Chaperon and adjuvant activity of hsp70: different natural killer requirement for cross-priming of chaperoned and bystander antigens

Heat shock proteins (HSP) convey both chaperoned propeptide and danger signal to dendritic cells (DC). However, few studies have compared the two activities. Using a murine inducible hsp70 secreted by cells distinct from those providing the tumor antigens, we showed that hsp70 exerts efficacious adjuvant effects toward DC cross-priming. Hsp70 induces DC maturation and phagocytosis of cellular debris both in vitro and in vivo, which are conducive to CTL response to chaperoned and nonchaperoned antigens. Whereas the ability of hsp70 to induce cross-presentation of chaperoned peptides is natural killer (NK) independent, the adjuvant activity requires NK cells at the site of DC-hsp70 interaction to induce CTL response and therapeutic effect against lung metastases. However, although bystander activity provides equal CTL induction, the best therapeutic efficacy rests on cell vaccine secreting hsp70 that combines chaperoned antigen and danger signal within the same cell.     

Vaccination for malignant melanoma: recent developments

The identification of tumor-associated antigens recognized by cellular or humoral effectors of the immune system has opened new perspectives for cancer immunotherapy. Different categories of cancer-associated antigens have been described as targets for CD8+ T cells in vitro and in vivo: (1) 'cancer-testis' (CT) antigens expressed in different tumors and normal testis; (2) melanocyte differentiation antigens; (3) point mutations of normal genes; (4) antigens that are overexpressed in malignant tissues, and (5) viral antigens. Clinical trials with antigenic peptides have been initiated to induce specific immunological responses in vivo. Immunological and clinical parameters for the assessment of peptide-specific reactions have been defined: DTH, CD8+ T cell, autoimmune and tumor regression responses. Preliminary results show that tumor-associated peptides alone elicit specific DTH and CD8+ T cell responses associated with tumor regression after intradermal vaccination. Granulocyte macrophage colony-stimulating factor has been shown to enhance peptide-specific immune reactions by amplification of dermal antigen-presenting dendritic cells. Complete tumor regressions have been observed after the induction of CD8+ T cell responses by peptide immunization. Based on these results, active immunotherapy with tumor-associated antigens may be a promising approach for patients in adjuvant treatment situations, who are at high risk for tumor recurrence. Recently, a strategy utilizing spontaneous antibody responses to tumor-associated antigens (SEREX) has led to the identification of a new CT antigen, NY-ESO-1. NY-ESO-1-specific spontaneous humoral and cellular immune responses were found in approximately 50% of patients with NY-ESO-1-positive tumors. Clinical studies have been initiated to evaluate the immunological effects of immunization with NY-ESO-1 peptides in cancer patients with detectable or absent immunity against NY-ESO-1.     

Therapeutic cancer vaccines

Therapeutic cancer vaccines target the cellular arm of the immune system to initiate a cytotoxic T-lymphocyte response against tumor-associated antigens. Immunotherapy offers one of the few therapeutic options that reproducibly leads to a subset of patients with long-term remissions (seemingly cures) of widely metastatic disease. Therapeutic cancer vaccines tested in clinical trials have included inactivated tumor cells administered in immunological adjuvants or after genetic modification to increase their immunogenicity. Other forms are heat shock protein vaccines and anti-ganglioside antibodies. Tumor-associated antigenic peptides have been fully characterized for some cancers. Finally, strategies to directly expand antitumor T lymphocytes and adoptively transfer them to patients with cancer have been developed and shown to induce objective tumor regressions.     

Epitope selection and development of peptide based vaccines to treat cancer

Cytotoxic T lymphocytes recognize peptides that associate with class I major histocompatibility complex molecules. Since cytotoxic T cells have the capacity to recognize and destroy tumor cells, identification of epitopes recognized by these cells in tumor-associated antigens would allow the production of compounds for the treatment of cancer. Here we review some of the approaches being explored to identify tumor-associated antigens and to develop peptide-based vaccines that induce cytotoxic T lymphocytes against specific tumors.     

Turning tumor cells in situ into T-helper cell-stimulating, MHC class II tumor epitope-presenters: immuno-curing and immuno-consolidation

Immunological control or cure of tumors depends on initiating a robust T helper cell response to MHC class II epitopes of tumor-associated antigens. T helper cells regulate the potency of cytotoxic T lymphocyte and antibody responses. We have developed a novel approach to stimulate T helper cells by converting tumor cells into MHC class II molecule-positive, antigen presenting cells. Furthermore, using antisense methods, we suppress expression of the Ii protein, that normally blocks the antigenic peptide binding site of MHC class II molecules during synthesis in the endoplasmic reticulum. In such gene-engineered tumor cells, the MHC class II molecules pick up antigenic peptides, which have been transported into the endoplasmic reticulum for binding to MHC class I molecules. All nucleated cells create such "surveys of self" to detect viral or malignant transformation. Our method extends that survey of self to MHC class II endogenous tumor-associated antigens. Simultaneous presentation of tumor antigens by both MHC class I and II generates a robust and long-lasting antitumor immune response. Injecting murine tumors with genes, which induce MHC class II molecules and suppress Ii protein, cures a significant number of animals with renal and prostate tumors. We have developed analogous human gene vectors that are suitable for most patients and cancers, because they are monomorphic and active in all HLA-DR alleles. We review our findings, and analyze remaining issues for preclinical study and the design of clinical trials.     

Role of nitric oxide in heat shock protein induced apoptosis of gammadeltaT cells

Activation induced cell death (AICD) has been proposed to serve as a mechanism to limit T lymphocyte proliferation induced by antigenic stimulation. Heat shock proteins (hsp60 and hsp70) expressed on oral tumor cells serve as ligands for peripheral blood gammadeltaT lymphocytes. Tumor cell lysis by gammadeltaT lymphocytes is mediated via recognition of hsp expressed on tumor cells. In the present study, we report that upon stimulation with hsp, gammadeltaT lymphocytes isolated from oral cancer patients undergo AICD as confirmed by DNA ploidy, annexin V staining and confocal microscopy. In cocultures of gammadeltaT lymphocytes and tumor cells, addition of antihsp60 and antihsp70 MAb, but not anti-Fas MAb (ZB4), inhibited DNA fragmentation of gammadeltaT lymphocytes. Flow cytometric analysis revealed a down regulation of Fas expression on gammadeltaT lymphocytes upon incubation with hsp60 and hsp70. Increased expression of iNOS was observed in hsp-stimulated gammadeltaT lymphocytes. Addition of monomethyl L-arginine monoacetate, competitive inhibitor of NOS, inhibited nitric oxide (NO) production and apoptosis of gammadeltaT lymphocytes induced by hsp60 and hsp70. The NO-induced apoptosis of gammadeltaT lymphocytes involves activation of caspase-9 and loss of mitochondrial membrane potential. The present study explains a novel strategy adopted by tumor cells to evade immune recognition by gammadeltaT lymphocytes.     

肝癌热休克蛋白—抗原肽复合物的构建及诱导CTL反应的初步研究

目的：研究体外构建热休克蛋白-抗原肽复合的方法,观察其在体外的抗肿瘤作用。方法：用经43℃热处理1小时的人肝细胞悬液,经过裂解液裂解,60%-80%饱和硫酸铵沉淀,用SephadexG-100柱制备,取分子量为70KD组份,Westen blot进行性质鉴定。应用多肽解离液处理该组份,SephadexG-25柱过滤获得未结合多肽的蛋白分子,使其在体外与肝癌抗原肽SLIVHLNEV结合,构建成热休克蛋白-抗原肽复合物。应用此复合物树突状细胞,激活同源外周血T淋巴细胞产生肿瘤特异性杀伤T淋巴细胞（CTL）,应用MTT法检测其对T2细胞及肿瘤细胞的杀伤活性。结果：所得蛋白经电泳及Western blot进行蛋白分子量及性质鉴定为热休克蛋白70。SephadexG-25柱双分离法证实应用上述方法成功构建了肝癌热休克蛋白70-抗原肽复合物,用该复合物负荷树突状细胞在体外可以诱导出较强的肝癌抗原肽物特异性CTL,可以杀伤负荷有该肽的T2细胞及递呈该肽的肿瘤细胞系。结论：体外构建的热休克蛋白-抗原肽复合物可以增强抗原肽诱导CTL反应能力,热休克蛋白是良好的T细胞免疫佐剂,有可能在肿瘤疫苗治疗中发挥重要作用。     

Equivalent induction of telomerase-specific cytotoxic T lymphocytes from tumor-bearing patients and healthy individuals.

Although high frequencies of T lymphocytes specific for certain tumor-associated antigens have been detected in some cancer patients, increasing evidence suggests that these T cells may be functionally defective in vivo and fail to induce meaningful clinical responses. One strategy to overcome this limitation is to target novel antigens that are ignored during the natural antitumor immune response but are nevertheless capable of triggering effector T-cell responses against tumors after optimal presentation by antigen-presenting cells. Here, we show that the telomerase catalytic subunit (hTERT)-a nearly universal tumor antigen identified by epitope deduction rather than from patient immune responses-is immunologically ignored by patients despite progressive tumor burden. Nevertheless, HLA-A2-restricted CTLs against hTERT are equivalently induced ex vivo from patients and healthy individuals and efficiently kill human tumor cell lines and primary tumors. Thus, telomerase-specific T cells from cancer patients are spared functional inactivation because of immunological ignorance. These findings support clinical efforts to target the hTERT as a tumor antigen with broad therapeutic potential.     

Hyperthermia classic article commentary: ‘Re-induction of hsp70 synthesis: An assay for thermotolerance’ by Gloria C. Li and Johnson Y. Mak,International Journal of Hyperthermia1989;5:389-403

Of the many heat shock proteins (HSPs), hsp70 appears to correlate best with heat resistance, either permanent or transient. We have investigated various approaches to quantify the concentration of hsp70, and examined the relationship between hsp70 and cells’ thermal sensitivity during the development and decay of thermotolerance in model systems. Specifically, experiments were performed to determine the possibility of using the rate of synthesis of hsp70 after a second test heat shock to predict the kinetics of thermotolerance in tumor cells in vitro and in animal tumor models. We found that the cells’ ability to re-initiate hsp70 synthesis in response to the test heat shock inversely correlated with retained thermotolerance. These data suggest the level of hsp70 in thermotolerant cells regulates the rate of synthesis of additional hsp70 in response to the subsequent heat challenge. Furthermore, the results showed that the rate of re-induction of hsp70 synthesis after a test heat shock can be used as a rapid measure of retained thermotolerance. This study suggests an approach for quantifying the level of retained thermotolerance during fractionated hyperthermia.     

Cancer immunotherapy in clinical oncology

The identification of tumor-associated antigens recognized by cellular or humoral effectors of the immune system has opened new perspectives for cancer therapy. Different groups of cancer-associated antigens have been described as targets for cytotoxic T lymphocytes (CTLs) in vitro and in vivo: 1) cancer-testis (CT) antigens, which are expressed in different tumors and normal testis; 2) melanocyte differentiation antigens; 3) point mutations of normal genes; 4) antigens that are overexpressed in malignant tissues; and 5) viral antigens. Clinical studies with peptides derived from these antigens have been initiated to induce specific CTL responses in vivo. Immunological and clinical parameters for the assessment of peptide-specific reactions have been defined, i.e., delayed-type hypersensitivity (DTH), CTL, autoimmmune, and tumor regression responses. Preliminary results demonstrate that tumor-associated peptides alone elicit specific DTH and CTL responses leading to tumor regression after intradermal injection. Granulocyte-macrophage colony-stimulating factor (GM-CSF) was proven effective in enhancing peptide-specific immune reactions by amplification of dermal peptide-presenting dendritic cells. Long-lasting complete tumor regressions have been observed after induction of peptide-specific CTLs. However, in single cases with disease progression after an initial tumor response, either a loss of the respective tumor antigen targeted by CTLs or of the presenting major histocompatibility complex (MHC) class I allele was detected as a mechanism of immune escape under immunization. Based on these observations, cytokines to enhance antigen and MHC class I expression in vivo are being evaluated to prevent immunoselection. Recently, a strategy utilizing spontaneous antibody responses to tumor-associated antigens (SEREX) has led to the identification of a new CT antigen, NY-ESO-1, which is regarded as one of the most immunogenic antigens known today inducing spontaneous immune responses in 50% of patients with NY-ESO-1-expressing cancers. Clinical studies involving antigenic constructs that induce both antibody and CTL responses will show whether these are more effective for immunotherapy of cancer.     

Multiple antigenic peptides of human heparanase elicit a much more potent immune response against tumors

Peptide vaccination for cancer immunotherapy requires an ideal immune response induced by epitope peptides derived from tumor-associated antigens (TAA). Heparanase is broadly expressed in various advanced tumors. Accumulating evidence suggests that heparanase can serve as a universal TAA for tumor immunotherapy. However, due to the low immunogenicity of peptide vaccines, an ideal immune response against tumors usually cannot be elicited in patients. To increase the immunogenicity of peptide vaccines, we designed three 4-branched multiple antigenic peptides (MAP) on the basis of the human leukocyte antigen (HLA)-A2-restricted cytotoxic T lymphocyte (CTL) epitopes of human heparanase that we identified previously as antigen carriers. Our results show that MAP vaccines based on the HLA-A2-restricted CLT epitopes of human heparanase were capable of inducing HLA-A2-restricted and heparanase-specific CTL in vitro and in mice. Moreover, compared with their corresponding linear peptides, heparanase MAP vaccines elicited much stronger lysis of tumor cells by activating CD8(+) T lymphocytes and increasing the releasing of IFN-γ. However, these heparanase-specific CTLs did not lyse heparanase-expressing autologous lymphocytes and dendritic cells, which confirm the safety of these MAP vaccines. Therefore, our findings indicate that MAP vaccines based on CTL epitopes of human heparanase can be used as potent immunogens for tumor immunotherapy because of advantages such as broad spectrum, high effectiveness, high specificity, and safety.     

树突状细胞融合瘤苗在肿瘤免疫中的研究进展

树突状细胞(DC)是诱导初始免疫应答反应的专职抗原提呈细胞,它能捕获抗原并加工处理成小分子多肽,通过MHC Ⅰ类和Ⅱ类分子提呈给T细胞和B细胞.Dc与肿瘤细胞融合所获得的融合瘤细胞,在共刺激信号存在的条件下,能够加工处理细胞内许多已知和未知的肿瘤相关抗原(TAA),并通过MHC Ⅰ类和Ⅱ类分子提呈.目前,DC融合瘤苗在动物和临床研究中取得了许多进展,已成为肿瘤免疫治疗的热点之一.     

Re-induction of hsp70 synthesis: an assay for thermotolerance

Of the many heat shock proteins (HSPs), hsp70 appears to correlate best with heat resistance, either permanent or transient. We have investigated various approaches to quantify the concentration of hsp 70, and examined the relationship between hsp70 and cells' thermal sensitivity during the development and decay of thermotolerance in model systems. Here, experiments were performed to determine the possibility of using the rate of synthesis of hsp70 after a second test heat shock to predict the kinetics of thermotolerance. Specifically, we studied the relationship between the retained thermotolerance in a murine tumor cell line SQ-1 and a human tumor cell line, HCT-8, after fractionated heat doses and the cells' ability to re-initiate synthesis of hsp70 in response to an additional test heat dose in vitro. Monolayers of cells were exposed to a first heat treatment (e.g., 41 degrees C, 4 h) and then incubated at 37 degrees C for 0-72 h. At various times after the first heat treatment, cells were either challenged with a 45 degrees C, 45 min heat shock to assess the residual thermotolerance by colony formation, or labelled with [35S]methionine before or after an additional test heat dose (e.g. 43.5 degrees C, 15 min). We found that the cells' ability to re-initiate hsp70 synthesis in response to the test heat shock inversely correlated with retained thermotolerance. Our data suggest the level of hsp70 in thermotolerant cells regulates the rate of synthesis of additional hsp70 in response to the subsequent heat challenge. Furthermore, the results showed that the rate of re-induction of hsp70 synthesis after a test shock can be used as a rapid measure of retained thermotolerance. This study suggests an approach for quantifying the level of retained thermotolerance during a course of fractionated hyperthermia.     

Antitumor immune responses mediated by adenoviral GDEPT using nitroreductase/CB1954 is enhanced by high-level coexpression of heat shock protein 70

Gene-directed enzyme prodrug therapy (GDEPT) is a promising approach to local management of cancer through targeted chemotherapy. Killing localized tumors by GDEPT in a manner that induces strong antitumor cellular immune responses might improve local management and allow benefit in disseminated cancer. Here we evaluated the combination of nitroreductase (NTR)/CB1954 GDEPT with high-level expression of heat shock protein 70 (HSP70, a stress protein that can shuttle cytosolic peptides into antigen-presenting cells) for induction of antitumor immunity using adenovirus gene delivery in an aggressive and nonimmunogenic BALB/c syngeneic 4T1 breast cancer model. The mechanism of cell death and spectrum of stress proteins induced are likely to be important determinants of the resulting immune responses. We showed that NTR/CB1954 treatment of 4T1 cells gave both apoptotic and nonapoptotic killing. In vivo killing of 4T1 cells expressing NTR gave weak antitumor immunity and very limited induction of stress proteins including HSP70. High-level coexpression of HSP70 during NTR/CB1954-mediated killing of 4T1 cells in vivo gave much greater protection from tumor challenge (67% long-term survivors compared to 17%) and induced 4T1-specific cytotoxic T-cell responses. The enhancement of antitumor responses resulting from HSP70 coexpression was similar to that conferred by coexpression of GM-CSF.     

Influence of drug-induced apoptotic death on processing and presentation of tumor antigens by dendritic cells

Here we have studied the effects of apoptotic cell death induced by chemotherapic agents on tumor phagocytosis by dendritic cells (DC) and presentation of the relevant antigen to T lymphocytes. Annexin-V-FITC (Ann-V) and propidium iodide (PI) staining was used to assess early apoptotic (Ann-V(+)/PI(-)) vs. late apoptotic/secondary necrotic (Ann-V(+)/PI(+)) death after a 24 hr observation of untreated and drug-treated gastric carcinoma cells. After treatments, the HLA-A*0201(+) tumor cell line KATO III was exposed for 24 hr to allogeneic, HLA-related GM-CSF, IL-4-driven immature (i) DC. Tumor-loaded iDC were tested for IL-12 release in an ELISA assay, incubated with the DC-maturating factor TNF-alpha and used as stimulators for autologous T lymphocytes. Generation of antitumor T response against KATO cells was evaluated in an anti-MHC class I MAb-blocked Interferon-gamma ELISPOT assay. After treatment with Cis-platin (cis), all dying cells were in early apoptosis, whereas secondary necrosis was the prevalent death pattern observed after epirubicin (epi) and doxorubicin (doxo). Doxo and epi increased tumor expression of heat shock protein (hsp) 70 and uptake of tumor cell components by DC, whereas cis treatment had no effect on hsp70 and was associated with poor tumor uptake by DC. Significant upmodulation of IL-12 was observed by DC that had taken up the doxo- and epi-treated tumors (p< 0.005 and p< 0.01, respectively). Increased IFN-gamma release was also observed after stimulation of T lymphocytes with DC loaded with doxo- and epi-treated (p< 0.02 and p< 0.005, respectively) but not with cis-treated DC. These data show that the products of early apoptosis cannot efficiently cross-activate MHC class I-restricted anti-tumor lymphocytes even in the presence of DC maturating factors, whereas secondary necrosis is associated with robust T cell response.     

Heat shock protein induced TCR gammadelta gene rearrangements in patients with oral cancer

We analyzed the T cell receptor (TCR) gammadelta gene repertoire in peripheral blood and tumor compartment of oral cancer (OC) patients before and after stimulation with heat shock proteins (hsp), which are known ligands for gammadelta T cells. Clonal TCR gamma and delta gene rearrangements in lymphocytes from tumor compartment and peripheral blood were studied using TCR Vgamma and Vdelta gene primers in PCR followed by heteroduplex analysis. Vgamma gene segments derived from VgammaI or VgammaII gene families were most dominantly expressed in peripheral blood lymphocytes (PBL) as compared to tumor infiltrating lymphocytes (TIL) of OC patients. Of the rearranged TCR delta alleles Vdelta1-Jdelta1 and Vdelta2-Jdelta1 gene rearrangements were the most predominant in PBL and TIL of OC patients respectively. Stimulation of gammadelta T cells with hsp 60/70 demonstrated a selective clonal expansion of Vgamma9-Vdelta2 (VgammaII family) subset indicating that, this expanded population of cells could be responsible for eliciting an immune response against oral tumor cells.     

Inducible Hsp70 as target of anticancer immunotherapy: Identification of HLA-A*0201-restricted epitopes

The design of a broad application tumor vaccine requires the identification of tumor antigens expressed in a majority of tumors of various origins. We questioned whether the major stress-inducible heat shock protein Hsp70 (also known as Hsp72), a protein frequently overexpressed in human tumors of various histological origins, but not in most physiological normal tissues, constitutes a tumor antigen. We selected the p391 and p393 peptides from the sequence of the human inducible Hsp70 that had a high affinity for HLA-A*0201. These peptides were able to trigger a CTL response in vivo in HLA-A*0201-transgenic HHD mice and in vitro in HLA-A*0201+ healthy donors. p391- and p393-specific human and murine CTL recognized human tumor cells overexpressing Hsp70 in a HLA-A*0201-restricted manner. Tetramer analysis of TILs showed that these Hsp70 epitopes are targets of an immune response in many HLA-A*0201+ breast cancer patients. Hsp70 is a tumor antigen and the Hsp70-derived peptides p391 and p393 could be used to raise a cytotoxic response against tumors of various origins.     

Re-induction of hsp70 synthesis: An assay for thermotolerance

Of the many heat shock proteins (HSPs), hsp70 appears to correlate best with heat resistance, either permanent or transient. We have investigated various approaches to quantify the concentration of hsp70, and examined the relationship between hsp70 and cells' thermal sensitivity during the development and decay of thermotolerance in model systems. Here, experiments were performed to determine the possibility of using the rate of synthesis of hsp70 after a second test heat shock to predict the kinetics of thermotolerance. Specifically, we studied the relationship between the retained thermotolerance in a murine tumor cell line SQ-1 and a human tumor cell line, HCT-8, after fractionated heat doses and the cells' ability to re-initiate synthesis of hsp70 in response to an additional test heat dose in vitro. Monolayers of cells were exposed to a first heat treatment (e.g., 41°C, 4 h) and then incubated at 37°C for 0–72 h. At various times after the first heat treatment, cells were either challenged with a 45°C, 45 min heat shock to assess the residual thermotolerance by colony formation, or labelled with [³⁵S]methionine before or after an additional test heat dose (e.g. 43.5°C, 15 min). We found that the cells' ability to re-initiate hsp70 synthesis in response to the test heat shock inversely correlated with retained thermotolerance. Our data suggest the level of hsp70 in thermotolerant cells regulates the rate of synthesis of additional hsp70 in response to the subsequent heat challenge. Furthermore, the results showed that the rate of re-induction of hsp70 synthesis after a test shock can be used as a rapid measure of retained thermotolerance. This study suggests an approach for quantifying the level of retained thermotolerance during a course of fractionated hyperthermia.     

Re-induction of hsp70 synthesis: An assay for thermotolerance

Of the many heat shock proteins (HSPs), hsp70 appears to correlate best with heat resistance, either permanent or transient. We have investigated various approaches to quantify the concentration of hsp70, and examined the relationship between hsp70 and cells' thermal sensitivity during the development and decay of thermotolerance in model systems. Here, experiments were performed to determine the possibility of using the rate of synthesis of hsp70 after a second test heat shock to predict the kinetics of thermotolerance. Specifically, we studied the relationship between the retained thermotolerance in a murine tumor cell line SQ-1 and a human tumor cell line, HCT-8, after fractionated heat doses and the cells' ability to re-initiate synthesis of hsp70 in response to an additional test heat dose in vitro. Monolayers of cells were exposed to a first heat treatment (e.g., 41 °C, 4 h) and then incubated at 37°C for 0–72 h. At various times after the first heat treatment, cells were either challenged with a 45 °C, 45 min heat shock to assess the residual thermotolerance by colony formation, or labelled with [³⁵S]methionine before or after an additional test heat dose (e.g. 43.5°C, 15 min). We found that the cells' ability to re-initiate hsp70 synthesis in response to the test heat shock inversely correlated with retained thermotolerance. Our data suggest the level of hsp70 in thermotolerant cells regulates the rate of synthesis of additional hsp70 in response to the subsequent heat challenge. Furthermore, the results showed that the rate of re-induction of hsp70 synthesis after a test shock can be used as a rapid measure of retained thermotolerance. This study suggests an approach for quantifying the level of retained thermotolerance during a course of fractionated hyperthermia.     

Distinct pattern of HSP72 and monomeric laminin receptor expression in human lung cancers infiltrated by γ/δ T lymphocytes

We have previously demonstrated that γ/δ T lymphocytes may participate in the host immune response against lung adenocarcinomas. Here we show that, in about one-fourth of human lung cancers, γ/δ T cells represented a significant proportion of freshly isolated tumor-infiltrating lymphocytes. Moreover, these cells selectively expand in vitro upon culture in the presence of IL-2, thus suggesting a prior activation in vivo. Finally, when we evaluated the expression of heat shock proteins and of a panel of tumor-associated antigens in lung cancers infiltrated by γ.δ vs. α/β T cells, we found that the former displayed a distinct antigenic pattern, characterized by over-expression of HSP72 and of the 67-kDa high-affinity laminin receptor, which might account for γ/δ T-cell recognition. © 1994 Wiley-Liss, Inc.