab against OMHP in patients with IgAN. Furthermore, We have shown that patients with IgAN have a specific increase in the production of IgA-Ab against OMHP via polyclonal activation against these, with switching of production from one isotype to another, e.g. from IgM to IgA, and that a significant relationship between IgA-Ab against OMHP and renal lesions exists in patients with IgAN. On the basis of the above findings, we attempted to clarify the immune response between OMHP and lymphocytes in tonsils from patients with IgAN or other glomerular diseases (OGDs). We have shown that lymphocytes from patients with IgAN have a significantly higher stimulation index to OMHP (thymidine incorporation in tonsillar lymphocytes with OMHP/thymidine incorporation in unstimulated tonsillar lymphocytes) and a significantly higher level of IgA-Ab and IgA1-Ab against OMHP in culture supernatants than lymphocytes from OGDs. We have also found positive correlations between concentrations of IgA- and IgG- Abs, between IgA- and IgM-Abs, and between IgG- and IgM-Abs against OMHP after PMHP stimulation by measuring Abs against in supernatants from OMHP-stimulated lymphocytes. These results suggest that the immune response between OMHP and lymphocytes in tonsils plays a role in pathogenesis of IgAN.

The present study investigated the preventive effect of Saiboku-to, a Chinese herbal medicine, upon chemically induced carinogenesis in mice, and analyzed the mechanism of effect. Saiboku-to was orally administered to C3H/HeJ mice in a daily dose of 100mg/kg or 1000mg/kg in each of two groups. Six weeks after initiating Saiboku-to administration, mice were subcutaneously injected with a tumorigenic dose (1mg/mouse) of 3-methylcholanthrene (MC) and tumor development was observed for 25 weeks. In control mice, the incidence of tumor was 40% 10 weeks after MC injection. However, the tumor incidence in Saiboku-to treated mice was only a few percent 10 weeks after MC injection. A tumor incidence of 40% in Saiboku-to treated mice was reached about 18 weeks after MC injection, showing the delaying effect of Saiboku-to on carcinognesis. Therefore, a delaying effect on carcinogenesis was observed in Saiboku-to treated mice, although all mice had tumors within 23 weeks after MC injection. The mechanism of the delay in carcinogenesis was investigated by depletion of NK cells, T cells and macrophages in vivo, respectively. The delay of carcinogenesis disappeared after using anti-asialo-GM1 antibody, anti-thymocyte antibody, and carrageenan in Saiboku-to treated mice, showing that NK cells, T cells, and macrophages play an important role in delaying by Saiboku-to administration in experimental carcinogenesis. Moreover, prior treatment for 6 weeks with Saiboku-to significantly enhanced the NK cell activity of splenic cells and the cytotoxity and lysosomal enzyme activities (acid Phosphatase, beta-galactosidase) of peritoneal macrophages in normal mice. These augmentations were suppressed using anti-asialo GM1 antibody or carrageenan in vivo. Thus, Saiboku-to showed prophylactic effect on chemical carcinogenesis, suggesting activa-tion of the immune status of the host.

The suppressive effects of OK-432 and Ge-132 on 3-methylcholanthrene (MC) carcinogenesis have been confirmed. To clarify the mechamism of this suppression, we investigated the effects of anti-asialo GM1 antibody (AGM1), anti-thymocyte antibody (Thy) and carrageenan (CAR), which eliminate NK cells, T cells and macrophages, respectively, on the growth of MC induced sarcoma. The suppressive effect of Ge-132 in mice was abolished by prior AGM1, Thy or CAR treatment ; moreover, these compounds enhanced the development of tumors. In mice treated with OK-432, AGM1 or CAR enhanced the development of tumors, as well ; however, Thy treatment did not alter tumor incidence. Prior treatment with AGM1 reduced the percentage of large granular lymphocytes and NK activity, which were increased by OK-432 or Ge-132 treatment ; however, Thy or CAR did not alter these parameters. These results indicate that NK cells and macrophages play an important role in the defense against MC induced sarcoma in OK-432-treated mice, while T cells as well as NK cells and macrophages do so in Ge-132-treated mice.

We identified T cell and T cell subsets in cancer tissues obtained from 74 cases of uterine cervical cancer and regional lymph nodes by using immunohistochemistry. The following results were obtained. （1） There was a difference in the pattern of T cells between squamous cell carcinoma and adenocarcinoma, that is, T cells infiltrating cancer tissues densely touched the foci in squamous cell carcinoma while only sparsely in adenocarcinoma. In general, T cell infiltration in adenocarcinoma was less than that in squamous cell carcinoma. （2） CD8 positive cells were found among the cancer cells in the cancerous foci. （3） Although a large number of T cells were recognized in stage O to stage IIb, there were few in stage IIIb. There was a predominance of CD8, which existed much more commonly than CD4. （4） The CD4/8 ratio in lymph nodes with metastasis was significantly lower than in those without metastasis. （5） Cases with a large number of infiltrating T cells tended not to have lymph node metastasis. In particular, lymph nodes with CD8-predominance had significantly less lymph node metastasis compared to those with CD4-predominance. （6） A small number of IL-2R positive cells were identified in cancer tissues and lymph nodes, being related to the number of infiltrating T cells. （7） Patients with MHC class I had a large number of infiltrating T cells and CD8 predominance in most cases. （8） Five-year survival in groups with heavy and moderate infiltration was 100%, while that in groups with slight and extremely slight infiltration was 86.7% and 50.0%, respectively. The five- year survival rates for cases of CD8-dominance, equality and CD4-dominance were 100%, 83.3% and 71.4%, respectively. As mentioned above, we recognized in cancerous foci and lymph nodes that differences in histologic types and clinical stage caused an alteration of the pattern of the T cell and its subsets, thus confirming the existence of an immune response in cancer tissues. Finally, we determined the effect of T cell infiltration on prognosis and found that the number of T cells in cancer tissue was one of the factors that influenced the prognosis.

Histamine not only mediates immediate allergic reactions, it also regulates cellular immune responses. H4R is the most recently identified histamine receptor (HR). In the present study, we examined the in vitro effect of histamine and H4R agonists on the responses of human T cells to purified protein derivative from Mycobacterium tuberculosis (PPD) and to Cry j1, the major allergen of Cryptomeria japonica pollen. Dimaprit, clobenpropit and clozapine, which are H4R agonists, dose-dependently blocked both PPD-induced interferon-gamma and Cry j1-induced interleukin-5 production by both peripheral blood mononuclear cells (PBMCs) and antigen-specific T-cell lines. However, the addition of thioperamide, an H3R/H4R antagonist, as well as a mixture of d-chlropheniramine, famotidine and thioperamide, did not reverse the inhibition. Pretreatment of PBMCs with SQ22536 and 8-bromoadenosine-3',5'-cyclic monophosphorothioate, Rp-isomer, had varying abilities to reverse the inhibitory effects of H4R agonists, except for clobenpropit. Moreover, the addition of H4R agonists induced annexin-V expression on PBMCs, especially in CD19(+) and CD4(+) cells. cDNA microarray analysis revealed that, among 16,600 genes tested, increased expression following treatment with clozapine was seen in 0 x 8% of the genes, whereas decreased expression was seen in 3 x 0% of the genes. These results suggest that H4R agonists inhibit antigen-specific human T-cell responses, although H4R does not appear to be important for this effect. In addition, the present study indicated that there may be orphan receptors or HR subtypes which can bind dimaprit, clobenpropit and clozapine, and that can exert an inhibitory effect on antigen-specific cellular responses via a cAMP/cAMP-dependent protein kinase-dependent, apoptotic pathway.

近交系WHT/Htマウス可移植性扁平上皮癌の腫瘍抗原に対するin vivo免疫反応について検討を行った.同腫瘍細胞の可溶化抗原,放射線不活化腫瘍細胞及び腫瘍生細胞を抗原として同系マウスに摂取しその免疫原性をImmunoprotection testで検索した.可溶化抗原では腫瘍の増殖抑制は認められなかったが,放射線不活化腫瘍細胞と,腫瘍生細胞を移植し後に,腫瘍を切除した免疫マウスで腫瘍の増殖抑制が認められ,腫瘍抵抗性が得られた.腫瘍切除による免疫マウスでは平均30%のマウスが腫瘍を拒絶した.腫瘍切除によって得られた免疫マウスの脾細胞の抗腫瘍活性をWinn assayにて検討した.免疫マウスの脾細胞は腫瘍の増殖を抑制し抗腫瘍活性がみられた.この脾細胞をanti-Thy 1.1.+C, anti-Lyt 1.2.+Cおよびanti-Lyt 2.2.+Cで処理すると抗腫瘍活性は消失した.担癌マウスの脾細胞を用いてWinn assayを行った結果,担癌初期から後期にかけて腫瘍の増殖を促進する傾向がみられた.腫瘍抗原により正の免疫応答が誘導され,抗腫瘍免疫能を示す細胞はT cellで,そのsubsetはLyt-1+ 2+ 3+である.担癌マウスでは担癌初期から後期にかけて徐々に負の免疫応答が優位になる

Surface marker analysis of tumor cells was performed in 50 patients with non-Hodgkin's lymphoma. Twenty eight were B cell lymphoma and 22 were T cell lymphoma. In B cell lymphoma, lymphoma cells reacted with CD19, CD20, CD21, anti-HLA DR, and partly CD10 monoclonal antibodies, and had monoclonal surface immunoglobulin. In T cell lymphoma, cells reacted with CD2, CD3, CD5, CD4 or 8, and partly CD7 monoclonal antibodies. The proportion of T cell lymphoma in this analysis was thought to be higher than that in eastern Japan. Remission rate of therapy was no difference between T and B cell lymphoma. Concerning to the circulating lymphocytes, in CD4 positive T lymphoma, patients were divided to three groups based on CD4/CD8 ratio. In low CD4/CD8 ratio group, CD4 positive rate was depressed. While, in high CD4/CD8 ratio group, patients had high CD4 positive rate. Kappa-lambda analysis was the useful method to detect monoclonal B cell population. Kappa-lambda analysis on peripheral lymphocytes in B cell lymphoma revieled that D values of active CSIII/IV group were significantly higher than those of active CSI/II group and CR group. D values were elevated during active period, and lowered to normal range during remission. Therefore, D values in kappa-lambda analysis corresponded with clinical stage of the patients with B cell lymphoma.

A variety of monoclonal antibodies （MoAbs） were generated, directed to three distinct antigen species, designated L-1, L-2 and L-3, respectively, present on human T cells or on their subsets. Whereas L-1 was expressed on most human thymus and T cells, and was identified to be a human homologue of mouse Lyt-1 antigen with a molecular weight （MW） of 70 killodaltons （KD）, L-2 was expressed on a T cell subset which contained 30-40% of peripheral T cells, and was thought to correspond to a human homologue of mouse Lyt-2, 3 antigens consisting of 32 KD MW components. L-3 was expressed on a T cell subset negative for L-2, and was regarded as a helper/inducer T cell antigen, previously reported for Leu-3 or OKT4. Precise serological analysis using binding-inhibition assays with fluorescein- or radio-labeled MoAbs demonstrated that these MoAbs developed in this study defined a number of different antigenic determinants present on L-1, L-2 and L-3 antigen molecules, respectively. Thus, two MoAbs （5B3 and 7C6） detected two distinct determinants existing on the same L-1 molecule, one of which seemed to be identical to that defined by Leu-1 MoAb. At least four distinct determinants were found on L-2 antigen molecules with 7 MoAbs directed to this molecular species, and one out of 4 determinants appeared identical to that detected by OKT8 MoAb. Among these 7 MoAbs, two （3D6 and 4A2） failed to react with L-2 antigen present on a number of cultured leukemic T cell lines, though these two MoAb clearly reacted with the L-2 positive thymus and T cells in a lesser number than that recognized by the other 5 L-2 MoAbs. We also produced 3 MoAbs against the L-3 antigen molecule, all of which recognized a single determinant identical or closely linked to that determined by OKT4A MoAb. The possible functional role of T cell antigens or their antigen determinants defined by MoAbs as described above was then studied by measuring the effects of these MoAbs on a variety of human T cell responses including proliferative reaction and cytotoxic T cell （CTL） induction in autologous mixed lymphocyte cultures （AMLC） stimulated with autologous Epstein-Barr virus-transformed B （EBV-B） cells as well as on T cell-mediated cytotoxicity. Whereas the L-1 MoAb tested had little effect on human T cell functions as described above, L-2 and L-3 MoAbs exerted their inhibitory effects on T cells either proliferating in AMLC or cytotoxic to autologous EBV-B cells. Thus, L-2 MoAb, despite their different reactivities with antigen determinants on the L-2 molecule, L-2 MoAb had significant inhibitory effects on cytotoxic reactions mediated by L-2 positive CTL and probably on CTL induction. L-3 MoAb, on the other hand, significantly suppressed the proliferation of T cells ?in AMLC as well as cytotoxicity mediated by L-3 positive CTL. These data suggest that L-2 and L-3 antigen molecules have important roles in antigen recognition and manifestation of the effector function mediated by human T cells.

Background: Head and neck squamous cell carcinoma (HNSCC) is a major cause of cancer-related morbidity and mortality worldwide. Epidermal growth factor receptor (EGFR)-targeted therapy is an attractive strategy alternative to conventional cancer treatments for HNSCC, but its efficacy remains controversial. T-cell-based immunotherapy has been proposed as a novel therapeutic approach to improve the clinical outcome for HNSCC. In this study, we report human epidermal receptor (HER) family epitopes that induced CD4 T-cell responses to HNSCC. The results provide support for a novel strategy to treat HNSCC by combining EGFR-targeted therapy with T-cell-based immunotherapy. Methods: We evaluated the capacity of predicted CD4 T-cell peptide epitopes from EGFR to induce antitumour immune responses in vitro. In addition, EGFR inhibitors were evaluated for their ability to augment tumour MHC class II expression in HNSCC cell lines and subsequently increase T-cell recognition. Results: Among several predicted peptide epitopes, EGFR875–889 elicited CD4 T-cell responses that were restricted by HLA-DR4, DR15, or DR53 molecules, indicating that the peptide functions as a promiscuous T-cell epitope. The peptide-reactive T cells responded to autologous dendritic cells loaded with EGFR-expressing tumour cell lysates, indicating that these epitopes are naturally processed. In addition, the CD4 T cells were capable of directly recognising and killing HNSCC cells expressing EGFR and the appropriate HLA class II molecule. T cells reactive with the EGFR875–889 epitope could be detected in the blood of HNSCC patients. EGFR875–889-reactive CD4 T cells were also able to recognise several peptide analogues derived from homologous regions of EGFR family members, HER-2, HER-3 and c-MET. Finally, we examined the effects of EGFR tyrosine kinase inhibition or EGFR-blocking antibodies on CD4 T-cell tumour reactivity. Treatment of tumour cells with the EGFR inhibitors enhanced tumour recognition by EGFR875–889-reactive T cells presumably due to the upregulation of HLA-DR expression in the HNSCC cells. Conclusion: We identified novel CD4 T-cell EGFR epitopes and amongst these, EGFR875–889 functions as a promiscuous helper T-cell epitope that can elicit effective antitumour T-cell responses against tumours expressing HER family members and c-MET. These observations should facilitate the translation of T-cell-based immunotherapy into the clinic for the treatment of HNSCC and provide a rational basis for EGFR inhibition, immune-targeted combination therapy.