Sylvia Miescher

Separation of tumor-infiltrating lymphocytes from tumor cells in human solid tumors: A comparison between velocity sedimentation and discontinuous density gradients

The separation of viable tumor-infiltrating lymphocytes (TIL) from surgical biopsies of human solid tumors was achieved by velocity sedimentation at unit gravity or by discontinuous density gradients. The two methods were adapted to small volumes and cell numbers not exceeding 1 X 10(8). The recovery, purity and composition of the TIL-enriched fractions were comparable in the two methods. Density gradients were more rapid, simpler and more practical for preparation under sterile conditions of TIL from clinical material than velocity sedimentation. Lymphocytes in the TIL-enriched fractions obtained by either of the methods were poorly responsive to mitogens. This poor responsiveness is a characteristic of the human TIL and seems to be related to effects exerted by tumor cells.

Clonal analysis and in situ characterization of lymphocytes infiltrating human breast carcinomas

SummaryT lymphocytes were isolated from tumor biopsies in 13 patients with breast carcinomas. Immunohistology with monoclonal antibodies confirmed the presence of mononuclear cell infiltrates composed primarily of T lymphocytes in all tumors studied. While the proportion of T lymphocytes expressing the T4 or the T8 surface marker varied from tumor to tumor as determined by morphometric analysis, T8+ cells were more numerous than T4+ cells in 8/12 breast tumors studied. Relatively few T cells (<10% in 11/12 tumors) were in an activated state as judged by the surface expression of HLA-DR antigens or the receptor for interleukin-2 (IL-2). In 1 case 20% of the infiltrating mononuclear cells were expressing the IL-2 receptor. The tumor infiltrating lymphocytes (TIL) recovered from 10 tumors were cloned in a microculture system that permits proliferation of nearly 100% of normal peripheral blood T lymphocytes (PBL-T). In contrast to normal and autologous PBL-T, frequencies of proliferating T lymphocyte precursors (PTL-P) were depressed (<0.01) in 7/10 TIL preparations indicating a decreased responsiveness of TIL to phytohemagglutinin at the single-cell level. The frequency of PTL-P was noticeably higher in 2 cases (0.03 and 0.09) and close to normal in 1 case (0.39).A total of 170 clones were expanded in vitro and analyzed for different functional capabilities. Most of these clones expressed the T4+/T8-phenotype (73%) and strikingly 53% of these T4+/T8− clones were cytolytic in a lectin-dependent assay, a functional subset which is uncommon among normal PBL-T. Some clones (10%) lysed allogeneic breast tumor cells (MCF7). Only 15% of the clones displayed natural killer activity. Among the cytolytic clones, 17 of 31 tested were also IL-2 producers irrespective of the T4 or T8 phenotype. Our results show that human mammary carcinomas contain many infiltrating T cells with cytolytic potential. Interestingly, among the proliferating cytolytic T cell clones (56% of the microcultures), many expressed the T4+/T8− phenotype. These findings may indicate that the in situ cytolytic reaction (against unknown antigens) is associated preferentially with class II antigens.

Sparse distribution of γ/δ T lymphocytes around human epithelial tumors predominantly infiltrated by primed/memory T cells

SummaryEvidence from the mouse system has suggested that T lymphocytes accumulating in non-lymphoid tissue, in particular epithelia, may preferentially express the T cell receptor (TCR) γδ. In this study, we characterize the T cell receptor αβ or γδ phenotype of lymphocytes infiltrating human tumors of epithelial origin using monoclonal antibodies (mAb) for immunohistology and flow cytometry on cells extracted by enzyme digestion. This report shows that the majority of CD3+ tumor-infiltrating lymphocytes are TCR αβ+ but a small percentage of TCR γδ can be clearly defined scattered throughout the tumor tissue with apparently no microanatomical selection. So far there has been little evidence for an accumulation of activated T cells in human tumor tissues as defined by mAb against molecules appearing transiently during the acute phase of activation. Now mAb are available that can identify primed or memory T cells such as mAb UCHL-1 recognizing the CD45RO antigen. Here we show that CD3+ tumor-infiltrating lymphocytes have a statistically significant accumulation of primed T cells, as compared to the autologous peripheral blood lymphocytes, suggesting their immune stimulation by tumor cells.

Production of tumor necrosis factor-α by naive or memory T lymphocytes activated via CD28

Abstract While it is well established that activated T cells can produce tumor necrosis factor alpha (TNF-α), it is less clear whether this function is confined to a given subset, e.g., memory cells. To approach this question, we investigated the production of TNF-α by human peripheral blood T lymphocytes activated with anti-CD28 mAb since this activation pathway is known to potentiate cytokine production. Under the culture conditions used, the amount of TNF-α produced was markedly enhanced compared to that obtained after activation with immobilized anti-CD3 mAb. The enhancement of TNF-α production was already apparent after incubation of T cells for 6 hr. Up to 5 ng/ml of TNF-α was measured on Day 2 in supernatants of cultures of 104 T lymphocytes. To determine the source of the cells producing high amounts of TNF-α, T lymphocytes were separated into two subpopulations, namely naive cells (expressing the CD45RA isoform) and memory cells (expressing the CD45RO isoform). While both subpopulations proliferated equally well after stimulation with anti-CD28 mAb, up to 90% of the TNF-α produced under these conditions originated from memory T cells. These results thus document that T cell activation via CD28 results in a marked increase in TNF-α production without affecting the functional disparity that exists between naive and memory T cells.

Effect of Transforming Growth Factor Beta on the EL4 Thymoma Variant EL4/6.1: Dissociation of Inhibition of Proliferation from Expression of IL-1 and IL-2 Receptors

In order to further characterize the action of transforming growth factor beta (TGF-beta) on lymphoid cells, we investigated the effects of porcine TGF-beta 1 and -2 on the IL-1 sensitive EL4/6.1 thymoma cell line. The proliferation of EL4/6.1 thymoma cells was inhibited by TGF-beta 1 and TGF-beta 2 (1 ng/ml) to a similar degree, the population doubling time was increased by 50-60%, total inhibition was not achieved. This decrease of proliferation was associated with an increase of the number of cells in the G0/G1 compartment of the cell cycle. TGF-beta-mediated inhibition could not be overcome by adding exogenous rIL-1 nor was the binding capacity for IL-1 reduced. In addition, TGF-beta did not interfere with the induction of IL-2 receptors by a combination of Ionomycin+PMA+IL-1. The data suggest that TGF-beta mediated inhibition of thymocyte/lymphocyte proliferation is not associated with an inhibition of the expression or the induction of expression of IL-2 or IL-1 receptors.

Stimulation of FACS-analysed CD4+ and CD8+ human tumour-infiltrating lymphocytes with ionomycin + phorbol-12,13-dibutyrate does not overcome their proliferative deficit.

Human tumour‐infiltrating lymphocytes (TIL) were prepared by enzyme digestion from a series of different tumours and were purified on a fluorescence‐activated cell sorter (FACS II) according to their CD4+ and CD8+ phenotype. CD4+ and CD8+ TIL were stimulated separately in a low density microculture system with phytohacmagglutinin (PHA) or with ionomycin plus phorbol‐12, 13‐dibutyrate (PDBu). The PHA‐induced proliferation of TIL was highly decreased when compared with control peripheral blood lymphocytes. A decreased proliferation of TIL was also observed when cells were stimulated with ionomycin plus PDBu, a combination which is thought to circumvent early events associated with lymphocyte activation. Some TIL were also plated in limiting dilution where they showed decreased frequencies of proliferating T cell precursors. The data suggest that one component of the inhibition of TIL must be acting ‘ownstream’ of the early events of lymphocyte activation.

Cytotoxic Tumor Infiltrating Lymphocytes in Nasopharyngeal Carcinoma

Tumor Infiltrating Lymphocytes (TIL) are particularly abundant in nasopharyngeal carcinoma (NPC) and participate into the “lympho-epithelial” histological pattern characteristic of this tumor. The malignant cells are of epithelial origin and it has been shown recently (1,2) that NPC tumors in nude mice constitutively express class II Major Histocompatibility Complex (MHC) antigens and produce Interleukine-1 (IL-1), a lymphokine which plays an important role in T cell migration and activation. The expression of such immuno-regulatory molecules by the malignant epithelial cells may play a key role in the striking lymphocytic infiltrate of NPC (T. TURSZ et al., this volume). The TIL are not malignant, do not contain EBV genomes and their majority belongs to the T cell lineage as shown by immunohistological studies (3,4).

Isolation, Functional Properties and Clonal Analysis of Tumor-Infiltrating Lymphocytes from Human Brain Tumors

Despite the numerous reports of decreased or defective systemic cell mediated immunity in glioma patients (1–7) there is evidence of a local immune response to the tumor. Thus mononuclear cell (MNC) infiltrates have been demonstrated within the parenchyma of human glial tumors in 30–60 % of cases reported in the literature (8, 9). Other studies have correlated the intensity of MNC infiltrates with tumor histology and survival in glioma patients (10–12). Von Hanwehr and co-workers (9) found that T lymphocytes constituted the major fraction of the infiltrates in gliomas. However the nature and functional capabilities of these cells and their potential cytolytic role in preventing tumor growth remain poorly understood. To resolve these questions it is necessary to isolate the tumor infiltrating lymphocytes (TIL) from the tumor tissue. To this end TIL from 7 human brain tumors were isolated using enzyme digestion and density gradient centrifugation. The resulting TIL fractions were then cloned in a limiting dilution assay (13) that allows virtualy all peripheral blood resting T cells to undergo clonal expansion. The frequency of proliferating T lymphocyte precursors (PTL-P) can then be calculated thus giving an indication of immunocompetent T cells present in the original infiltrate compared to patient and normal peripheral blood. In addition the clones obtained can be expanded to allow for functional assays in vitro. This report demonstrates that TIL cloned from human gliomas and grown in the presence of interleukin-2 do exhibit cytolytic activity in vitro in both allogeneic and autologous systems.