Function Of Tumor-infiltrating Lymphocytes Research Articles

Helicobacter pylori secreted peptidyl prolyl cis, trans-isomerase drives Th17 inflammation in gastric adenocarcinoma

Helicobacter pylori infection is characterized by an inflammatory infiltrate, consisting mainly of neutrophils and T cells. This study was undertaken to evaluate the type of gastric T cell response elicited by the secreted peptidyl prolyl cis, trans-isomerase of H. pylori (HP0175) in patients with distal gastric adenocarcinoma. The cytokine profile and the effector functions of gastric tumor-infiltrating lymphocytes (TILs) specific for HP0175 was investigated in 20 patients with distal gastric adenocarcinoma and H. pylori infection. The helper function of HP0175-specific TILs for monocyte MMP-2, MMP-9, and VEGF production was also investigated. TILs cells from H. pylori infected patients with distal gastric adenocarcinoma produced Interleukin (IL)-17 and IL-21 in response to HP0175. HP0175-specific TILs showed poor cytolytic activity while expressing helper activity for monocyte MMP-2, MMP-9 and VEGF production. These findings indicate that HP0175 is able to drive gastric Th17 response. Thus, HP0175, by promoting pro-inflammatory low cytotoxic TIL response, matrix degradation and pro-angiogenic pathways, may provide a link between H. pylori and gastric cancer.

A novel iNKT-mediated vaccine maneuver overcomes PD-1-mediated T cell exhaustion in cancer (165.13)

Abstract Our immune system plays an important role in preventing cancer. However, tumor-derived immuno-suppression hinders effective anti-tumor immunity. T cell dysfunction or exhaustion in tumor-bearing hosts is one such mechanism. Numerous studies have identified PD-1 and Tim-3 as markers of exhausted T cells, and antibodies blocking PD-1 and Tim-3 have been shown to partially restore T cell function. We have found that both Tim-3 and PD-1 are expressed on CD8+ tumor-infiltrating lymphocytes (TILs) in an animal model of B cell lymphoma and that these cells represent the predominant fraction of T cells infiltrating tumors. We find that iNKT stimulation using the ligand, alpha-galactosylceramide, combined with TLR9 stimulation using the ligand, CpG, can down-regulate Tim-3 and PD-1 expression in TILs, restore TIL function as measured by proliferation and production of TNF and IFNg in response to tumor, and induce long-lasting therapeutic immune responses in mice with large advanced (>1cm2) tumors.

Abstract 5408: CD73 as a target in cancer immunotherapy

Abstract It is increasingly being recognised that the tumor microenvironment modulates the effector functions of tumor-infiltrating lymphocytes and consequently suppresses anti-tumor immunity. One immunosuppressive component of the tumor microenvironment is elevated levels of adenosine. The conversion of ATP into adenosine occurs in a stepwise manner essentially through the enzymatic activity of CD39 (NTPDase I) (ATP>AMP) and CD73 (ecto-5′-nucleotidase- AMP>adenosine). CD73 is a glycosylphosphatidyl-inositol (GPI)-linked cell surface enzyme constitutively expressed on endothelial cells, foxp3+ Tregs and subsets of leukocytes, and is considered as the rate-limiting enzyme in the production of extracellular adenosine. We have recently demonstrated that one of the mechanisms contributing to the immunosuppressive accumulation of extracellular adenosine in tumors is the expression of CD73 by tumor cells, but CD73 expression on foxp3+ Tregs is also important for their suppression of anti-tumor immunity. We now report on the role of host-derived CD73 in de novo tumor development in mice. Combinations of anti-CD73 with agonist and T cell checkpoint blockade antibodies demonstrate significant beneficial effects in experimental and de novo models of tumorigenesis. In humans using large cohorts we have shown that CD73 is highest in triple negative breast cancer (TNBC) and expression correlates with an invasion marker and the lack of ER signalling. CD73 expression is also associated with a worse prognosis in TNBC irrespective of treatment and predicts response to anthracycline therapy. The development of CD73 as a target for cancer immunotherapy will be further discussed. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5408. doi:1538-7445.AM2012-5408

Tumor-infiltrating lymphocytes contain a higher proportion of FOXP3 + T lymphocytes in cervical cancer

The subpopulations and functions of tumor-infiltrating lymphocytes (TILs) from cervical cancer (CC) are altered. Dysfunction of TIL could be partially because of the inhibition by regulatory T (T(reg)) cells. FOXP3 is the control gene for the T(reg) cells. We investigated the distribution of TILs and FOXP3(+) cells in CC (n=10) and cervical intraepithelial neoplasia (n=8) tissues. Double-immunofluorescence and confocal-based image quantitative microscopic analysis were used to calculate the number of cluster of differentiation (CD)4(+)CD25(+)FOXP3(+) T(reg) cells around the tumor cells. The CD4(+)CD25(+)FOXP3(+) phenotype of T(reg) cells was accumulated around the tumor cells. CC contains a significantly higher proportion of the FOXP3(+) T cells than in cervical intraepithelial neoplasia (p<0.001). Moreover, CC with lymph node metastasis has a higher proportion of the FOXP3(+) T cells than that without lymph node metastasis (p<0.05). The increased accumulation of T(reg) cells suggests that T(reg) cells are important in the immunopathogenesis of CC.

Lovastatin Inhibits T-cell Proliferation While Preserving the Cytolytic Function of EBV, CMV, and MART-1-specific CTLs

Statin treatment has been shown to reduce graft-versus-host disease while preserving graft-versus-tumor effect in allogeneic stem cell transplantation. Herein, we investigated whether lovastatin treatment affects the function of human cytolytic T lymphocytes (CTLs). Upon T-cell receptor stimulation, lovastatin significantly inhibited the proliferation of both CD4+ and CD8+ T cells from healthy donors whereas their intracellular cytokine production including interferon-γ and tumor necrosis factor-α remained the same with a slight decrease of interleukin-2. Moreover, the specific lysis of target cells by CTL lines derived from patients and normal donors specific for Epstein-Barr virus-encoded antigen latent membrane protein-2 or cytomegalovirus-encoded antigen pp65 was uncompromised in the presence of lovastatin. In addition, we evaluated the effect of lovastatin on the proliferation and effector function of the CD8+ tumor-infiltrating lymphocytes (TILs) derived from melanoma patients specific for MART-1 antigen. Lovastatin significantly reduced the expansion of antigen-specific TILs upon MART-1 stimulation. However, the effector function of TILs, including the specific lysis of target cells and secretion of cytokine interferon-γ, remained intact with lovastatin treatment. Taken together, these data demonstrated that lovastatin inhibits the proliferation of Epstein-Barr virus, cytomegalovirus, and MART-1-specific CTLs without affecting cytolytic capacity. The differential effect of lovastatin on the proliferation versus cytotoxicity of CTLs might shed some light on elucidating the possible mechanisms of graft-versus-host disease and graft-versus-tumor effect elicited by alloimmune responses.

Down-regulation of the antigen processing machinery is linked to a loss of inflammatory response in colorectal cancer

Antitumor inflammatory response is known to inhibit tumor growth in colorectal carcinoma. The density and functionality of tumor-infiltrating lymphocytes (TIL) is regulated by the antigen processing machinery through regulator proteins such as transporters associated with antigen processing (TAP) and major histocompatibility complex (MHC) class I antigen. We aimed to investigate the in vivo association of those factors and their impact on prognosis in colorectal cancer. TAP1, TAP2 and MHC class I antigen expression, inflammatory infiltrate and TIL (CD4(+), CD8(+), and CD20(+)) were assessed by immunohistochemistry in 336 sporadic colorectal carcinomas. The factors were correlated with each other and with clinic-pathological parameters and patient outcome. We found TAP1 and TAP2 expression to be significantly associated with MHC class I antigen expression (TAP1: r = 0.363, P < .001; TAP2: r = 0.393, P < .001). Increased density of CD8(+) TIL was predominantly found in TAP1, TAP2 and MHC class I antigen-positive cases. Increased density of CD4(+) TIL was linked with TAP1 and TAP2, but not with MHC class I antigen. High CD4(+) and CD8(+) cell count but not TAP1, TAP2 and MHC class I antigen expression had favorable prognostic impact in colorectal cancer (P = .003 and P = .003, respectively). In conclusion, our data show that the expression of key components of the antigen processing machinery is tightly linked to the density of TIL, which are positive prognostic factors in colorectal cancer in vivo. This implies that modulation of these factors may help to enhance antitumor inflammatory response which in turn may improve patient prognosis.

NK cell-mediated cytotoxicity modulation by A2 adenosine receptor agonist in different mammalian species

Adenosines, endogenous purine nucleosides, appear in the extracellular space under metabolically stressful conditions associated with ischemia, inflammation, and cell damage. Their activity on innate immunity is prevalently inhibitory and can develop both in infectious and neoplastic diseases. During cancer development, tumor cells that release high concentrations of adenosines can impair the function of tumor-infiltrating lymphocytes and assist tumor growth by neo-angiogenesis. We evaluated the influence of A(2) adenosine receptor (A(2)AR) agonist on cytotoxic-cell response comparing human with other mammalian species (rodents, pigs, goats), both in healthy and in cancer conditions. The A(2)AR agonist developed dose-dependent inhibition of the cytotoxic activity of immune effector cells in all studied species. However, variability of the response was observed in relation to the species and the target cells that were used. Altogether, our data indicate that the A(2)AR plays a central role in adenosine-mediated inhibition of immune response to tumors.

Boosting antitumor responses of T lymphocytes infiltrating human prostate cancers

Immunotherapy may provide valid alternative therapy for patients with hormone-refractory metastatic prostate cancer. However, if the tumor environment exerts a suppressive action on antigen-specific tumor-infiltrating lymphocytes (TIL), immunotherapy will achieve little, if any, success. In this study, we analyzed the modulation of TIL responses by the tumor environment using collagen gel matrix–supported organ cultures of human prostate carcinomas. Our results indicate that human prostatic adenocarcinomas are infiltrated by terminally differentiated cytotoxic T lymphocytes that are, however, in an unresponsive status. We demonstrate the presence of high levels of nitrotyrosines in prostatic TIL, suggesting a local production of peroxynitrites. By inhibiting the activity of arginase and nitric oxide synthase, key enzymes of L-arginine metabolism that are highly expressed in malignant but not in normal prostates, reduced tyrosine nitration and restoration of TIL responsiveness to tumor were achieved. The metabolic control exerted by the tumor on TIL function was confirmed in a transgenic mouse prostate model, which exhibits similarities with human prostate cancer. These results identify a novel and dominant mechanism by which cancers induce immunosuppression in situ and suggest novel strategies for tumor immunotherapy.

Study of the Expression of FasL and of Apoptosis in Gastric Epithelial Dysplasia and Gastric Adenocarcinomas

Purpose: This study was to observe whether the apoptotic function of tumor-infiltrating lymphocytes (TIL) is induced in human gastric epithelial dysplasia and gastric adenocarcinoma according to the role of FasL expression. Materials and Methods: A total of 56 gastric epithelial dysplasia and gastric adenocarcinoma patients were enrolled in this study: 9 cases of gastric epithelial dysplasia, 18 cases of early gastric carcinomas (EGC) and 29 cases of advanced gastric carcinomas (AGC). Immunohistochemical staining was performed for FasL and CD45, and the terminal deoxynucleotidyl transferase mediated dUTP nick end labelling (TUNEL) method was used to detect cell death in tumor-infiltrating lymphocytes. Results: 1) Positive reactions of FasL to neoplastic cells were (8/9) in gastric epithelial dysplasia, (15/18) in EGC, and (22/29) in AGC. 2) Expression of TIL was decreased in the FasL positive region and was increased in the FasL negative region, and significant expression of TIL was observed in the AGC group (P=0.001). 3) Expression of apoptotic TIL was very similar to the FasL expression, and expression was observed in gastric epithelial dysplasia group. 4) Expression of apoptotic TIL was increased in the FasL positive region and decreased in the FasL negative region, and significant apoptotic expression was observed in the gastric epithelial dysplasia and EGC groups (P=0.0420, P=0.0263, respectively). Conclusion: These results suggest that FasL is a prevalent mediator of immune privilege in epithelial dysplasia and cancer of the stomach.

Overexpression of caltractin gene in tumor-infiltrating lymphocytes.

Differential display is a technique which relies on the polymerase chain reaction to identify messenger RNA differences between related tissue samples. We have employed an improved differential display technique, called fluorescent differential display (FDD), to identify the genes that are differentially expressed in normal and malignant mammary tissues. From FDD fingerprints, we identified changes in intensity of approximately 3% (185 bands) of a total of 5, 837 bands. Each of these 185 bands represented a differentially expressed gene, and we focused our attention on the expression of the gene for caltractin, a member of the calcium-binding EF-hand protein superfamily. Northern blot analysis revealed that the level of mRNA for caltractin was higher in breast carcinoma than in corresponding normal tissue in all cases tested (5/5). Moreover, high-level expression of the gene for caltractin was also recognized in other malignant tumors, such as hepatocellular carcinoma (HCC), gastric cancer and leiomyosarcoma. The results of in situ hybridization showed strong stainings for caltractin mRNA in tumor-infiltrating lymphocytes (TIL), but not in malignant tumor cells. Our data suggests that the caltractin gene might be associated with the function of TIL.

In situ cytokine production by breast cancer tumor-infiltrating lymphocytes

Human breast cancers progressively grow despite the presence of extensive lymphocytic infiltration and specific antitumor immune recognition, thereby calling into question the competency of breast tumor-infiltrating lymphocytes (TIL). The function of breast TILs in vivo and their possible role in the suppression of an antitumor immune response are largely unknown. The cytokines produced in situ by lymphocytes in 89 breast carcinomas and 14 benign breast lesions were assessed using immunohistochemistry. The majority of tumor and benign breast samples contained T-cell infiltrates, which were disclosed using an anti-CD3 antibody stain. The percentage of tumor samples in which > or =3% of the lymphocytes were producing cytokines was as follows: interleukin (IL)-2 45%, IL-4 36%, tumor necrosis factor-alpha (TNF-alpha) 28%, transforming growth factor-beta 1 (TGF-beta 1) 20%, IL-10 11%, interferon-gamma (IFN-gamma) 4%, and granulocyte-macrophage colony-stimulating factor (GM-CSF) 3%. Production of IL-2, IL-4, and TGF-beta 1 by TILs in breast cancers exceeded that detected in benign breast lesions (p < 0.005). Significantly more tumor samples contained lymphocytes producing IL-2, IL-4, TGF-beta 1, and TNF-alpha than IFN-gamma and GM-CSF (p < 0.002 for each comparison). One or more of the potentially immunoinhibitory cytokines-IL-4, IL-10, or TGF-beta 1-were produced by lymphocytes in 44% of the specimens. No significant associations were seen between lymphocyte production of a particular cytokine and disease-free survival (median follow-up 43 months). Immunohistochemical techniques can be used to detect cytokine secretion by TILs in preserved tissue. The relative lack of secretion of IFN-gamma and GM-CSF, rather than a deficiency of IL-2, may explain why the antitumor immune response to breast cancer is impaired.

In vivo trafficking of adoptively transferred interleukin-2 expanded tumor-infiltrating lymphocytes and peripheral blood lymphocytes. Results of a double gene marking trial.

Adoptive immunotherapy with tumor-infiltrating lymphocytes (TIL) and IL-2 appears to produce dramatic regressions in patients with metastatic melanoma and renal cancer. However, the in vivo mechanism of TIL function is not known. We conducted an UCLA Human Subject Protection Committee, Recombinant DNA Advisory Committee, and FDA-approved clinical trial using genetically-marked TIL to test the hypothesis that these cells have unique, tumor-specific in vivo trafficking patterns. TIL and PBL (as a control effector cell population) were isolated and expanded in parallel in vitro in IL-2-containing medium for 4-6 wk. During the expansion, TIL and PBL were separately transduced with the amphotropic retroviral vectors LNL6 and G1Na. Transduced TIL and PBL were coinfused into patients and their respective numbers measured in tumor, peripheral blood, and normal tissues; integrated provirus could be quantitated and distinguished by DNA PCR. Nine patients were treated (six melanoma, three renal) and received between 4.5 x 10(8) and 1.24 x 10(10) total cells. Both "marked" TIL and PBL could be detected circulating in the peripheral blood, in some patients for up to 99 d after infusion. Marked TIL and/or PBL could be detected in tumor biopsies in six of nine patients as early as day 6 and as late as day 99 after infusion. No convincing pattern of preferential trafficking of TIL vs. PBL to tumor was noted. Moreover, concurrent biopsies of muscle, fat, and skin demonstrated the presence of TIL/PBL in comparable or greater numbers than in tumor in five patients. The results of this double gene marking trial provide interesting insights into the life span and trafficking of adoptively transferred lymphocytes, but do not support the hypothesis that TIL specifically traffic to tumor deposits.

Variable expression of CD3‐ζ chain in tumor‐infiltrating lymphocytes (TIL) derived from renal‐cell carcinoma: Relationship with til phenotype and function

It has been reported that in mice and in humans, tumor-infiltrating lymphocytes (TIL) may exhibit a defect in CD3-zeta-chain expression. Therefore, the level of CD3-zeta was analyzed in TIL derived from patients with renal-cell carcinoma, and its correlation with TIL phenotype and function was assessed. We identified 4 out of 13 tumor-infiltrating lymphocytes derived from renal-cell carcinoma, with a significant decrease in CD3-zeta-chain expression as compared with control peripheral-blood lymphocytes. This defect was never found after culturing TIL with IL2. In one case, the low expression of zeta chain observed in TIL on day 0 was reversed when TIL were cultured with IL2. The zeta-chain level did not seem to predict the growth of TIL in response to IL2. All the TIL, irrespective of the level of zeta-chain expression, exhibited lower proliferative response when stimulated with PHA or anti-CD3 MAb in comparison with normal peripheral-blood mononuclear cells. Nevertheless, in this limited series of patients, a correlation was observed between the level of zeta-chain expression and T-cell infiltration (p = 0.02). After TIL stimulation with PHA or anti-CD3, in contrast to IL2 or IFN-gamma production, a trend towards a relationship between TNF alpha induction and the level of zeta-chain expression was observed.

Culture of tumour-infiltrating lymphocytes from melanoma and colon carcinoma: removal of tumour cells does not affect tumour-specificity.

The therapeutic potential of adoptive therapy using tumour-infiltrating lymphocytes (TIL) has been demonstrated in a number of clinical trials. However, freshly isolated tumour-infiltrating lymphocytes (TIL) are often impaired in their proliferative and cytotoxic responses, which limits their use in immunotherapy. Several hypotheses with regard to the poor effector function of TIL have been postulated, including the production of immunosuppressive factors by tumour cells. In a previous paper we reported the efficient expansion of immunoreactive TIL from a variety of solid tumours by stimulation with a combination of monoclonal antibodies (mAbs) against CD3 and CD28. In the present study we analysed whether this protocol would be improved by the removal of tumour cells at the start of the culture. We tested a highly immunogenic tumour, melanoma, and a poorly immunogenic tumour, colon carcinoma. Removal of tumour cells highly improved anti-CD3/CD28 stimulated expansion of TIL from colon carcinoma, resulting in a significantly higher percentage of potentially tumour-specific CD8-positive T-cells and a reduced CD4/CD8 ratio compared to expansion in the presence of tumour cells. In contrast, expansion and CD4/CD8 ratio of melanoma-derived TIL was not significantly influenced by the removal of autologous tumour cells. CD3/CD28-stimulated melanoma TIL cultured in the absence of tumour cells showed specific lysis of autologous tumour cells comparable to melanoma TIL cultured in high-dose IL2. However, no cytotoxicity could be detected in colon TIL irrespective of the culture conditions used. On the other hand, 3/8 colon carcinoma TIL cultures and 9/12 melanoma-derived TIL cultures showed IFN gamma secretion upon stimulation with autologous tumour cells. We conclude that stimulation of TIL with a combination of mAbs to CD3 and CD28 in the absence of tumour cells induces efficient expansion of potentially tumour-specific cells from a highly and a poorly immunogenic tumour. Removal of tumour cells does not have a negative influence on the generation of tumour-specific T cells, while cell yield improves. Therefore, for large-scale cultures this protocol can efficiently induce the outgrowth of tumour-specific TIL, at the same time providing a useful source of autologous tumour cells that can be stored and used to direct or test antitumour specificity.

Pulmonary Macrophages Suppress the Proliferation and Cytotoxicity of Tumor-infiltrating Lymphocytes

Adoptive immunotherapy with interleukin-2 and tumor-infiltrating lymphocytes (TIL) is rarely effective in primary lung cancer. We hypothesize that pulmonary macrophages (PM), which are increased substantially in the lungs of smokers, might suppress TIL function. The addition of PM into the TIL cytotoxicity assay produced a concentration-dependent suppression of TIL cytotoxicity with up to 71% inhibition of autologous tumor killing at the 1:1 PM:TIL ratio. Inhibition was not target-specific, as killing of NK-sensitive (K562), NK-resistant (M14), and autologous tumor targets were equally suppressed. Nor was inhibition specific for lung TIL, as similar inhibition was observed with melanoma and renal TIL. Using a model system, we demonstrated that both CD3+ antigen-specific and CD56+ nonspecific lymphocytes are susceptible to the suppressive effects of the PM. Direct co-incubation of PM and TIL for 4 to 44 h resulted in progressive suppression of TIL proliferation and cytotoxicity. TIL cytotoxicity remained suppressed even if PM were removed from the co-culture after 24 h, but was restored if the separated TIL were re-incubated in interleukin-2. These results suggest that PM may locally regulate the proliferative and cytotoxic function of adoptively transferred TIL.

Resident research award: Tumor necrosis factor α selectively enhances growth and cytotoxic activity of tumor infiltrating lymphocytes from human colorectal cancer

Adoptive immunotherapy with tumor-infiltrating lymphocytes (TIL) and interleukin-2 (IL2) can induce regression of tumor metastases in animal models and in human metastatic malignant melanoma. We investigated the potential of colorectal cancer TIL as a source of killer cells and the effect of tumor necrosis factor α (TNFα) in combination with IL2 on their cytotoxic activity. Tumor-infiltrating lymphocytes were isolated from surgical specimens using a mechanical and enzymatic dissociation process. Autologous lamina propria mononuclear cells (LPMC) were used as control. Tumor-infiltrating lymphocytes and LPMC were cultured in the presence of IL2 with/without TNFα (1000 U/ml each) for 5 to 8 weeks. Cytotoxicity (% lysis) was tested against Daudi target cells in a 4-hr 51Cr-release assay. The combination of IL2 and TNFα resulted in a significantly greater-fold expansion of TIL than IL2 alone ( P < 0.01). Lamina propria mononuclear cells expanded less than TIL, and TNFα had an inhibitory effect on their growth ( P < 0.05). Tumor-infiltrating lymphocytes and LPMC showed comparable cytotoxicity when cultured with IL2 alone. However, the addition of TNFα augmented the killer activity of TIL while inhibiting that of LPMC ( P = 0.035). These results indicate that TNFα selectively increases the IL2-induced growth and cytotoxic function of colorectal cancer TIL, but not those of gut mucosal lymphoid cells, suggesting that TIL and LPMC differ in their response to TNFα. Therefore, this combination of cytokines may hold more promise than single agents for the immunotherapy of colorectal cancers with TIL.

Clonal analysis of tumor‐infiltrating lymphocytes from human primary and metastatic liver tumors

Phenotypic and functional characteristics of tumor-infiltrating lymphocytes (TIL) obtained from human primary and metastatic liver tumors were studied. Lymphocytes isolated from 18 tumors and autologous (A) peripheral blood (6 cases) were phenotyped by 2-color flow cytometry and cloned in a limiting dilution system, which allows virtually all normal T lymphocytes to proliferate; 70-80% of fresh TIL were T cells (i.e., CD3+), and the ratio of CD4+/CD8+ cells was 1.2 in both primary and metastatic liver tumors. TIL contained significantly more CD56+ (NKHI+) cells, half of which were CD3+CD56+, CD3+CD25+ cells and CD3+HLA-DR+ cells, than A-PBL. The frequencies of proliferating T-cell precursors (PTL-p) and cytolytic T-lymphocyte precursors (CTL-p) reactive with K562, allogeneic tumor cells and autologous tumor cells, were determined. Mean PTL-p frequencies for TIL from hepatocellular carcinomas, cholangiocarcinomas and metastatic liver tumors were 0.52 (0.22-0.83), 0.10 (0.05-0.16) and 0.16 (0.01-0.30), respectively. The frequency of CTL-p with natural-killer-like activity was lower in TIL than in A-PBL. The frequency of CTL-p for autologous tumor cells in fresh TIL isolated from primary liver tumors was 0.02-0.13 and 12/81 clones were reactive against autologous tumor. In contrast, only 1/66 TIL clones obtained from colon carcinomas metastatic to liver showed autotumor reactivity. No clones reactive with autologous tumor were obtained from peripheral blood of patients with liver cancer. These data indicate that substantial differences in anti-tumor functions of TIL between primary and metastatic liver tumors exist, which can be detected at a clonal level.

Systemic and in-situ natural killer activity in tumour-bearing rats

Single-cell suspensions prepared by enzymatic disaggregation of an immunogenic 3-methylcholanthrene-induced sarcoma (Mc40A) contain a significant proportion of infiltrating leucocytes (approximately 42%), comprising T lymphocytes, macrophages and non-phagocytic FcR+ lymphoid-like cells. Tumour-infiltrating lymphocytes (TIL) were isolated and purified by successive passage over Sephadex G-10 columns and their cytotoxic activity in vitro compared with that of lymphoid cells from normal rats and from tumour-bearers at different times after implantation. For this purpose, surviving target cells were quantified by incorporation of the gamma-emitting analogue of methionine, 75Sel-methionine, in a 48-h assay which detected both cytotoxic and cytostatic effects. The reactivity of TIL, which was consistently demonstrable from 11 days after tumour transplantation, was essentially similar to that of normal splenic lymphocytes in magnitude and specificity. Reciprocal cytotoxicity tests using TIL and cultured targets from an antigenically unrelated tumour of similar aetiology (Mc57) showed that the manifestation of TIL cytotoxicity was determined, not by the tumour of origin, but by the susceptibility of the target cells. Evidence that the effector function of TIL was mediated in part by natural killer (NK) cells was derived from concurrent experiments using human myeloid cells (K562) as targets in an 18h 51Cr-release assay. In this system the level of NK activity was critically dependent on the numbers of tumour cells in the TIL population; contamination in excess of 2% gave rise to dose-dependent inhibition of NK function. The results show that within a progressively growing tumour known to possess rejection antigens, NK reactivity was detected in the absence of a demonstrable tumour-specific cytotoxic component.