Biomedical subjects

D Santoli

Publications and source records attributed to D Santoli.

At least 19 recordsLinked to original sources

PubMed2001

Towards global analysis of mammalian proteomes using sample prefractionation prior to narrow pH range two-dimensional gels and using one-dimensional gels for insoluble and large proteins.

The number of unique protein species in proteomes from a single mammalian cell type is not well defined but is likely to be at least 10000-20000. Since standard-size two-dimensional gels typically resolve only about 1500 to 3000 spots, they merely analyze a small portion of these proteomes. In addition, all insoluble proteins and typically proteins > 100 kDa are seldom resolved on two-dimensional (2-D) gels. The current study demonstrates the feasibility of an overall strategy for more comprehensive quantitative comparisons of complex proteomes derived from physiological fluids or mammalian cell extracts. A key feature of this approach is to prefractionate samples into a few well-resolved fractions based on the proteins' isoelectric points (pIs) using microscale solution isoelectric focusing. These fractions are then separated on narrow pH range two-dimensional gels approximately +/- 0.1 pH unit wider than the prefractionated pool. When this prefractionation approach is applied to complex mammalian proteomes, it improves resolution and spot recovery at high protein loads compared with use of parallel narrow pH range gels without prefractionation. The minimal cross-contamination between fractions allows quantitative comparisons in contrast to most alternative prefractionation methods. In addition, complementary data can be obtained by parallel analysis of the solubilized fraction on high-resolution large-pore-gradient one-dimensional gels followed by mass spectrometric identification to analyze proteins between 100 and approximately 500 kDa. Similarly, insoluble proteins can be analyzed using large-pore gels for large proteins and 10-12% one-dimensional sodium dodecyl sulfate (SDS) gels for smaller proteins. Together, these strategies should permit more reliable quantitative comparisons of complex mammalian proteomes where detection of at least 10000 protein spots is needed in order to analyze the majority of the unique protein species.

Animals↗

PubMed2000

The human leukemic T-cell line, TALL-104, is cytotoxic to human malignant brain tumors and traffics through brain tissue: implications for local adoptive immunotherapy.

Preclinical studies with the human MHC nonrestricted cytotoxic T-cell leukemic line, TALL-104, were performed in anticipation of its use in cellular immunotherapy trials for primary malignant brain tumors. In this study, we have: (a) quantitated the in vitro brain tumor cell lysis; (b) measured the cytokine secretion upon coincubation of TALL-104 cells with brain tumor cells; (c) investigated the effect of dexamethasone on brain tumor cell cytolysis by TALL-104 cells; (d) explored the effects of lethal irradiation and cryopreservation on TALL-104 cell viability and lytic efficacy; and (e) estimated the damage TALL-104 cells induce to murine normal and tumor brain cells and their trafficking patterns in both normal and tumor-bearing rat brain upon intracranial infusion. In vitro coincubation of TALL-104 cells with human brain tumor cells, explants, and cell lines resulted in significant lysis of them, but normal brain cells were spared. Lysis of tumor at 4 h was unaffected by dexamethasone or lethal irradiation. Secretion of tumor necrosis factor-alpha, tumor necrosis factor-beta, IFN-gamma, or granulocyte/macrophage-colony stimulating factor upon TALL-104 cell coincubation with brain tumor cells variably occurred without always correlating with lysis. In vivo experiments using irradiated TALL-104 cells, placed at multiple times into normal cannulated rat brain, produced focal sterile abscesses at the instillation site but no widespread allergic encephalitic reaction. Cells morphologically consistent with TALL-104 cells specifically trafficked from the site of instillation through the neuropil, occasionally into the contralateral brain, and egressed at perivascular and leptomeningeal spaces. In vivo experiments with cannulated rats bearing 9L gliosarcoma showed a preferential localization of the TALL-104 cells in tumor compared with normal brain. Taken together, these data support the concept that TALL-104 cells can be used as a novel nontoxic and efficacious paradigm for cellular immunotherapy trials in human primary malignant brain tumors.

Animals↗

PubMed2000

N-(n-Benzylpiperidin-4-yl)-2-[18F]fluorobenzamide: a potential ligand for PET imaging of breast cancer.

N-(N-Benzylpiperidin-4-yl)-2-[(18)F]fluorobenzamide (2), a potential ligand for PET imaging of sigma receptor, has been found to be a potential agent for detection of breast cancer. In vivo studies in severe combined immunodeficient (SCID) mice bearing MDA-MB231 tumors showed that the uptake of compound 2 in these tumors was high (3.8%/g); the ratios of tumor/muscle and tumor/blood were 6.2 and 7.0, respectively, at 1 h postinjection. Pretreatment of SCID mice with haldol increased the uptake of compound 2 in blood, muscle, and other well-perfused organs while decreasing its uptake in tumors. The ratios of tumor/muscle and tumor/blood decreased from 6.2 and 7.0 to 1.3 and 1.1, respectively, at 1 h postinjection. At 2 h postinjection, the ratios of tumor/muscle and tumor/blood decreased from 4.9 and 7.8 to 1.4 and 1.4, respectively. The tumor uptake of compound 2 in SCID mice bearing primary tumor explants from a human breast cancer patient was lower than that in MDA-MB231 tumors (1.66%/g versus 3.78%/g), and the ratios of tumor/muscle and tumor/blood were 3.5 and 3.7, respectively, at 1 h postinjection. These results suggest that compound 2 may be a potential ligand for PET imaging of breast cancer.

Animals↗

PubMed2000

Antitumor activity of a human cytotoxic T-cell line (TALL-104) in brain tumor xenografts.

Malignant glioma in adults and primitive neuroectodermal tumors/medulloblastomas in children are the most common malignant primary brain tumors that either respond poorly to current treatment or tend to recur. Adoptive therapy with TALL-104 cells-an IL-2-dependent, major histocompatibility complex nonrestricted, cytotoxic T-cell line-has demonstrated significant antitumor activity against a broad range of implanted or spontaneously arising tumors. This study investigates distribution of systemically and locally administered TALL-104 cells and their efficacy in effecting survival of a rat model of human brain tumor. In vitro, TALL-104 cells showed significant cytotoxic activity when added to human glioblastoma cell lines U-87 MG, U-251 MG, and A1690; the medulloblastoma cell lines DAOY, D283 Med, and D341 Med; and the epidermoid cancer cell line A431. In brain tumor-bearing rats, the amount of fluorescent dye-labeled TALL-104 cells in brain increased after they were given by intracarotid injection as compared with i.v. cell administration. However, TALL-104 cells rapidly decreased to low levels within 1 h after intracarotid injection. This finding suggests that TALL-104 cells given systemically may not invade brain or tumor tissues, but rather may remain in the vascular system, making this approach less efficient for brain tumor treatment. In a model of athymic rats engrafted with human A431 carcinoma brain tumor, repetitive local administration of TALL-104 cells directly into the tumor bed resulted in a significant increase in survival time compared with control animals. Therefore, local therapy with TALL-104 cells may be a novel and highly effective treatment approach for malignant brain tumors.

Animals↗

PubMed2000

Phase I trial of TALL-104 cells in patients with refractory metastatic breast cancer.

The human cytotoxic T-cell line TALL-104 displays antitumor effects in animals with implanted and spontaneous malignancies. A Phase I trial was conducted to determine toxicity of TALL-104 cell therapy in women with metastatic refractory breast cancer. Fifteen patients with metastatic infiltrating ductal (n = 12), lobular (n = 2), or medullary (n = 1) carcinoma received escalating doses of lethally irradiated TALL-104 cells (three patients/group received 10(6), 3 x 10(6), 10(7), 3 x 10(7), and 10(8) cells/kg) for 5 consecutive days (induction course). Patients without progressive disease received monthly maintenance 2-day infusions at the same dose level. Mild grade I/II toxicity developed in 11 patients regardless of cell dose. One grade IV toxicity consequent to hepatic tumor necrosis occurred in a patient given 10(8) cells/kg, 3 weeks after the induction course. Nine patients progressed within 1 month from induction, and five patients had stable disease for 2-6 months. One patient (at 3 x 10(7)/kg) had improvement of liver metastases and ascites, and a second patient (at 10(6)/kg) experienced a dramatic relief in bone pain. Increases in blood natural killer cell activity and levels of IFN-gamma, interleukin-10, and activation markers (soluble interleukin-2 receptor and soluble intercellular adhesion molecule-1) were often seen. Only one patient developed anti-HLA class I antibody responses against TALL-104 cells; specific CTL activity developed in three patients during induction and in four patients during the maintenance boosts. In conclusion, TALL-104 cells were well tolerated by patients with metastatic breast cancer at the doses and regimen tested. The clinical responses observed in this preliminary trial demonstrate that further investigation of TALL-104 cell therapy is warranted.

Adult↗

PubMed1999

Effects of development of host immunity on the biodistribution of xenogeneic MHC non-restricted cytotoxic T cells: implications for adoptive cell therapy of cancer.

The human MHC non-restricted cytotoxic T cell line TALL-104 has potent anti-tumor effects in dogs with spontaneous tumors. This study was designed to examine the effects of the development of host immune responses on the baseline organ distribution of TALL-104 cells in healthy dogs. 111In-oxine labeled TALL-104 cells (107 cells/kg) were infused systemically in three dogs, either on day 1, 3, or 5 of a 5-day injection cycle; two dogs received two more injections of the labeled cells at monthly intervals, whereas the third dog received free 111In-oxine, 3 months after the first 5-day infusion. Analysis of blood and plasma cell clearances and imaging studies indicated a progressively faster clearance of the cells from the blood and organs after multiple daily injections as well as at the time of each monthly boost when host immune responses against the xenogeneic cells had developed. These findings have important therapeutic implications for the design of effective TALL-104 cell administration schedules in clinical trials.

Animals↗

PubMed1999

Biodistribution of human MHC non-restricted TALL-104 killer cells in healthy and tumor bearing mice.

The human cytotoxic T cell line TALL-104 displays potent anti-tumor effects in animals with spontaneous and induced malignancies. We investigated the biodistribution of TALL-104 cells in tumor bearing and healthy mice. 111In-labeled TALL-104 cells, injected intravenously, localized primarily in the lungs for the first 2 h, and redistributed to liver, spleen, and kidneys in the following 24 h both in immunodeficient and immunocompetent mice. TALL-104 cells consistently accumulated in the tumor and at sites of metastases. In animals injected with free 111In-oxine, most of the radioactivity remained in the blood pool with no significant organ accumulation. These data support the tumor homing properties of TALL-104 cells, information which is crucial to their therapeutic efficacy in forthcoming clinical trials.

Animals↗

PubMed1999

Adjuvant treatment of canine osteosarcoma with the human cytotoxic T-cell line TALL-104.

The human cytotoxic T-cell line TALL-104 has been used successfully to treat cancer in experimental mouse models with implanted tumors and in dogs with spontaneously occurring malignancies. This study investigated the efficacy of TALL-104 cells given in an adjuvant setting to dogs with appendicular osteosarcoma after surgery and chemotherapy. Of the 23 dogs enrolled in the study, 20 had undergone amputation of the affected limb, and 3 had undergone limb salvage surgery. After surgery, all dogs but one received cisplatin (CDDP) chemotherapy (60 mg/m2 i.v. every 21 days x 1-4 cycles). Four dogs also received one to six cycles of CDDP before limb amputation. After CDDP therapy, dogs without overt metastasis received gamma-irradiated (40 Gy) TALL-104 cells systemically (10(8)/kg) for 5 consecutive days, followed by 2-day monthly boosts (at the same dose) for a total of 9 months. Of the 23 dogs treated, 9 survive disease-free at 12.1-29.5 months after surgery, 11 died of metastatic disease between 5 and 21.5 months, 1 experienced a relapse in the lung 9.5 months after surgery but is still alive without further treatment at 13 months, 1 developed severe discopathy at 4 months after surgery, and 1 developed progressive neuropathy at 5.9 months after surgery. The overall median survival time is 11.5 months, and the median disease-free interval is 9.8 months. Our cell therapy results compare favorably with historical median survival times (up to 9 months) and disease-free intervals (up to 7.5 months) of dogs with osteosarcoma receiving standard therapy (surgery and chemotherapy) and support the effectiveness of adjuvant TALL-104 cell administration in preventing or delaying disease recurrence in these dogs.

Adoptive Transfer↗

PubMed1998

Role of CD38 and its ligand in the regulation of MHC-nonrestricted cytotoxic T cells.

Human CD38 is a type II transmembrane glycoprotein that regulates lymphocyte adhesion, proliferation, and cytokine production. The mAb Moon-1 recognizes a ligand for CD38 (CD38L) and specifically inhibits CD38-mediated cell adhesion. To analyze the role of CD38 and its ligand in MHC-nonrestricted T cell activation, we examined the effects of Moon-1 and the anti-CD38 mAb IB4 on the effector functions of the IL-2-dependent T cell line TALL-104 (CD3/TCR-alphabeta+, CD8+, CD56+) and of LAK cells (90% CD3+). TALL-104 cells were almost 100% reactive with both mAbs, whereas the reactivity of LAK cells for IB4 and Moon-1 ranged from 10 to 60% among different donors. From 78 to 94% of the cytotoxic CD8+/CD56+ LAK subset was CD38L+. Like mAb OKT3 (anti-CD3), and at variance with IB4, Moon-1 drastically enhanced the cytotoxicity of TALL-104 and CD8+ LAK cells against a resistant tumor target. Granule exocytosis did not appear to play a role in Moon-1-induced cytotoxicity. Moreover, neither IB4 nor Moon-1 induced [Ca2+]i mobilization in LAK and TALL-104 cells. Whereas stimulation of CD3 and CD38 resulted in a dramatic induction of cytokine (granulocyte-macrophage-CSF, IFN-gamma, TNF-alpha, and TNF-beta) release by both TALL-104 and LAK cells, ligation of CD38L was not followed by cytokine production in TALL-104 cells. Thus, cytotoxicity and cytokine release are independently regulated, at least in this system. These data demonstrate that CD38 and its ligand can regulate some T cell functions using signaling pathways distinct from those of CD3.

ADP-ribosyl Cyclase↗

PubMed1998

Growth characteristics and metastatic properties of human breast cancer xenografts in immunodeficient mice.

We evaluated the growth and metastatic potential of two human breast cancer cell lines and 16 patient-derived biopsy specimens, representing the most common histological types of breast carcinomas, upon subcutaneous implantation into severe combined immunodeficient (SCID) mice. The method of engraftment we used, based on implantation of intact tissue specimens and complete immunosuppression of the host, provided an easier system to grow human breast carcinoma specimens in mouse models and resulted in a 50% success rate of tumor take. No correlation was found between growth in SCID mice and pathological diagnosis, grading, or estrogen/progesterone receptor expression by the tumor biopsy specimen. Serial passage of the tumor fragments in SCID mice resulted in increased metastasis rates and more rapid emergence of a palpable tumor mass. A tumor from a patient with infiltrating ductal carcinoma, which grew aggressively and metastasized in 100% of the female SCID mice, was also successfully engrafted in 100% of nonobese diabetic (NOD)/SCID female mice, but systemic spread was minimal. Fragments of the same tumor grew in only 33% of male SCID mice with very limited metastases. A strong correlation (r = 0.997) was observed between tumor burden and the presence of soluble (serum) interleukin-2 receptor, a marker associated with a subset of human breast tumors. All together, these data indicate the usefulness of SCID/human breast tumor xenografts for measuring tumor progression and evaluating novel therapeutic approaches to breast cancer.

Adenocarcinoma↗

PubMed1997

Toxicological and immunological evaluation of the MHC-non-restricted cytotoxic T cell line TALL-104.

The human MHC-non-restricted cytotoxic T cell line TALL-104 has been shown to display potent antitumor effects in several animal models with spontaneous and induced malignancies. In view of its potential future use in cancer therapy, we investigated the tolerability and target-organ toxicity of these cells in various animal species. The acute toxicity of TALL-104 cell administrations was evaluated in: (a) healthy immunocompetent mice and immunodeficient (SCID) mice bearing human tumors using multiple (up to 15) intraperitoneal (i.p.) injections, and (b) healthy dogs, tumor-bearing dogs, and healthy monkeys using multiple (up to 17) intravenous (i.v.) injections. TALL-104 cells were gamma-irradiated (40 Gy) prior to administration to mice and dogs, but administered without irradiation in monkeys. Cell doses ranged from 5 x 10(7)/kg to 10(10)/kg for each injection. All regimens were well tolerated, the main clinical signs observed being transient gastrointestinal effects. Moderate and transient increases in liver transaminase levels were observed in all animal species. Discrete and transient leukocytosis with neutrophilia was also noted in dogs and monkeys after i.v. injections of TALL-104 cells. Histological analysis revealed foci of hepatic necrosis with lympho-/mono-/granulocytic infiltration in immunocompetent mice injected i.p. with 5 x 10(9)-10(10) cells/kg. In the same mice, the colon showed an increased number of muciparous cells and alterations in the villi structure: these alterations were completely reversed by 72 h after the last injection, while liver alterations reversed more slowly (1 week). No delayed or chronic toxicity was observed in any of the animals even when non-irradiated TALL-104 cells were administered: both immunocompetent mice and healthy dogs were found to be grossly and histopathologically normal when sacrificed (1 year and 1 month after the last TALL-104 injection respectively). TALL-104 cells did not persist in these hosts. In addition, monkeys showed no molecular signs of TALL-104-cell-induced leukemia in their blood 1 year after the last cell injection. Despite immunosuppression, most of the tumor-bearing dogs as well as the healthy dogs and monkeys developed both humoral and cellular immune responses against TALL-104 cells. The data derived from these preclinical studies suggest that administration of high doses of irradiated TALL-104 cells is well tolerated and would be unlikely to induce severe toxicity if applied in clinical trials to the treatment of patients with refractory cancer.

Animals↗

PubMed1997

Successful treatment of canine malignant histiocytosis with the human major histocompatibility complex nonrestricted cytotoxic T-cell line TALL-104.

The human MHC nonrestricted cytotoxic T-cell line TALL-104 exerts potent antitumor effects in animal models with both induced and spontaneous cancers. The present report documents the ability of systemically delivered TALL-104 cells to induce durable clinical remissions in four of four dogs with malignant histiocytosis (MH). The animals received multiple i.v. injections of lethally irradiated (40 Gy) TALL-104 cells at a dose of 10(8) cells/kg, with (two dogs) or without (two dogs) cyclosporin A, followed by monthly boosts. No significant clinical or laboratory toxicities developed during cell therapy; interestingly, a strong correlation was found between the dogs' clinical and immunological responses. One dog with advanced disease (intrathoracic involvement) refractory to chemotherapy achieved a complete remission (CR) within 2 months of the first TALL-104 cell infusion. This dog died 14 months later of unrelated causes: histological analysis of its organs postmortem revealed no evidence of neoplasia, thus confirming the achievement of CR also at the pathological level. The other three dogs with MH that at diagnosis had multiple s.c. and cutaneous lesions and lymphadenopathy, but no visceral involvement, were treated with TALL-104 cells as single agent (no chemotherapy was administered). Two of these dogs achieved a CR soon after cell therapy, and the third dog had two long-lasting partial responses; CR in this dog was later achieved by combined administration of chemotherapy and cell therapy. None of the three dogs that received cell therapy at diagnosis developed visceral disease in the approximately 9-22 months of follow-up. The clinical responses experienced by all four MH cases to TALL-104 cell therapy suggest the high responsiveness of this canine tumor to these xenogeneic effectors and their therapeutic potential even in the most aggressive forms of the disease.

Animals↗

PubMed1997

Cell therapy of a highly invasive human breast carcinoma implanted in immunodeficient (SCID) mice.

Although enormous progress has been made in the detection and treatment of localized (nonmetastatic) breast cancer, there has been relatively moderate progress toward the effective treatment of advanced disease. This study investigates the antitumor efficacy of a potent MHC nonrestricted cytotoxic human T cell line (TALL-104) upon transfer into a clinically relevant mouse model of metastatic breast cancer. Fragments from a surgical specimen of a patient with infiltrating ductal carcinoma were implanted s.c. in the flank region of severe combined immunodeficient (SCID) mice. One hundred % of the animals developed a local tumor mass that metastasized to subaxillary and inguinal lymph nodes, bones, lungs, liver, kidneys, ovaries, and brain, very closely mimicking the human disease. Multiple i.p. transfers of gamma-irradiated (nonproliferating) TALL-104 cells into mice bearing low tumor burden (the primary tumor mass weighed only 150 mg) completely arrested local tumor growth and prevented systemic spread into local lymph nodes and distant organs. Remarkably, cell therapy administered in an advanced disease stage (when the tumor weighed 2 g) induced a significant or total regression of established metastasis with no obvious effects on the primary tumor mass. Profound antitumor effects against both local and systemic disease were instead seen in mice that received cell therapy after surgical excision of the primary tumor. The implications of these data in adjuvant breast cancer therapy are discussed.

Aged↗

PubMed1996

Antitumor efficacy of a human major histocompatibility complex nonrestricted cytotoxic T-cell line (TALL-104) in immunocompetent mice bearing syngeneic leukemia.

The human MHC nonrestricted cytotoxic T-cell line TALL-104 displays potent tumoricidal activity both in vitro and in animals bearing either spontaneous or induced tumors. In the present study, we used B6D2F1 mice engrafted with the syngeneic pre-B leukemia cell line 7OZ as a model system to investigate the mechanisms by which TALL-104 cells display antitumor activity in an immunocompetent host. In vitro studies indicated that 7OZ cells are resistant to TALL-104 cell-induced necrotic death, as measured in 51Cr release assays, but can be killed by the xenogeneic effectors via apoptotic mechanisms. Adoptive transfer experiments showed that 70% of cyclosporin A-treated mice that received multiple i.p. injections of gamma-irradiated (nonproliferating) TALL-104 cells at an early or intermediate stage of disease did not develop any clinical or molecular signs of leukemia. If the same treatment was initiated at an advanced disease stage, it doubled the survival of the animals. Importantly, 100% of the treated mice that remained disease-free in a 4-month follow-up period rejected a further challenge of high-dose 7OZ cells; these mice had developed tumor-specific Immoral and cellular responses. In another set of experiments, marrows from leukemic mice containing </= 10% 7OZ cells were purged ex vivo with irradiated TALL-104 cells and engrafted into sublethally irradiated B6D2F, mice; up to 80% of the recipients failed to develop leukemia, and 40% of them were protected against a 7OZ rechallenge 4 months later. TALL-104 purging efficacy was lower if the marrows were infiltrated by >/= 30%. Ex vivo purging with a human lymphokine-activated killer cell preparation in the same conditions proved to be less effective. The overall data indicate the potent antileukemic effects of TALL-104 cells in vivo and provide evidence that xenogeneic effectors can induce tumor regression via a dual mechanism of direct tumoricidal activity and recruitment of endogenous antitumor immunity.

Animals↗

PubMed1996

Phase I clinical trial with a human major histocompatibility complex nonrestricted cytotoxic T-cell line (TALL-104) in dogs with advanced tumors.

The human TALL-104 cell line is endowed with a uniquely potent MHC nonrestricted tumoricidal activity across several species. In view of the potential applicability of TALL-104 cells as an anticancer agent, this study was conducted to evaluate the possible toxicity and efficacy of this new cell therapy in a superior animal model with spontaneous tumors. Nineteen canine cases with advanced, refractory malignancies of various histological types were entered in the study. All dogs had failed all other available treatments and had very limited life expectancy. Cyclosporin A was administered p.o. (10 mg/kg/day) starting from the day before TALL-104 cell administration throughout the treatment to prevent rejection of the xenogeneic effectors. Lethally irradiated (40 Gy) TALL-104 cells (10(8)/kg) were administered systemically following two treatment schedules. In the first schedule, the cells were given every other day for 2 weeks in a row and then once a week for 3 additional weeks; in the second schedule, TALL-104 cells were administered daily for a total of 5 days. None of the 19 cases showed significant clinical or laboratory toxicity; in addition, none of the dogs had to be withdrawn from the study because of immediate adverse reactions to the infusions. The severe side effects usually associated with classical lymphokine-activated killer therapy in association with high doses of interleukin 2, such as "capillary leak syndrome," were absent in this study. Remarkably, TALL-104 therapy induced various degrees of antitumor effects in 7 of the 19 dogs, including 1 complete response (continuing at +13 months), three partial responses (duration of 2 months, 3 months, and continuing at +2 months), and three transient responses. Clinical responses and immunological parameters correlated well in each case. Taken together, these data indicate that systemic administration of lethally irradiated TALL-104 cells in the absence of exogenous interleukin 2 may be regarded as a safe and promising adjuvant type of treatment for advanced cancer patients.

Animals↗

PubMed1996

Use of a lethally irradiated major histocompatibility complex nonrestricted cytotoxic T-cell line for effective purging of marrows containing lysis-sensitive or -resistant leukemic targets.

Improved marrow purging protocols are needed in autologous bone marrow transplantation (BMT) to achieve complete eradication of minimal residual disease. This study investigates the potential of a human major histocompatibility complex (MHC) nonrestricted killer T-cell line (TALL-104) as a new marrow purging agent in a clinical setting. TALL-104 cells can be irradiated without losing cytotoxic activity against tumor targets in vitro. In vivo, the irradiated killers can be adoptively transferred into immunodeficient and immunocompetent leukemia-bearing mice, and reverse their disease even in advanced stages. The present study shows that gamma-irradiated TALL-104 cells, cultured for 18 hours with marrows from healthy donors, do not impair the viability and long-term growth of committed and pluripotent hematopoietic progenitors. However, as determined by polymerase chain reaction (PCR) and colony assays, TALL-104 cells could completely purge marrows containing up to 50% lysis-susceptible myelomonocytic leukemia cells (U937). When marrows were admixed with a pre-B leukemia cell line (ALL-1), which is fairly resistant to TALL-104 cell lysis in longterm 51Cr-release assays but can be totally growth inhibited by TALL-104 cells in proliferation assays, residual ALL-1 cells were detectable by PCR after TALL-104 purging. However, importantly, these PCR+ marrows were devoid of tumorigenic activity when transplanted into the human hematopoietic microenvironment of human severe combined immunodeficient (SCID) chimeras. These data indicate the strong potential of the TALL-104 cell line in future marrow purging strategies against lysis-susceptible and -resistant leukemias.

Animals↗

PubMed1995

Clonal expansion of germline B-lineage acute lymphoblastic leukemia in severe combined immunodeficient mice.

CD19+ B lineage acute lymphoblastic leukemias (ALLs) with unrearranged Ig and TCR genes are designated germline B lineage ALLs. We used CDR3 PCR to determine whether pediatric germline B lineage ALLs contain minor subclones with rearranged Ig H V genes. In six of seven cases there were no PCR detectable CDR3 rearrangements. One case with a smear pattern on CDR3 PCR contained multiple unique CDR3 sequences at frequencies of 1-2 per 2,600, suggesting that polyclonal B cells were present at low frequency. To verify that the germline patterns were from leukemic cells and evaluate in vivo subclone differentiation, a germline B lineage ALL with the t(4;11) translocation was propagated in severe combined immunodeficient SCID) mice. The Ig and TCR genes in the leukemic cells recovered from mouse tissues were germline by Southern blot analysis except for single rearrangements that suggested subclone evolution at the Ig H and lambda loci in addition to the germline population. No CDR3 sequences were detected, indicating that the observed Ig H gene rearrangement most likely was a DJ joining. This study suggests that the transformed cell in germline B lineage ALL represents an early pro-B cell with limited tendency to further differentiate.

Adolescent↗

PubMed1995

A revertant TCR gamma delta + cell clone which has lost MHC nonrestricted cytotoxic activity but retains redirected killing upon stimulation of the CD3 receptor.

A variant nonkiller (K-) T-cell clone derived from the MHC nonrestricted killer (K+) cell line TALL-103/2 (CD3/TCR gamma delta +) was studied to determine whether its lytic defects were at the tumor-binding or post-binding level. The two TALL-103/2 clones were found to display similar mRNA expression of TCR/CD3 complex epsilon, zeta, gamma, and delta chains, and the same mRNA and protein levels of SRC-like protein tyrosine kinase and kinase activity. However, the K- cells express much less surface CD45 RA and contain smaller intracytoplasmatic cytotoxic granules with a lower expression of the TIA-1 protein. Although the K- cells do not undergo granule exocytosis upon contact with a susceptible (K562) target, they can be triggered to degranulate and display redirected killing by activation of the CD3 receptor. Moreover, OKT3 induces PPI turnover and Ca2+ mobilization in both the K+ and K- cells, whereas K562 cells induces PPI turnover only in the K+ clone. The overall data indicate that, although the K- cells have significant post-binding defects that prevent them from killing MHC nonrestricted targets, they can fully utilize their lytic machinery upon specific activation of the CD3 pathway.

Animals↗