Autologous Cytolytic T Lymphocytes Research Articles

Adoptively transferred human lung tumor specific cytotoxic T cells can control autologous tumor growth and shape tumor phenotype in a SCID mouse xenograft model

BackgroundThe anti-tumor efficacy of human immune effector cells, such as cytolytic T lymphocytes (CTLs), has been difficult to study in lung cancer patients in the clinical setting. Improved experimental models for the study of lung tumor-immune cell interaction as well as for evaluating the efficacy of adoptive transfer of immune effector cells are needed.MethodsTo address questions related to the in vivo interaction of human lung tumor cells and immune effector cells, we obtained an HLA class I + lung tumor cell line from a fresh surgical specimen, and using the infiltrating immune cells, isolated and characterized tumor antigen-specific, CD8+ CTLs. We then established a SCID mouse-human tumor xenograft model with the tumor cell line and used it to study the function of the autologous CTLs provided via adoptive transfer.ResultsThe tumor antigen specific CTLs isolated from the tumor were found to have an activated memory phenotype and able to kill tumor cells in an antigen specific manner in vitro. Additionally, the tumor antigen-specific CTLs were fully capable of homing to and killing autologous tumors in vivo, and expressing IFN-γ, each in an antigen-dependent manner. A single injection of these CTLs was able to provide significant but temporary control of the growth of autologous tumors in vivo without the need for IL-2. The timing of injection of CTLs played an essential role in the outcome of tumor growth control. Moreover, immunohistochemical analysis of surviving tumor cells following CTL treatment indicated that the surviving tumor cells expressed reduced MHC class I antigens on their surface.ConclusionThese studies confirm and extend previous studies and provide additional information regarding the characteristics of CTLs which can be found within a patient's tumor. Moreover, the in vivo model described here provides a unique window for observing events that may also occur in patients undergoing adoptive cellular immunotherapy as effector cells seek and destroy areas of tumor growth and for testing strategies to improve clinical effectiveness.

Derivation of Tumor-specific Cytolytic T-Cell Clones from Two Lung Cancer Patients with Long Survival

We derived lung carcinoma cell lines from tumor material resected from a patient with small-cell lung cancer (SCLC) and from a patient with non-small-cell lung cancer (NSCLC). The patient with NSCLC was vaccinated with irradiated autologous tumor cells. The two patients enjoyed an exceptionally favorable clinical evolution and are currently without signs of cancer 10 and 8 yr after their diagnoses, respectively. Autologous mixed lymphocyte-tumor cell cultures (MLTC) were produced with blood lymphocytes stimulated with irradiated autologous tumor cells. The first patient's SCLC cells, which carried a small amount of human leukocyte antigen (HLA) class I molecules, were incubated with interferon-gamma (IFN-gamma) before being used as stimulator cells. A cytolytic T-lymphocyte (CTL) clone was derived that specifically lysed the IFN-gamma-treated SCLC cells but did not lyse untreated tumor cells or autologous lymphoblasts. Clones of autologous tumor-specific CTL, directed against the NSCLC cells of the other patient, were also obtained. These tumor cells carried a higher level of HLA class I molecules and were lysed by the CTL without incubation with IFN-gamma. Altogether, these results indicate that SCLC and NSCLC cancer cells can be recognized by autologous CTL, and might therefore be susceptible to specific immunotherapy.

Autologous strain-specific cytolytic T lymphocyte responses directed against human immunodeficiency virus type 1 Env.

The Env glycoprotein of human immunodeficiency virus is critical for the pathogenesis of the acquired immunodeficiency syndrome, and has been the prime target for candidate HIV-1 vaccines. Cytolytic T lymphocytes (CTLs) may be important for the immunologic control of HIV infection and HIV-1 Env-specific cytolytic T cells have been isolated from infected individuals and seronegative recipients of HIV-1 vaccines. Most prior studies have used assays that detect Env-specific CTLs directed against standard laboratory viral variants. These studies may be limited because the Env proteins of these laboratory strains (for example, LAI and MN) may differ significantly from the Env proteins from primary HIV-1 strains, and a single amino acid change can abrogate the recognition of HIV-1 Env by some CTL clones. Therefore, this study measured CTL activity directed against HIV-1 Env representing the infected individual's (autologous) HIV-1 viral variants. For two HIV-1-infected individuals, recombinant vaccina viruses expressing cloned HIV-1 env genes were constructed. Using an in vitro stimulation method, strain-specific CTL activity directed against autologous HIV-1 Env was detected in both individuals. From one subject, strain-specific CTL clones directed against autologous and HIV-1LAI Env were characterized. Therefore, some infected individuals have Env-specific CTLs directed against autologous strains of HIV-1. Detection and characterization of autologous Env-specific CTL activity may have important implications relative to the current HIV-1 vaccine development strategies focusing on Env derived from laboratory strains of HIV-1.

The expression of mouse gene P1A in testis does not prevent safe induction of cytolytic T cells against a P1A‐encoded tumor antigen

Tumor antigen P815AB is recognized by cytolytic T lymphocytes (CTL) on mouse mastocytoma P815. This antigen is encoded by P1A, a gene activated in several tumors but silent in normal tissues except for testis and placenta. Notwithstanding the expression of P1A in testis, we found that male mice mounted P815AB-specific CTL responses as efficiently as females. The responding males remained fertile and no autoimmune lesions were observed in their testes. By immunohistochemistry with a rabbit antiserum directed against the P1A protein, we identified spermatogonia as the testicular cells expressing P1A. The absence of MHC class-1 molecules on spermatogonia could be one of the mechanisms of protection against testicular autoimmunity, as the antigenic peptide should not be displayed at the cell surface. Human genes MAGE, BAGE and GAGE, which also code for tumor antigens recognized by autologous CTL, are not expressed in normal tissues other than testis. The results obtained in mice with antigen P815AB suggest that immunization of human males with such antigens will not generate autoimmune side-effects. Although P1A is strongly expressed in placenta, we also found that gestation did not prevent generation of CTL responses against antigen P815AB, and that such CTL responses did not affect gestation outcome. We identified labyrinthine trophoblasts as the placental cells expressing P1A. Again, the absence of MHC class-1 molecules on these cells provides a plausible explanation for placental protection, although other mechanisms may also play a role. Int. J. Cancer, 70:349–356, 1997. © 1997 Wiley-Liss, Inc.

The expression of mouse gene P1A in testis does not prevent safe induction of cytolytic T cells against a P1A-encoded tumor antigen.

Tumor antigen P815AB is recognized by cytolytic T lymphocytes (CTL) on mouse mastocytoma P815. This antigen is encoded by P1A, a gene activated in several tumors but silent in normal tissues except for testis and placenta. Notwithstanding the expression of P1A in testis, we found that male mice mounted P815AB-specific CTL responses as efficiently as females. The responding males remained fertile and no autoimmune lesions were observed in their testes. By immunohistochemistry with a rabbit antiserum directed against the P1A protein, we identified spermatogonia as the testicular cells expressing P1A. The absence of MHC class-I molecules on spermatogonia could be one of the mechanisms of protection against testicular autoimmunity, as the antigenic peptide should not be displayed at the cell surface. Human genes MAGE, BAGE and GAGE, which also code for tumor antigens recognized by autologous CTL, are not expressed in normal tissues other than testis. The results obtained in mice with antigen P815AB suggest that immunization of human males with such antigens will not generate autoimmune side-effects. Although P1A is strongly expressed in placenta, we also found that gestation did not prevent generation of CTL responses against antigen P815AB, and that such CTL responses did not affect gestation outcome. We identified labyrinthine trophoblasts as the placental cells expressing P1A. Again, the absence of MHC class-I molecules on these cells provides a plausible explanation for placental protection, although other mechanisms may also play a role.

A new gene coding for an antigen recognized by autologous cytolytic T lymphocytes on a human renal carcinoma

Previous reports have described antigens that are recognized on human melanoma cells by autologous cytolytic T lymphocytes (CTL). The genes coding for a number of these antigens have been identified. Here we report the cloning of a gene that codes for an antigen recognized by autologous CTL on a human renal carcinoma cell line. This antigen is presented by HLA-B7 and is encoded by a new gene that we have named RAGE1. No expression of RAGE1 was found in normal tissues other than retina. RAGE1 expression was found in only one of 57 renal cell carcinoma samples, and also in some sarcomas, infiltrating bladder carcinomas, and melanomas. This represents the first identification of an antigen recognized by autologous CTL on a renal tumor.

The Majority of Autologous Cytolytic T-Lymphocyte Clones Derived from Peripheral Blood Lymphocytes of a Melanoma Patient Recognize an Antigenic Peptide Derived from Gene Pmel17/gp100

Anti-melanoma cytolytic T-lymphocyte (CTL) clones were derived from peripheral blood lymphocytes of HLA-A2 melanoma patient LB265 after stimulation with the autologous tumor cell line LB265-MEL, which showed high expression of melanocyte-lineage specific genes. Of 55 CTL clones, 46 recognized HLA-A2-restricted antigens. These 46 CTL clones were studied for their ability to specifically release tumor necrosis factor in the presence of COS cells cotransfected with the HLA-A2 gene and the cDNA of either tyrosinase, Melan-A/MART1, Pmel17/gpl00, gp75/TRP1, or MSH receptor. Six CTL clones recognized the Melan-A/MART1 antigen, whereas the remaining 40 CTL clones recognized a Pmel17/gp100 antigen. These 40 anti-PmelI7/gpl00 CTL clones were all able to lyse T2 cells pulsed with the antigenic peptide YLEPGPVTA, as previously reported. The T-cell receptor beta chain hypervariable region was sequenced and found to be identical in the 15 CTL clones analyzed. Taken together, these data show a high frequency of Pmell7/gp100-specific T cells in autologous antitumor CTL clones derived from peripheral blood of a melanoma patient.

Differences in the antigens recognized by cytolytic T cells on two successive metastases of a melanoma patient are consistent with immune selection

We have studied the patterns of antigens recognized by autologous cytolytic T lymphocytes (CTL) on two melanoma cell lines derived from metastases that were removed from patient LB33 at several years distance. Cell line LB33-MEL.A was obtained after surgery in 1988. A large number of CTL clones directed against LB33-MEL.A was obtained with blood lymphocytes collected from the patient in 1990. In vitro selection of melanoma cells that were resistant to these CTL clones indicated that at least five different antigens were recognized on LB33-MEL.A by autologous CTL. Four of these antigens were found to be presented by HLA-A28, B13, B44 and Cw6, respectively. The patient remained disease-free until 1993 when a metastasis was detected and was used to obtain cell line LB33-MEL.B. This cell line proved resistant to lysis by all the CTL clones directed against the LB33-MEL.A cells and showed no expression of HLA class I molecules except for HLA-A24. Using LB33-MEL.B cells to stimulate blood lymphocytes collected from the patient in 1994 we derived CTL clones that lysed these cells. All these CTL clones recognized a new antigen presented by HLA-A24. These results suggest that in patient LB33 the melanoma cells may have lost the expression of several HLA molecules under the selective pressure of an anti-tumor CTL response.

BAGE: a new gene encoding an antigen recognized on human melanomas by cytolytic T lymphocytes

Several tumor antigens are recognized by autologous cytolytic T lymphocytes (CTL) on human melanoma MZ2-MEL. Some of them are encoded by genes MAGE-1 and MAGE-3, which are not expressed in normal tissues except in testis. Here, we report the identification of a new gene that codes for another of these antigens. This gene, named BAGE, codes for a putative protein of 43 aa and seems to belong to a family of several genes. The antigen recognized by the autologous CTL consists of BAGE-encoded peptide AARAVFLAL bound to an HLA-Cw 1601 molecule. Gene BAGE is expressed in 22% of melanomas, 30% of infiltrating bladder carcinomas, 10% of mammary carcinomas, 8% of head and neck squamous cell carcinomas, and 6% of non-small cell lung carcinomas. Like the MAGE genes, it is silent in normal tissues with the exception of testis. Because of its tumor-specific expression, the BAGE-encoded antigen may prove useful for cancer immunotherapy.

Analysis of antigens recognized on human melanoma cells by A2‐restricted cytolytic t lymphocytes (CTL)

We have pursued our analysis of potential tumor-rejection antigens recognized on human melanoma by autologous cytolytic T lymphocytes (CTL). We reported previously that 3 distinct antigens (A,B,C) were recognized on melanoma cell line SK29-MEL in association with HLA-A2. Selection for melanoma-cell variants resistant to anti-A CTL revealed that antigen A consists of at least 2 determinants (Aa, Ab) which can be lost separately. Genetic linkage between Aa and Ab was suggested by concomitant loss of Aa and Ab in an immunoselected tumor-cell variant. This variant was also resistant to an autologous CTL clone restricted by HLA-B45, indicating that this CTL may also recognize a determinant of antigen A. Of 11 allogeneic HLA-A2 melanoma cell lines that were tested, 5 expressed both Aa and Ab, 1 expressed only Aa, and 1 only Ab. None of them was lysed by anti-B or anti-C CTL clones. A CTL clone derived from another HLA-A2-melanoma patient was found to have exactly the same lytic pattern as the anti-Ab CTL of the first patient. This suggested that it may be possible to elicit an anti-Ab response in many HLA-A2 patients. We conclude that there are at least 2 distinct antigens presented in association with HLA-A2 that are common to many melanomas and therefore constitute promising targets for specific immunotherapy.

Lysis of human pancreatic adenocarcinoma cells by autologous hla‐class I‐restricted cytolytic T‐lymphocyte (CTL) clones

From the primary site of a pancreatic adenocarcinoma (patient BE) a permanent cell line (MZ-PC-2) was established in tissue culture. In the course of mixed lymphocyte-tumor-cell cultures (MLTC) with autologous blood-derived lymphocytes, we isolated CTL clones that lysed autologous tumor cells but not autologous EBV-transformed B cells (EBV-B) and not K562. Pre-treatment of MZ-PC-2 cells with IFN-gamma was required to obtain significant lysis in 4-hr cytotoxicity assays. IFN-gamma was superior to IFN-alpha in that respect. Among MLTC responder lymphocytes, tumor-reactive CTL proliferated more strongly in response to MZ-PC-2 cells treated with IFN-gamma than to untreated tumor cells. Three CTL clones derived from MLTC were chosen for further analysis. They were CD3+, CD8+, TCR-alpha/beta+ and behaved identically in all functional aspects tested. They all expressed the same TCR-beta chain, indicating that they descended from a common precursor lymphocyte and were directed against the same antigen. According to antibody-inhibition experiments, BE-CTL recognized their targets via an HLA-B molecule carrying the Bw6 supertypic determinant. Irrespective of pre-incubation with IFN-gamma, low levels of tumor-cell lysis, or none, were seen when MZ-PC-2 cells were kept in medium supplemented with autologous serum or serum pooled from healthy volunteers instead of FCS. Lysability was restored when TNF-alpha was added to human serum. Serum-free medium was found to enhance the susceptibility of MZ-PC-2 cells to lysis by autologous CTL.

Antigens recognized on a melanoma cell line by autologous cytolytic T lymphocytes are also expressed on freshly collected tumor cells

Peripheral blood lymphocytes of a melanoma patient were stimulated in vitro with a permanent cell line derived from the autologous tumor. Stable cytolytic T lymphocyte (CTL) clones were obtained that lysed the melanoma cell line and did not lyse autologous Epstein-Barr virus-transformed B lymphocytes or K-562 cells. These CTL clones were directed against two distinct antigens on the melanoma line. In view of the possibility that these antigens could be culture artefacts, we tested the stimulatory ability of tumor cells that had been freshly collected from metastatic relapses on the CTL clones. A considerable CTL proliferation was observed and it appeared to be specific. We conclude that the antigens recognized by the autologous CTL clones on the permanent melanoma cell line were expressed by the tumor cells in the patient.

Lysis of human melanoma cells by autologous cytolytic T cell clones. Identification of human histocompatibility leukocyte antigen A2 as a restriction element for three different antigens.

From the peripheral blood of the melanoma patient (AV), we derived cytolytic T lymphocyte (CTL) clones that lysed the autologous tumor line SK-MEL-29, but not autologous EBV-B cells, K562, and other tumor targets. By immunoselection experiments it was shown that the CTL clones recognized at least three different antigens on the autologous tumor cells. We demonstrate here that these melanoma antigens are presented to the CTL in association with HLA-A2. First, HLA-A2-reactive pregnancy sera as well as an mAb against HLA-A2 inhibited the CTL lysis. Second, immunoselected melanoma subclones that were resistant to lysis by CTL clones against the three antigens described were found to lack expression of HLA-A2. By sensitizing the patient's lymphocytes against an HLA-A2- melanoma clone, we established a new series of CTL clones recognizing autologous AV melanoma cells. However, efficient lysis was only seen when target cells were pretreated with IFN-gamma. The lytic activity of these CTL was selectively inhibited by an mAb against a common HLA-B determinant. These results indicate that in addition to HLA-A2, other class I antigens are involved in the recognition of AV melanoma cells by autologous CTL.