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In allogeneic hematopoietic cell transplantation (allo-HCT), the immune recognition of host antigens by donor T lymphocytes leads to a beneficial graft-versus-leukemia (GvL) effect as well as to life-threatening graft-versus-host disease (GvHD). Genetic modification of T lymphocytes with a retroviral vector (RV) expressing the herpes simplex virus-thymidine kinase (TK) suicide gene confers selective sensitivity to the prodrug ganciclovir (GCV). In patients, the infusion of TK+ lymphocytes and the subsequent administration of GCV resulted in a time-wise modulation of antihost reactivity for a GvL effect, while controlling GvHD. Because activation required for genetic modification with RV may reduce antihost reactivity, we investigated the requirements for maximizing the potency of human TK+ lymphocytes. Whereas T-cell receptor triggering alone led to effector memory (EM) TK+ lymphocytes, the addition of CD28 costimulation through cell-sized beads resulted in the generation of central memory (CM) TK+ lymphocytes. In a quantitative model for GvHD using nonobese diabetic/severely combined immunodeficient mice, CM TK+ lymphocytes were more potent than EM TK+ lymphocytes. GCV administration efficiently controlled GvHD induced by CM TK+ lymphocytes. These results warrant the clinical investigation of CM suicide gene-modified human T lymphocytes for safe and effective allo-HCT.

Anp32e/Cpd1, a member of the acidic nuclear phosphoprotein (Anp)32 family, is characterized by the presence of an amino terminal domain containing four leucine-rich repeats and a carboxyl-terminal low-compositional complexity acidic region. In previous studies performed to understand the biological role of Anp32e/Cpd1, we showed a predominant presence of Anp32e/Cpd1 in the nucleus. However, when Anp32e/Cpd1 is in the cytoplasm, it co-localizes spatially with protein phosphatase 2A (PP2A) near cell membranes, far from the synapses. In the present work, we show that Anp32e/Cpd1 is also present as a membrane-bound 74/76-kDa protein with a widespread distribution in the brain. We reveal that the expression, synthesis and half-life of this high-molecular-weight form of Anp32e/Cpd1 are spatially and temporally correlated with the cerebellar synaptogenesis period. We demonstrate that synaptic Anp32e/Cpd1 co-localizes, interacts and inhibits PP2A activity, and that phosphorylation of Anp32/Cpd1 is required for the Anp32e-PP2A interaction. Also, subcellular localization was shown with electronic microscopy. Finally, we examine Anp32e/Cpd1 and PP2A distribution in two ataxic mutant models, weaver and staggerer, and show that their co-localization in Purkinje cell dendrites depends on parallel fibre/Purkinje cell contacts. Based on these observations, we propose that Anp32e/Cpd1 mediates synaptogenesis process by modulating PP2A activity.

Herein we describe the characteristic features of the Anp32 family represented by the cerebellar leucine-rich repeat protein (Lanp) and the cerebellar developmental-regulated protein 1 (Cpd1). The Anp32 family consists of 32 evolutionarily-conserved proteins and is included within the superfamily of leucine-rich repeat (LRR) proteins characterized by the presence of tandem arrays of a LRR, a structural motif implicated in the mediation of protein-protein interactions. We describe three novel human Anp32 proteins, reveal the evolutionary relationships of the members of the Anp32 family, provide insights into their biochemical and structural properties, and review their macromolecular interactions, substrate specificities, tissue distribution/expression patterns, and physiological and pathological roles. Recent findings indicate a conserved role of members of the Anp32 family during evolution in the modulation of cell signalling and transduction of gene expression to regulate the morphology and dynamics of the cytoskeleton, cell adhesion, neural development or cerebellar morphogenesis.

The PTEN (phosphatase and tensin homolog deleted on chromosome 10) tumor suppressor gene codifies a lipid inositol 3'-phosphatase that negatively regulates cell survival mediated by the phosphatidyl inositol 3' kinase (PIP3-kinase)--protein kinase B/Akt signaling pathway. Recently, PIP3-kinase was involved in axon polarization, but PTEN functions in dendrites are uncertain. Using amino-terminal antibodies against the catalytic domain, we found a 34 kDa fragment of PTEN protein detected only in mouse brain tissue, present in neuron dendrites and spines of cerebral cortex, cerebellum, hippocampus and olfactory bulb. The PTEN-fragment reaches the synaptic fraction with a positive temporal correlation with synaptic stabilization in postnatal cerebellum and brain. In the weaver mutant mice, PTEN was absent only in the Purkinje cells dendrites that cannot receive the granule cells synaptic input. Furthermore, the activated p-Akt/PKB was present in axons but not in dendrites of mature neuron cells. P-Akt was also altered by the weaver mutation maintaining the inverse correlation with the PTEN-fragment in Purkinje cell dendrites. In contrast, the expression of this fragment was not affected by the staggerer mutation. Together, these results suggest that synaptogenesis is a necessary process for polarization in PIP3 pathway mediated by the PTEN catalytic-fragment into dendrites of CNS neurons.

Cystic fibrosis (CF), a disease caused by mutations in the cystic fibrosis transmembrane regulator (CFTR) chloride channel, is associated in the respiratory system with the accumulation of mucus and impaired lung function. The role of the CFTR channel in the regulation of the intracellular pathways that determine the overexpression of mucin genes is unknown. Using differential display, we have observed the differential expression of several mRNAs that may correspond to putative CFTR-dependent genes. One of these mRNAs was further characterized, and it corresponds to the tyrosine kinase c-Src. Additional results suggest that c-Src is a central element in the pathway connecting the CFTR channel with MUC1 overexpression and that the overexpression of mucins is a primary response to CFTR malfunction in cystic fibrosis, which occurs even in the absence of bacterial infection.

1. Tau, which is a microtubule-associated protein, with mRNA targeted to the axon and growth cone, is involved in axonal elongation. During postnatal development in mouse, Tau expression in cerebellar granule cells is reduced afte the second postnatal week. The aim of this work was to study the regulation of the rate of the synthesis of Tau protein during the period of granule cell axonal growth in mouse cerebellum. 2. We found four [35S]methionine-labeled isoforms of Tau synthesized postnataly. Their levels remain constant from postnatal day 9 to 12 (P9-P12), and decreased by P20. 3. The rate of Tau synthesis showed differences with the rate of synthesis of total proteins. They also differ from proteins phosphatases 2A and 2B, both associated with the regulation of Tau function. In addition, the turnover of newly synthesized Tau increased at P20, compared with P9 and P12. 4. These results imply a specific developmental regulation of mRNA translation of Tau, and indicate that, after the period of synapse formation is complete, and therefore axonal growth has finished (P20), only a limited number of new Tau molecules are synthesized. This might reflect that, after synapse formation is complete, newly synthesized Tau molecules are not longer needed.

This study evaluates the effects of insulin-like growth factor (IGF)-1 receptor (IGF-1R) down-regulation in stimulated T lymphocytes by investigating the expression of early activation proteins CD69, CD25, and interleukin (IL)-2. We found that IGF-1 does not modify CD69 expression but increases transcription and protein synthesis of CD25 and IL-2. The lowest level of IGF-1R detected after 15 min of activation suggested that the effects of IGF-1 occur at the initiation of cell activation. The activation of IGF-1R was confirmed by IGF-1R phosphorylation and increased phosphorylation of microtubule-associated protein kinase. We also detected the alternative IGF-1 transcripts Ea, with paracrine/autocrine regulation, and Eb, with endocrine regulation, in Jurkat cells and in quiescent T lymphocytes, and we detected IGF-1 protein in the culture medium after stimulation. These data suggest that the proliferative effects of IGF-1 on T lymphocytes include both autocrine/paracrine and endocrine processes.

The adenomatous polyposis coli (APC) tumor suppressor protein is involved in the Wnt/wingless pathway, modulating beta-catenin activity. We report the development of a highly specific, chemically synthesized oligobody (oligonucleotide-based synthetic antibody), directed against the N-terminal region of APC. Using this reagent, we found that within 16 h of disrupting HT-29 cell-cell contacts by harvesting cells with trypsin/EDTA treatment and replating, APC was translocated from the cytoplasm to the nucleus. Five days after plating the cells, when the cells had returned to their normal confluent phenotype and cell-cell contacts were reestablished, APC returned to the cytoplasm. These results suggest that APC functions as part of a "sensor" system, and responds to the loss of cell-cell contacts by moving to the nucleus, and returning to the cytoplasm when the contacts are fully restored., (Copyright 2001 Academic Press.)

Oligonucleotide aptamers obtained using the SELEX procedure can recognize different molecules with high affinity. However, for proteins, this recognition is limited to native conformations and the specificity has not been clearly demonstrated by methods such as Western blotting, immunohistochemistry or immunoprecipitations. Using a library of oligonucleotides and a selection strategy based on high specificity instead of high affinity, we have reported previously the preparation of polyclonal oligobodies, reagents that recognize the protein PP2A in a very specific way. Here we report a method to obtain monoclonal oligobodies. The oligobody developed specifically recognized both native and denatured states of the protein CPD1 used as a model system. We further demonstrate the specificity of the monoclonal oligobody using Western blots, immunohistochemistry, and immunoprecipitation, procedures previously limited only to antibody-based detection. In addition, a confocal microscopy is shown that was obtained using an oligobody made by chemical synthesis using an oligonucleotide synthesizer, being this the first "synthetic antibody" reported.

Using synthetic peptides and a combinatorial library of 56 mer random oligonucleotides, we have developed reagents that behave as "synthetic antibodies". The results obtained with the protein phosphatase 2A as a model system are shown here. The specificity of these reagents, named "oligobodies", has been demonstrated by Western blot analysis and immunohistochemistry. The oligobodies have enormous advantages compared to antibodies: their production is independent of the immune system, they can be prepared in a few days and there is no need for a purified target protein. These reagents can be produced even if the corresponding protein was never isolated or purified, since only a partial DNA sequence from a database provides enough information to make them.

Tumor cells have been shown recently to escape immune recognition by developing resistance to Fas-mediated apoptosis and acquiring expression of Fas ligand (FasL) molecule that they may use for eliminating activated Fas+ lymphocytes. In this study, we report that tumor-specific T lymphocytes isolated from tumor lesions by repeated in vitro TCR stimulation with relevant Ags (mostly represented by normal self proteins, such as MART-1/Melan A and gp100) can develop strategies for overcoming these escape mechanisms. Melanoma cells (and normal melanocytes) express heterogeneous levels of Fas molecule, but they result homogeneously resistant to Fas-induced apoptosis. However, CD4+ and CD8+ CTL clones kill melanoma cells through Fas/FasL-independent, granule-dependent lytic pathway. In these lymphocytes, Ag/MHC complex interaction with TCR does not lead to functional involvement of FasL, triggered, on the contrary, by T cell activation with nonspecific stimuli such as PMA/ionomycin. Additionally, melanoma cells express significant levels of FasL (detectable on the cell surface only after treatment with metalloprotease inhibitors), although to a lesser extent than professional immune cells such as Thl clones. Nevertheless, antimelanoma CTL clones resist apoptosis mediated by FasL either in soluble form or expressed by Thl lymphocytes or FasL+ melanoma cells. These results demonstrate that CD4+ and CD8+ antimelanoma T cell clones can be protected against Fas-dependent apoptosis, and thus be useful reagents of immunotherapeutic strategies aimed to potentiate tumor-specific T cell responses.

The susceptibility of Th1 and Th2 cell clones to apoptosis following HIV-gp120/CD4 cross-linking and TCR activation was investigated. We show that only Th1 clones are susceptible to HIV-gp120-sensitized apoptosis, although both types of clones express similar levels of CD4 and bind similar amounts of recombinant gp120. Both types of clones, however, undergo apoptosis induced by CD95 cross-linking with agonistic monoclonal antibody (MoAb). Apoptosis induced by gp120 in the Th1 clones is inhibited by either an anti-CD95 neutralizing MoAb or an anti-CD95L neutralizing MoAb as well as by a specific interleukin-1 beta converting enzyme (ICE) inhibitor. When triggered to apoptosis by gp120, Th1 but not Th2 clones express both cell-associated and soluble CD95L. The CD95L produced by Th1 clones induces cell death, inhibitable by anti-CD95 neutralizing MoAb, of CD95 positive Jurkat cells. These data suggest that, like activation-induced apoptosis, HIV-gp120 sensitized apoptosis in Th1 clones occurs via CD95/CD95L interaction and that lack or insufficient production of CD95L is responsible, at least in part, for the resistance of Th2 clones to such apoptosis.

The aim of our work was to study apoptosis as a possible mechanism of CD4+ lymphocyte depletion in AIDS patients and to test whether IL-12 could limit this phenomenon. As an in vitro model, we used a human IL-2-dependent Th1 clone from an uninfected individual. We found that CD4 cross-linking, obtained either by mouse anti-CD4 mAb plus goat anti-mouse or by recombinant gp120 plus anti-gp120 mAb, followed by activation with immobilized anti-CD3 or anti-TCR mAb, induced apoptosis at early times (15-25% apoptotic cells at 4 hr), whereas IL-2 deprivation required longer times (20-40 hr) to induce apoptosis. Both CD4 cross-linking and IL-2 deprivation-induced apoptosis appeared to be PTK-dependent and were inhibited by either IL-2 or IL-12. Our data suggest that in vivo CD4/gp120 interactions could directly prime the apoptosis of Th1 lymphocytes and that IL-2 and IL-12 could be used to prevent this phenomenon.

Adherent lymphokine-activated killer (A-LAK) activity has been recently differentiated in recombinant interleukin-2 (rIL-2) activated PBL. A pilot study on A-LAK + rIL-2 injection into the post-surgical cavity of glioblastoma-operated patients is ongoing. Preliminary data support the feasibility of this technique, which may improve the antitumor response of the host.

To see whether cancer patients possess CD3+ CD4+ lymphocytes able to proliferate in response to autologous tumor cells (Auto-Tu), this lymphocyte subset was isolated either by positive or negative selection, both methods resulting in highly enriched CD4+ populations. Unseparated and isolated CD3+ CD4+ lymphocytes were then assayed for proliferating activity in the presence or absence of various amounts of Auto-Tu, with or without recombinant interleukin-2 (IL-2) (1.5-15 U/ml) and DR+ adherent cells or E- lymphocytes as autologous accessory cells (Auto-AC). Isolated CD3+ CD4+ lymphocytes were stimulated by Auto-Tu alone in only 1 out of 12 cases. CD3+ CD4+ cells failed to proliferate significantly in response to low doses of IL-2 alone but the addition of Auto-Tu caused stimulation in 8 out of 12 cases (67%). The further addition of Auto-AC to Auto-Tu + IL-2 resulted in enhanced response of isolated CD3+ CD4+ lymphocytes in 6 out of 8 cases tested. When reactivities to Auto-Tu in the presence of IL-2 and IL-2 + Auto-AC were considered together, positive responses of CD3+ CD4+ lymphocytes were seen in 11 out of 12 cases (92%). On the other hand, unseparated lymphocytes were stimulated by Auto-Tu alone in none out of 12 cases. Unseparated lymphocytes, however, responded to IL-2 in 11 out of 12 cases; such a response was increased by the addition of Auto-Tu in only 2 cases. Moreover, the IL-2 proliferation of unseparated lymphocytes was suppressed in 4 and in 3 out of 12 cases tested when Auto-Tu or Auto-Tu + Auto-AC were added respectively. These data indicate that lymphocytes of cancer patients contain CD3+ CD4+ cells that are usually unable to proliferate in response to Auto-Tu only. This proliferation, however, occurs when low doses of exogenous IL-2 are present and can be further amplified by the addition of Auto-AC. No response of CD4+ cells is observed in the presence of DR+ Auto-AC + IL-2 except in 2 out of 7 cases tested (28%), suggesting an Auto-Tu-restricted reactivity of CD3+ CD4+ lymphocytes in the majority of cases.

In this study 15 consecutive melanoma patients were treated with two courses of bolus recombinant interleukin 2 (rIL2) and rIL2 plus in vitro-generated lymphokine-activated killers (LAK), respectively. The immunological monitoring performed after 4 days of rIL2 or rIL2 plus LAK, indicate that the in vivo peripheral blood lymphocyte (PBL), activation (spontaneous proliferation, tumor cytotoxicity, number of DR+ PBL, obtained after the second cycle of rIL2 plus LAK is significantly higher than after the first cycle of rIL2 alone. During the 5-day interval between the two courses, PBL activation returns to baseline levels and no evidence for increased sensitivity of PBL to rIL2 is present. To further confirm this, two additional patients were studied, in whom rIL2 was administered by continuous i.v. infusion. In these two patients the in vitro versus in vivo PBL activation could be directly and simultaneously compared by using in vitro the same concentration of rIL2 reached and maintained in the patients' sera. The PBL activation induced in vivo by a cycle of rIL2 alone was significantly less (about 10 times) than that obtained in vitro with a comparable rIL2 concentration. Thus, the infusion of in vitro highly activated PBL could explain the increased in vivo lymphocyte activation of the second cycle of rIL2 plus LAK over the first cycle of rIL2 alone.

A phase-I study of the recombinant, non-mutagenized interleukin 2 (rIL2, BioleukinTM) was performed in 12 melanoma patients (Pts). From 100 to 800 micrograms/m2 of rIL2 were administered by i.v. bolus injection, TID for 4-8 days. Side-effects included fever, malaise, low serum K+ and Ca++ values, electrocardiographic abnormalities, leukopenia and thrombocytopenia. No major organ toxicity and no significant fluid retention were observed at the administered doses. Treatment induced a rapid depletion of peripheral blood lymphocytes (PBL) with a rebound (2-6 times the pre-treatment values), 24-48 hr after rIL2 discontinuation. PBL obtained between the 5th treatment day and the 2nd post-treatment day showed: (a) enhanced proliferation (II/12 Pts) with stimulation indexes of 6-52; (b) increased cytotoxicity against autologous tumor cells (2/2 Pts), allogeneic melanomas (5/7 Pts), the Daudi (5/6 Pts) and K562 cell lines (7/12 Pts); and (c) increased expression of IL2 receptors (8/12 Pts) and of DR antigens (6/12 Pts). Lymphocytes collected 1-2 days after treatment and activated in vitro with rIL2 showed a more rapid development of tumor cytotoxicity, with an earlier loss of activity. Spontaneous proliferation, autologous or allogeneic tumor cytotoxicity and expression of IL2 receptors obtained after in vivo treatment with rIL2 were significantly weaker than those induced during in vitro stimulation. No major objective responses were detected in these patients.

The sensitivity of three different human and murine doxorubicin (Dx)-sensitive or -resistant pairs of tumor cells to recombinant interleukin 2 (rIL2)-activated lymphocytes was studied. In two pairs of these sublines (LoVo human colon carcinoma and B16 mouse melanoma sublines), resistance to Dx was induced in vitro, while in the third pair (9229 human metastatic melanoma clones), Dx resistance was spontaneously present in clone 9229.24. Dx-resistant cells were efficiently lysed by rIL2-activated lymphocytes in a short-term 51Cr release assay; in some experiments a trend toward higher lysis of Dx-resistant cells was present. We then tested the tumor cell growth-inhibitory activity of rIL2-activated lymphocytes in the human tumor clonogenic assay after lymphocyte-tumor coculture. Complete inhibition of tumor cell growth was obtained with five of six sublines or clones (both Dx sensitive and resistant) after 3 to 6 days of coculture at effector lymphocyte/target tumor cell ratios of 5 to 50/1; a maximum 99% inhibition was observed with the melanoma clone 9229.4 even after coculture for 6 days at an effector lymphocyte/target tumor cell ratio of 50/1. By using lower effector lymphocyte/target tumor cell coculture ratios (1, 5, 25/1), it was shown that all the three Dx-resistant cell types were significantly more affected by activated lymphocytes than their Dx-sensitive counterparts. The LoVo/DX subline was also more lysed than its Dx-sensitive counterpart LoVo/H subline by an antitumor monoclonal antibody in a complement-mediated cytotoxicity assay, despite the fact that both sublines expressed a similar amount of antigen on the cell surface. These data indicate that Dx-resistant cancer cells are more susceptible to the lysis by rIL2-activated lymphocytes than their Dx-sensitive counterparts and that a complete inhibition of their clonogenic potential can be obtained in vitro.

Six short term-cultured melanoma cell lines and one small cell lung cancer cell line were treated in vitro with the alkylating agent mafosfamide. The sensitivity of the surviving cells to in vitro lysis by recombinant interleukin 2-activated autologous and allogeneic lymphocytes was then investigated. In no case did chemo-surviving tumor cells appear less sensitive to lymphocyte-mediated lysis than untreated counterparts. In three of seven cases (two of which were derived from the same patient), chemo-selected cells were even more sensitive to cytotoxic lymphocytes, a difference not explained by a different distribution of neoplastic cells in the various cell cycle phases. We also studied the inhibitory activity of activated lymphocytes on the clonogenic potential of chemo-surviving tumor cells by the human tumor clonogenic assay. Inhibitions of tumor cell growth in the two patients tested were 100 and 94%, respectively; the activity of lymphocytes was dependent on the coculture time and the effector/target cell ratio. These data indicate that in vitro treatment with mafosfamide does not select cells resistant to the action of activated lymphocytes and that, given the right experimental conditions, these immune effectors can completely lyse tumor cells.

A new procedure for activation of peripheral blood lymphocytes (PBL) with recombinant interleukin 2 (rIL2) is described. PBL obtained by leukapheresis were subjected to NH4Cl (ACK) treatment to clear erythrocyte contamination; Ficoll separation was not performed. PBL were subsequently seeded in 10-floor multitrays (Cell FactoryTM, CF), gasified and incubated at 37 degrees C for 3-4 days in a humidified 5% CO2 atmosphere. This procedure achieved an activation (evaluated as cytotoxicity and proliferation) comparable with that obtained by culturing PBL in small flasks. Optimal activation of PBL was achieved in CF even in the presence of granulocyte contamination of up to 40%. It was also possible to freeze, thaw and recover most of the frozen cells and their cytotoxic activity. With this procedure therefore large quantities of lymphokine activated killer cells (LAK) can be easily produced to be used in adoptive immunotherapy trials.

A phosphoprotein of 65 kDa, as determined by SDS-gel electrophoresis, has been isolated from yeast crude extracts. This phospho form copurifies with phosphoenolpyruvate carboxykinase in the enzyme purification procedure worked out in our laboratory (Tortora, P., Hanozet, G.M. and Guerritore, A. (1985) Anal. Biochem. 144, 179-185). Moreover, both proteins bind strongly to 5'AMP-Sepharose 4B in the presence of Mn2+, whereas a substantially lower binding occurs if Mn2+ is replaced by Mg2+. This binding pattern is consistent with the well-known Mn2+-dependence of yeast phosphoenolpyruvate carboxykinase. These data suggest that the 65-kDa protein might be a phosphorylation product of the native enzyme. Furthermore, although the phospho form is not immunoprecipitated by anti-phosphoenolpyruvate carboxykinase antibodies, addition of Protein A-Sepharose CL-4B to crude extracts preincubated with the antibodies results in the binding to the resin of the phospho form, thus providing immunological evidence for its identification as a modified form of native enzyme. The same 65-kDa phosphoprotein is detectable in extracts from cells grown in the presence of [32P]Pi, as well as in cell extracts incubated with [gamma-32P]ATP. Moreover, digestion of the phosphoprotein with BrCN or with Staphylococcus aureus V8 proteinase, yields two and three fragments, respectively, which appear parallel to digestion products of phosphoenolpyruvate carboxykinase, again supporting the proposed identification. Finally, analysis of the phosphorylated amino acids in the 65-kDa protein shows that phosphoserine is the only labelled phosphoamino acid.

Peripheral blood lymphocytes (PBL) and tumor-infiltrating lymphocytes (TIL) were isolated from six cancer patients and cultured in the presence of 100 units/ml of recombinant interleukin 2 (IL2). Both IL2-stimulated PBL (IL2-PBL) and IL2-stimulated TIL (IL2-TIL) lysed fresh and short-term cultured autologous tumor cells in four and six cases, respectively. In four out of six patients IL2-TIL showed a slightly higher tumor cytotoxicity than IL2-PBL without lysing autologous normal PBL or TIL. Like IL2-PBL, IL2-TIL also killed allogeneic fresh and cultured targets of different histotypes, suggesting a lack of autologous tumor cytotoxic specificity. TIL cultured for 3 weeks in IL2 maintained their killing activity against autologous and allogeneic tumor targets. Phenotypic analysis of uncultured TIL showed a predominance of CD3+ T cells (approximately 70%) with CD4+ (approximately 60%) and CD8+ (20%) lymphocyte subsets, whereas less than or equal to 3% of CD16+ natural killer cells were present. TIL but not PBL contained 12%-19% of lymphocytes which expressed activation markers such as DR and TAC. The culture of both TIL and PBL in IL2 for 2-3 weeks induced an increase in the percentage of CD8+ and a decrease in CD4+ and augmentation of Leu 19+, DR+, and TAC+ cells. These results indicate that IL2-TIL can lyse autologous tumor cells slightly better than IL2-PBL, although such an effect was also evident against allogeneic neoplastic targets.

This article surveys the available data on the sensitivity of drug-resistant tumor cells to recombinant interleukin 2 (rIL2)-activated lymphocytes (LAK). In our own study, three different experimental systems were used: 1. in vitro treatment of tumor cells with an anticancer drug followed by the use of surviving cells as targets of LAK; 2. use of pairs of drug-resistant and drug-sensitive cell sublines; 3. analysis of several tumor clones obtained from the same tumor. The antitumor activity of LAK was evaluated both by the 51Cr release and the human tumor clonogenic assay (HTCA). In all the experimental systems used, drug-resistant tumor cells were found to be significantly lysed by LAK, with a consistent trend towards a higher susceptibility than their drug-sensitive counterparts. A positive correlation between the sensitivity to LAK and the ID50 for doxorubicin (Dx) was found in 44 melanoma clones analyzed, suggesting that spontaneously drug-resistant clones have a higher sensitivity to LAK than the drug-sensitive clones. Drug-resistant cells were also more sensitive to antibody and complement-mediated lysis, whereas the higher lysis of drug-resistant tumor cells exerted by LAK was maintained in a lectin dependent cytotoxicity assay. These data offer a rationale for combining chemotherapy with adoptive immunotherapy in the treatment of cancer. Moreover, studying the reasons for the higher LAK sensitivity of drug-resistant tumor cells may provide insights into the mechanisms by which tumor cells can resist LAK action.