Fresh Autologous Tumor Research Articles

Cancer and immunity

The development of modern tumor immunology based on animal tumor models demonstrated the presence of tumor-specific antigens and the ability of tumor-specific immunity to protect against tumor cell challenge. However, neither of these characteristics are readily attributed to many human cancers. Although in certain animal models induced by virus or carcinogen, antigen-specific T cell (CTL) play an important role against tumor cell killing, the role of specific CTL is not so clear in human cancer.Recently, Brimm et al reported that peripheral blood leukocytes from cancer patients could be activated in culture in preparation of interleukin-2 (IL-2), resulting in the development of effector cells cytotoxic for autologous fresh solid tumor cells. The IL-2-mediated activation of PBL to become cytotoxic to fresh autologous solid tumor cells probably reflects a common and non-specific activation mechanism of the host. As the anomalous killer cells distinct from CTL and NK cell, lectin-activated killer, alloantigen-activated killer and lymphokine-activated killer are included.These cell killings were examined to the PBL of cancer patients.Lymphokine-activated killers represent a unique and fundamental cytotoxic effector system that may play a role in immune surveillance against NK resistant solid tumor cells, and may have a possible role in the adoptive immunotherapy of tumors.The generation of an immune response requires not only the appropriate interaction of cells from the monocytic and lymphoid lineages but also the release of appropriate cytokine by these cell populations. Evidemce is accumulating that many of the functions attributed to lymphocytes and monocytes are actually mediated by the cytokines they release. IL-2 is a lymphokine that is released by T cells after appropriate stimulation. IL-2 production activity in peripheral T cells of cancer patients was examined using IL-2 dependent T CTLL cell lines. The action of IL-2 is of the essential growth factor supporting the in vitro proliferation of T suppressor/cytotoxic cells, lymphokine-activated and NK cells. Furthermore, IL-2 enhances the functional activity of both lymphokine-activated and NK cells.The adopttve immunotherapy using IL-2 will be effective to cancer patients. Tumor necrosis factor (TNF) in one of monokine which has marked anti-tumor activity in vitro and in vivo experiments.Recently human recombinant IL-2 and TNF have been made in my country. The availability of molecular-cloned IL-2 and TNF offers the great advantages to the patients.

Intrapleural administration of OK432 in cancer patients: augmentation of autologous tumor killing activity of tumor-associated large granular lymphocytes.

Ten patients with carcinomatous pleural effusions were treated with single intrapleural (i.pl.) injections of the streptococcal preparation OK432 on day 0 and the effects of i.pl. OK432 on the lysis of fresh or cryopreserved autologous tumor cells isolated from the pleural effusions were observed on day 7. In eight patients tumor cells in the effusions had decreased or disappeared by day 7. The other two patients, however, had no clinical evidence of therapeutic benefit from i.pl. OK432. Effusion tumor cells were relatively resistant to lysis by autologous lymphocytes when tested in a 4-h 51Cr-release assay. Positive reactions were recorded for blood and effusion lymphocytes in two of ten untreated patients. Injection of OK432 i.pl. resulted in an induction or augmentation of cytotoxicity against autologous tumor cells and K562 in the effusions of seven of ten subjects by day 7. In contrast, autologous tumor killing activity of blood lymphocytes was not always modified by i.pl. OK432. Purification of large granular lymphocytes (LGL) by discontinuous Percoll gradient centrifugation enriched autologous tumor killing activity, with no reactivity in LGL-depleted, small T lymphocytes. Significant lysis of autologous tumor cells was observed with effusion LGL from seven of ten untreated patients. Seven days after i.pl. OK432 injection, effusion LGL expressed enhanced cytotoxicity against autologous effusion tumor cells, whereas T cells were still not cytotoxic to autologous tumor cells on day 7. The frequency of LGL among effusion lymphocytes was not altered by i.pl. OK432. Adherent effusion cells were not involved in lysis of autologous effusion tumor cells in either untreated or OK432-treated patients. In vitro treatment of blood and effusion lymphocytes with OK432 induced an enhancement of autologous tumor-killing activity in patients who subsequently responded to i.pl. OK432 treatment. OK432 augmented in vitro autologous tumor killing activity of LGL, whereas T cells failed to lyse autologous tumor cells even after in vitro activation with OK432. These results indicate that i.pl. administration of OK432 to cancer patients will result in an augmentation of autologous tumor killing activity of LGL in the pleural effusions, and that this could be responsible for the antitumor activity of i.pl. OK432 therapy.

The human mixed lymphocyte-tumor cell interaction test. I. Positive autologous lymphocyte proliferative responses can be stimulated by tumor cells as well as by cells from normal tissues.

Co-culture of cancer patients' nonadherent peripheral blood lymphocytes with irradiated autologous fresh tumor cells, termed the mixed lymphocyte-tumor interaction (MLTI) test, resulted in significant stimulation of 3H-Tdr in corporation on day 6 in 19 of 37 autologous combinations. The MLTI test was performed in a microtiter wells (0.2 ml) and a variety of solid tumor cells (sarcomas and carcinomas) were used. Tumor cells were dissociated from the fresh biopsy tissue by nontrypsin enzymatic digestion (deoxyribonuclease, hyaluronidase, and collagenase) and the tumor cells enriched by depletion of macrophages using adherence procedures. Occasionally, further tumor cell purification was achieved by separation of cells on the basis of size on dis-continuous gradients. Positive MLTI resulted in stimulation as high as 20-fold over the backgrounds of PBL and tumor cells cultured alone. Mean positive MLTI was SI of 7.7. The negative MLTI were not a reflection of generalized immunosuppression, because tumor cell preparations that did not stimulate autologous PBL did stimulate allogeneic PBL. In an additional patient, PBL not responding in the autologous MLTI did respond to allogeneic tumors. MLTI using cryopreserved cells reproduced the MLTI results using fresh cells in 11 of 16 tests; the other five tests were all positive in the fresh MLTI and negative when using cryopreserved cells. Despite reports from many other groups it appears that positive MLTI were not tumor-specific. In 14 experiments we were able to simultaneously test the proliferative response to autologous tumor as well as to an autologous normal tissue (lung, liver, colon, and bowel). In eight of these experiments positive responses were obtained with tumor stimulators and in seven of these, positive proliferation was also obtained with normal tissue.

Phase I study of the adoptive immunotherapy of human cancer with lectin activated autologous mononuclear cells.

In previous in vitro studies, the authors showed that phytohemagglutinin (PHA) stimulated peripheral blood lymphocytes (PBL) from cancer patients to generate cells that were lytic for fresh autologous tumor but not for lymphocytes or lymphoblasts. Thus, after IRB approval, a phase I clinical protocol was instituted in cancer patients who had failed all other therapy to determine the toxicity and effects, in vivo, of the infusion of large numbers of such PHA activated autologous PBL. Ten patients were treated on the protocol, six with sarcoma, one with melanoma, and three with colorectal cancer. Up to a total of 1.7 X 10(11) PBL were obtained from 7 to 15 successive leukaphereses, the cells from each leukapheresis being incubated in vitro in medium containing PHA and human AB serum for 2 days and then reinfused following the next leukapheresis 2 days later. Toxicity encountered included fever and chills in 10/10 patients, headaches in 5/10, nausea and vomiting in 3/10, and requirement for erythrocyte transfusion in 8/10. No evidence for autoimmune disease, abnormal serum chemical or coagulation studies, or pulmonary emboli was found. 111Indium trafficing studies showed distribution of infused cells mainly to the spleen and liver, with some accumulation in the lungs and tumor especially after repeated infusions. In 9/10 patients, activated PBL were detected in the peripheral circulation by the sixth leukapheresis. Evidence for this was found by assaying the incorporation of tritiated thymidine (3H-Tdr) into, and lysis of fresh tumor cells by, unstimulated PBL from successive leukaphereses. No tumor regression was seen in these patients with bulk disease. These studies demonstrated that large numbers of PHA-activated PBL can be safely obtained and infused into humans, achieving an increase in the number of circulating activated cells with evidence of migration of cells to tumor, lungs, liver and spleen. Further studies of the use of activated lymphocyte infusion in conjunction with chemotherapy in humans are in progress.

Lysis of Fresh Human Tumor Cells by Autologous Peripheral Blood Lymphocytes and Pleural Effusion Lymphocytes Activated by OK432<xref ref-type="fn" rid="FN2">2</xref>

Lymphocytes from peripheral blood (PBL) and from pleural effusions (PEL) of cancer patients were tested for cytotoxicity against tumor cells freshly isolated from carcinomatous pleural effusion of the same patient. Significant lysis of autologous tumor cells was recorded for 4 of 28 PBL samples and for 5 of 28 PEL cases when investigated in a 4-hour 51Cr release assay. In vitro treatment of lymphocytes for 20 hours with the streptococcal preparation OK432 resulted in an induction or augmentation of cytotoxicity against autologous tumor cells in 21 of 28 PBL and PEL specimens. OK432-induced cytotoxicity required active cell metabolism, RNA and protein syntheses, but not DNA synthesis of lymphocytes. Supernatants of OK432-stimulated lymphocytes, and interferon and interleukin 2 failed to induce autologous tumor killing. Nylon wool-nonadherent lymphocytes were involved in both spontaneous and OK432-induced lysis of fresh autologous tumor cells. OK432-activated lymphocytes from normal donors and cancer patients caused lysis of fresh allogeneic tumor cells and also K562 cells.

Lysis of fresh human solid tumor cells by autologous lymphocytes activated in vitro by allosensitization.

We have previously demonstrated that cancer patients' peripheral blood lymphocytes (PBL) allosensitized against single or pool normal donor PBL are capable of lysing fresh autologous tumor cells in a 4-h 51Cr-release assay. In this report, we present further investigations into this phenomenon. These alloactivated killer cells (A-AK cells) lysed autologous and allogeneic tumors and allogeneic but not autologous PBL. Furthermore, cold target inhibition studies demonstrated that autologous and allogeneic tumors were lysed by the same effector cells. Multiple metastases from the same patient were equivalently lysed by these A-Ak cells. The presence of adherent cells and proliferation of the precursors were necessary to generate A-AK cells, although the effector cell itself was radioresistant and nonadherent. The effects of allosensitization were enhanced by the addition of lectin-free interleukin-2 preparations to the in vitro culture by partial depletion of adherent cells prior to sensitization. The A-Ak effector cell was OKT3+, OKT8+, OKT4-, OKM1- and could be generated by just 3 days of allosensitization. The precursors for A-Ak cells could be separated from NK cells on percoll gradients and lysis could be generated from thoracic duct lymphocytes, a population devoid of NK cells. The phenotype of the majority of the precursor cells was OKT3+, OKT4-. Theses alloactivated PBL could be expanded in crude or lectin-free interleukin-2 without loss of cytotoxicity for fresh autologous tumor cells. Activated T cells represent a population of on-NK cells with broad lytic specificity for fresh tumor cells. Such cells may be of value in the adoptive immunotherapy of human solid tumors and may play a role in immune surveillance.

Characterization of the lysis of fresh human solid tumors by autologous lymphocytes activated in vitro with phytohemagglutinin.

Human peripheral blood lymphocytes (PBL), obtained from patients with a variety of malignancies, when incubated in vitro with phytohemagglutinin (PHA), lysed fresh autologous tumors during a short-term 51Cr-release assay. These PHA-activated killer (PAK) cells produced maximum lysis of tumor cells by 4 to 8 hr of co-cultivation. PHA incubation induced the generation of cells lytic for autologous and allogeneic tumors but not autologous or allogeneic PBL. Cold target inhibition studies demonstrated that autologous and allogeneic tumors of various histologic types all shared the determinants recognized by PAK cells. Some adherent cells were necessary for generation of these PAK, but higher numbers were suppressive. Augmentation of tumor cell lysis was seen when adherent cells were partially removed before PHA activation. The PAK effector cell was OKT3+, OKT8+. The precursor cell of the PAK was separated from natural killer (NK) cells on Percoll gradients and was generated from thoracic duct lymphocytes, a population devoid of NK cells. Furthermore, the phenotype of the majority of the precursor cells was OKT3+, OKM1-. Activation by PHA for 2 days was found to be an efficient and convenient method for generating lymphoid cells lytic for fresh autologous human tumor. The biologic and possible therapeutic role of these cells is currently being investigated.

Lysis of human solid tumors by autologous cells sensitized in vitro to alloantigens.

Human lymphocytes sensitized in vitro to allogeneic single-donor cells as well as to pools of allogeneic donor cells were evaluated for their cytotoxic potential against autologous fresh solid tumor cells, autologous tissue-cultured tumor and fibroblasts, fresh peripheral blood lymphoid cells (PBL), and Con A blasts of PBL. Using 4-hr 51Cr release assays, pool-sensitized effector cells lysed autologous tissue-cultured tumor and fibroblast targets in 4 of 4 experiments. Similarly, in 2 of 2 experiments with single-donor allosensitized effectors, autologous tissue-cultured tumor was lysed. No lysis of fresh PBL was seen. Significant lysis of fresh autologous tumor target cells was seen in 10 of 12 experiments with pool-sensitized effectors, though, again, no lysis of autologous PBL was observed. No lysis of autologous Con A blasts was seen in 2 of 2 experiments. Fresh tumor cells, PBL, and Con A blasts appeared equally lysable, since they were equally lysed by effector cells specifically sensitized to alloantigens present on these cells. Cold target inhibition studies demonstrated that cultured tumor and fibroblasts but not PBL shared the determinants recognized by pool-sensitized cells.