Infiltrating lymphocytes in undifferentiated nasopharyngeal cancer lack metallothionein expression.

Metallothionein (MT) is a metal-binding protein with functional roles in cell growth, repair and differentiation. MT is reported to be differentially expressed in lymphocytes of malignant gastrointestinal lesions. The level of MT protein was examined by immunohistochemical analysis at light microscopic and ultrastructural level in infiltrating lymphocytes from 20 cases of undifferentiated nasopharyngeal carcinoma (NPC). MT expression was found to be absent in the infiltrating lymphocytes of NPC and in reactive lymphocytes of lymphoid hyperplasia in nasopharyngeal tissues. Ultrastructural examination confirmed the absence of MT immunoreactivity in the lymphoid infiltrate of NPC. On the other hand, malignant lymphoblasts of diffuse large cell lymphoma, showed MT-immunopositivity by immunoelectron microscopy. This study demonstrates a lack of MT expression in the lymphoid stroma of undifferentiated NPC, a further characteristic of its non-neoplastic nature.

Detection of chromosomal aberrations in tumor cells and tumor infiltrating lymphocytes by molecular cytogenetics in patients with gynecological cancer.

Conventional cytogenetic studies of tumor cells from patients with breast or ovarian cancer have shown multiple chromosomal abnormalities including chromosomes 7, 12, and 17. This study was designed to analyze the cytogenetic features of tumor cells and tumor infiltrating lymphocytes (TILs) by using a combination of magnetic activated cell sorting (MACS) and fluorescence in situ hybridization (FISH). Tumor cell, peripheral blood (PB), and TIL samples from 37 patients (20 ovarian tumors, 13 breast cancers, 3 uterine sarcoma, 1 carcinoma of the filamentary tube) were analyzed for the presence of numerical aberrations of chromosomes 7, 12, and 17. All of the tumor cells showed a high frequency of numerical aberrations of chromosomes 7, 12, and 17, especially trisomies or tetrasomies. There was no statistically significant difference in the incidence of chromosomal abnormalities in tumor tissue and effusions, or between primary and relapsed disease in patients with breast or ovarian tumors. However, tumor cells from patients with solid metastatic disease had significantly higher numbers of aberrations of chromosome 7 in the primary tumor than in tumors from patients without metastases (P = 0.049), suggesting that chromosome 7 is frequently involved in the progression of disease. Monosomies and trisomies of chromosomes 7 and 12 also occurred at a low percentage of TILs without any statistically significant difference between primary and relapsed tumors. The presence of these aneuploidies might be responsible for treatment failures in the immunotherapy of gynecological cancer.

Molecular characterisation of tumour infiltrating lymphocytes in oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is often associated with a lymphocytic infiltrate that is believed to represent an in vivo immune reaction to the tumour cells. In this study, the tumour-infiltrating lymphocytes (TIL) associated with primary OSCC were characterised molecularly in six newly-diagnosed patients to determine the nature of the immune response to the primary tumour. The primary tumours in three of the six patients were associated with a moderate to dense CD8+ T cell infiltrate whilst the cellular infiltrate in the other primary tumours was sparse. The CD3+ T cells were also HLA-DR+. In all cases, there were few CD56+ cells, suggesting that TIL were predominantly T cells bearing the alpha/beta T cell receptors (TCR). The TCR Vbeta repertoire of TIL in these six cases was analysed. TCR Vbeta gene usage by TIL was heterogeneous. A restricted usage of TCR Vbeta genes by TIL was evident in two tumours associated with a dense CD3+ T cell infiltrate. In one of these, there was histological evidence of tumour cell destruction by TIL. Further analysis of the predominant TCR Vbeta gene family used by TIL in this individual showed a unique in-frame nucleotide sequence in 100% of the transcripts. This recurrent transcript was not detected in the peripheral blood of this patient, indicating a local T cell clonal expansion in the vicinity of the tumour. Overall, these results suggest that activation and clonal expansion of T cells occurs and may play a role in local tumour destruction in OSCC.

[Ultrastructural study of tumor infiltrating lymphocyte in tissues of laryngeal squamous cell carcinoma].

By transmission electron microscopy tumor infiltrating lymphocyte (TIL) in laryngeal squamous cell carcinoma tissues of 14 cases was studied. The results showed that the infiltrating lymphocyte in varying degrees eristed in laryngeal squamous cell carcinoma tissues. Most of them were present in a motionless state, and the others showed the feature of activated metrocyte and closely contracted with cancer cells and resulted in distinct morphological changes both the cancer cells and themselves. Therefore, this study provided an important morphological evidence for TIL as killer and inhibiter to the growth of cancer cells.

Growth and immunogenicity of human glioma in severe combined immunodeficiency-human mice.

The severe combined immunodeficiency (SCID) mouse was investigated as a model system to study the growth and immunogenicity of human gliomas. Human glioma cell lines U-251MG, KNS-42, SF-188, A-172, and T-98G were injected subcutaneously into SCID mice. The cell lines U-251MG and KNS-42 grew as large, subcutaneous masses; SF-188, A-172, and T-98G did not grow. Glioma-immune system interactions were studied by the transplantation of human peripheral blood lymphocytes into tumor-bearing SCID mice. In the resultant SCID-human (SCID-hu) mice, transplanted lymphocytes infiltrated into the subcutaneously growing tumors and expressed the surface markers of human B, T, and natural killer cells. The SCID-hu mouse is a potentially powerful model to study the basic tumor biology of some human gliomas and also represents a useful screening system to evaluate experimental immunotherapies for brain tumors.

Interleukin 3 enhances cytotoxic T lymphocyte development and class I major histocompatibility complex "re-presentation" of exogenous antigen by tumor-infiltrating antigen-presenting cells.

We show that interleukin 3 (IL-3) enhances the generation of tumor-specific cytotoxic T lymphocytes (CTLs) through the stimulation of host antigen-presenting cells (APCs). The BALB/c (H-2d) spontaneous lung carcinoma line 1 was modified by gene transfection to express ovalbumin as a nominal "tumor antigen" and to secrete IL-3, a cytokine enhancing myeloid development. IL-3-transfected tumor cells are less tumorigenic than the parental cell line, and tumor-infiltrating lymphocytes isolated from these tumors contain increased numbers of tumor-specific CTLs. By using B3Z86/90.14 (B3Z), a unique T-cell hybridoma system restricted to ovalbumin/H-2b and implanting the tumors in (BALB/c x C57BL/6)F1 (H-2d/b) mice, we demonstrate that the IL-3-transfected tumors contain an increased number of a rare population of host cells that can process and "re-present" tumor antigen to CTLs. Electron microscopy allowed direct visualization of these host APCs, and these studies, along with surface marker phenotyping, indicate that these APCs are macrophage-like. The identification of these cells and their enhancement by IL-3 offers a new opportunity for tumor immunotherapy.

T cell receptor Vbeta repertoire of tumour-infiltrating lymphocytes in oral squamous-cell carcinoma.

Oral squamous cell carcinoma (OSCC) are frequently infiltrated by tumour infiltrating lymphocytes (TILs) which may demonstrate specific anti-tumour cytotoxicity. We have used a panel of family-specific primers in reverse-transcriptase (RT)-polymerase chain reactions (PCR) to determine the TCR Vbeta repertoire of TILs in the primary and metastatic tumours in patients with OSCC. We observed a diverse and heterogenous usage of TCR Vbeta genes by TILs in the primary tumours, a scenario not unlike normal oral mucosal lymphocytes. In one patient with a longstanding history of OSCC, the repertoire was restricted. Restricted TCR Vbeta gene usage also occurred in TILs of metastatic tumours. Within individual patients, discrepancies of TCR Vbeta gene usage were also observed between TILs in the primary OSCC and those in metastatic lymph nodes. Work is ongoing to determine the clonality and functional significance of these TILs.

Analysis of T cell receptor alpha beta variability in tumor-infiltrating lymphocytes in primary and metastatic melanoma.

The T cell receptor (TCR) alpha beta variable (V) gene family usage of tumor-infiltrating lymphocytes (TIL) in different primary human malignant melanomas and corresponding metastatic lesions were characterized using a recently developed method using the reverse transcription coupled polymerase chain reaction (RT-PCR). This semiquantitative RT-PCR method could be adapted to analysis of formalin-fixed, paraffin-embedded histopathological samples of primary tumor material and demonstrated to be reproducible and to be useful for the assessment of V alpha- and V beta-gene family usage in tumor samples. The TIL in primary tumors were observed to preferentially express certain TCR V alpha- and V beta-gene families: V alpha 4, and V beta 8 were highly expressed in several of the primary tumors analyzed using this method. With respect to V alpha 22 and V beta 8, the preferential expression of these V-gene families was demonstrated to be due in situ clonal expansion of T cells by means of cloning and sequencing of the CDR3 regions (V-J or V-D-J, respectively) corresponding to the RT-PCR products from one of the primary tumors. The observed preferential usage of certain TCR V alpha and V beta-genes strongly suggest the in situ clonal expansion of specific populations of T cells in accordance with recent results from others. These clonal T cell populations probably react with certain melanoma-associated peptides presented by specific HLA molecules. The preferential usage of certain V alpha- and V beta-gene families observed in several tumors further supports the involvement of a limited number of shared melanocyte or melanoma-associated peptides. Since the HLA status of the patients is obviously important to interpret these results, some of the patients were typed for HLA-A1 and -A2, the two most well-characterized restriction elements for melanoma-associated antigens, either serologically or by a newly developed RT-PCR method which similarly could by applied directly to the tumor material. In TIL in primary melanomas, a possible correlation was suggested between HLA-A2 and the preferential usage of the TCR V-gene families V alpha 4, V alpha 5, V alpha 22 and V beta 8, whereas the V beta 3-gene family appeared to be expressed together with HLA-A1. The V-gene families which were highly expressed in the primary tumors were generally not, or only very weakly, expressed in the corresponding metastases and vice versa, possibly reflecting a substantial change in the phenotype of the metastatic melanoma target cells. Continued studies of larger patient materials will be necessary to extend and validate these conclusions and of obvious interest for the further analysis of the T cell response in melanoma.

Analysis of T cell receptor alpha beta variability in lymphocytes infiltrating melanoma primary tumours and metastatic lesions.

The T cell receptor (TCR) alpha beta variable (V) gene family usage of tumour-infiltrating lymphocytes (TIL) in four different primary human malignant melanomas and their corresponding metastatic lesions was characterized using a recently developed method based on the reverse-transcription-coupled polymerase chain reaction (RT-PCR). All patients were typed for HLA-A1 and -A2, either serologically or by a newly developed RT-PCR method. Two of these patients expressed HLA-A2, one the HLA-A1 haplotype and one further patient was heterozygous HLA-A1/-A2. The prognostic parameters for all four patients indicated that rapid progression of the disease was to be expected. However, only two of the patients showed rapid progression, while the remaining two patients are still alive after more than 3 years. In TIL in primary melanomas, a possible correlation was suggested between HLA-A2 and the preferential usage of the TCR V gene families V alpha 4, V alpha 5, V alpha 22 and V beta 8, whereas the V beta 3 gene family appeared to be expressed together with HLA-A1. Other highly expressed V gene families, apparently not restricted to either HLA-A1 or -A2, were V alpha 1 (expressed in three of four primary tumours) and V alpha 21 (expressed in two of four tumours). We found no evidence suggesting any correlations between the haplotypes HLA-A1 and -A2 and preferential V gene family expression in the metastatic lesions, and the only common feature was V alpha 8, which was found to be highly expressed in two out of three subcutaneous metastases. The V gene families, which were highly expressed in the primary tumour were generally not, or only very weakly, expressed in metastases and vice versa, possibly reflecting a change in the phenotype of the metastatic melanoma target cells. With regards to patient 0368, it was possible to obtain and study material from two subcutaneous metastases. The first metastasis was excised more than a year after the primary tumour, showing a completely different V region repertoire. The second metastasis was excised at surgery 2 years after primary surgery and likewise showed a dramatic shift in comparison to the first subcutaneous metastasis. Although the present study only included a small number of patients, it suggests that the estimation of V gene expression, if applied to a larger amount of patient material, might make it possible to substantiate further the suggested correlations between the T cell response against the tumour, HLA and antigen expression.(ABSTRACT TRUNCATED AT 400 WORDS)

T lymphocytes infiltrating renal cell carcinoma have a reduced expression of transferrin receptor.

T-cell responses have been reported to be impaired in cancer patients, and lymphocytes infiltrating human tumors (T-TIL) appear to be more affected than those in the peripheral blood. T-TIL display a poor proliferative response when compared to peripheral blood T (T-PBL) cells that show a strong response to all stimuli. Here we report that T-TIL from patients with renal cell carcinoma (RCC) also have a defect in transferrin receptor (TfR) expression that is not present in T-PBL cells. Immunocytometry studies (dual staining for CD3 epsilon and TfR) demonstrated that autologous T cells from the peripheral blood but not from the tumor expressed TfR following stimulation with IL2, anti-CD3 or PHA. Expression of TfR correlated with the capacity of T cells from the blood and tumor to proliferate. Gene expression studies using reverse transcriptase-polymerase chain reaction (RT-PCR) demonstrated that TfR mRNA levels in T-TIL were undetectable or low relative to T-PBL following stimulation. The failure to detect TfR mRNA in T-TIL after stimulation was not due to a shift in kinetics of mRNA accumulation since TfR mRNA was not detectable at any of the times tested (4, 12, 24 and 36 hr). The defect in TfR gene expression is selective since IL2R alpha gene expression was induced in T-TIL. Because IL2 binding to its receptor results in TfR expression, the defect in TfR induction in T-TIL appears to be distal to IL2R alpha expression. Our studies illustrate another alteration in T-TIL that is not observed in T cells from the peripheral blood. The absence of TfR gene expression may contribute to the poor proliferative response of T cells from the tumor.

Analysis of T cell receptor variability in fresh tumor-infiltrating lymphocytes from human head and neck cancer.

In this study, we analyzed T cell receptor (TCR) gene rearrangements in tumor-infiltrating lymphocytes (TIL) freshly obtained from 15 patients with head and neck cancer using the reversely transcribed polymerase chain reaction (RT-PCR) method. These TILs showed preferential expression of V alpha 10, V alpha 8 and V alpha 1, detected in 13 (87%), 11 (73%), and 9 cases (60%), respectively. The TCRV beta gene revealed diversity without preferential usage. The head and neck region is exposed to bacteria and viruses, so it is possible that the tumor site can become infected and accumulate T cells involved in infection and inflammation. Therefore, we also investigated TCR gene usage in T cells infiltrating in chronic sinusitis mucosa to address the question of whether the V alpha 1, V alpha 8, and V alpha 10 subfamilies are characteristic in TIL from squamous cell carcinoma of head and neck. TCR V alpha 10 gene usage was also the most common in V alpha segment in T cells infiltrating the sinus mucosa, but V alpha 1 and V alpha 8 were not detected in the T cells in sinusitis. These results indicate that the V alpha 10 subfamily, the preferred T cell population in both TIL and T cells in inflammatory disease, might be involved mainly in inflammation or infection. On the other hand, V alpha 1 and V alpha 8 appear to be relatively specific populations for antitumor immunity in head and neck cancer.

Ligation of CD3 triggers transmembrane proximity between LFA-1 and cortical microfilaments in a cytotoxic T cell clone derived from tumor infiltrating lymphocytes: a quantitative resonance energy transfer microscopy study.

We have explored the transmembrane associations of leukocyte function associated antigen-1 (LFA-1) in response to T cell receptor ligation using resonance energy transfer (r.e.t.) microscopy to detect receptor to microfilament proximity. R.e.t. was detected using both imaging and photon counting techniques. T cells were labeled with fluorescein-conjugated F(ab')2 fragments of an anti-LFA-1 monoclonal antibody. Cells were incubated at 37 degrees C on unmodified glass surfaces and surfaces coated with anti-CD3 or anti-H-9 antibodies. Microfilaments of fixed cells were labeled with rhodamine-phalloidin. R.e.t. was not affected on unmodified (blank) or irrelevant antibody-treated (H-9) surfaces. However, both fluorescence images and photon count rates were significantly enhanced when cells bound to anti-CD3-coated surfaces. This enhancement was not due to a general effect of T cell activation on transmembrane cytoskeletal proximity since CD45-phalloidin r.e.t. was not affected by CD3 ligation. These experiments provide direct physical evidence that ligation of the CD3 complex specifically increases the proximity of LFA-1 and microfilaments, which may be relevant to T cell mediated adherence reactions.

Some remarks on adherence reactivity of autologous PHA--transformed lymphocytes on human glial tumor cells in vitro.

In 16 patients with primary supratentorial and in 1 with cerebellar tumor among them 5 multiforme glioblastomas, 3 malignant astrocytomas, 6 astrocytomas of other subtypes and 1 mixed glioma (oligo-astrocytoma) the peripheral blood was drawn and lymphocytes were separated from it. Out of the removed part of the tumors about 10 cultures in each case were prepared and cultivated at least 14 days. When the growth zone was well developed the cultures were used for further studies. All samples of the separated lymphocytes activated with PHA were cultivated for 72 h. So prepared lymphocytes were added to the tumor culture in vitro and observed for 24 h. After that time the not adhered lymphocytes were removed and the remaining tissue cultures with adhered lymphocytes were fixed and stained and the number of lymphocytes was counted. It was found that 1 h after the addition of lymphocytes the number of lymphocytes was very high, though a great part of them did not adhere to the tumor tissue. After 24 h the number of adhered lymphocytes was small or minute. Taking into consideration the results obtained it seems that the low efficiency of therapy of gliomas with autologous lymphocytes in vivo can result from the very weak direct contact with tumor cells. The influence of lymphocytes can be very limited so more as the tumor cells can secret biological active substances like PGE2, which counteract the cytotoxic activity of lymphocytes. For that reason the number of lymphocytes can be of significant value as counterbalance to that properties of the tumor cells. Taking into account even secretion of tumor cells of the same biological active substances such as PGE2 which counteract the cytotoxic activity of lymphocytes for the efficient activity of lymphocytes the number of them may be of significant value.

Unrestricted T-cell receptor V-region gene repertoire in tumor-infiltrating lymphocytes from human breast carcinomas.

BACKGROUND: Several lines of evidence indicate that a more favorable prognosis is correlated with the degree of lymphocyte infiltration within breast carcinomas. The characterization of these T-cell infiltrates appears necessary to explore the possible existence of an in situ immunologic response of the host against neoplastic cells. Previous studies have indeed suggested that the T-cell receptor (TCR) repertoire of tumor-infiltrating T-lymphocytes (TIL) should be restricted if they act specifically against tumor-related antigens. METHODS: To address this question, the expression of variable (V) region genes of the TCR-alpha and beta chains in intratumoral lymphocytes from five breast carcinoma biopsy specimens and one normal mammary gland specimen was analyzed using the polymerase chain reaction (PCR). The neoplastic samples included one medullary carcinoma and four ductal infiltrating carcinomas selected on the basis of a rich lymphoid stromal component. Primers specific for 18 different V alpha and 21 V beta families were used for PCR. RESULTS: In every case, TIL showed an unrestricted pattern of TCR V-region gene expression, similar to the repertoire observed in peripheral blood lymphocytes and in the normal breast tissue. CONCLUSIONS: According to these nonquantitative PCR experiments, the apparent lack of selection of a limited number of T-cell specificities in the affected tissues does not favor the existence of an in vivo lymphoid recruitment based on TCR recognition of neoplastic antigenic determinants. Further studies, however, leading to an accurate quantification of immunologically relevant T-cell clones and including larger series of cases still are necessary before it will be possible to draw a final conclusion.

Autopsy report of primary CNS B-cell lymphoma indistinguishable from multiple sclerosis: diagnosis with the immunoglobulin gene rearrangements analysis.

We report a case of primary CNS B-cell lymphoma indistinguishable from multiple sclerosis (MS). MRI of the head showed the spontaneous disappearance of the white matter lesions and the progressive cerebral atrophy. The brain biopsy failed to make a diagnosis of CNS lymphoma but rather suggested MS. Although the primary CNS lymphoma was suspected at autopsy, the immunohistochemical study showed the CNS-infiltrating lymphoid cells comprising both T-cells and B-cells. Analysis of the immunoglobulin and T-cell receptor gene rearrangements first provided evidence of primary CNS B-cell lymphoma.

Induction of interleukin-2 receptor by tumor necrosis factor alpha on cultured ovarian tumor-associated lymphocytes.

We have recently reported that autologous tumor-specific cytotoxic T lymphocyte (CTL) lines and clones can be developed from lymphocytes infiltrating ovarian malignant ascites (TAL). In this study, we investigated the biological effects of tumor necrosis factor alpha (TNF alpha) in the induction, expansion, long-term proliferation and lytic function of CD8+ TAL. TNF alpha up-regulated the IL-2 receptor (IL-2R) alpha chain (Tac antigen) on the surface of CD3+ CD8+ CD4- TAL, enhanced the proliferation of autologous tumor-specific CTL, and potentiated their lytic function in long-term cultures. Furthermore, in the induction and expansion phase of CD8+ TAL, the presence of TNF alpha was associated with a selective increase in CD8+ IL-2R+ (Tac+) cells, and subsequent decrease in CD4+ IL-2R+ (Tac+) cells. These results suggest that the observed facilitation of the outgrowth of CD8+ cells in TAL cultures may be due, at least in part, to the up-regulation of IL-2R, and indicate the usefulness of TNF alpha in the analysis of signalling in autologous tumor-reactive CTL.

Oligoclonal T lymphocytes infiltrating human lung cancer tissues.

To clarify the nature of tumor-infiltrating lymphocytes (TILs), we investigated the possible clonality of the T cells in TILs freshly isolated from human primary lung cancer tissues by assessing the rearrangement pattern of the T-cell receptor (TCR) gene beta locus using Southern blotting. First, in phenotypic analysis, TILs represented different populations among corresponding peripheral blood lymphocytes (PBLs) with an increased proportion of CD20+ (B) cells as well as a decreased proportion of CD16+ (natural killer) cells, and a variable CD4/CD8 ratio. Considering the central role of T cells in immune responses, we analyzed TCR beta gene rearrangement patterns in TILs and corresponding PBLs from 12 patients. In 10 of the 12 cases, TILs showed one or more TCR gene rearrangement bands with a predominance of the C beta 2 gene, in which 2 types of common rearranged band were observed among the cases with different clinical profiles in terms of histological types and disease stage, with bands at about 9.5 kb in 7 and at 11.5 kb in 8 patients. On the other hand, predominant rearranged bands were hardly detected in corresponding PBLs except in 2 cases. From these results, we conclude that TILs in lung cancer tissues frequently contain oligoclonal T-cell populations, which were probably sensitized by relatively common antigens at the tumor sites.

Ultrastructure of IL2-stimulated tumor-infiltrating lymphocytes showing cytolytic activity against tumor cells.

Tumor-infiltrating lymphocytes (TIL) obtained from tumor tissue and pleural effusion of breast carcinoma were cultured with interleukin-2 (IL2) and thus activated. The ultrastructure of TIL stimulated by IL2 to kill various breast carcinoma cells was then investigated. Freshly isolated TIL cultured with autologous tumor cells for 48 h without IL2 were small, round and showed neither binding to nor killing of tumor cells. TIL stimulated to proliferate by IL2 became effector cells and showed cytotoxicity against tumor cells. Ultrastructurally, the effector TIL resembled large granular lymphocytes, and adhered to tumor cells through interdigitation or close apposition of the two plasma membranes accompanied by spot-like close membrane contacts. At the site of each spot-like contact, there was a 5-nm intercellular space. The morphology of the TIL processes did not differ from those of LAK and other CTL or NK cell processes during contact, invagination or the killing of target cells. The granules in TIL were considered to participate in the cytotoxic effect. Phenotypically heterogeneous TIL, CD8+/CD57- and CD8+/CD57+, adhered to autologous tumor cells and MCF7 (human breast carcinoma cell line). However, it was unclear which cell or cells acted as the effector for tumor-cell killing.

Retrovirus-mediated gene transfer into CD4+ and CD8+ human T cell subsets derived from tumor-infiltrating lymphocytes and peripheral blood mononuclear cells.

Studies were undertaken to test the susceptibility of individual T cell subpopulations to retroviral-mediated gene transduction. Gene transfer into human tumor-infiltrating lymphocytes (TIL) or peripheral blood mononuclear cells (PBMC) was carried out by transduction with an amphotropic murine retroviral vector (LNL6 or N2) containing the bacterial neoR gene. The presence of the neoR gene in the TIL population was demonstrated by Southern blot analysis, detection of the enzymatic activity of the gene product and by the ability of transduced TIL to proliferate in high concentrations of G418, a neomycin analog that is toxic to eukaryotic cells. The presence of the neoR gene in TIL did not alter their proliferation or interleukin-2 dependence compared to nontransduced TIL. The differential susceptibility of CD4+ and CD8+ lymphoid cells to the retro-virus-mediated gene transfer was then tested. Transduction of heterogeneous TIL cultures containing both CD4+ and CD8+ cells resulted in gene insertion into both T cell subsets with no preferential transduction frequency into either CD4+ or CD8+ cells. In other experiments highly purified CD4+ and CD8+ T cell subpopulations from either TIL or PBMC could be successfully transduced with the neoR gene as demonstrated by Southern blot analysis and detection of the gene product neophosphotransferase activity. No such activity of vector DNA could be detected in controls of nontransduced cells. In these highly purified cell subsets the distinctive T cell phenotypic markers were continually expressed after transduction, G418 selection and long-term growth. Clinical trials have begun in patients with advanced cancer using heterogeneous populations of CD4+ and CD8+ gene-modified TIL.