Lymphokine-activated killer (LAK) cell activity in tumor-infiltrating lymphocytes from non-small cell lung cancer

Tumor-infiltrating lymphocytes (TILs) are often seen in non-small cell lung cancers (NSCCs). Their functional role in the pathogenesis of lung cancer is unknown. The authors studied TILs in 27 patients with NSCC and determined the following: (1) the immunologic phenotype as defined by monoclonal antibodies against various surface markers, (2) activation state as indicated by interleukin-2 (IL-2) receptor expression and the kinetics of proliferation response to IL-2, and (3) the ability to develop lymphokine-activated killer (LAK) type cytotoxicity against both natural killer (NK)-resistant tumor cell targets (M14) and fresh autologous tumor cells. The authors' results show TILs from NSCCs to be a heterogeneous population composed of T-cells, B-cells, monocytes, and NK cells in frequencies similar to those found in peripheral blood lymphocytes (PBLs). TILs demonstrated increased IL-2 receptor expression and a more rapid proliferative response to IL-2 than PBLs, implying activation of TILs by the tumor milieu. Finally, TILs generated cytotoxicity against NK-sensitive (K562) and NK-resistant (M14) cell line targets consistently after in vitro treatment with IL-2 but were less consistent in their ability to lyse fresh autologous tumor cells and less effective than PBL LAK cells in lysing all targets. Comparison with LAK cells generated from normal volunteers suggests that decreased killing of autologous tumor cells only partially results from an inherent resistance to lysis by fresh NSCC targets. It appears, therefore, that tumor cells taken from NSCCs are not readily killed by the immune cells that infiltrate the tumor stroma and that this failure does not result from nonspecific immune deficiency in TILs.     

Augmentation of cytotoxicity of tumor-infiltrating lymphocytes by biological response modifiers.

Lymphocyte infiltration into a tumor has been regarded as an expression of host immunity against cancer, but tumor-infiltrating lymphocytes (TIL) have little or no cytotoxicity. This study examined two different approaches to augment this low cytotoxicity. Firstly, biological response modifiers (OK-432, PSK) were injected into gastric cancer intralesionally. Intralesional injection of OK-432 or PSK significantly augmented the cytotoxicity of TIL. By the injection of OK-432, the ratio of OKT8-, Leu7-positive cells were increased in the TIL subset. In the second approach, TIL of gastric or pulmonary cancer patients were cultured with interleukin-2 (IL-2) in vitro. Co-culturing with IL-2 augmented the low cytotoxicity of TIL, and broad-reactive lymphokine-activated killer (LAK) cells were generated from TIL.     

Suppression of lymphokine-activated killer cell generation by tumor-infiltrating lymphocytes

A functional analysis of tumor-infiltrating lymphocytes (TILs) from renal cell carcinoma (RCC) and malignant melanoma was performed. TILs were expanded in recombinant interleukin-2 (50 U/ml) in Iscoves medium. Phenotypic and functional (cytolytic vs regulatory) analyses were carried out with the fresh and expanded TIL populations after 4 weeks in culture. Only one TIL population from an RCC case (out of six cases studied) was CD8+ and demonstrated MHC class I-restricted tumor-specific cytotoxicity against the autologous RCC target. TIL populations from the other five cases became predominantly CD4+ and they neither killed the respective autologous tumor cells nor killed the NK-sensitive target K-562 cells. When studied for other functions, two CD4+ TIL populations were found to suppress the lymphokine-activated killer cell response by peripheral blood lymphocytes (PBL) in coculture. Of these two, a TIL population from an RCC case (MJ TIL) was used to study the cellular and molecular mechanisms of suppression. The MJ TIL synthesized a supernatant factor that blocked activation of resting PBL as measured by the induction of high-affinity IL-2 receptor (IL-2R) when stimulated by phytohemagglutinin but did not down-regulate the fully expressed IL-2R on activated T cells. The suppression of high-affinity IL-2R induction on T cells did not result from tumor necrosis factor-alpha and beta or from transforming growth factor-beta as these cytokines were not detected in the cell-free supernatant from the MJ TIL culture. The supernatant factor also suppressed IL-2-mediated enhancement of cytotoxicity by natural killer (NK) cells without demonstrating direct toxic effect on the NK cells. Thus, when TIL are used for adoptive immunocytotherapy, it may be useful to fully characterize them functionally, in vitro.     

肿瘤浸润性淋巴细胞的分离、培养及其表型分析

本文运用不连续密度离心的方法从鼠B16黑色素瘤体中分离到肿瘤浸润性淋巴细胞(TIL),并对TIL的体外培养的条件及细胞性质进行了研究。TIL能在含IL-2的培养基中长期培养,且可不断扩增至一定数量,经液氮冻存的TIL仍可复苏、存活.在含IL-2的培养基中,TIL的扩增能力强于LAK细胞.生长TIL为T淋巴细胞.TIL的细胞表型随培养时间而有变化,即培养初期至30天内以Lyt-2~+细胞(Tc/Ts)占多数,以后则以L3T4~+细胞(T_H)为主.本文建立的TIL的分离、培养技术可靠、易行,为开展TIL的肿瘤过继免疫疗法的研究提供了条件.     

Tumor-specific human CD4+ regulatory T cells and their ligands: implications for immunotherapy

Regulatory T cells play an important role in the maintenance of immunological self-tolerance by suppressing immune responses against autoimmune diseases and cancer. Little is known, however, about the nature of the physiological target antigens for CD4(+) regulatory T (Treg) cells. Here we report the identification of the LAGE1 protein as a ligand for tumor-specific CD4(+) Treg cell clones generated from the tumor-infiltrating lymphocytes (TILs) of cancer patients. Phenotypic and functional analyses demonstrated that they were antigen-specific CD4(+) Treg cells expressing CD25 and GITR molecules and possessing suppressive activity on the proliferative response of naive CD4(+) T cells to anti-CD3 antibody stimulation. Ligand-specific activation and cell-cell contact were required for TIL102 Treg cells to exert suppressive activity on CD4(+) effector cells. These findings suggest that the presence of tumor-specific CD4(+) Treg cells at tumor sites may have a profound effect on the inhibition of T cell responses against cancer.     

Adoptive cell therapy with autologous tumor infiltrating lymphocytes and low-dose Interleukin-2 in metastatic melanoma patients

ABSTRACT: BACKGROUND: Adoptive cell therapy may be based on isolation of tumor-specific T cells, e.g. autologous tumor infiltrating lymphocytes (TIL), in vitro activation and expansion and the reinfusion of these cells into patients upon chemotherapy induced lymphodepletion. Together with high-dose interleukin (IL)-2 this treatment has been given to patients with advanced malignant melanoma and impressive response rates but also significant IL-2 associated toxicity have been observed. Here we present data from a feasibility study at a Danish Translational Research Center using TIL adoptive transfer in combination with low-dose subcutaneous IL-2 injections. METHODS: This is a pilot trial (Clinical trials.gov identifier: NCT00937625) including patients with metastatic melanoma, PS [LESS-THAN OR EQUAL TO]1, age <70, measurable and progressive disease and no involvement of the central nervous system. Six patients were treated with lymphodepleting chemotherapy, TIL infusion, and 14 days of subcutaneous low-dose IL-2 injections, 2 MIU/day. RESULTS: Low-dose IL-2 considerably decreased the treatment related toxicity with no grade 3--4 IL-2 related adverse events. Objective clinical responses were seen in 2 of 6 treated patients with ongoing complete responses (30+ and 10+ months), 2 patients had stable disease (4 and 5 months) and 2 patients progressed shortly after treatment. Tumor-reactivity of the infused cells and peripheral lymphocytes before and after therapy were analyzed. Absolute number of tumor specific T cells in the infusion product tended to correlate with clinical response and also, an induction of peripheral tumor reactive T cells was observed for 1 patient in complete remission. CONCLUSION: Complete and durable responses were induced after treatment with adoptive cell therapy in combination with low-dose IL-2 which significantly decreased toxicity of this therapy.     

Selective lysis of the autologous tumor by delta TCS1+ gamma/delta+ tumor-infiltrating lymphocytes from human lung carcinomas.

Two distinct non-overlapping populations of TcR1+ (gamma/delta) T cells have been described: the first, bearing the disulfide-linked gamma/delta heterodimer, is predominant in the peripheral blood; the second, expressing the non-disulfide-linked form of TcR1, is mostly confined to epithelial tissues (lung, gut, skin). TcR1+ lymphocytes may be cytotoxic and could be involved in anti-tumor immunity, especially against tumors at epithelial sites. Freshly derived tumor-infiltrating lymphocytes (TIL) obtained from two patients with lung cancer were enriched in CD3+WT31- cells. The percentage of this subset substantially increased upon culture in the presence of interleukin 2. These cells were TcR1+ as demonstrated by immunofluorescence and immunoprecipitation. In one case only 40% of this population reacted with delta TCS1 mAb, that recognizes the non-disulfide-linked form of TcR1, and co-expressed the CD8 antigen. Cultured TcR1+ TIL were able to kill fresh autologous tumor cells, K-562 and, to a lesser extent, some natural killer-resistant cell lines and allogeneic lung tumor cells in 4-h 51Cr-release cytotoxicity assays. The fractionated delta TCS1+ TIL lysed only autologous tumor cells and K-562, whereas the lytic activity against all the other targets was confined to the delta TCS1- subset. Moreover, the autotumor cytotoxicity was inhibited by anti-HLA class I but not by anti-CD1c or anti-LFA-1 mAb, suggesting that killing of the autologous tumor cells and non-major histocompatibility complex-restricted cytotoxicity are mediated by different mechanisms.     

Functional characterization of T lymphocytes propagated from human lung carcinomas

Tissue fragments from biopsies of six patients with malignant tumors of the lung were cultured in interleukin 2 (IL-2). Cultures of proliferating lymphocytes were isolated from all cases. Tumor cell lines (small cell carcinoma and adenocarcinoma) were established in parallel cultures from two of these patients. Lymphocytes that proliferated in vitro were virtually all mature T lymphocytes (greater than 95% T3+, T11+). The T8+ subset accounted for an average of 70% while T4+ cells averaged 20% of the cells in culture. HNK-1 antigen was presented on 23% of cells. Seventy-four percent of cells expressed Ia (HLA-DR) antigens. B cells did not proliferate under these conditions. In all cases the cells lysed K562 targets and were active in lectin-mediated cytolysis against human lymphoblasts. All cultures produced lymphokines (IL-2 and IFN-gamma) when stimulated with PHA. Lymphocytes grown from a tissue specimen with adenocarcinoma were capable of killing autologous tumor cells in vitro. Specific cytotoxicity has been maintained by these cultured lymphocytes for greater than 6 months. IL-2 activated peripheral blood cells in this case showed little specific cytotoxicity for autologous tumor cells. Lymphocytes from another specimen of adenocarcinoma also lysed this tumor, but cells from the other four specimens did not. Lymphocytes propagated from the specimen of small cell undifferentiated cancer did not lyse autologous tumor cells. These data show that primary lung tumors contain activated T cells which will respond to IL-2 in vitro. These tumor-infiltrating lymphocytes have demonstrable function, which can include cytolytic activity against autologous lung tumor.     

MTT比色法检测IL－2和杀伤细胞活性的研究

本文采用ＭＴＴ比色法测定了ＬＡＫ和ＴＩＬ细胞培养上清ＩＬ－２活性及ＬＡＫ细胞介导对体外培养的白血病细胞株的杀伤活性。结果表明ＬＡＫ细胞和ＴＩＬ细胞培养上清有较高的ＩＬ－２活性，可用于支持培养ＩＬ－２依赖株的生长；ＬＡＫ细胞对体外培养的白血病细胞株有明显的杀伤作用。其杀伤效应随效靶比例增加而增高；同时亦证明：活的白血病细胞数与ＭＴＴ甲赞形成产物呈直线相关性，提示通过ＭＴＴ法测定ＯＤ值能较好地反映活细胞数目及细胞毒效应。     

Selective expansion of a specific anti-tumor CD8+ cytotoxic T lymphocyte clone in the bulk culture of tumor-infiltrating lymphocytes from a melanoma patient: cytotoxic activity and T cell receptor gene rearrangements

In vitro culture of metastatic melanoma fragments with 150 units of recombinant interleukin 2 resulted in the successive expansion of CD4+ and then CD8+ tumor-infiltrating lymphocytes (TIL) throughout a 2-month period. TIL cultured for 43 days and consisting of 95% CD8+ and 10% CD4+ T lymphocytes were cloned by limiting dilution (LD). Thirteen CD8+ and thirty-one CD4+ clones were obtained, indicating that the frequency of clonogenic CD8+ proliferative T lymphocytes was much lower than that of their CD4+ homologues. When LD was performed in the presence of autologous melanoma cells the frequency of CD8+ clones was increased by factor 4. The DNA from TIL of day 43 bulk culture and of six CD8+ clones was hybridized with T cell receptor (TcR) beta and gamma probes. Identical configuration of the nonfunctional gamma and functional beta TcR genes was found in "bulk culture" and cloned TIL. The CD8+ clones therefore derived from a clonal population of CD8+ cells which had expanded in vitro before the LD. All the CD8+ clones tested were strongly cytotoxic for autologous melanoma cells but did not kill autologous fibroblasts or concanavalin A blasts, or any of the 10 allogeneic tumor targets tested, including 5 melanomas, 2 breast cell lines, 1 neuroblastoma, K-562 and the Epstein-Barr virus-transformed cell line used as a feeder. Furthermore, specific killing was inhibited by monoclonal antibodies against CD3, CD8, TcR alpha/beta and against class I major histocompatibility complex antigens indicating that these cytotoxic T lymphocyte clones recognized autologous tumor cells through the TcR, in an HLA class I-restricted manner. These data show that it is feasible to obtain tumor-specific cytotoxic T lymphocytes from melanoma TIL with a simple culture technique and that a single clone could be expanded to more than 10(10) cells which should allow testing of immunotherapeutic potential of these cells by adoptive transfer into melanoma patients.     

Interleukin-2 activated microglia engulf tumor infiltrating T cells in the central nervous system

Interleukin-2 (IL-2) has been utilized to treat cancer patient. However, recent studies disclosed that IL-2 induces T cell death. To clarify IL-2 induced T cell apoptosis at tumor sites in the central nervous system (CNS), we utilized an intracranial implantation of IL-2 cDNA transduced murine tumor cells and examined freshly recovered tumor infiltrating T lymphocytes (TIL) with a magnetic beads separation method. CD8(+) TIL recovered from the IL-2 therapy model had three times more apoptosis than a control group, tumor weights at day 12 decreased (0.016 versus 0.041 g/mouse) and the number of TIL per gram of tumor tissue increase more than six times by IL-2 therapy (5.69x10(6) versus 33.7x10(7) cells per mouse). In addition, both activation marker expressions (CD25 and CD69) and cytokine message levels (interferon gamma, and tumor necrosis factor alpha) on CD8(+) TIL decrease in the IL-2 therapy model. Moreover, we detected higher CD8beta message levels in purified tumors associated with F4/80(+) cells from the IL-2 model than the control by a one-step RT-PCR method. Finally, we observed many CD8beta(+) TIL surrounded by numerous infiltrating F4/80(+) cells in the tumor tissues of the IL-2 therapy model by immuno-fluorescence microscopic analysis. Our data show that IL-2 sensitization of apoptosis induction for CD8(+) TIL occurred and the apoptotic T cells were eliminated by F4/80(+) microglia in the CNS. Moreover, this is the first report describing in situ elimination of TIL by F4/80(+) phagocytic cells in the CNS.     

Immunological effects of BCG as an adjuvant in autologous tumor vaccines

The role of Bacillus Colmette-Guérin (BCG) as an adjuvant in autologous tumor vaccines was examined. In nine patients with renal cell cancer, irradiated tumor cells alone (wild-type, WT) or with BCG were inoculated intradermally into contralateral thighs. Seven to 10 days later, the draining vaccine-primed lymph nodes (WT-VPLN and BCG-VPLN) were excised. BCG increased the number of harvested VPLN cells by 10-fold (mean +/- SE = 61.8 +/- 20.6/x10(-7)/patient). BCG-VPLN had significantly greater percentages of CD3(+) and CD4(+) T cells compared to WT-VPLN. Both groups of VPLN cells were activated in vitro with anti-CD3 or anti-CD3/CD28 mAbs followed by expansion in IL-2. Anti-CD3/CD28 activation resulted in greater expansion of CD4(+) T cells compared to anti-CD3. After activation, VPLN cells were stimulated with irradiated autologous tumor targets and cytokines (IFN-gamma, GM-CSF, IL-10) released into the supernatants were measured 24 h later. Anti-CD3/CD28-activated BCG-VPLN cells were found to have a greater release of IFN-gamma compared with that of WT-VPLN cells, which was not observed significantly with IL-10 or GM-CSF. BCG resulted in increased VPLN cell yield as well as enhanced type 1 (IFN-gamma release) immune responses of VPLN cells to autologous tumor without upregulating type 2 (IL-10 release) responses. Anti-CD3/CD28 was superior to anti-CD3 activation in this cellular response.     

Antigen(s)-specific tumour-infiltrating lymphocytes from tumour induced by human herpes virus-6 (HHV-6) DNA transfected NIH 3T3 transformants.

Tumour infiltrating lymphocytes (TIL) have recently been shown to mediate potent therapeutic effects in certain malignancies in mice and in humans. To understand the mechanism of TIL immunotherapy it would be advantageous to generate tumour-specific TIL and to study a defined system of TIL and target cells in which the tumour epitope(s) recognized by TIL might be identified. We have established tumourigenic cell lines by transfection of NIH 3T3 cells with the entire genome of human herpesvirus-6 (HHV-6) and its small fragment (about 5% of the viral DNA sequence). Injection of these cells into nude mice produced tumours termed G-2T and 14-2T, respectively. Cell lines derived from these tumours when injected in NIH Swiss mice produced tumours, G-2TS and 14-2TS, respectively. We have generated TIL from G-2TS tumour that can kill G-2TS tumour cells in vitro but not other related tumours (14-2TS or MCA-106). These TIL can be expanded between 2-6.5 every 3-5 days. The TIL proliferated in tissue culture in response to recombinant interleukin-2 and interleukin-4 and maintained their tumor specificity for up to 6 months in vitro. Their phenotype was Thy 1.2+, Lyt-2+ and L3T4-. The availability of such tumour-specific stable TIL lines and specific viral-transformed targets will provide an opportunity to characterize the tumour-associated antigen critical for the specific cytotoxicity in this system and thereby to clarify the mechanism of this promising immunological approach to cancer therapy.     

Defective induction of T-cell help and natural killing following anti-CD3 stimulation of autoimmune lymphocytes

Anti-CD3 monoclonal antibody (MoAb) stimulates T cells in normal peripheral blood to proliferate and develop cytotoxic activity against NK-sensitive tumor cell lines. We now find that anti-CD3 MoAb also generates cytotoxic activity against a cell line (MEL-21) resistant to classical NK cell killing. After activation in vitro with anti-CD3 MoAb for 18 h, normal peripheral blood mononuclear cells (PBMNC) develop more HLA-DR-positive helper than suppressor T cells, manifest a functional helper effect as measured by increased IgG synthesis (P less than 0.01), as well as kill MEL-21 target cells. PBMNC from rheumatoid arthritis (RA) patients respond normally but mononuclear cells from rheumatoid arthritis synovial fluid (RASF) respond poorly. PBMNC from systemic lupus erythematosus (SLE) patients also respond poorly to anti-CD3 stimulation. Thus, the ability of anti-CD3 to stimulate IgG production and generate enhanced natural cytolytic activity are defective in both RASF and SLE lymphocytes.     

In vitro and in vivo action of cyclosporin A on the induction of human interleukin-2 receptor alpha and beta chains.

To compare in vitro and in vivo cyclosporin A (CyA) effects on early events involved in human T cell activation, lymphocytes obtained from healthy donors and from diabetic patients undergoing CyA therapy were studied for their interleukin 2 (IL-2) responsiveness, surface IL-2 receptor (IL-2R) expression and IL-2R mRNA accumulation, following stimulation with mitogen or anti-CD3 monoclonal antibody. T cells recovered from eight in vivo CyA-treated patients and stimulated in vitro for 4 h with mitogen or anti-CD3 (in the absence of CyA) showed significant (50-60%) inhibition of Tac mRNA accumulation, as assessed and quantified by scanning densitometry. Conversely, these cells showed no modification in their expression of membrane alpha (p55, Tac) or beta (p70) chains of IL-2R in binding experiments performed with both iodinated anti-Tac and IL-2 following 18 h stimulation with either mitogen or anti-CD3. Normal lymphocytes treated in vitro with CyA showed significant inhibition of alpha chain IL-2R expression both at the mRNA and the membrane level. At variance, expression of the IL-2R beta chain was unaffected; a significant number of high-affinity IL-2 binding sites was still detectable after in vitro CyA treatment. These results suggest that: (1) a residual immunosuppressive effect of CyA on T cell activation may be evidenced in in vivo treated cells by measuring very early events triggered following short-term stimulation; (2) CyA activity on T cell activation seems similar in vivo and in vitro; and (3) the described approach would be potentially useful to monitor the individual in vivo immunosuppressive capacity of CyA.     

Th17/Treg及其相关细胞因子在结肠癌中的表达并与肿瘤分期的相关性

研究结肠癌患者肿瘤组织中Th17细胞、Treg细胞及患者外周血中相关细胞因子的表达水平,探讨其表达与肿瘤分期的相关性及可能机制。运用流式细胞分析(FACS)技术检测30例结肠癌肿瘤组织及癌旁正常组织中Th17细胞及Treg细胞的比例;采用逆转录聚合酶链反应技术(RT-PCR)检测20例结肠癌患者外周血中Th17、Treg相关细胞因子IL-23和IL-10的表达水平。结果显示结肠癌肿瘤组织中Th17细胞和Treg细胞的比例明显高于癌旁正常组织(P<0.05),进展期肿瘤组织中Treg细胞的比例高于早期(P<0.05),而Th17细胞的比例较早期无明显差异(P>0.05),进展期肿瘤组织中Th17/Treg细胞的比例比早期偏低(P<0.05)。结肠癌患者外周血中IL-23、IL-10的mRNA水平升高,与健康对照组差异明显(P<0.05),且进展期与早期结肠癌IL-10mRNA的表达水平差异显著(P<0.05),而IL-23mRNA在两组间无明显差异(P>0.05)。随着结肠癌病程的进展,肿瘤组织内Th17细胞及Treg细胞的比例逐渐升高,且Treg细胞比Th17细胞升高更加明显。相关细胞因子IL-23和IL-10在患者外周血中的变化趋势和Th17、Treg细胞在肿瘤组织中的变化趋势相一致,提示Th17、Treg细胞在结肠癌的表达可能与肿瘤免疫微环境中相关的细胞因子调节有关。     

Phenotype, cytokine production and cytolytic capacity of fresh (uncultured) tumour-infiltrating T lymphocytes in human renal cell carcinoma

We investigated the phenotype and functional capacities of tumour-infiltrating lymphocytes (TIL), freshly isolated from primary renal cell carcinoma (RCC) specimens (n = 20). Three-colour flow cytometry immunophenotyping revealed that RCC TIL consist mainly of CD3⁺ T cells, with a clear predominance of CD4⁻CD8⁺ over CD4⁺ CD8⁻ T cells, and a marked population of CD4⁺ CD8⁺ T cells. Natural killer (NK) cells were also strongly represented (> 25% in 15 of 20 tumour samples), while B cells constituted a minor TIL subset (< 5% in 18 of 20 tumour samples). More importantly, the T and NK cells within the tumour displayed a significantly higher expression of the early activation marker CD69 than their counterparts in adjacent normal renal tissue and in peripheral blood. Expression of CD54 and of HLA-DR was also elevated on CD3⁺ TIL, and HLA-DR expression was further vigorously up-regulated following ex vivo stimulation with anti-CD3, all suggesting enhanced immune activity within the tumour microenvironment. CD3⁺ CD4⁺ TIL displayed a normal capacity to up-regulate CD25 expression and to secrete both Th1-type (IL-2, tumour necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ)) and Th2-type (IL-4, IL-5 and IL-10) cytokines upon triggering with anti-CD3. Furthermore, cytokine production was susceptible to modulation by CD28 costimulation. CD3⁺ CD8⁺ TIL, on the other hand, consistently demonstrated a poor up-regulation of CD25 upon triggering with anti-CD3, and displayed poor ex vivo cytolytic activity in an anti-CD3-redirected 4-h cytotoxicity assay against murine P815 cells. Collectively, our findings indicate that the CD3⁺ CD4⁺ TIL in RCC have normal functional capacities, whereas the proportionally major CD3⁺ CD8⁺ TIL are functionally impaired. The relevance of these findings to the in vivo local immune response in RCC is discussed.     

Biased activation of human T lymphocytes due to low extracellular pH is antagonized by B7/CD28 costimulation

As T cell response to tumor-associated antigens may be impaired by the acidic microenvironment typical of solid tumors, we assessed the effect of extracellular pH (pH(e)) on the activation and proliferation of human T lymphocytes and generation of the cytotoxic response. T lymphocytes stimulated with anti-CD3 mAb or PHA at low pH(e) were unable to secrete IL-2 and IFN-gamma and their ability to progress through the cell cycle was impaired. T lymphocytes also displayed up-regulation of IFN-gammaR2 chain and CTLA-4 expression, rendering them sensitive to negative regulatory signals. Agonistic mAb against CD28, but not against CD2, completely restored cytokine production and cell cycle progression, but down-regulated IFN-gammaR2 and CTLA-4 expression. The anti-CD28mAb rescued the CTL response of allogeneic anti-tumor cultures generated at low pH(e). Following anti-CD28 mAb treatment, T cells synthesized cyclooxygenase-2 (Cox-2) protein, which is involved in the early phases of T cell activation. This rescue of T cell activation was independent of the inducible 6-phosphofructo-2-kinase (iPFK-2) pathway, which stimulates proliferation in hypoxic and acidic conditions. The restoration of proliferative and cytotoxic T cell responses by CD28-triggering provides insight into the mechanisms by which B7 enhances the T cell anti-tumor response in vivo.