KRASG12C mutation-induced TOPK overexpression contributes to tumour progression in non-small cell lung cancer.

KRAS mutation is the most frequent type of genetic mutation in non-small cell lung cancer (NSCLC), especially in lung adenocarcinoma. However, KRAS mutation can affect many biological processes and the mechanisms underlying KRAS mutation-mediate carcinogenesis in NSCLC have not been fully understood. In this research, we found that KRASG12C mutation was associated with the upregulation of T-LAK cell-originated protein kinase (TOPK), which is a well-known serine/threonine MAPK-like protein kinase implicated in tumorigenesis. The overexpression of TOPK significantly promoted the malignant phenotype of A549 cells, and TOPK silencing impaired the malignant phenotype with KRASG12C mutation. Moreover, we demonstrated that TOPK level was regulated by MAPK/ERK signalling and the transcription factor Elk1. TOPK was also found to promote the activation of NF-κB signalling in A549 cells with KRASG12C mutation via facilitating the phosphorylation of TAK1. In the in vivo tumorigenesis model, the administration of TOPK inhibitor OTS514 enhanced the anticancer effect of 5-FU, and the combinatory use of OTS514 and KRASG12C inhibitor AMG510 showed synergistic anti-tumour effect. These results suggest that KRAS-TOPK axis contributes to the progression of NSCLC and targeting this axis could synergize with anticancer effect of the existing chemotherapeutics.

Topical application of TOPK inhibitor OTS514 suppresses psoriatic progression by inducing keratinocytes cell cycle arrest and apoptosis.

T-LAK cell-oriented protein kinase (TOPK) potently promotes malignant proliferation of tumour cells and is considered as a maker of tumour progression. Psoriasis is a common inflammatory skin disease characterized by abnormal proliferation of keratinocytes. However, the role of TOPK in psoriasis has not been well elucidated. This study aims to investigate the expression and role of TOPK in psoriasis, and the role of TOPK inhibitor in psoriasis attenuation. Gene Expression Omnibus datasets derived from psoriasis patients and psoriatic model mice were screened for analysis. Skin specimens from psoriasis patients were collected for TOPK immunohistochemical staining to investigate the expression and localization of TOPK. Next, psoriatic mice model was established to further confirm TOPK expression pattern. Then, TOPK inhibitor was applied to investigate the role of TOPK in psoriasis progression. Finally, cell proliferation assay, apoptosis assay and cell cycle analysis were performed to investigate the potential mechanism involved. Our study showed that TOPK was upregulated in the lesions of both psoriasis patients and psoriatic model mice, and TOPK levels were positively associated with psoriasis progression. TOPK was upregulated in psoriatic lesions and expressed predominantly by epidermal keratinocytes. In addition, TOPK levels in epidermal keratinocytes were positively correlated with epidermal hyperplasia. Furthermore, topical application of TOPK inhibitor OTS514 obviously alleviated disease severity and epidermal hyperplasia. Mechanismly, inhibiting TOPK induces G2/M phase arrest and apoptosis of keratinocytes, thereby attenuating epidermal hyperplasia and disease progression. Collectively, this study identifies that upregulation of TOPK in keratinocytes promotes psoriatic progression, and inhibiting TOPK attenuates epidermal hyperplasia and psoriatic progression.

Xanthohumol inhibits non-small cell lung cancer via directly targeting T-lymphokine-activated killer cell-originated protein kinase.

Xanthohumol is a principal prenylated chalcone isolated from hops. Previous studies have shown that xanthohumol was effective against various types of cancer, but the mechanisms, especially the direct targets for xanthohumol to exert an anticancer effect, remain elusive. Overexpression of T-lymphokine-activated killer cell-originated protein kinase (TOPK) promotes tumorigenesis, invasion and metastasis, implying the likely potential for targeting TOPK in cancer prevention and treatment. In the present study, we found that xanthohumol significantly inhibited the cell proliferation, migration and invasion of non-small cell lung cancer (NSCLC) in vitro and suppressed tumor growth in vivo, which is well correlated with inactivating TOPK, evidenced by reduced phosphorylation of TOPK and its downstream signaling histone H3 and Akt, and decreased its kinase activity. Moreover, molecular docking and biomolecular interaction analysis showed that xanthohumol was able to directly bind to the TOPK protein, suggesting that TOPK inactivation by xanthohumol is attributed to its ability to directly interact with TOPK. The findings of the present study identified TOPK as a direct target for xanthohumol to exert its anticancer activity, revealing novel insight into the mechanisms underlying the anticancer activity of xanthohumol.

Heterotypic cell-in-cell structures in colon cancer can be regulated by IL-6 and lead to tumor immune escape.

Heterotypic CICs (cell-in-cell structures) have been found between tumor cells and various immune cells in a variety of cancer tissues. The frequency of CICs has been found to correlate with tumor malignancy in some studies but not in others. Herein, we examined in depth the CICs observed in colon cancer to determine their potential significance in disease progression. Heterotypic CICs were observed by histochemistry between epithelial cells and lymphocytes in an expanded spectrum of colon tissue from colitis to cancer and in vitro studies were performed using the colonic tumor cell line HCT8 and human peripheral blood lymphocytes. Our data revealed that the CICs formed by colonic epithelial cells and infiltrated lymphocytes not only positively correlated with tumor malignancy but also were upregulated by the inflammatory cytokine IL-6. In addition, we observed that colon cancer cells could initiate autophagy for survival after cytotoxic lymphocyte internalization and that IL-6 could also be involved in this process to promote the death of lymphocytes in CIC structures. Furthermore, certain changes were observed in tumor cells after experiencing CICs. Our findings suggest that CICs formed by colon cancer cells and lymphocytes contribute to tumor escape from immune surveillance, which could be facilitated by IL-6, and might represent a previously undescribed pathway for tumor cells to adapt and evade host immune defense.

Inhibiting ALK-TOPK signaling pathway promotes cell apoptosis of ALK-positive NSCLC.

T-LAK cell-oriented protein kinase (TOPK) is a potential therapeutic target in tumors. However, its role in anaplastic lymphoma kinase (ALK)-positive non-small cell lung cancer (NSCLC) has not been reported. Here, we found that TOPK was highly expressed in ALK-positive NSCLC. Additionally, ALK was identified as another upstream kinase of TOPK by in vitro kinase assay screening. Then, it was proven that ALK phosphorylated TOPK at Y74 in vitro and ex vivo, and the pathways downstream of ALK-TOPK were explored by phosphoproteomic analysis. Subsequently, we demonstrated that inhibiting TOPK enhanced tumor sensitivity to alectinib (an ALK inhibitor). The combination of alectinib and HI-032 (a TOPK inhibitor) suppressed the growth and promoted the apoptosis of ALK-positive NSCLC cells ex vivo and in vivo. Our findings reveal a novel ALK-TOPK signaling pathway in ALK-positive NSCLC. The combination of alectinib and HI-032 might be a promising therapeutic strategy for improving the sensitivity of ALK-positive NSCLC to targeted therapy.

Alloreactivity and anti-tumor activity segregate within two distinct subsets of cytokine-induced killer (CIK) cells: implications for their infusion across major HLA barriers.

Donor-derived cytokine-induced killer (CIK) can be infused as adoptive immunotherapy after hematopoietic cell transplant (HCT). Promising results were recently reported in HLA-identical HCT, where mild grafts versus host (GVH) events were observed. To extend this strategy across major HLA barriers (e.g. HLA-haploidentical HCT), further studies on CIK cells' alloreactivity are needed. We hypothesized that alloreactivity and anti-tumor activity of CIK cells segregate within two different cell subsets and could consequently be separated according to CD56 and CD3 expression. We tested CIK cells expanded from seven patients who underwent HCT as treatment of metastatic colorectal cancer. We found that CIK cells maintained their alloreactivity across major HLA barriers when tested as bulk population; after CD56-positive selection, anti-tumor activity was restricted to the CD3+/CD56+ cell fraction and alloreactivity versus HLA-mismatched PBMC was restricted to the CD3+/CD56- cell fraction. Bulk CIK cells from engrafted patients did not exhibit alloreactivity in response to host- or donor-derived PBMC, confirming their low potential for GVH across minor HLA barriers. Moreover, we tested if CIK cells expanded from engrafted patients after HCT were as effective as donor-derived ones and could be considered as an alternative option. The expansion rate and tumor cell killing was comparable to that observed in sibling donors. In conclusion, depletion of CD3+/CD56- cells might reduce the risk of GVH without affecting the tumor-killing capacity and could help extending CIK infusions across major HLA barriers. Engrafted patients after HCT could also be considered as an effective alternative option to donor-derived CIK cells.

Probable Mechanisms Involved in Immunotoxin Mediated Capillary Leak Syndrome (CLS) and Recently Developed Countering Strategies.

Antibody-toxin fused agents or immunotoxins, are a newly engineered class of cytotoxic agents consisting of a bacterial or plant toxin moiety hooked up either to a monoclonal antibody or a specific growth factor. Nevertheless, acquiring a full potency in clinic is mostly restricted due to the Capillary leak syndrome (CLS), a serious immune provoked, life-threatening side effect, subsequent to the endothelial damage, resulting in fluid escape from the bloodstream into tissues including lungs, muscle and brain, developing organ failure and eventually death. Proposed underlying mechanisms include direct damage to endothelial cells, acute inflammation, Lymphokine-activated killer (LAK) cells engagement, alteration in cell-cell/cell-matrix connectivities and cytoskeletal dysfunction. Very poor biodistribution and heterogeneous extravasation pattern in tumor site result in accumulation of ITs close to the extravasation site, gradual toxin release and initiation of nearby endothelial cells lysis, secretion of pro-inflammatory cytokines, development of acute inflammation and engagement of Lymphokine-activated killer (LAK) cells. Intrinsic immunogenicity of applied toxin moiety is another important determinant of CLS incidence. Toxins with more intrinsic immunogenicity possess more probability for CLS development. Recently, development of new generations of antibodies and mutated toxins with conserved cytotoxicity has partly tapered risk of CLS development. Here, we describe probable mechanisms involved in CLS and introduce some of the recently applied strategies for lessening incidence of CLS as much as possible.

Tracking of green fluorescent protein (GFP)-labeled LAK cells in mice carrying B16 melanoma metastases.

In adoptive immunotherapy, in vivo trafficking of adoptively transferred cells, including their accumulation at tumor sites, remains to be further investigated. Tracking of these cells by visualization is useful to clarify antitumor mechanisms and develop new modalities to enhance antitumor capacities. In the present study, an in vivo tracking study was performed using an adoptive transfer model of lymphokine-activated killer (LAK) cells induced from green mice into C57/BL6 mice with B16 melanoma metastases. Green mice are green fluorescent protein (GFP) transgenic mice originating from C57/BL6 mice. All of the tissues, except for erythrocytes and hair, express green fluorescence under excitation light. Although LAK cells in combination with IL-2 potently suppressed pulmonary metastases with survival prolongation, very few LAK cells accumulated in tumor tissues compared to those localized in the spleen, as visualized by fluorescent microscopy and quantitated by flow cytometry. The present method using transfer of green mice-derived cells into parental tumor-bearing mice is simple because there is no need for in vitro labeling and is feasible for the in vivo tracking of effector cells in an adoptive immunotherapy model.

Antitumor effects of a bispecific antibody targeting CA19-9 antigen and CD16.

Bispecific murine monoclonal antibodies that target tumor and Fc gamma RIII (CD16) can promote relevant tumor lysis by large granular lymphocytes. For these antibodies to be clinically useful, their properties should be maintained in vivo, where competing human immunoglobulin, shed target antigen, and shed CD16 may be encountered. At a minimum, bispecific antibody antitumor effects should be preserved in whole blood. Furthermore, potentiation of tumor lysis should be reflected by demonstrating the ability of bispecific antibody-retargeted effector cells to infiltrate and mediate lysis of organized tumor. If these characteristics are demonstrated, and there is evidence of in vivo efficacy of bispecific antibody-based therapy in a relevant animal model, further clinical development of such antibodies would be warranted. In this report the ability of CL158 bispecific antibody supernatants to mediate lysis of SW948 tumor growing in monolayer is shown to be preserved in the presence of interleukin 2-activated whole blood. When SW948 cells were grown in vitro as multicellular human tumor spheroids, incubation with interleukin 2-activated lymphocytes (LAK cells) and CL158 led to structural and widespread necrosis. This was dependent on CL158 and resistant to competition by pooled human immunoglobulin or interleukin 2-exposed whole blood. These effects were not promoted by the monospecific antibodies produced by the parent clones of CL158 and were not observed when the IgG2a variant of CA19-9 antibody, which mediates conventional antibody-dependent cellular cytotoxicity, was used instead of its bispecific derivative. To examine the efficacy of bispecific antibody-based treatments on in vivo tumor, scid mice bearing early s.c. SW948 xenografts were treated with interleukin 2 for 5 consecutive days, supplemented by three i.v. injections of 10(7) human LAK cells and various antibodies. Treatment of mice bearing SW948 tumors with LAK cells did not retard tumor growth, but when CL158 was added, significant delays in tumor growth were observed. Tumor growth delay required treatment with both LAK cells and the bispecific antibody. Treatment with the IgG2a variant of CA19-9 antibody, alone or with LAK cells, had no effects on tumor growth. Although the mechanisms of these antitumor effects require further study, it is clear that human LAK cell treatment of animals bearing early, established s.c. tumors is enhanced by the addition of bispecific antibodies with relevant binding characteristics. When compared with the IgG2a isotype variant of CA19-9 monoclonal antibody, this bispecific antibody offers the advantages of preservation of activity in physiological conditions, infiltration and disruption of organized tumor in vitro, and antitumor effects in a relevant xenograft model.(ABSTRACT TRUNCATED AT 400 WORDS)

Lymphokine-activated killer cells induced from decidual lymphocytes reduce the angiogenic activity of trophoblasts by enhancing the release of soluble fms-like tyrosine kinase-1 from trophoblasts: an implication for the pathophysiology of preeclampsia.

T helper (Th)1 cytokine-predominating status and compromised placental vasculature is thought to be central to the pathogenesis of preeclampsia. However, it remains to be clarified how these two phenomena relate to each other. We have reported that lymphokine-activated killer (LAK) cells induced from decidual mononuclear cells (DMCs) with interleukin (IL)-2 expressed in preeclamptic placenta reduced the angiogenic activity of cytotrophoblasts (CTs). The objective of this study was to examine how LAK cells reduced the angiogenic activity of CTs. We investigated the angiogenesis-related molecules released from cultured CTs obtained from first trimester placenta that had been pretreated with either non-activated DMCs or LAK cells from DMCs. The amounts of vascular endothelial growth factor (VEGF), placenta growth factor (PlGF) and their antagonist, soluble fms-like tyrosine-kinase-1 (sFlt-1) released in CT culture media were measured using ELISA. CTs pretreated with LAK cells released more sFlt-1 compared with those pretreated with non-activated lymphocytes, and CTs pretreated with non-activated lymphocytes released more sFlt-1 compared with those without pretreatment. The release of total VEGF and free PlGF from CTs was not altered by pretreatment with DMCs. Thus, in preeclamptic placenta, LAK cells induced from DMCs by co-existing IL-2 may react to the invading CTs and enhance the release of sFlt-1 from CTs without any change of VEGF or PlGF secretion. This might result in the reduction of actual angiogenic potential of the VEGF system in decidua and the placental vascular system might be compromised, which may lead to the development of preeclampsia.

Antileukemia activity of a natural killer cell line against human leukemias.

We describe here the in vitro and in vivo antileukemia activity of a recently described natural killer (NK) cell line (NK-92), which has features of human activated NK cells. The cytotoxic activity of rhIL2-dependent cultured NK-92 cells against primary patient-derived leukemic target cells [12 acute myelogenous leukemias (AMLs), 7 T acute lymphoblastic leukemias (T-ALLs), 14 B-lineage-ALLs, and 13 chronic myelogenous leukemias (CMLs)], human leukemic cell lines (K562, KG1, HL60, Raji, NALM6, TALL-104, CEM-S, and CEM-T) and normal bone marrow cells was measured in 51Cr-release assay (CRA). The patient-derived leukemias could be subdivided into three groups based on their sensitivity to NK-92 cells: insensitive (< or =19% lysis), sensitive (20-49% lysis), and highly sensitive (> or =50% lysis) at an E:T ratio of 9:1. Of 46 patient-derived samples, 24 (52.2%) were sensitive or highly sensitive to NK-92-mediated in vitro cytotoxicity (6 of 12 AMLs, 7 of 7 T-ALLs, 5 of 14 B-lineage-ALLs, and 6 of 13 CMLs). NK-92 cells were highly cytotoxic against all of the eight leukemic cell lines tested in a standard 4-h CRA. Normal human bone marrow hematopoietic cells derived from 18 normal donors were insensitive to NK-92-mediated cytolysis. In comparison with human lymphokine-activated killer cells, normal NK cells, and T cells, NK-92 cells displayed more powerful antileukemia activity against a patient-derived T-ALL as well as K562 and HL60 cells, both in in vitro CRA and in a xenografted human leukemia SCID mouse model. The NK-92 cells did not induce the development of leukemia in SCID mice after i.v., i.p., or s.c. inoculation. In adoptive transfer experiments, SCID mice receiving i.p. inoculations of human leukemias derived from a T-ALL (TA27) and an AML (MA26) that were highly sensitive to the cytolysis of NK-92 cells in vitro, as well as a pre-B-ALL (BA31) that was insensitive to the in vitro cytolysis of NK-92 cells, were treated by administration of NK-92 cells with or without rhIL2 (2 x 10(7) NK-92 cells i.p.; one dose or five doses). Survival times of SCID mice bearing the sensitive TA27 and MA26 leukemias were significantly prolonged by adoptive cell therapy with NK-92 cells. Some of the animals who received five doses of NK-92 cells with or without rhIL2 administration were still alive without any signs of leukemia development 6 months after leukemia inoculation. In contrast, survival of mice bearing the insensitive BA31 leukemia were not affected by this treatment. This in vitro and in vivo antileukemia effect of NK-92 cells suggests that cytotoxic NK cells of this type may have potential as effectors of leukemia control.

Modulation of leukemic cell sensitivity to lymphokine-activated killer cytolysis: role of intercellular adhesion molecule-1.

The role of CD11/CD18 leukocyte adhesion molecules and their ligands in mediating non-major histocompatibility complex (MHC) restricted lymphocyte cytotoxicity is controversial. In order to examine the role of target cell intercellular adhesion molecule-1 (ICAM-1; CD54), a ligand of lymphocyte function-associated antigen (LFA-1) (CD11a/CD18), we exposed the human leukemia cell line, HL-60, to a variety of agents implicated in modulating ICAM-1 expression and/or sensitivity to lymphocyte cytolysis. Exposure of HL-60 cells to retinoic acid (RA), interferon (IFN)-alpha, IFN-beta, and IFN-gamma induced protection from lymphokine-activated killer (LAK) cytolysis. Only RA and IFN-gamma induced ICAM-1 expression. Tumor necrosis factor and vitamin D3, which also induced ICAM-1 expression, increased HL-60 sensitivity to LAK lysis. Granulocyte-macrophage colony-stimulating factor also increased sensitivity to LAK lysis; ICAM-1 was not induced. The state of cellular differentiation and expression of class I and II MHC antigens also did not correlate with sensitivity to LAK cytolysis. Exposure of untreated HL-60 cells and HL-60 cells expressing ICAM-1 to monoclonal antibody (mAb) versus ICAM-1 did not modulate LAK sensitivity. Exposure of LAK cells to mAb versus LFA-1 partially inhibited cytolysis; mAb versus CD18 inhibited cytolysis more completely. HL-60 cells were resistant to natural killer lysis; exposure to the various experimental agents did not alter sensitivity. We conclude that leukemic cell sensitivity to LAK cytolysis can be modulated by a variety of agents. Although our results suggest a role for leukocyte CD11/CD18 adhesion molecules in LAK cytolysis, the poor correlation between ICAM-1 expression and sensitivity to LAK lysis suggest that interactions other than LFA-1/ICAM-1 conjugation may be more central to the processes involved.

Effects of insulin and transferrin on the generation of lymphokine-activated killer cells in serum-free medium.

We have developed a serum-free medium designated RDSF for the generation of LAK cells based on RD6F medium, which was originally developed as a serum-free medium for the growth of myeloma and hybridoma cells. The cytotoxic activity of LAK cells generated in RDSF against Raji, K562 and oral cancer cells, is 3-4 times that of LAK cells generated in medium containing 10% human type AB serum. RDSF medium consisted of nutrient mixture supplemented with transferrin, 2-aminoethanol, 2-mercaptoethanol, sodium selenite and interleukin-2. In this study, we have found that insulin which has been shown to be the most important polypeptide hormone in serum-free media for animal cells, inhibited the generation of cytotoxic activity of LAK cells cultured from peripheral blood lymphocytes. In addition, we found that transferrin was an essential component for the growth and generation of LAK cells in serum-free culture. These results suggest that RDSF may be useful in adoptive immunotherapy for cancer as well as for studying factors involved in the growth and differentiation of LAK cells.

Highly oncolytic adherent lymphocytes: therapeutic relevance for leukemia.

We have generated and characterized a highly oncolytic adherent lymphocyte subset (A-LAK) from eight leukemic patients with non-lymphocytic leukemia (NLL) in remission and one NLL patient in relapse. Our studies demonstrated that A-LAK was superior in its oncolytic activity (tested in a 3-h 51Cr release assay) to conventionally prepared (LAK) and non-adherent (NA) IL-2 cultures. No activity was observed by this highly oncolytic subset against normal bone marrow (BM). A-LAK also displayed highest proliferative activity in 7-11 day cultures (5- to 58-fold expansion) in comparison to LAK (0.7- to 2.7-fold) or NA (1.0- to 2.6-fold) cultures. Analysis of phenotype of unseparated, NA and adherent (A-LAK) lymphocytes 24 h after IL-2 activation showed that the A-LAK was composed predominantly of high intensity (bright) CD11a+ (LFA-1) lymphocytes (75 +/- 4.8%) when compared to the other two populations (12 +/- 2.1%). Similarly, A-LAK contained higher proportion of CD11b (CR3 receptor)-positive lymphocytes (39 +/- 2.1%) than unseparated and NA lymphocytes (11 +/- 1.4%). Double marker phenotypic studies showed that A-LAK cultures were heterogeneous and distribution of individual lymphocyte subsets differed among NLL patients. While in A-LAK culture of some patients the CD56+, CD3- natural killer (NK) cell subset was predominant, CD3+, CD56- lymphocyte subset was prevalent in others. Highest A-LAK lytic activity was always correlated with highest NK cell content. Characterization studies (using the complement-depletion technique) showed that independently of the distribution of lymphocytes in A-LAK cultures, CD16+, CD56+, CD3- NK cell subset displayed highest oncolytic effect. CD5+ subset also participated in cytotoxic function. These observations indicated that A-LAK may represent a new therapeutic approach to treatment of leukemia.

Irreversible cancer cell-induced functional anergy and apoptosis in resting and activated NK cells.

The interaction of natural killer (NK) cells with target cells, such as K562, results in NK functional inactivation and apoptosis. The role of NK-activating cytokines, IL-2, IL-12, and IL-15, in the regulation of NK inactivation and programmed cell death by target cells was examined. Purified natural killer cells were obtained from human peripheral blood and either co-incubated with K562 target cells and cytokines or the NK cells were pretreated with cytokines for 18 h prior to co-culture with K562 cells. Sorted NK cells were examined for cytotoxic activity and NK co-cultured with K562 were examined for cytokine secretion, phenotyping and DNA fragmentation. The cytotoxic activity was inhibited and was not alleviated by cytokine treatment. Whereas the cytokine treatment maintained NK cell viability for several days, NK cell viability was decreased significantly in the presence of K562 target cells. Downregulation of CD16 and upregulation of CD69 on NK cells were induced by K562 target cells and no modulation of these antigens was observed with cytokine treatment. A subpopulation of target-treated NK cells succumbed to cell death by apoptosis and cell death was not rescued by the activating cytokines. These findings demonstrate that target-induced functional inactivation and apoptosis of NK cells were not rescued by the activating cytokines IL-2, IL-12, and IL-15 regardless of whether the NK cells were pretreated with cytokines prior to exposure to K562 or the cytokines were added to the NK-K562 mixtures. These results also suggest that signals triggered by the target cells and resulting in NK cell anergy and apoptosis override cytokine-mediated signals for activation, cell proliferation, and survival.

Phenotype and serine esterase production of human cardiac allograft-infiltrating lymphocytes.

Human cardiac allograft-infiltrating lymphocytes were studied by in vitro expansion in interleukin-2. Of 28 graft-infiltrating lymphocyte cultures from 17 recipients, 17 were comprised predominantly of CD4+ T cells and 10 predominantly CD8+ T cells; one culture had equal numbers of CD4+ and CD8+ cells. The mean percentages (+/- SE) of T-cell subsets for all cultures were as follows: CD4+, 49% +/- 29%; CD8+, 42% +/- 31%. No correlation was observed between the culture phenotype and histologic findings, length of time from transplantation, or number (or class) of mismatched HLA antigens. N-alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester serine esterase (BLT-SE) is an enzyme associated with intracellular cytotoxic T-cell granules and with target-cell destruction. Sixteen cultures were tested for BLT-SE activity and had significantly increased enzyme activity as compared to untreated peripheral blood mononuclear cells from healthy control subjects (p < 0.002), or interleukin-2-treated control cells (p < 0.05). A low percentage (0.4% +/- 0.2%) of cells in the graft-infiltrating lymphocyte cultures expressed the phenotypic marker NKH-1, suggesting that the source of BLT-SE in these cultures was not natural killer or lymphokine-activated T cells. Elevated BLT-SE was observed in five of ten cultures containing predominantly CD4+ cells and five of six cultures containing predominantly CD8+ T cells. Mixed phenotype graft-infiltrating lymphocyte cultures depleted of either CD4+ or CD8+ T cells retained BLT-SE activity. Thus both CD4+ and CD8+ graft-derived T cells can produce this enzyme although much greater variability in enzyme production was seen for CD4+ T cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanism of interferon gamma-induced protection of human gliosarcoma cells from lymphokine-activated killer lysis: division of lymphokine-activated killer cells into natural killer- and T-like cells.

The mechanism by which interferon gamma (IFN-gamma) decreases the susceptibility of the established cultured gliosarcoma line Gl-1 to lymphokine-activated killer (LAK) lysis was analyzed. The results of monolayer depletion and lectin-dependent cellular cytotoxicity assays by LAK cells revealed that the resistance to LAK lysis of IFN-gamma-treated Gl-1 cells is manifested at the stage of LAK cell target recognition alone. We have also divided LAK cells into populations of phenotypically natural killer (NK)- and T-like cells with monoclonal antibodies and complement, respectively. We have used these cells to examine the mechanism of IFN-gamma-induced protection of Gl-1 cells from LAK lysis in cold target inhibition, monolayer depletion, and direct binding assays. The results revealed that NK-like cells do not recognize IFN-gamma-treated Gl-1 cells as efficiently as they do untreated targets, whereas T-like cells show the opposite tendency. In conclusion, we have demonstrated that the IFN-gamma induced protection of tumor cells from LAK lysis is predominantly regulated by the target recognition of NK-like cells. On the other hand, IFN-gamma-treated tumor cells may bind to T-like cells but fail to trigger them to initiate further stages for lysis as effectively as NK-like cells.

Histological changes in glioblastoma after intratumoral injection of autologous lymphocytes and human lymphoblastoid interferon.

Seven glioblastomas were studied between 1 and 6 months after intratumoral injection of autologous lymphocytes and human lymphoblastoid interferon. Morphological study showed a great number of lymphocytes within the tumor tissue, and interactions between lymphocytes and glioblastoma cells, suggesting a killing phenomenon. These data support the potential usefulness of adoptive immunotherapy in patients with glioblastoma by means of intratumoral administration of activated lymphoid cells.

Lymphokine-activated killer cells can kill target cells not only by early killing but also by late killing, and the late killing level against autotumor cell line.

Generally, lymphokine-activated killer (LAK) cells kill target cells early after the LAK cells adhere to them. In this study, we describe that LAK cells can also kill at a later time, such as 24-96 h. LAK cells were generated from a cancer patient and healthy volunteers. As target cells, the patient's autotumor cell line H41 was used. When LAK cells were added to the target cells in a culture well, the LAK cells killed the target cells by cell-cell adhesion within 1-4 h (early killing), but not all cells were killed. The LAK cells were then removed. However, the remaining cells ultimately died 24-96 h later (late killing). The late killing was different from the early killing because numerous granules and vacuoles appeared in the cytoplasm. The late killing was not induced by adding supernatant of the LAK cell culture, suggesting that LAK-target cell contact may be necessary for the killing. The cell injury was inhibited by 3-methyladenine (lysosome inhibitor). It suggests that the vacuoles may be caused by activated lysosome. The patient's LAK cells induced late killing at high levels. There was a high percentage of CD8(+)CD16(+) cells in the peripheral blood lymphocytes (PBL). This subset induced late killing more effectively than the CD8(-)CD16(+) subset. Killing was more conspicuous against H41 than against allogeneic cell line T98G. This type of killing is noteworthy for understanding of killing mechanism of LAK cells.

Perilymphatic injections of recombinant interleukin-2 (rIL-2) partially correct the immunologic defects in patients with advanced head and neck squamous cell carcinoma.

Patients with advanced head and neck squamous cell carcinoma (HNSCC) are severely immunocompromised. In virtually all such patients who have been studied, reduced numbers of circulating CD3+ T-cell-receptor (TCR)alpha/beta+ T lymphocytes, a reduction of natural killer (NK) activity, and a poor induction of lymphokine-activated killer (LAK) cell activity (following in vitro treatment with recombinant interleukin-2 [rIL-2]) have been detected. Recently, however, it has been demonstrated that perilymphatic injections of low doses of rIL-2 may induce a local reduction of tumor masses in these patients. The present study, a cooperative pilot effort on the clinical effects of this route of administration, showed an activation of the lytic machinery in lymphocytes belonging to the T-cell lineage, as well as a potentiation of NK activity in the peripheral blood. These findings demonstrated that the severe immunodeficiency of HNSCC patients may be at least partially corrected by in vivo administration of rIL-2.