Lysis of autologous human melanoma cells by in vitro allosensitized peripheral blood lymphocytes

Summary Peripheral blood lymphocytes (PBL) of melanoma patients were sensitized in vitro with lymphocytes of a single donor or with a pool of lymphocytes of 5–20 different donors. After 6–7 days, the cytotoxic activity of the sensitized PBL was tested against cultured autologous tumor cells and lymphocytes in a ⁵¹Cr-release assay. Tumor lysis was observed in 13 of 16 cases in which patients' PBL (Pt-PBL) were stimulated by a pool of allogeneic lymphocytes and in five out of seven cases when single sensitization was performed. In no case was lysis of autologous normal lymphocytes or blasts seen. Cultivation of Pt-PBL with irradiated autologous tumor cells never led to the induction of lymphocytes cytotoxic to melanoma cells. Lysability by pool-activated autologous Pt-PBL of fresh cryopreserved tumor cells was compared to that of short-term cultured tumor cells, and no significant differences were observed. Cold-target inhibition experiments indicated that the cytotoxicity of Pt-PBL was tumor-restricted since only autologous melanoma cells but not lymphocytes were able to inhibit the reaction. These results indicate that activation of Pt-PBL is necessary in order to elicit or amplify their antitumor activity.     

Autologous tumor cell killing activity of tumor-associated lymphocytes in patients with head and neck carcinomas

In order to select the most cytotoxic effector cells for adoptive immunotherapy, lymphokine activated killer (LAK) cells, tumor infiltrating lymphocytes (TILs) and autologous mixed lymphocyte tumor cell culture (MLTC) cells derived from peripheral blood mononuclear cells (PBMC) in the same subject with head and neck carcinomas were prepared. The autologous tumor cell killing activity and cell surface phenotypes of each of the three effector cells were studied. MLTC cells cultured with interleukin-2 (IL-2) showed the strongest cytotoxic activity among these three different effector cells. Although TILs had suppressed killing activity immediately after isolation, after successive cultivations with IL-2, a cytotoxic activity against autologous tumor cells stronger than that of LAK cells appeared. Both IL-2 stimulated MLTC cells and TILs showed an enrichment of CD8 positive and CDU negative cells in a CD3 positive subpopulation.Abbreviations CD cluster differentiation - IL-2 interleukin-2 - LA lymphokine activated - LAK lymphokine activated killer - MLTC mixed lymphocyte tumor cell culture - NK natural killer - PBMC peripheral blood mononuclear cells - TILs tumor infiltrating lymphocytes     

Regulation of cellular immune response against autologous human melanoma. II. Mechanism of induction and specificity of suppression

The cytotoxic immune response in the peripheral blood lymphocytes (PBL) against an autologous malignant melanoma cell line, PJ-M, was found to be down-regulated in in vitro co-culture (IVC) selectively by unfractionated resident lymph node lymphocytes (derived from a lymph node infiltrated with the PJ-M melanoma cells) and T4+ as well as T8+ fractions of the resident lymph node-derived lymphocytes. In this study, the mechanism involved in, and the specificities of, cytotoxic immune response in this autologous system were examined at population and clonal levels. Resident lymph node lymphocytes were isolated from both involved and uninvolved lymph nodes from the same patient. Resident lymphocytes from both sources regulated the generation of cytotoxic immune response when both types of resident lymph node lymphocytes were further sensitized against the PJ-M cells in IVC and were expanded in interleukin 2 (IL 2). An IL 2-dependent homogeneous lymphocyte line (I-10:1) bearing the phenotype of a helper T cell (T4+) and a T4+ clone (I-10.3) of the I-10:1 line, established by limiting dilution culture, also down-regulated the generation of cytotoxic immune effector cells in the PBL in IVC against the PJ-M targets. The IL 2-dependent T4+ inducer line I-10:1 generated a functionally differentiated T8+ suppressor population(s) that, in turn, could abrogate cytotoxic response in fresh PBL in IVC against PJ-M cells. The inducer line I-10:1 and its subclone I-10.3 suppressed the generation of cytotoxic effector cells in the PBL in IVC selectively against the autologous PJ-M cells. Generation of cytotoxic allo-response in IVC was unaffected by the inducer lines. These results provide further evidence for the involvement of the regulatory network in cytotoxic immune response in an autologous human tumor system, and suggest a potential explanation for cytotoxic unresponsiveness against autologous melanoma cells.     

Regulation of cellular immune response against autologous human melanoma. I. Evidence for cell-mediated suppression of in vitro cytotoxic immune response

The potential existence of down-regulation of cytotoxic immune response against an autologous human melanoma line was investigated as a possible explanation for cytotoxic unresponsiveness against the autologous melanoma cells. The melanoma cell line, PJ-M, was established and lymph node resident lymphocytes (LNL) were isolated from a lymph node which was partially infiltrated with the melanoma cells. Autologous peripheral blood lymphocytes (PBL) were sensitized in in vitro co-culture (IVC) against radiated PJ-M cells in the presence or absence of PJ-M-sensitized LNL and enriched suppressor (OKT8+) or inducer (OKT4+) LNL populations, and were assayed for cytotoxicity in a 4-hr 51Cr-release microcytotoxicity assay. Significant cytotoxic response against PJ-M could be generated in the PBL, but not in the LNL. The addition of sensitized, unfractionated LNL, OKT8+, or OKT4+ LNL populations abrogated cytotoxic response in the PBL against PJ-M. The suppression of cytotoxic response was induced selectively against the PJ-M targets, because IVC of PBL in the presence of the sensitized LNL did not affect the generation of polyclonal cytotoxic alloreactivities, nor did they abrogate the generation of cytotoxic response against allogeneic targets in IVC against the corresponding allogeneic targets. These results suggest the possibility that cytotoxic immune response against the autologous melanoma cells might have been suppressed by the individual's own immunoregulatory circuit.     

Expansion of tumor-specific cytolytic T lymphocytes using in vitro restimulation with tumor-specific transplantation antigen

Tumor-specific T lymphocytes (CTL) induced by in vivo immunization of C3H/HeJ mice with the syngeneic methylcholanthrene (MCA)-induced fibrosarcoma MCA-F were expanded in vitro by restimulation with 1-butanol-extracted, isoelectrophoretically purified, tumor-specific transplantation antigen (TSTA) in combination with purified rat interleukin-2 (IL-2) and fresh, syngeneic, 2000-R-irradiated, adherent splenic antigen-presenting cells (APC). The cultured immune T-cell population, containing 40–55% Lyt 2⁺ and 40–60% L3T4⁺ cells, displayed TSTA-specific proliferative and cytotoxic activities in vitro. The expanded T cells appear to recognize butanol-extracted TSTA in association with specific H-2 class I antigens, as revealed by the benefit of syngeneic over allogeneic cells as APC and by the adverse effect of depletion using anti-H-2K, but not anti-Ia, monoclonal antibodies. In adoptive transfer assays in vitro, expanded T cells specifically neutralize homotypic, but not heterotypic, tumor growth in vivo. Based upon the effects of depletion of T-lymphocyte subpopulations using monoclonal antibodies, the Lyt 2⁺ cytotoxic T lymphocytes (CTL) appear to display greater in vivo neutralizing activity than L3T4⁺ T cells. Thus in vitro stimulation of in vivo-immunized T cells, using butanol-extracted TSTA in combination with IL-2 and syngeneic APC, expands tumor-specific CTL.     

Adoptive immunotherapy of advanced melanoma patients with interleukin-2 (IL-2) and tumor-infiltrating lymphocytes selected in vitro with low doses of IL-2

Freshly isolated tumor-infiltrating lymphocytes (TIL) from stage IV melanoma patients were cultured for 2 weeks with low doses of interleukin-2 (IL-2; 120 IU/ml), to select potentially for tumor-specific lymphocytes present in the neoplastic lesion, followed by high doses (6000 IU/ml) to achieve lymphocyte expansion. TIL were serially analyzed for their expansion, phenotype and cytotoxic activity against autologous and allogeneic tumor cells. A preferential lysis of autologous melanoma cells was obtained in long-term cultures of 7/13 cases (54%), while the remaining ones showed a major-histocompatibility-complex-unrestricted, lymphokine-activated-killer(LAK)-like activity at the time of in vivo injection. Sixteen patients with metastatic melanoma were infused with TIL (mean number: 6.8×10⁹, range: 0.35 × 10⁹–20 × 10⁹) and IL-2 (mean dose: 130 × 10⁶ IU, range: 28.8 × 10⁶–231 × 10⁶ IU); 1 complete and 3 partial responses were observed in 12 evaluable patients (response rate 33%). In all responding patients, injected TIL showed an in vitro preferential lysis of autologous tumor cells, while in no cases were TIL with LAK-like activity associated with a clinical response. The mean autologous tumor cytotoxic activity of TIL at the time of in vivo injection was significantly higher in responding patients in comparison to nonresponding ones, suggesting that a marked and preferential cytolysis of autologous tumor cells is associated with the therapeutic efficacy of TIL.     

Target level blocking of T-cell cytotoxicity for human malignant melanoma by monoclonal antibodies

Cytotoxic T lymphocytes (CTL) for autologous malignant melanoma in culture of a patient AV were induced by restimulation of PBL (peripheral blood leukocytes) with AV melanoma cells in vitro and subcultured in interleukin 2 (IL-2) conditioned media. Monoclonal antibodies detecting six antigenic systems on melanoma cell surfaces were tested for blocking activity on the effector function of subcultured cytolytic T lymphocytes for autologous melanoma cells. The monoclonal antibodies R₂₄ (γ3), specific for the G_D3 disialoganglioside on melanoma cell surfaces and I₂₄ (γM), detecting a similar antigenic determinant, blocked autologous T lymphocytotoxicity for malignant melanoma cells on the target level. The effector function of alloantigen activated cytolytic T lymphocytes generated by coculture of allogeneic PBL with Epstein-Barr virus (EBV) transformed AV B lymphocytes, was blocked by monoclonal antibody R₂₄ when tested against AV melanoma targets, but not when tested against AV B lymphocyte targets. It is concluded that blocking by mAb R₂₄ occurs in this system as a nonspecific effect, unrelated to the specific target antigen recognition by cytotoxic T lymphocytes. Steric hindrance or antibody induced membrane changes may account for the blocking effect of monoclonal antibody R₂₄.     

Cellular origin of cytotoxic effectors and secondary educated lymphocytes in human mixed leukocyte reaction

Human lymphocytes sensitized in vitro during a mixed leucocyte reaction (MLR) against an allogeneic-stimulating cell respond by blast transformation and generation of specific cytotoxic effector cells. Both proliferation and cytotoxicity are maximum on Days 6 and 7 of culture. On Day 14, no more dividing cells or cytotoxic cells are detected in such primary cultures. Restimulation by the specific priming cell triggers a secondary proliferative response and rapid reappearance of specific cytotoxic effector cells. The velocity sedimentation cell separation method which separates cells according to their size was applied to human lymphocytes sensitized in vitro during an MLR on Day 7 of culture. Blast cells were separated from nondividing small lymphocytes. It was shown that: (1) cytotoxic effectors generated at the peak of a primary response are exclusively present in the isolated blast population; (2) highly cytotoxic secondary effector cells are induced to reappear mainly from the blast-derived population upon restimulation; and (3) secondary educated proliferative cells mainly derive from the blast population. Conversely, the blast-depleted small lymphocyte population is operationally depleted of cells able to respond by proliferation to the priming cell while responding normally against third party control cells. HLA-D region specificity of the secondary proliferative response is suggested.     

Assessment of the number of local cytotoxic T lymphocytes required for degradation of micrometer-size tumor spheroids

Adoptive immunotherapy with human cytotoxic T lymphocytes (CTL) is a promising cancer treatment. Previously we showed that human CTLs against various types of tumors can be efficiently produced by coculturing peripheral blood cells with target cells. The aims of this study were to simulate the interaction of CTLs and micrometer-size tumor tissues in vitro and to assess the required number of CTLs at local tumor sites for degradation of a tumor. Allogeneic CTLs against a human transitional cell carcinoma cell line and autologous CTLs against a renal cell carcinoma cell derived from a surgical specimen were generated. The cytotoxic activities of CTLs against tumor cells in monolayer culture and tumor spheroids formed in U-bottom 96-well culture plates were assessed. Both allogeneic and autologous CTLs showed greater destructive activity than lymphokine activated killer (LAK) cells against target tumor spheroids. CTLs inoculated at E/T ratios of 0.1 to 1 coexisted with the tumor spheroid for 5 to 6 days and then increased in number with apparently lethal activity against the tumor spheroid. In contrast to CTLs, the increase in LAK cell numbers was scarcely observed, and the proliferated LAK cells did not show cytotoxicity against the tumor spheroid. These observations suggest that, when a small number of CTLs reach a local tumor site, they can destroy micrometer-size tumors after considerable local proliferation. This revised version was published online in August 2006 with corrections to the Cover Date.     

The human lymphokine-activated killer cell system. V. Purified recombinant interleukin 2 activates cytotoxic lymphocytes which lyse both natural killer-resistant autologous and allogeneic tumors and trinitrophenyl-modified autologous peripheral blood lymphocytes

Culture of human peripheral blood lymphocytes (PBL) in purified natural or recombinant interleukin 2 in the absence of exogenous antigen or mitogen causes the differentiation of nonlytic precursor cells into lymphokine-activated killers (LAK). A titration of purified Jurkat IL-2 (BRMP, FCRC, NIH) IL-2 showed that the relatively low concentration of 5 U/ml was optimal for LAK activation. When the responding PBL were pretreated with either mitomycin C or gamma irradiation, LAK activation did not occur, indicating that proliferation, in addition to differentiation, is required. The spectrum of target cells susceptible to LAK lysis in a 4-hr chromium-51-release assay includes fresh NK-resistant tumor cells and trinitrophenyl (TNP)-modified autologous PBL. Unmodified PBL are not lysed. Cold target inhibition studies indicated that LAK lysis of autologous TNP-PBL is totally inhibited by fresh tumors cells, and that tumor lysis is inhibited by TNP-PBL. Additionally, allogeneic tumors totally inhibit lysis of autologous tumor cells in other cold target studies. These results demonstrate that the lytic activity expressed by LAK is not HLA restricted, is not limited to tumor cells, and is "polyspecific" as indicated by the cross-reactive recognition of multiple target cell types in these cold target inhibition studies.     

Comparison of recombinant-interleukin-2-activated peripheral blood and tumor-infiltrating lymphocytes of patients with epithelial ovarian carcinoma: Cytotoxicity,growth kinetics and phenotype

Summary We have compared the growth and tumordirected cytotoxic efficacy of recombinant-interleukin-2-(rIL-2)-activated peripheral blood (PBL) and tumor-infiltrating lymphocytes (TIL) from patients with epithelial ovarian carcinoma. These studies demonstrated that TIL and PBL displayed similar levels of cytotoxicity and a broad range of target cell killing, as exemplified by their reactivity against autologous and allogeneic ovarian tumors as well as against tumor cell lines. No specificity of autologous tumor cell killing was manifested by TIL. Even though TIL of some patients showed higher proliferative activity (especially at the later times in rIL-2 culture) this was not a general phenomenon. In fact, in one case TIL did not proliferate at all, and in the other case the PBL proliferated more actively. While the cultures were composed primarily of CD3⁺ lymphocytes, the major cytotoxic cells displayed the CD56⁺ and CD16⁺ phenotype. Addition of OKT3 mAb to rIL-2 cultures resulted in an increased proliferative index, but showed only a minor effect on the cytotoxic potential of cultured lymphocytes. The therapeutic potential of rIL-2-activated TIL and PBL is discussed.Recipient of the Florence Maude Thomas Cancer Research Professorship     

Addition of interleukin-2in vitro augments detection of lymphokine-activated killer activity generatedin vivo

Summary Thein vivo administration of repetitive weekly cycles of interleukin-2 (IL-2) to patients with cancer enhances the ability of freshly obtained peripheral blood lymphocytes (PBL) to lyse both the natural-killer(NK)-susceptible K562 and the NK-resistant Daudi targets. Lysis of both targets is significantly augmented by inclusion of IL-2 in the medium during the cytotoxicity assay. This boost is much greater for cells obtained following thein vivo IL-2 therapy than for cells obtained prior to the initiation of therapy or for cells from healthy control donors. In addition to direct lytic activity, the PBL obtained followingin vivo IL-2 show a rapid increase in lymphokine-activated killer (LAK) activity with more prolongedin vitro IL-2 exposure, indicating that LAK effectors primedin vivo respond with secondary-like kinetics to subsequent IL-2in vitro. Lymphocytes from healthy control individuals, cultured in IL-2 under conditions attempting to simulate thein vivo IL-2 exposure, function similarly to PBL obtained from patients following IL-2, in that low-level LAK activity was significantly boosted by inclusion of IL-2 during the cytotoxic assay and the cells also responded with secondary-like kinetics to subsequent IL-2in vitro. The augmentation of the LAK effect was also dependent on the dose of IL-2 added during the 4-h⁵¹Cr-release cytotoxicity assay, with higher doses of IL-2 having a more pronounced effect. While continuous infusion of IL-2 induces a greater cytotoxic potential per milliliter of blood obtained from patients, the peak serum IL-2 levels attained are greater with bolus IL-2 infusions. These pharmacokinetic results, together with the IL-2 dose dependence of LAK activity generatedin vivo shown in this report, suggest that a combination of treatment with bolus IL-2 infusions superimposed on continuous IL-2 infusion may transiently expose IL-2 dependent LAK cells, activatedin vivo, to higher concentrations of IL-2, facilitating theirin vivo cytotoxic potential.This work was supported by NIH contract NO1 CM-47669-02, NIH grants CA-32685, RR-031086, NO1 CM-47669-03, and American Cancer Society grant CH-237     

Lymphokine-activated suppressor (LAS) cells in patients with gastric carcinoma

Summary T-cell-growth-factor (TCGF) activated peripheral blood lymphocytes (PBL), cultured for 14 days, showed killer cell activities against natural-killer resistant Daudi cells in a 4 h ⁵¹Cr-release assay. However, the effector cells obtained from patients with nonresectable carcinoma exhibited very much lower cytotoxicity to tumor cells. To analyze the mechanism of depression, we have attempted to examine suppressor cell activities of the TCGF-activated PBL. The assay for the suppressor cell activities was made by in vitro inhibition of cell-mediated cytotoxicity by incubating radiolabeled target tumor cells with lymphokine-activated killer (LAK) cells and TCGF-activated PBL. LAK cells were induced by cultivation with recombinant interleukin-2. TCGF-activated PBL, obtained from four out of ten patients with resectable carcinoma and nine out of ten patients with nonresectable carcinoma, significantly suppressed the LAK cell activities. However, this suppression was not observed in TCGF-activated PBL from ten normal healthy control subjects. TCGF-activated PBL with immunosuppressive reactivity were named lymphokine-activated suppressor (LAS) cells. To investigate the phenotypic characterization of TCGF-activated PBL, the cells were analyzed by two-color flow cytometry. TCGF preferentially expanded CD8⁺CD11^– cells and decreased the growth of CD8⁺CD11⁺ cells in both normal healthy control subjects and gastric cancer (resectable and nonresectable) patients. Dominantly expressed CD8⁺CD11^– cells on TCGF-activated PBL in patients — especially those with nonresectable gastric carcinoma — showed strong LAS cell activity, irrespective of the presence of killer cell activities of CD8⁺CD11^– cells in TCGF-activated PBL from normal healthy control subjects. The results suggested the generation of CD8⁺CD11^– LAS cells from cancer patients, and revealed that CD8⁺CD11^– T-cells contained killer and/or suppressor cell function. In addition, it was found that the TCGF-activated PBL from gastric cancer patients were associated with an increased proportion of CD4⁺Leu8⁺, HLA-DR⁺CD8⁺ and HLA-DR⁺CD25⁺ cells.Abbreviations LAK lymphokine-activated killer - TCGF T-cell growth factor - PBL peripheral blood lymphocytes - rIL-2 recombinant interleukin-2 - LAS lymphokine-activated suppressor This study was supported by a grant-in-aid for scientific research (no. 62570307) from the Ministry of Education, Science and Culture, Japan     

Adoptive immunotherapy with peripheral blood lymphocytes cocultured in vitro with autologous tumor cells and interleukin-2

A clinical trial of adoptive immunotherapy was carried out with peripheral blood lymphocytes (PBL), cocultured in vitro with autologous tumor cells and interieukin-2 (IL-2), in 14 patients with advanced melanoma. PBL from these patients were cocultured with irradiated autologous tumor cells for 7 days, which was followed by expansion in IL-2-containing medium. These lymphocytes were returned to the patient along with intravenous IL-2 at doses up to 2×10⁶ IU m^–2 day^–1. A dose of 300 mg/m² cyclophosphamide was administered to each patient intravenously 4 days prior to each treatment. Following coculture, the lymphocytes were primarily CD3⁺ T cells and they expressed varied degrees of cytotoxicity against autologous melanoma cells. In 9 patients the activated cells were al least 80% CD4⁺ and in 2 cases they were mostly CD8+. Some of the activated cells exhibited suppressor or helper activity in a functional regulatory coculture assay. No major therapeutic response was observed in this study. Minor responses were observed in 2 patients. Toxicities were those expected from the IL-2 dose administered.This work has been supported by an American Cancer Society Institutional Research Grant (ACS-IRG 91-230), by the University of Connecticut Clinical Research Center (grant 0021), and by the Hartford Hospital Research Fund (grant 1017-20-018). Dr. Sporn is a recipient of American Cancer Society Clinical Oncology Career Development Award 90-230     

Use of human leukocyte antigen-mismatched allogeneic lymphokine-activated killer cells and interleukin-2 in the adoptive immunotherapy of patients with malignancies

Clinical effects and side effects were studied in the adoptive immunotherapy of patients bearing malignant diseases using human leukocyte antigen (HLA)-mismatched allogeneic lymphokine-activated killer (LAK) cells. Allogeneic LAK cells were induced from peripheral blood lymphocytes (PBL) of normal donors by means of initial stimulation with pokeweed mitogen (PWM). Six of 15 patients applied in the adoptive immunotherapy showed clinical effects such as partial or complete regression of pulmonary metastasis, pleural effusion and primary tumor. All pulmonary metastatic lesions were eliminated in one case by this adoptive immunotherapy combined with chemotherapy. Generally toxic effects were chillness, fever and general fatigue which were reversible, and no allergic side effects occurred even though allogeneic LAK cells were injected frequently except one patient who showed preshock like symptom accompanied with leukocytopenia and continuous hypotension immediately after infusion but was finally rescued. In the patients who received more than 10¹¹ of allogeneic LAK cells, anti-HLA class I antibodies appeared without any evidence of autoantibody. However, immunological side effects were never experienced after injection of allogeneic LAK cells even when the anti-HLA class I antibodies appeared in the patients. Taken together, allogeneic LAK cells could be considered as alternative therapy for patients with malignancies who could not supply sufficient materials of autologous LAK cells.Abbreviations PWM pokeweed mitogen - LAK lymphokine-activated killer - IL-2 interleukin 2 - PEL peripheral blood lymphocytes - TIL tumor-infiltrating lymphocytes - GVHD graft-versus-host disease - HLA human leukocyte antigen     

In vitro generation of effector cells cytotoxic to autologous targets from chronic myeloid leukemia patients in remission

Summary Peripheral blood lymphocytes (PBL) from chronic myeloid leukemia (CML) patients in remission were stimulated in vitro, in a 3-cell assay with autologous leukemic cells or autologous bone marrow (BM) cells alone, or each in combination with allogeneic PBL. The responder cells were used as effectors in a 4-h ⁵¹Cr release cytotoxicity assay using autologous targets such as leukemic cells, BM cells, phytohemagglutinin-induced lymphoblasts, and allogeneic K562 (erythroblastoid leukemic cell line) target cells. Sensitization of lymphocytes from CML patients with either autologous leukemic cells or BM cells generated cytotoxic cells (CTCs) capable of killing both the targets. These results suggested that in CML, the PBL may have been sensitized to myeloid maturation-related antigens in vivo, which, on secondary stimulation in vitro, may result in differentiation of CTCs cytotoxic to immature myeloid cells, either from autologous leukemic cells or autologous BM. The inability of PBL from patients with oral cancers to lyse autologous BM cells upon in vitro stimulation, supported this possibility. Clonogenic assays conducted to assess the colony forming potential of BM cells which had interacted with CTCs indicated that there was about 37% reduction in committed granulocyte stem cell colony formation without an appreciable change in committed granulocyte/monocyte stem cell units and clusters. Therefore, since the BM toxicity of the CTCs is not very high, these cells may have a potential clinical use in CML.     

Adoptive immunotherapy of malignant diseases with IL-2-activated lymphocytes

Lymphokine activated killer cells (LAK cells) or interleukin 2 (IL-2)-activated killer cells were induced by recombinant IL-2 (TGP-3) for clinical adoptive immunotherapy of malignant diseases. After incubation of peripheral blood lymphocytes (PBL) with IL-2 and normal human plasma for 1-2 weeks LAK cells were obtained that showed a maximum cytotoxicity against target cells, and did not need a toxic dose of IL-2 to enhance or maintain their cytotoxicity. Both autologous and allogeneic LAK cells were used in five clinical cases without any immune side effects, and were effective in three cases.     

Methods for amplifying the induction and expression of cytotoxic response in vitro to syngeneic and autologous freshly-isolated solid tumors of mice

Summary Spontaneously arising tumors are frequently poorly immunogenic and exhibit a limited capacity to induce cytotoxic effector lymphocytes. In the present study, various approaches have been used to amplify the induction and expression of cytotoxic responses in vitro toward freshly isolated, autologous, and syngeneic solid neoplasms of spontaneous origin in mice. Cytotoxic lymphocytes were generated in one-way mixed lymphocyte-tumor cell cultures (MLTC) consisting of splenocytes or lymph node cells from normal and from tumor-bearing mice co-cultured with inactivated tumor cells. Optimal culture conditions have been established for the number of responder (R) cells, the method of inactivation of the stimulating (S) tumor cells, the responder/stimulator (R/S) cell ratio, and the duration of sensitization. Under optimal sensitization conditions only weak cytotoxic responses, as measured by the ⁵¹Cr-release assay, were generated. The antitumor cytotoxic activity could be augmented 2- to 12-fold by using each of the following procedures: (a) addition of crude or of partially purified interleukin-2 (IL-2) to the sensitization cultures; (b) depletion of nylon-adherent cells from the responding cell population; (c) enrichment of large lymphoblasts from the sensitized effector cell population by Percoll density gradient; and (d) treatment of mice donating the responder lymphocytes with low doses of either cyclophosphamide, adriamycin, or indomethacin. Although the highly reactive effector cells generated under the improved conditions also reacted appreciably with unrelated tumor target cells, only low levels of cytotoxicity could be demonstrated against normal target cells. The antitumor cytotoxic cells in sensitized splenocyte cultures were exclusively Thy1⁺, Lyt1^–2⁺, whereas in lymph node cell cultures some cytotoxicity was also exerted by Thy1⁺, Lyt1⁺2⁺ cells.     

Tumor-infiltrating lymphocytes from nonrenal urological malignancies

Summary Tumor-infiltrating lymphocytes (TIL) were isolated from 15 of 20 surgical specimens of transitional cell carcinoma of the urinary bladder, prostate cancer, testicular cancer, Wilms tumor and adrenal cancer. Expansion was carried out in four different culture conditions, each containing 1000 U/ml interleukin-2: RPMI medium with or without 20% (by volume) of lymphokine-activated killer cell (LAK) supernatant and AIM V medium with or without 20% LAK supernatant. The resultant cell populations were then assayed for cytotoxicity against a variety of autologous and allogeneic tumor targets and phenotypic analysis was performed with fluorescein-labeled monoclonal antibodies. TIL growth was unrelated to the initial percentage of lymphocytes or tumor cells present in the enzymatically dispersed specimens or whether fresh or cryopreserved tissue was utilized. Better growth was seen in AIM V than in RPMI medium (P = 0.013); the beneficial effect of the addition of LAK supernatant to RPMI was indicated (P = 0.065), and the addition of LAK supernatant to AIM V did not improve the ability to culture TIL (P = 0.5) from these cancers. TIL in long-term culture were predominantly CD3⁺. The ratio of CD4⁺/CD8⁺ cells varied with time in culture and culture medium, but most cultures eventually became CD4⁺. Cells bearing B cell, natural killer cell, and macrophage markers disappeared early in culture. Overall 14/15 TIL samples were lytic against one of the autologous and allogeneic targets tested, but specific lysis against the autologous tumor from which it was derived was seen in only one TIL culture originating from a bladder cancer. Our results suggest that TIL can be expanded to therapeutic levels from a variety of urological malignancies and that their potential role in future therapy should be further explored.     

Tumour-reactive lymphocytes stimulated in mixed lymphocyte and tumour culture

Summary Lymphocytes from cancer patients were stimulated in mixed culture with autologous tumour (MLTC) or pooled allogeneic lymphocytes (MLC). Both protocols induced increased uptake of ³H-thymidine at 5 days and the appearance of lymphoblasts. Blasts were isolated on discontinuous Percoll gradients and either expanded as bulk cultures or cloned directly under limiting dilution conditions in the presence of conditioned medium containing IL-2. Results with MLTC-blast-CTC have been reported elsewhere. MLC-activated cultures lysed autologous tumour but not autologous lymphoblasts. Lysis of some allogeneic tumours, lymphoblasts from members of the inducing pool, and K562 was also apparent. MLC activated cultures did not undergo restimulation in response to autologous tumour or lymphocytes but were restimulated by leukocytes from pool members.MLTC clones showed autologous tumour-specific cytotoxic activity or cross-reactive proliferative responses with tumours of the same site and histology. The majority of MLC clones cytotoxic for autologous tumour were also specific and did not lyse allogeneic tumour, K562, or lymphoblasts from the inducing pool. Two clones lysed autologous tumour and pool members. None of the clones tested proliferated in response to autologous tumour following MLC activation but some were responsive to pool members and one clone was restimulated by autologous monocytes. No association was found between clone phenotype and function. The implication of these data is that the effector cells with activity against autologous tumour induced in MLC arose largely by transstimulation of in vivo-activated tumour reactive lymphocytes by IL-2 release rather than expansion of NK-like effectors or sharing of antigenic specificities between tumour and allogeneic lymphocytes. Since MLC activation of cancer patients lymphocytes does not induce proliferative responses to autologous tumour it is unlikely to be a useful procedure in preparing cells for immunotherapy protocols. Abbreviations used in this paper: PBL, peripheral blood lymphocytes; TIL, tumour infiltrating lymphocytes; MLTC, mixed lymphocyte tumour culture; IL-2, interleukin-2; MLC, mixed lymphocyte culture; LSM, lymphocyte separation medium; BSS, balanced salt solution; HuSe, human serum; PBS, phosphate-buffered saline; CTC, cultured T cells; PHA, phytohaemagglutinin; CM, cultured medium; NK, natural killer; FcR, receptor for the Fc portion of IgG