CD4-CD8- T cell receptor alpha beta T cells: generation of an in vitro major histocompatibility complex class I specific cytotoxic T lymphocyte response and allogeneic tumor rejection

The generation of an in vitro major histocompatibility complex class I specific response of CD4-CD8- T cell receptor (TCR) alpha beta cytotoxic T lymphocytes (CTL) and their allogeneic tumor rejection were investigated. Inocula of BALBRL male 1 were rejected in C57BL/6 (B6) mice treated with minimum essential medium (MEM) (control), anti-L3T4 (CD4) monoclonal antibody (mAb) or anti-Lyt-2.2 (CD8) mAb and CTL against the tumor were generated in vitro. No rejection and no induction of CTL were observed in B6 mice treated with anti-L3T4 (CD4) plus anti-Lyt-2.2 (CD8) mAb. CTL with the classical Thy-1+ CD3+CD4-CD8+ TCR alpha beta phenotype were generated in mixed lymphocyte tumor cell culture (MLTC) spleen cells from B6 mice treated with MEM (control) or anti-L3T4 (CD4) mAb, whereas CTL with an unusual Thy-1+CD3+CD4-CD8- TCR alpha beta phenotype were generated in MLTC spleen cells from anti-Lyt-2.2 (CD8) mAb-treated B6 mice. Both types of CTL were reactive with both H-2Kd and Dd (Ld) class I antigen. These findings suggest that when CD4+ cells were blocked by anti-L3T4 (CD4) mAb, CD8+ CTL mediated rejection, and when CD8+ cells were blocked by anti-Lyt-2.2 (CD8) mAb, CD4+ cells were capable of mediating rejection, although less efficiently than CD8+ cells, by inducing CD4-CD8- TCR alpha beta CTL. The finding that adoptive transfer of CD4 and CD8-depleted MLTC spleen cells, obtained from anti-Lyt-2.2 (CD8) mAb-treated B6 mice that had rejected BALBRL male 1, resulted in rejection of BALBRL male 1 inoculated into B6 nu/nu mice confirmed the above notion. CTL clones with the CD4-CD8- TCR alpha beta phenotype specific for Ld were established.     

Clonal expansion of CD8+ cytotoxic T lymphocytes against human T cell lymphotropic virus type I (HTLV-I) genome products in HTLV-I-associated myelopathy/tropical spastic paraparesis patients.

Short-term culture of peripheral blood mononuclear cells (PBMC) derived from patients with human T cell lymphotropic virus type I-associated myelopathy (HAM)/tropical spastic paraparesis resulted in dominance by DR+ activated CD8+ T cells. Variations in the T cell receptor (TCR) V alpha and V beta chains in these cells were analyzed, and in all 10 patients examined, 2-3 V gene families were dominant in both TCR V alpha and V beta. In five patients we examined, cultured lymphocytes contained cytotoxic lymphocytes for p40tax (patients HAM2, 3, 7, and 8) or env protein (patient HAM4) of human T lymphotropic virus type I. In patients HAM2 and HAM8, cultured lymphocytes contained a large proportion of V beta 8+ CD8+ and/or V beta 12+ CD8+ cells. The sequence of V beta 8+ and V beta 12+ cDNA revealed that they were oligoclonal with identical or similar sequences in each patient. Elimination experiments with monoclonal antibodies for TCR V beta 8 and V beta 12 showed that they were CD8+ cytotoxic T lymphocytes (CTL) for p40tax. In addition, flow cytometry and sequencing analysis of uncultured PBMC revealed that in HAM2, V beta 8+ CTL and their precursors account for 7% and V beta 12+ CTL and their precursors account for 18% of total CD8+ cells. This indicates the presence of two markedly expanded clones in vivo. No common dominant TCR V alpha or V beta were observed among 10 HAM patients analyzed.     

T cell receptor (TCR) structure of autologous melanoma-reactive cytotoxic T lymphocyte (CTL) clones: tumor-infiltrating lymphocytes overexpress in vivo the TCR beta chain sequence used by an HLA-A2- restricted and melanocyte-lineage-specific CTL clone

HLA-A2+ melanomas express common melanoma-associated antigens (Ags) recognized in vitro by autologous cytotoxic T lymphocytes (CTL). However, it is not known whether tumor Ags can drive in vivo a selective accumulation/expansion of Ag-specific, tumor-infiltrating T lymphocytes (TIL). Therefore, to evaluate this possibility, 39 CTL clones isolated from several independent mixed lymphocyte tumor cultures (MLTC) of TIL and peripheral blood lymphocytes (PBL) of an HLA- A2+ melanoma patient and selected for T cell receptor (TCR)-dependent, HLA-restricted tumor lysis, were used for analysis of TCR alpha and beta chain structure by the cDNA polymerase chain reaction (PCR) technique with variable gene-specific primers followed by sequencing. Despite absence of oligoclonality in fresh TIL and PBL, as well as in T cells of day 28 MLTC (day of cloning), sequence analysis of TCR alpha and beta chains of TIL clones revealed a dominance of a major category of melanoma-specific, HLA-A2-restricted T cells expressing a V alpha 8.2/J alpha AP511/C alpha and V beta 2.1/D beta 1/J beta 1.1/C beta 1 TCR. The same TCR was also found in 2 out of 14 PBL clones. The other PBL clones employed a V alpha 2.1 gene segment associated with either V beta 13.2, 14, or w22. Clones A81 (V alpha 2.1/J alpha IGRJ alpha 04/C alpha and V beta 14/D beta 1/J beta 1.2/C beta 1) and A21 (V alpha 8.2/J alpha AP511/C alpha and V beta 2.1/D beta 1/J beta 1.1/C beta 1), representative of the two most frequent TCR of PBL and TIL, respectively, expressed different lytic patterns, but both were HLA-A2 restricted and lysed only HLA-A2+ melanomas and normal melanocytes, thus indicating recognition of two distinct HLA-A2-associated and tissue-related Ags. Finally, by the inverse PCR technique, the specific TCR beta chain (V beta 2.1/D beta 1/J beta 1.1/C beta 1) expressed by the dominant TIL clone was found to represent 19 and 18.4% of all V beta 2 sequences expressed in the fresh tumor sample and in the purified TIL, respectively, but < 0.19% of V beta 2+ sequences expressed in PBL. These results are consistent with the hypothesis that a clonal expansion/accumulation of a melanocyte-lineage-specific and HLA-A2-restricted T cell clone occurred in vivo at the site of tumor growth.     

Grafting primary human T lymphocytes with cancer-specific chimeric single chain and two chain TCR

Primary human activated T lymphocytes were genetically grafted with chimeric T cell receptors (TCR). Three domain single chain (sc-) TCR as well as two chain (tc-) TCR gene constructs were derived from the melanoma-specific cytotoxic human T cell (CTL) clone 82/30, and linked to the CD3-zeta signaling element. Chimeric TCR alpha and beta receptor genes were structurally designed to prevent pairing with endogenous TCR alpha and beta chains in order to prevent the generation of unpredictable immune specificities. After transduction of polyclonally activated human peripheral blood lymphocytes with retroviral vectors harboring the chimeric receptor genes, genetically engineered cells specifically recognized and responded to MAGE-A1POS/HLA-A1POS cells. Importantly, each type of transduced T lymphocytes that bound specifically to peptide/MHC complexes also showed specific antitumor reactivity as well as lymphokine production. Genetically engineered primary human T lymphocytes expressing chimeric sc- or tc-TCR therefore hold promise for disease-specific therapies.     

T cell receptor genes in a series of class I major histocompatibility complex-restricted cytotoxic T lymphocyte clones specific for a Plasmodium berghei nonapeptide: implications for T cell allelic exclusion and antigen-specific repertoire

We report here the first extensive study of a T cell repertoire for a class I major histocompatibility complex (MHC)-restricted cytotoxic T lymphocyte (CTL) response. We have found that the T cell receptors (TCRs) carried by 28 H-2Kd-restricted CTL clones specific for a single Plasmodium berghei circumsporozoite nonapeptide are highly diverse in terms of V alpha, J alpha, and J beta segments and aminoacid composition of the junctional regions. However, despite this extensive diversity, a high proportion of the TCRs contain the same V beta segment. These results are in contrast to most previously reported T cell responses towards class II MHC-peptide complexes, where the TCR repertoires appeared to be much more limited. In our study, the finding of a dominant V beta in the midst of otherwise highly diverse TCRs suggests the importance of the V beta segment in shaping the T cell repertoire specific for a given MHC-peptide complex. As an additional finding, we observed that nearly all clones have rearranged both TCR alpha loci. Moreover, as many as one-third of the CTL clones that we analyzed apparently display two productive alpha rearrangements. This argues against a regulated model of sequential recombination at the alpha locus and consequently raises the question of whether allelic exclusion of the TCR alpha chain is achieved at all.     

H-2-restricted cytolytic T lymphocytes specific for HLA display T cell receptors of limited diversity.

We previously showed that H-2Kd-restricted cytotoxic T lymphocyte (CTL) clones specific for a single nonapeptide derived from the Plasmodium berghei circumsporozoite (PbCS) protein displayed T cell receptors (TCRs) of highly diverse primary structure. We have now analyzed the TCR repertoire of CTLs that recognize a peptide derived from the human class I major histocompatibility complex (MHC) molecule HLA-Cw3 in association with the same murine class I MHC molecule H-2Kd. We first sequenced the TCR alpha and beta genes of the CTL clone Cw3/1.1 and, based on this genomic analysis, the TCR alpha and beta cDNA junctional regions of 23 independent H-2Kd-restricted CTL clones specific for HLA-Cw3. The results show that the TCR chains display very limited heterogeneity, both in terms of V alpha, J alpha, V beta, and J beta segments, and in terms of length and sequence of the CDR3 alpha and beta loops. The TCR repertoire used in vivo was then analyzed by harvesting CTL populations from the peritoneal cavity of immune mice. The peritoneal exudate lymphocytes (PELs) displayed HLA-Cw3-specific cytolytic activity in the absence of any stimulation in vitro. Remarkably, most of these freshly isolated PELs expressed TCRs that shared the same structural features as those from HLA-Cw3-reactive CTL clones. Thus, our results show that a peptide from HLA-Cw3 presented by H-2Kd selects CTLs that bear TCRs of very limited diversity in vivo. When taken together with the high diversity of the TCRs specific for the PbCS peptide, these findings suggest that natural tolerance to self peptides presented by class I MHC molecules may substantially reduce the size of the TCR repertoire of CTLs specific for antigenic peptides homologous to self.     

Cytotoxic T lymphocytes form an antigen-independent ring junction

Immunological synapses are organized cell-cell junctions between T lymphocytes and APCs composed of an adhesion ring, the peripheral supramolecular activation cluster (pSMAC), and a central T cell receptor cluster, the central supramolecular activation cluster (cSMAC). In CD8(+) cytotoxic T lymphocytes, the immunological synapse is thought to facilitate specific killing by confining cytotoxic agents to the synaptic cleft. We have investigated the interaction of human CTLs and helper T cells with supported planar bilayers containing ICAM-1. This artificial substrate provides identical ligands to CD4(+) and CD8(+) T cells, allowing a quantitative comparison. We found that cytotoxic T lymphocytes form a ring junction similar to a pSMAC in response to high surface densities of ICAM-1 in the planar bilayer. MICA, a ligand for NKG2D, facilitated the ring junction formation at lower surface densities of ICAM-1. ICAM-1 and MICA are upregulated in tissues by inflammation- and stress-associated signaling, respectively. Activated CD8(+) T cells formed fivefold more ring junctions than did activated CD4(+) T cells. The ring junction contained lymphocyte function associated antigen-1 and talin, but did not trigger polarization and granule translocation to the interface. This result has specific implications for the mechanism of effective CTL hunting for antigen in tissues. Abnormalities in this process may alter CTL reactivity.     

Activated anti-tumor immunity in cancer patients after high intensity focused ultrasound ablation

T cell-mediated immune responses represent the main cellular antitumor immunity in cancer patients. Recent studies have shown that that both surgical procedure and radiation therapy could cause the functional suppression of lymphocyte-mediated cellular immunity. The purpose of current study is to evaluate whether high intensity focused ultrasound (HIFU) might change a systemic antitumor immunity, particularly T lymphocyte-mediated immunity in cancer patients. A total of 16 patients with solid malignancies were treated with HIFU. Among them, six patients had osteosarcoma (Enneking stage, II(B)4, III(B) 2), five had hepatocellular carcinoma (TNM stage, III 3, IV 2), and five had renal cell carcinoma (TNM stage, III 2, IV 3). Using flow cytometry technique, T lymphocyte and subset, B lymphocyte and natural killer cell (NK) in the peripheral blood were measured in these patients on the day before HIFU and 7 to 10 d after HIFU. The statistical significance of any observed difference is evaluated by Student's t-test. The results showed a significance increase in the population of CD4(+) lymphocytes (p < 0.01) and the ratio of CD4(+) /CD8(+) (p < 0.05) in the circulation of cancer patients after HIFU treatment. The abnormal levels of CD3(+) lymphocytes returned toward the normal range in two patients, CD4(+)/CD8(+) ratio in 3, CD19(+) lymphocytes in one and cytotoxic NK in one, respectively, in comparison to control values. It is concluded that HIFU could enhance a systemic antitumor cellular immunity in addition to local tumor destruction in patients with solid malignancies.     

T cell stimulation by staphylococcal enterotoxins. Clonally variable response and requirement for major histocompatibility complex class II molecules on accessory or target cells

Staphylococcal enterotoxins (SE) are the most potent mitogens for T lymphocytes known; concentrations of less than 10(-9) M are sufficient for T cell activation. The mechanism of T cell activation by SE is unknown. We have used cloned human cytotoxic and proliferative T lymphocytes to dissect the molecular mechanism of T cell activation by SE. With rare exceptions, all TCR alpha/beta chain-expressing T cell clones of CD4+ or CD8+ phenotype, as well as CD4-8- TCR alpha/beta chain negative chain-expressing T lymphocyte clones, respond with proliferation and/or cytotoxicity to SE. For triggering of all these clones, the presence of autologous or allogeneic MHC class II molecules on accessory or target cells is necessary. This requirement for class II antigens is not due to an immunological recognition of processed SE, since inhibition of antigen processing has no influence on the T cell response to SE. SE acts on the T cells directly since (a) they stimulate a rise in intracellular calcium concentration in T cell lines or purified T cells, and (b) accessory cells can be replaced by phorbolesters in the proliferative activation of resting T cells by SE. Furthermore, the T cell response to SE shows extensive clonal heterogeneity. These results suggest that SE are functionally bivalent mitogens binding highly selectively to HLA class II molecules and the TCR. Thus, compared with other polyclonal T cell activating agents, activation with SE most closely mimicks the physiological way of MHC-restricted antigen recognition by T lymphocytes.     

外周血单核细胞来源树突状细胞体外扩增及诱导抗乳腺癌免疫的研究

目的研究外周血来源树突状细胞(DC)的体外扩增及诱导特异性抗乳腺癌细胞的免疫效应。方法(1)从外周血中分离单个核细胞后,获得单核细胞(monocyte,Mo)。粒-单集落刺激因子(GM-CSF)和白介素4(IL-4)诱导分化扩增培养7d后,应用流式细胞术进行表型分析。(2)诱导单核细胞分化的第3天加入人乳腺癌细胞株3A0的冻融抗原培养4d后,获得负载肿瘤抗原的成熟DC;将致敏DC与从外周血中分离的同种异体T淋巴细胞共培养3d,获得细胞毒T淋巴细胞(CTL);四甲基偶氮唑蓝法检测CTL及上清液对人乳腺癌细胞株3A0、人胚肾细胞株293T(对照细胞)、人肝癌细胞株HCCC-9810的细胞毒作用。结果(1)外周血来源Mo在GM-GSF和IL-4作用下,第7天后可分化生成成熟的DC,高表达DC特异性抗原CDla、CD80(B7-1)、CD86(B7-2)HLA-DR、CD83。(2)DC可负载并递呈肿瘤抗原,激活同种异体T淋巴细胞,诱导肿瘤特异性CTL产生。不同浓度CTL及上清液对乳腺癌细胞3A0有特异性杀伤、抑制作用(P<0.05)。结论外周血中Mo可体外分化扩增为成熟的功能性DC,并诱导出特异性杀伤乳腺癌细胞的免疫效应。     

Transfer of a TCR gene derived from a patient with a marked antitumor response conveys highly active T-cell effector functions

The genes for the alpha and beta chains of a highly reactive anti-MART-1 T-cell receptor were isolated from T-lymphocytes that mediated in vivo regression of tumor in a patient with metastatic melanoma. These genes were cloned and inserted into MSCV-based retroviral vectors. After transduction, greater than 50% gene transfer efficiency was demonstrated in primary T-lymphocytes stimulated by an anti-CD3 antibody. The specificity and biologic activity of TCR gene-transduced T-cells was determined by cytokine production after coculture of T-cells with stimulator cells pulsed with MART-1 peptide. The production of interferon-gamma and granulocyte macrophage-colony stimulating factor (GM-CSF) was comparable to highly active MART-1 specific peripheral blood lymphocytes (PBL) in the amount of cytokine produced and transduced cells recognized peptide pulsed cells at dilutions similar to cytotoxic T lymphocyte (CTL) clones. Human leukocyte antigen (HLA) class I restricted recognition was demonstrated by mobilization of degranulation marker CD107a, by cell lysis, by cytokine production, and by proliferation in the presence of HLA-A2-positive but not HLA-A2-negative melanoma cell lines. Similar data was obtained when tumor-infiltrating lymphocytes (TIL) were transduced with the TCR genes, converting previously nonreactive cells to tumor reactive cells. TCR-transduced T-cells are thus attractive candidates for evaluation in cell transfer therapies of patients with cancer.     

Adoptive Transfer of Human Papillomavirus E7-specific CTL Enhances Tumor Chemoresponse Through the Perforin/Granzyme-mediated Pathway

Adoptive cytotoxic T lymphocyte (CTL) therapy has an important implication in treating cancer patients. Here, we investigate whether adoptive transfer of human papillomavirus (HPV) E7-specific CTL can enhance tumor chemoresponse using an established cervical cancer animal model. Cisplatin-based chemotherapy plus CTL therapy showed an improved therapeutic effectiveness, along with antitumor protective responses to a parental tumor cell rechallenge. Cisplatin treatment dose-dependently increased the expression of Fas, intercellular adhesion molecule (ICAM)-1, and major histocompatibility complex (MHC) class I antigens (Ags) on tumor cells in vitro. However, CTL-expressing FasL failed to improve antitumor activity in vitro and in animals, resulting from nonfunctional Fas expressed on tumor cells. In contrast, ethylene glycol tetraacetic acid (EGTA) treatment blocked increased sensitivity of cisplatin-treated tumor cells to CTL-mediated killing in vitro, suggesting an important role of the perforin/granzyme-mediated pathway for improved therapeutic effectiveness. This notion was further confirmed by perforin knockout animal studies. Thus, this study shows that (i) modulation of Ag (Fas, ICAM-1) expression by tumor cells has little effect on their increased sensitivity to CTL-mediated killing, (ii) improved therapeutic effectiveness is mediated mainly through the perforin/granzyme-mediated tumor killing pathway, and (iii) a combination of chemotherapy and adoptive E7-specific CTL transfer augments antitumor therapeutic activity in vivo. This finding may have important implications for treating HPV-associated cervical cancer.     

A novel beta 4, alpha 6 integrin-associated epithelial cell antigen involved in natural killer cell and antigen-specific cytotoxic T lymphocyte cytotoxicity

Efficient immune responses require interactions between cell adhesion molecules on lymphocytes and counter-receptors on antigen presenting cells or target cells. While target-specific receptors or ligands have not been identified for natural killer (NK) cells, cell adhesion molecules have been implicated in the interaction between NK cell effectors and tumor cell targets. Herein, we describe monoclonal antibodies (mAbs) against a carcinoma cell line that efficiently block the cytolytic activity of interleukin 2-activated NK cell lines and clones. L280 mAb reacts with secretory epithelial cells in normal human tissues, but does not react with hematopoietic cells or other tissue types. Biochemical analysis revealed that L280 mAb immunoprecipitates the beta 4, alpha 6 integrin, as well as a novel 98-kD glycoprotein, and probably reacts with a carbohydrate epitope on these molecules. Involvement of the L280 antigen in cellular immunity is not restricted to NK cell-mediated cytotoxicity. L280 mAb also efficiently inhibits alloantigen-specific cytotoxicity against Colo-205 cells mediated by human histocompatibility leukocyte antigen (HLA)-A2 alloantigen specific alpha beta-TCR+ and gamma delta-TCR+ cytotoxic T lymphocyte (CTL) clones. Additionally, we demonstrate that L280 mAb blocks cytotoxicity mediated by influenza peptide-specific HLA-restricted CTL clones. These data indicate that the antigen recognized by L280 mAb is important in both NK and CTL function, and that an as yet unidentified receptor for this epithelial antigen is present on both NK and T lymphocytes. The restricted expression of L280 antigen indicates that this molecule may be important in immune reactions in epithelial tissues.     

Interleukin 7 promotes long-term in vitro growth of antitumor cytotoxic T lymphocytes with immunotherapeutic efficacy in vivo

A major obstacle to the effective use of adoptive immunotherapeutic treatment of cancer is the difficulty of obtaining tumor-reactive lymphocytes in either sufficient numbers or with appropriate in vivo function to make such an approach feasible. Previous studies have shown that antitumor cytotoxic T lymphocytes (CTL) with in vivo efficacy can be generated in vitro from lymphoid cells obtained from lymph nodes that drain the anatomical site of a tumor. Results presented here demonstrate that inclusion of interleukin 7 (IL-7) into the medium in which such CTL are cultured can support their growth in vitro for prolonged periods of time in the absence of repeated stimulation with either tumor stimulator cells or tumor antigen. More importantly, antitumor CTL propagated in medium containing IL-7 have retained both their antigenic specificity and their ability to reject tumors in vivo subsequent to intravenous injection. Parallel cultures of antitumor CTL similarly cultured in medium containing only IL-2 could only be maintained for 5-6 wk, after which the number and proportion of viable cells that were recoverable from such cultures progressively decreased. Phenotypic analysis of CTL maintained after extended culture (i.e., 22 mo) in medium containing IL-7 demonstrated them to be CD3+4-8+ T cells. These cells were also found to express lymphocyte function associated 1, intercellular adhesion molecule 1, and Mel-14 cell interaction molecules. The data also demonstrate that these CTL do not require the presence of antigen-presenting cell populations to mount a proliferative response to tumor stimulator cells. Cells in these cultures were also demonstrated to produce IL-2 after stimulation with irradiated tumor cells, thereby indicating that these CTL have become independent of the requirement for CD4+ helper cells to survive and function either in vitro or in vivo. Collectively, the findings that IL- 7 can beneficially augment the generation, and propagate the long-term growth, of antitumor CTL from lymph nodes draining a tumor site may have profound implications for promoting the immunotherapeutic treatment of cancer in humans.     

Expression of specific cytolytic activity by H-2I region-restricted, influenza virus-specific T lymphocyte clones

Among murine class II major histocompatibility complex (MHC)-restricted cytotoxic T lymphocyte (CTL) clones specific for type A influenza virus, we have identified both noncytolytic clones and clones exhibiting H-2 I region-restricted cytolytic activity. After appropriate antigenic stimulation, both cytolytic and noncytolytic clones proliferated in the absence of exogenous interleukin 2. All of the clones possess the Thy-1.2+, Lyt-1+2-, L3T4+ phenotype. The class II MHC restriction of viral recognition by the CTL clones was mapped by proliferation using recombinant mouse strains and by inhibition of cytotoxic activity with monoclonal antibodies directed to class II MHC products and L3T4a. The restriction specificity of two CTL clones was unambiguously assigned to the E beta d chain by using L cell transfectant lines expressing E alpha kE beta d or E alpha kE beta k gene products. Analysis of the viral specificity of the cloned lines revealed subtype-specific and crossreactive patterns of viral antigen recognition; the pattern of viral antigen specificity exhibited by each clone in proliferation and cell-mediated cytotoxicity was identical. Each CTL clone also demonstrated antigen-dependent release of helper factor(s) that promoted in vitro primary anti-SRBC responses. Finally, the cytotoxic effector function of the class II MHC-restricted CTL clones was mediated by direct lysis of virus-infected cells, and not by secretion of a cytolytic lymphokine.     

Molecular basis for leukocyte integrin alpha(E)beta(7) adhesion to epithelial (E)-cadherin

Cadherins are expressed in tissue-restricted patterns and typically mediate homophilic adhesion. Cadherins also mediate lymphocyte adhesion, providing the opportunity for lymphocyte attachment to parenchymal cells. The best characterized example of lymphocyte adhesion to a tissue-specific cell adhesion molecule, as opposed to a vascular endothelial adhesion molecule, is the interaction between integrin alpha(E)beta(7) on intraepithelial lymphocytes and E-cadherin on epithelial cells. However, the molecular basis for an integrin-cadherin interaction is not well defined. Realization that the cadherin domain adopts a topology similar to the immunoglobulin (Ig) fold suggested that integrin recognition of E-cadherin might be similar to recognition of Ig superfamily ligands. Thus, we modeled domain 1 of human E-cadherin and studied the role of solvent-exposed loops that connect Ig-like core-forming beta strands. Mutational analyses localized the integrin alpha(E)beta(7) recognition site to the top of domain 1 at the face formed by the BC and FG loops, a site distinct from the region recognized in intercellular adhesion molecule (ICAM)-1, -2, and -3, mucosal addressin cell adhesion molecule 1 (MAdCAM-1), vascular cell adhesion molecule 1 (VCAM-1), and fibronectin by their integrin ligands. Moreover, the integrin alpha(E)beta(7) binding site is distinct from the homophilic binding site on E-cadherin. These studies provide a conceptual basis for integrin-cadherin binding and extend the model that an Ig-like fold can serve as a scaffold for recognition.     

B7H costimulates clonal expansion of, and cognate destruction of tumor cells by, CD8(+) T lymphocytes in vivo.

B7H/B7RP (hereby called B7H) is a new member of the B7 family of costimulatory molecules and interacts with inducible costimulatory molecule (ICOS). Its function for CD8 T cells has not been reported. We report here that expression of B7H on the tumor cells reduced tumorigenicity and induced immunity to subsequent challenge with parental tumor cells. The immune protection correlates with an enhanced cytotoxic T lymphocyte (CTL) response against P1A, the major tumor antigen expressed in the J558 tumor. To understand the mechanism of immune protection, we adoptively transferred transgenic T cells specific for tumor antigen P1A into mice that bore P1A-expressing tumors. We found that while the transgenic T cells divided faster in mice bearing the B7H(+) tumors, optimal B7H-induced clonal expansion of P1CTL required costimulation by B7-1 and B7-2 on the endogenous host antigen-presenting cells (APCs). Interestingly, when B7H(+) and B7H(-) tumors were coinjected, P1CTL selectively eliminated the B7H(+) tumor cells. Moreover, B7H expressed on the tumor cells made them highly susceptible to destruction by CTL in vivo, even if the CTL was administrated into mice with large tumor burdens. Tumors that recurred in the P1CTL-treated mice lost transfected B7H and/or H-2L(d), the class I molecule that presents the P1A peptide. Taken together, our results reveal that B7H costimulates clonal expansion of, and cognate destruction by CD8(+) T lymphocytes in vivo.     

A novel 80-kD cell surface structure identifies human circulating lymphocytes with natural killer activity

Human lymphocytes with natural killer (NK) activity, including most activated gamma/delta+ T lymphocytes, recognize and lyse tumor target cells without requiring recognition of major histocompatibility complex antigen. However, unlike gamma/delta+ T lymphocytes, NK cells do not express CD3/T cell receptor (TCR) molecules, and the receptors involved in cell-mediated cytotoxicity are unknown. To further delineate circulating NK cells, we developed monoclonal antibodies (mAbs) against the human NK leukemia YT2C2. We report the isolation of a mAb termed BY55, recognizing at the cell surface a novel 80-kD protein with restricted expression. In addition to the immunizing cell line, this mAb binds to circulating NK cells, gamma/delta+ cells, and a minor subset of alpha/beta+ T lymphocytes. Expression of the BY55 mAb- reactive epitope/molecule is regulated by activation, as short-term culture of peripheral blood lymphocytes (PBL) with phorbol ester induced its downmodulation. Furthermore, BY55 mAb reactivity was found neither with the NK nor with the TCR alpha/beta+ gamma/delta+ clones tested. Biochemical studies as well as phenotypic analysis revealed that this structure is different from all previously identified molecules on the lymphocyte cell surface. Interestingly, we found that BY55+ cells exert most NK activity obtained with fresh circulating lymphocytes. We report that within fresh E rosette-positive PBL only a subset of the CD16+, CD56+, and CD57+ cells coexpressed BY55 molecule, indicating that BY55 mAb defines a unique subset mediating NK activity of circulating PBL.     

A therapy modality using recombinant IL-12 adenovirus plus E7 protein in a human papillomavirus 16 E6/E7-associated cervical cancer animal model

Interleukin (IL)-12 has been reported to induce cellular immune responses for protection against tumor formation. Here we investigate the utility of adenoviral delivery of IL-12 as an adjuvant for a human papillomavirus E7 subunit vaccine in a mouse tumor challenge model. Direct intratumoral injection of AdIL-12 resulted in a significant suppression of tumor growth compared to the control group. Injection of E7 protein into either a tumor site or the distance site along with AdIL-12 further enhanced antitumor effects significantly higher than either AdIL-12 or E7 injection alone. This combined injection resulted in complete regression of 9-mm-sized tumor in 40% of animals as well as lasting antitumor immunity against tumor recurrence. We also evaluated immune responses induced by these injections. AdIL-12 plus E7 enhanced E7-specific antibody responses significantly higher than AdIL-12 or E7 injection. In particular, the production level of interferon (IFN)-gamma from E7-specific CD4(+) T cells was similar between AdIL-12 group and AdIL-12 + E7 group. However, IFN-gamma production from E7-specific CD8(+) T cells was the most significant when injected with AdIL-12 + E7. This was consistent with intracellular IFN-gamma staining levels of CD8(+) T cells, suggesting that AdIL-12 + E7 injection enhances antitumor immunity in the human papillomavirus (HPV) 16 tumor model through increased expansion of the cytotoxic T-lymphocyte (CTL) subset. This enhanced protection appeared to be mediated by CD8(+) T cells, as determined by in vivo T-cell subset deletion. Thus, these studies demonstrate that E7 vaccines can induce CTL responses responsible for antitumor effects in the presence of IL-12 delivered via adenovirus vectors. This likely provides one additional approach for immune therapy against cervical cancers.     

Long-term phenotypic, functional and genetic stability of cancer-specific T-cell receptor (TCR) alphabeta genes transduced to CD8+ T cells

In adoptive T-cell transfer as an intervention for malignant diseases, retroviral transfer of T-cell receptor (TCR) genes derived from CD8(+) cytotoxic T-lymphocyte (CTL) clones provides an opportunity to generate a large number of T cells with the same antigen specificity. We cloned the TCR-alphabeta genes from a human leukocyte antigen (HLA)-A(*)2402-restricted CTL clone specific for MAGE-A4(143-151). The TCR-alphabeta genes were transduced to 99.2% of non-TCR expressing SupT1, a human T-cell line, and to 12.7-32.6% of polyclonally activated CD8(+) T cells by retroviral transduction. As expected, TCR-alphabeta gene-modified CD8(+) T cells showed cytotoxic activity and interferon-gamma production in response to peptide-loaded T2-A(*)2402 and tumor cell lines expressing both MAGE-A4 and HLA-A(*)2402. A total of 24 clones were established from TCR-alphabeta gene-transduced peripheral blood mononuclear cells and all clones were functional on a transduced TCR-dependent manner. Four clones were kept in culture over 6 months for analyses in detail. The transduced TCR-alphabeta genes were stably maintained phenotypically, functionally and genetically. Our results indicate that TCR-transduced alphabeta T cells by retroviral transduction represent an efficient and promising strategy for adoptive T-cell transfer for long term.