Mutation analysis of PVRL1 in patients with non-syndromic cleft of the lip and/or palate in Guangdong.

Non-syndromic cleft of the lip and/or palate (NSCLP) is a very common birth defect; the poliovirus receptor-like 1 gene (PVRL1) has been identified as a genetic risk factor for NSCLP in patients from Norway, the Philippines, and South America. Given the considerable variation in allele frequencies across these geographical regions, this study explored the relationship between NSCLP and mutations of PVRL1 in patients from Guangdong, China. We recruited 171 NSCLP patients and 100 volunteers, and divided our samples into 2 groups: a sequencing group and a mass spectrometry group. In the sequencing group, we screened for mutations in exons 2 and 5 of PVRL1 by polymerase chain reaction and direct sequencing in 71 NSCLP patients and 100 volunteers. In the mass spectrometry group, we screened for amino acid mutations in α-spliced transcript codons 112, 131, and 395, and in the ß-spliced transcript codon 1082 using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis in 100 NSCLP patients and 100 volunteers. No mutations were detected in either PVRL1 exons 2 or 5 in the 71 NSCLP patients and 100 volunteers, nor did we find mutations of α-spliced transcript codons 112, 131, 395 and the ß-spliced transcript codon 1082 in any of the 100 NSCLP patients and 100 volunteers. Thus, mutations in exons 2 and 5 of PVRL1, and T334A, A391T, G1183A in the α-spliced transcript, and G1082T in the ß-spliced transcript do not participate in the development of NSCLP in patients from Guangdong.

Adoptive immunotherapy of newly induced murine sarcomas.

Two newly induced methylcholanthrene sarcomas of C57BL/6 mouse origin were selected for studies of the adoptive immunotherapy of established tumors. The MCA 105 and MCA 106 tumors, used during their first five transplant generations, possessed weakly immunogenic tumor-associated transplantation antigens as revealed by failure to elicit immunity to reject 10(6) tumor cells by tumor growth and excision. Specific immunity to reject a 10(6) tumor cell challenge could be elicited in less than 50% of mice by immunization with a mixture of viable tumor cells and Corynebacterium parvum. Adoptive transfer of spleen cells from properly immunized mice consistently mediated the regression of established MCA 105 and MCA 106 tumors. Following systemic administration of 10(8) immune cells into mice bearing palpable tumor, the tumor grew for at least 1 week and then completely regressed. The adoptive immunotherapy was immunologically specific for each of these tumors and was mediated by sensitized T-lymphocytes. Irradiation (1000 R) of the transferred cells abrogated their in vivo activity. With both tumors, successful therapy required prior immune suppression of the host. This latter finding suggested the existence of suppression mechanisms mediated by tumor-bearing mice although we have been unable to reconstitute this suppression by giving T-cell-depleted mice syngeneic spleen cells. The two new animal tumor models characterized in this study not only demonstrate the feasibility of adoptive immunotherapy to weakly immunogenic tumors but also provide unique opportunities for mechanistic studies of the specificity of adoptive immunotherapy.

Phenotype analyses and cellular mechanisms of the pre-effector T-lymphocyte response to a progressive syngeneic murine sarcoma.

Lymph nodes draining the progressively growing, weakly immunogenic, MCA 105 sarcoma contained tumor-sensitized but not fully functional pre-effector T-cells. These cells could further differentiate to acquire full antitumor effector function for adoptive therapy in an established in vitro sensitization (IVS) procedure. In this study, we utilized selective depletion with antibodies of lymphocyte subsets bearing the L3T4 (CD4) or Lyt-2 (CD8) antigen and of cells bearing the asialo-GM1 (ASGM-1) glycosphingolipid to identify the phenotype of pre-effector cells elicited during progressive tumor growth. Cells from lymph nodes draining a progressive MCA 105 tumor in the footpad were treated with antibodies plus complement prior to IVS. The antitumor efficacy of resulting IVS cells was assessed in adoptive therapy of 3-day established pulmonary MCA 105 metastases. Depletion of Lyt-2+ cells eliminated in vivo antitumor reactivity with concurrent elimination of in vitro cytotoxic activity against the MCA 105 tumor, whereas depletion of L3T4+ cells did not have an impact on either in vivo or in vitro antitumor reactivities. Treatment with ASGM-1 antiserum plus complement was also found to abrogate therapeutic efficacy. However, the in vitro cytotoxic activity was not affected. These results indicate that the pre-effector cells were Lyt-2+, L3T4-, and ASGM-1+. We next examined whether the sensitization of pre-effector cells in vivo required the participation of L3T4+ helper cells. To approach this, mice were depleted of L3T4+, Lyt-2+, or ASGM-1+ cells by antibody injections before tumor inoculation. Treatment with Lyt-2 monoclonal antibody abrogated the pre-effector cell response in the draining lymph nodes, as evidenced by failure to generate therapeutically effective cells following IVS. On the other hand, neither L3T4 nor ASGM-1 antibody treatment affected the generation of pre-effector cells. Thus, sensitization of Lyt-2+ pre-effector cells in response to progressive tumor occurred in the absence of L3T4+ helper cells.

Cellular interactions in effector cell generation and tumor regression mediated by anti-CD3/interleukin 2-activated tumor-draining lymph node cells.

Previous studies have demonstrated that progressive growth of the weakly immunogenic MCA 106 murine sarcoma stimulated, in the draining lymph nodes, the production of tumor-sensitized but not fully functional preeffector lymphocytes. These lymphocytes could develop into specific immune effector cells after sequential in vitro activation with anti-CD3 monoclonal antibody and interleukin 2 (IL-2). In this study, we analyzed cellular requirements for in vivo sensitization of preeffector cells, for generation of immune effector cells by the method of anti-CD3/IL-2 activation, and for adoptive immunotherapy mediated by activated cells. By selective depletion of T-cell subsets in vivo, we found that tumor regression after systemic adoptive immunotherapy required the collaboration of activated CD4+ and CD8+ cells. It was further demonstrated that CD8+ immune cells alone could mediate antitumor effects if exogenous IL-2 was provided in vivo. These results suggest that CD8+ cells served as immediate effector cells, whereas CD4+ immune cells provided a helper function via the secretion of IL-2. During in vitro anti-CD3/IL-2 activation, generation of effector cells depended on the collaborative interaction between previously sensitized CD4+ and CD8+ preeffector cells. At the stage of in vitro activation, the addition of IL-2 could not substitute the function of CD4+ cells. We next examined whether the sensitization of preeffector cells in the draining lymph nodes required cellular interactions between CD4+ and CD8+ T-cells. By in vivo depletion of T-cell subsets during tumor growth, we found that CD4+ cells were sensitized independently of CD8+ cells. More interestingly, in vivo sensitization of CD8+ preeffector cells also occurred independently in the absence of a CD4+ helper cell response. The lack of T-cell-T-cell interactions in vivo may explain the failure of effector cell generation during progressive tumor growth. Taken together, these results demonstrate that the anti-CD3/IL-2 activation defines an immune response distinct from many previously described mechanisms of antitumor immune responses.

Differences in the effects of host suppression on the adoptive immunotherapy of subcutaneous and visceral tumors.

A syngeneic transplantable sarcoma induced in C57BL/6 mice, MCA 105, was used in studies to examine host suppression on the adoptive immunotherapy of established intradermal and experimentally induced pulmonary and hepatic metastases. Fresh immune splenocytes were generated from mice immunized to the MCA 105 tumor by a mixture of viable tumor cells and Corynebacterium parvum. The adoptive immunotherapy of intradermal MCA 105 tumor with immune cells required prior immunosuppression of the recipient by sublethal irradiation with 500 R or T-cell depletion. The effect of whole-body sublethal irradiation appeared to eliminate a systemic host suppression mechanism, since partialbody irradiation involving the tumor-bearing area did not permit successful immunotherapy. Host irradiation was not required to achieve successful immunotherapy of experimentally induced pulmonary or hepatic metastases. In nonirradiated recipients bearing both intradermal and pulmonary tumors, host suppression did not affect the function of transferred immune cells to induce regression of pulmonary metastases. Thus, suppression of adoptive immunotherapy appears to be relevant to tumors confined to the skin and subcutaneous tissue but not to tumor in visceral sites, such as the lung and liver.

The 'marginal division': a new subdivision in the neostriatum of the rat.

Using a combination of anterograde and retrograde (Phaseolus vulgaris leucoagglutinin; PHA-L and wheat germ agglutinin conjugated horseradish peroxidase; WGA-HRP) tract-tracing methods and histochemical techniques, a new subdivision of the neostriatum, the marginal division, has been found in the rat brain. The marginal division is approximately 120 microns wide and is located at the caudal extent of the neostriatum and surrounds the rostral edge of the globus pallidus. The neuronal somata of the marginal division are mostly fusiform in shape, with their long axes running parallel to the border between the striatum and the globus pallidus. Histochemically, the marginal division is lighter in AChE staining, is more densely filled with Met-enkephalin-immunoreactive terminals, and has fewer choline acetyltransferase (ChAT)-immunoreactive neurons than does the rest of the neostriatum. Injections of PHA-L or WGA-HRP demonstrated that the projections of the marginal division differ from those of the main body of the striatum. The striatopallidal projection from the marginal division terminates in the caudal-most part of the globus pallidus which is rich in cholinergic neurons. In contrast, the projection from the main region of the neostriatum terminates in two bands in the globus pallidus, both of which are rostral to the area of termination of the fibres from the marginal division. The striatonigral fibres from the marginal division terminate in the caudal part of the substantia nigra pars reticulata whereas the rest of neostriatum projects to a more rostral region. Based on its cellular morphology, immunohistochemistry and projection pattern, we conclude that the marginal division of the striatum is a distinct subdivision of the neostriatum.

ABO hemolytic disease of the newborn.

The charts of newborn infants with positive direct Coombs' test were studied. Only cases in which the mother's blood was group O and the infant's group A or group B were studied. There was no difference between group A and group B infants in the frequency and severity of the hemolytic process caused by maternal antibodies. In group B infants, monospecific antibodies (anti-B) were associated with more severe hemolytic process than cross-reacting antibodies (anti-A,B). In group A infants there was no difference in the severity of the disease between monospecific antibodies (anti-A) and cross reacting antibodies (anti-A,B). Even though there was no significant difference in the sex distribution of affected infants, there was a higher number of boys in the more severely affected group.

Seroepidemiologic study of Cryptosporidium infection in children from rural communities of Anhui, China and Fortaleza, Brazil.

A cluster-sampling, cross-sectional study was conducted for assessing the prevalence of Cryptosporidium infection in children less than 16 years of age from three villages, Dondian, Linshan, and Fuziyin, in rural Anhui in eastern China. Among 320 apparently healthy children less than 10 years of age from Dondian who had stool specimens collected, cryptosporidial oocysts were found in stools of three children from Dondian, and no positive specimens were found in 239 children studied from Linshan. In addition, a total of 610 serum samples from children in these three villages were tested for specific IgG antibody to Cryptosporidium with an enzyme-linked immunosorbent assay (ELISA) and the prevalence rates were 42.3%, 51.7%, and 57.5%, respectively, in Dondian, Linshan, and Fuziyin. Seroprevalence increased progressively with age. No detectable antibody was found in infants between two and six months of age, and seropositivity steadily increased after one year of age. Among 36 sera from adults 15-60 years of age without diarrheal illness in Huanglu villages of rural Chaohu, 50% (18 of 36) were positive. As expected, a good correlation was found in the specific IgG antibody between the paired serum specimens from 30 matched mother-neonates who showed transplacental transfer of IgG. However, little or no IgM antibody was seen in the neonates even though several mothers had a positive anticryptosporidial IgM enzyme-linked immunoassay result. Forty randomly selected serum samples from children less than four years of age in a similarly impoverished semiurban community in Fortaleza, Brazil, where the majority of households also have pit toilets and shared community water supplies and 172 serum samples from patients one month to 29 years of age admitted to the University of Virginia Hospital without diarrhea were also examined. In Fortaleza, almost all children acquired antibody by their second year of life, demonstrating the high prevalence of this infection. In rural Anhui, only about half the children were infected by 5-7 years of age. The overall prevalence rate (16.9%) of seropositivity among children and young adults in Virginia was much lower than in China and Brazil. These results indicate that cryptosporidial infection is ubiquitous, and is highly endemic in these impoverished communities. The difference between China and Brazil may reflect earlier weaning, hygiene practices, poorer water or sanitation, multiple siblings in family and geographic environment in Brazil.

Anterograde and retrograde axonal transport of Phaseolus vulgaris leucoagglutinin (PHA-L) from the globus pallidus to the striatum of the rat.

Iontophoretic administration of PHA-L into the globus pallidus of rats resulted in the labeling of neuronal perikarya in the striatum as well as axons and terminals in the striatum, entopeduncular nucleus, subthalamus and substantia nigra. The labeled striatal perikarya were densely stained in Golgi fashion with virtually complete filling of the dendrites and spines. It is concluded that the striatal cells were filled by the retrograde transport of PHA-L and represent either striatopallidal cells, or striatonigral cells whose axons were interrupted as they passed through the injection site. The anterogradely labeled axon terminals in the striatum were observed in close apposition to the dendrites of the retrogradely labeled neurons suggesting the existence of synaptic contacts between the two groups of cells. This study demonstrates that PHA-L can be transported retrogradely as well as anterogradely following iontophoretic injections.

Differential expression of S100B and S100A6(1) in the human fetal and aged cerebral cortex.

S100B and S100A6 (calcylin) are two members of the S100 Ca(2+)-binding protein family and have been localized in the mammalian nervous system. However, information on their distribution in the human nervous system, especially in the developing human fetal brain, is scarce. In the present study, an immunocytochemical method was used to examine the spatio-temporal protein expression patterns of S100B and S100A6 in normal human fetal hippocampus, entorhinal cortex and occipital cortex. Normal aged adult human brain specimens were also included for comparison. From week 15 onwards, an increase with advancing gestation age in both the number and staining intensity of S100B positive, astrocyte-like cells was found in the pyramidal layer of the hippocampus, while both the molecular and polymorphic layers showed similar S100B immunoreactivities at all stages examined. A decrease in the immunoreactivities was found in the molecular layer of the aged adult hippocampus while other layers exhibited immunoreactivities similar to those of the late fetus. At week 15, the molecular, pyramidal and ganglionic/multiform layers of the entorhinal cortex also showed positive S100B immunoreactivities which were maintained throughout the rest of the gestation and in adult specimens. In the occipital cortex, the numbers of positive cells for all layers were about twofold higher than those found in the hippocampus and entorhinal cortex, and immunoreactivities detected in the granular layer increased from week 21, reaching a plateau at around week 27. S100B positive fibers were also found at week 30 but were not observed in aged adult specimens. S100A6 positive cells were on the whole fewer in number than those of S100B in the brain regions examined. The S100A6 immunoreactivities which were localized in some pyramidal neuron-like and some glial-like cells of the pyramidal and molecular layers of the hippocampus increased by midgestation and became weak in the late fetus and in aged adult specimens. Weakly stained S100A6 positive cells were also observed in the entorhinal cortex throughout the gestation and in aged adult cortex. S100A6 immunoreactivities were weak in the fetal occipital cortex. They were also localized in the glial-like cells of the aged adult occipital cortex. The differential spatio-temporal expression of S100B and S100A6 proteins suggests that the proteins play different roles in different brain regions during development and in adulthood.

The glucose oxidase-DAB-nickel method in peroxidase histochemistry of the nervous system.

A combination of the glucose oxidase-diaminobenzidine (DAB) method and the DAB-nickel method can successfully bring out details of immunoreactive structures in immunostained preparations. It is especially beneficial for visualizing fibers and terminals.

High density of zinc-containing and dynorphin B- and substance P-immunoreactive terminals in the marginal division of the rat striatum.

A distinct subdivision of the striatum has recently been described which is located at the caudomedial margin of the striatum, surrounding the rostrolateral edge of the globus pallidus. This "marginal division" has an internal organization and an efferent distribution which is distinct from the rest of the striatum. The striatum contains moderately high levels of zinc and the neuropeptides enkephalin, dynorphin and substance P. In the present study we have examined the distribution of histologically detectable zinc and of dynorphin B- and substance P-immunoreactivity in the marginal division of the striatum. Each of these substances was more dense within the confines of the marginal division than in the rest of the striatum. These data provide further evidence that the marginal division is a structurally distinct subdivision of the striatum.

Ultrastructural characteristics of substance P-immunoreactive terminals in marginal division of rat striatum.

In our previous work using immunocytochemical method combined with tract tracing techniques a new subdivision was described in the striatum of the rat. This "marginal division" is more densely filled with substance P, enkephalin and dynorphin B terminals than the rest of the striatum. In the present study, the synaptic organization of the substance P immunoreactive (SPIR) terminals in the marginal division of the rat striatum was studied using electron microscopy and immunocytochemistry for substance P (SP). Four major types of SPIR synapses were identified in the marginal division: axodendritic, axospinous, axo-axonal, and compound synapses. Axodendritic and axospinous synapses, in which the postsynaptic targets were small or large dendrites or spines, were the most common. A few axo-axonic synapses were observed as were several subtypes of compound synapses with more than two synaptic components. SPIR axon terminals formed the presynaptic components of all these synaptic types, but in one case an unlabeled bouton was observed making a synaptic connection onto a SPIR dendrite. Both symmetric and asymmetric SPIR synapses were observed in the marginal division. The vesicles in the SPIR presynaptic boutons were mostly pleomorphic although a few of them were round. The existence of asymmetric synapses, round synaptic vesicles and small postsynaptic dendrites distinguishes the ultrastructure of the marginal division from that of the other parts of the striatum. The complex characteristics of the synaptic organization in the marginal division implies that the SPIR terminals in the marginal division originate from a different source than those in the rest of the striatum. The complexity of the synaptic organization further suggests that the function of the marginal division is different from that of the rest of the striatum.

Ultrastructure of the marginal division in the rat striatum.

The discovery of a new subdivision in the striatum of the rat, the marginal division, has recently been reported. The marginal division is located at the caudal extent of the striatum, surrounding the rostrolateral border of the globus pallidus, and has different cellular morphology, immunohistochemistry and an efferent projection pattern from those of the main body of the striatum. In the present study, the ultrastructural organization of the marginal division was investigated. Most neuronal somata in the marginal division were fusiform in shape and had a large pale oval nucleus without in-foldings. There were four types of synapses in the marginal division: axo-somatic, axo-dendritic, axo-spinous and axo-axonic. Both symmetric and asymmetric synapses were observed on the somata, dendrites, or dendritic spines. Most of the symmetric synapses contained pleomorphic vesicles, whereas the asymmetric ones contained mainly round vesicles. Individual axo-axo-spinous synapses, which were first described in the striatum, were also observed in the marginal division. These ultrastructural characteristics distinguish the marginal division from the rest of the striatum.

[Experimental study on effect of Brucea javanica oil emulsion on rabbit intracranial pressure].

Using the method of intubation into lateral cerebral ventricle, the effect of Brucea javanica oil emulsion (BJOE) venous emulsion and oral emulsion on rabbits with normal and intracranial hypertension respectively were observed, to study whether BJOE could reduce intracranial pressure or not. The results shown that venous emulsion of BJOE had strong action against the elevation of intracranial pressure produced by SNP (P < 0.01) while oral emulsion had mild action against it, which was similar to the clinical observation exhibiting improvement of clinical manifestations after application of BJOE on intracranial hypertension caused by brain metastasis from lung cancer.

Generation of therapeutic T lymphocytes from tumor-bearing mice by in vitro sensitization. Culture requirements and characterization of immunologic specificity.

We have previously established an in vitro sensitization (IVS) procedure with which lymphocytes from tumor-bearing mice could be expanded and sensitized to acquire antitumor reactivity capable of mediating the regression of established pulmonary metastases from the weakly immunogenic MCA 105 murine sarcoma. Culture conditions required for the optimal generation of therapeutic effector cells were evaluated in the current study. Generation of effector cells by IVS required stimulation by intact tumor cells. Tumor cells killed by heat or disrupted by sonication were ineffective, but the antigenicity of tumor cells was not affected by gamma-irradiation. Long term established tumor cell lines could also serve as antigenic stimulator cells albeit with lower efficiency than fresh tumor cells. IL-2 was essential for cellular proliferation during IVS. The concentration of 1000 U/ml of IL-2 also induced nonspecific lymphokine-activated killer (LAK) activity. However, cytotoxic cells were generated during IVS in response to a broad range of IL-2 concentrations. At low IL-2 concentrations (2 to 10 U/ml), IVS cells were generated which displayed little or no LAK activity, had a greater therapeutic efficacy than those generated with high concentrations of IL-2 (100 to 1000 U/ml). Despite having high LAK activity, IVS cells, from cultures where IL-2 was added 3 or more days after initiation, had no therapeutic effect. Thus, the generation of therapeutic cells occurred independently of LAK cell production. Adoptive immunotherapy with IVS cells from MCA 105 tumor-bearing mice demonstrated cross-reactivity with the immunologically distinct MCA 106 but not the nonimmunogenic MCA 102 tumor. In contrast, IVS cells from MCA 106 tumor-bearing mice exhibited specific in vivo reactivity. In vitro cytotoxicity analyses revealed that IVS cells from MCA 105 and MCA 106 tumor-bearing mice were able to lyse both MCA 105 and MCA 106 target cells, but the reactivity toward inoculating tumors was highest. Considering previous findings that the MCA 105 and MCA 106 sarcomas possessed distinct tumor-specific transplantation Ag, the cross-reactivity observed in this study suggests that the immune response during progressive tumor growth may be different from that elicited in response to active immunization.

Lymphocytes generated by in vivo priming and in vitro sensitization demonstrate therapeutic efficacy against a murine tumor that lacks apparent immunogenicity.

The adoptive transfer of sensitized lymphocytes is an effective means to mediate the regression of established tumors. However, successful therapy can only be demonstrated in animal models where tumors are intrinsically immunogenic, capable of eliciting systemic immunity. To explore the potential of this therapeutic approach to tumors of less immunogenicity, we have selected and used a murine tumor, MCA 102, for the current study because all attempts to immunize syngeneic mice failed. We report here that inoculation of mice with a mixture of tumor cells and a bacterial adjuvant, Corynebacterium parvum led to the production of sensitized, but not fully functional, lymphocytes in the draining lymph nodes (LN). These cells, termed pre-effector cells, could nevertheless further differentiate to acquire full immunologic function by an established in vitro sensitization culture method. In adoptive immunotherapy experiments, transfer of as few as 1.5 X 10(7) in vitro sensitized cells not only reduced established pulmonary MCA 102 metastases but also prolonged survival and cured tumors in a majority of the treated animals. In order to elicit pre-effector cells in vivo, inoculation with both tumor cells and C. parvum was essential. Although a broad range of numbers of MCA 102 tumor cells appeared to be effective, generation of pre-effector cells was dependent on the dose of C. parvum. We have found that a C. parvum dose of 25 micrograms was optimal, whereas higher doses of the adjuvant had suppressive effects. Analysis of the kinetics of their appearance revealed that the generation of pre-effector cells was transient. They were detectable 7 days after in vivo priming followed by a rapid decline. Furthermore, pre-effector cells were detected only in the regional draining LN. No reactivity was demonstrable in the spleen, mesenteric LN, PBL, or bone marrow. Taken together, these results expand the scope of immunotherapy by demonstrating the feasibility of manipulating a limited and obscure immune response to the MCA 102 tumor for therapeutic efficacy.

Generation from tumor-bearing mice of lymphocytes with in vivo therapeutic efficacy.

Systemic transfer of sensitized lymphocytes can effectively mediate the regression of established tumors. However, virtually all prior experimental applications of this approach have utilized lymphocytes from animals that have been immunized to reject tumor challenge. A similar source of cells is not available in the human. With the use of a weakly immunogenic murine tumor, MCA 105, we demonstrate here that following in vitro sensitization (IVS) with viable tumor cells and interleukin 2, the nontherapeutic lymphoid cells from mice bearing a progressively growing tumor acquired antitumor reactivity capable of mediating the regression of established pulmonary metastases. Although the IVS system induced nonspecific lymphokine-activated killer-like cytotoxic activity from lymphoid cells of normal as well as tumor-bearing mice, therapeutically active cells could only be generated from cultures initiated with lymphoid cells from tumor-bearing animals, indicating that the IVS was a secondary in vitro immune response. Without other treatment, the IVS cells could mediate antitumor effects. However, low doses of exogenous interleukin 2 administration could enhance their therapeutic efficacy. By in vivo T cell subset depletion with monoclonal antibodies, the primary effector cells were identified as belonging to cytotoxic/suppressor T cell lineage expressing the Lyt-2 phenotype. In addition, these therapeutic effector cells could be further expanded in numbers in vitro with continuous stimulation by tumor cells in the presence of interleukin 2. Compared to the number of cells initiating the culture, as many as 126 times the number of cells were obtained after 9 days of IVS followed by in vitro expansion for an additional 5 days. Studies on the kinetics of the occurrence of the pre-effector lymphocytes during tumor growth revealed that they were readily obtained from draining lymph nodes of mice with a broad range of tumor burdens as well as durations of tumor growth. The ability to generate and expand, in vitro, therapeutically active lymphocytes from tumor-bearing hosts has important implications for cellular therapy of human cancers.

Activation by anti-CD3 of tumor-draining lymph node cells for specific adoptive immunotherapy.

Lymph nodes draining progressive tumors contain tumor-sensitized but not functional preeffector T lymphocytes. These cells can acquire antitumor reactivity after stimulation with tumor cells and interleukin-2 (IL-2). We demonstrated here that, in the absence of tumor cells, preeffector cells could be stimulated and expanded by sequential culture with anti-CD3 monoclonal antibody and IL-2. The adoptive transfer of such activated cells mediated immunologically specific reductions of established pulmonary metastases. The therapeutic effects could be enhanced by the administration of IL-2. This activation represents a secondary immune response because effector cells could be generated only from tumor-draining but not from normal or adjuvant-stimulated lymph nodes. Furthermore, treatment of advanced metastases with these cells resulted in prolongation of survival and cure of the disease. Thus, anti-CD3 may serve as a universal reagent for activating tumor-sensitized T lymphocytes for cancer therapy.