Phenotypic and functional characteristics of CD39high human regulatory B cells (Breg).

CD39 and CD73 are key enzymes in the adenosine (ADO) pathway. ADO modulates pathophysiological responses of immune cells, including B cells. It has recently emerged that a subpopulation of ADO-producing CD39+CD73+ B cells has regulatory properties. Here, we define the CD39high subset of these cells as the major contributor to the regulatory network operated by human B lymphocytes. Peripheral blood B cells were sorted into CD39neg, CD39inter and CD39high subsets. The phenotype, proliferation and IL-10 secretion by these B cells were studied by flow cytometry. 5'-AMP and ADO levels were measured by mass spectrometry. Agonists or antagonists of A1R, A2AR and A3R were used to study ADO-receptor signaling in B cells. Inhibition of effector T-cell (Teff) activation/proliferation by B cells was assessed in co-cultures. Cytokine production was measured by Luminex. Upon in vitro activation and culture of B cells, the subset of CD39high B cells increased in frequency (p < 0.001). CD39high B cells upregulated CD73 expression, proliferated (approximately 40% of CD39high B cells were Ki-67+ and secreted fold-2 higher IL-10 and ADO levels than CD39neg or CD39inter B cells. CD39high B cells co-cultured with autologous Teff suppressed T-cell activation/proliferation and secreted elevated levels of IL-6 and IL-10. The A1R and A2AR agonists promoted expansion and functions of CD39high B cells. CD39 ectonucleotidase is upregulated in a subset of in vitro-activated B cells which utilize ADO and IL-10 to suppress Teff functions. Proliferation and functions of these CD39high B cells are regulated by A1R- and A2AR-mediated autocrine signaling.

Methods for generation of genetically modified fibroblasts for immunotherapy of cancer.

Considerable evidence has accumulated indicating that cultured human or rodent tumor cells can be successfully transduced with cytokine genes and selected in the appropriate antibiotic-containing culture media. The selected transductants are generally able to secrete the cytokine coded for by the transduced gene, and in many cases, substantial levels (e.g., ng quantities) of the cytokine are produced. Using retroviral vectors, it has been possible to obtain stably transduced tumor cells with a variety of cytokine genes (1-4). These tumor cells have been used for immunotherapy of cancer in numerous animal models of tumor growth or metastasis, and more recently, in vaccination protocols in patients with cancer. One possible criticism that can be leveled at this type of vaccination approach is that cultured, genetically modified, and selected tumor cells might have phenotypic characteristics that are substantially different from those of unmodified tumor cells. Since retroviral vectors are often used for transduction, it is also possible that viral antigens expressed on transduced tumor cells contribute to the immune response generated as a result of vaccination. Also, primary cultures of human tumor cells are often difficult to establish and maintain.

Effects of anaerobic exercise on the immune system in eight- to seventeen-year-old trained and untrained boys.

OBJECTIVES: To determine the immunologic response to a brief bout of intense exercise in children and to determine the effects of prolonged activity and maturation level of the subjects on this response. STUDY DESIGN: We determined counts of leukocytes and their subsets, counts of lymphocytes and their subsets, and natural killer (NK) cell activity and cell number before and 3 and 60 minutes after a Wingate anaerobic test (WAnT) in 16 male swimmers (9 to 17 years of age) and 17 male nonswimmers (9 to 17 years of age). Subjects were also categorized by pubertal status based on Tanner staging and by level of physical activity. The Student t test and analysis of variance were used to determine statistical significance, with values expressed as mean +/- SEM. RESULTS: Three minutes after the WAnT, all children had increases in leukocytes (28%), lymphocytes (43%), and NK cells (395%) (p < 0.01). Swimmers had less baseline NK cell activity (54 +/- 6 cytolytic units) than nonswimmers (87 +/- 10 cytolytic units) after the WAnT (p < 0.01), although both groups showed an increase to similar levels of NK activity 3 minutes after exercise. Pubertal effects on these responses were not significant. CONCLUSIONS: Our results demonstrate transient leukocytosis, lymphocytosis, and increases in NK cell number and activity in 8- to 17-year-old boys after a brief bout of intense exercise. Formal athletic training appears to be associated with a lower baseline NK cell activity, and yet such activity is still within the normal range for this age group. Further investigations are necessary to determine the impact of such training on overall health and the ability to fight infection.

Generation of activated natural killer (A-NK) cells in patients with chronic myelogenous leukaemia and their role in the in vitro disappearance of BCR/abl-positive targets.

Activated natural killer (A-NK) cells, a subset of CD56(dim)CD3- lymphocytes, are obtained from PBMC of normal donors by adherence to plastic and culture in the presence of IL2. In this study we tested the feasibility of generating A-NK cells in patients with Ph+ chronic myeloid leukaemia (CML). Cultures obtained from patients with early chronic phase (ECP; n=7) contained a mean (+/-SD) of83 +/- 7% of CD3- cells, and those from patients with advanced chronic phase (ACP; n=7) contained 27+/-33% CD56+CD3- cells. In three patients with leukaemia in a blastic phase (BP) it was only possible to obtain one culture enriched in CD56+CD3- cells (81%). Cellular aggregates of myeloid cells and large granular lymphocytes were observed in early A-NK cell cultures. Paired freshly-adherent and cultured A-NK cells were tested for the presence of BCR/abl mRNA by RT-PCR. The BCR/abl+ cells were detected in all 12 preparations of the freshly adherent A-NK cells tested. In 6/12 the BCR/abl+ cells were no longer detectable by RT-PCR on day 14 of culture. Both proliferation and antileukaemic cytotoxicity were significantly higher (P=0.002 and P=0.029, respectively) in the BCR/abl- cultures than those in the six BCR/abl+ cultures. 5/6 BCR/abl- cultures were highly enriched in A-NK cells on day 14, and 1/6 contained predominantly CD56+CD3+ cells. Only 2/6 BCR/abl + cultures were enriched in A-NK cells on day 14, but they had poor cytotoxicity and a low proliferative index. Myeloid cells (CD33+) were more frequently detected in the BCR/abl+ than BCR/abl- A-NK cell cultures (P=0.028). These observations suggest that: (1) populations of benign A-NK cells can be generated from the peripheral blood of CML patients; (2) the ability to generate A-NK cells is impaired in patients with advanced CML; and (3) the ability to generate A-NK cells with antileukaemic activity correlates with the disappearance of BCR/abl+ cells from these cultures.

Production by human squamous cell carcinoma of a factor inducing activation and proliferation of immune cells.

OBJECTIVE: To examine supernatants (SNs) of human squamous cell carcinoma of the head and neck cell lines for soluble tumor-derived factors capable of inducing activation and proliferation of human immune cells. DESIGN: The SN of squamous cell carcinoma of the head and neck cell line PCI-50 was cultured in serum-free medium and tested for the ability to induce expression of activation antigens, proliferation, cytotoxicity against tumor cell targets and cytokine production by purified human natural killer (NK) and CD4+ T cells. RESULTS: Supernatant of PCI-50 promoted expression of the following activation markers on NK and T cells: CD25 (interleukin-2R-alpha), HLA-DR (major histocompatibility complex class II), CD54 (ICAM-1), CD71 (transferrin receptor), and CD69 (activation-inducing molecule). In addition, SN induced and significantly sustained (P < .01) proliferation of human unseparated peripheral blood lymphocytes and NK or T cells in culture. Purified human NK or T cells cultured in the presence of the SN and IL-2 (120 IU/mL) had significantly higher antitumor cytotoxicity than that mediated by NK or T cells cultured in AIM-V medium and IL-2. The SN induced cytokine (interferon gamma, tumor necrosis factor alpha, interleukin-6) production in purified NK or T cells. When the SN was fractionated by molecular weight-based filtration into fractions greater and less than 30 kd, the growth- and cytotoxicity-promoting activities were consistently detectable in the greater than 30-kd fraction. CONCLUSIONS: Culture SN of squamous cell carcinoma of the head and neck cell lines contain a soluble factor(s) capable of activating NK and CD4+ T cells and of promoting growth and antitumor cytotoxicity of these lymphocyte subsets in vitro.

Antitumor activities of subsets of human IL-2-activated natural killer cells in solid tissues.

Human NK cells can be separated into two functionally distinct subpopulations based on the ability to rapidly respond to IL-2 by adherence to solid surfaces. To determine functions of the NK cell subsets in solid tumor tissues, adherent (A) and nonadherent (NA) NK cells were evaluated for their ability to infiltrate multicellular tumor spheroids in vitro, to kill carcinoma (CA) cell targets in these spheroids, and to mediate antitumor activity in vivo. A-NK cells were less cytolytic than NA-NK cells against CA targets in single cell suspensions or in monolayers. However, A-NK cells showed a significantly better ability than NA-NK cells to infiltrate tumor tissues and kill tumor cells in spheroids of human squamous cell CA of the head and neck or breast CA. Perilesional delivery of human A-NK cells and IL-2 resulted in regression of established human squamous cell carcinoma of the head and neck tumors growing subcutaneously in immunosuppressed nude mice. Similarly, in a xenograft model of human gastric CA metastatic to liver of nude mice, a single intrasplenic injection of A-NK cells in combination with i.p. infusions of IL-2 significantly reduced the number of established hepatic metastases (p < 0.007) and prolonged survival of the mice (p < 0.003). In contrast, NA-NK cells were ineffective in either of the in vivo xenograft tumor models. These findings demonstrate that A-NK cells represent a biologically unique and important subset of NK cells that, in contrast to the rest of NK cells, function as effector cells in solid tumor tissues and, consequently, have a great antitumor therapeutic potential.

Proliferation of hematopoietic cell lines induced by a soluble factor derived from human squamous cell carcinomas of the head and neck.

The supernatant of a cell line of squamous cell carcinoma of the head and neck (SCCHN), PCI-50, was previously shown to induce activation, promote proliferation and increase antitumor cytotoxicity of freshly purified human natural killer (NK) cells and CD4+ T lymphocytes [Arch Otolaryngol Head Neck Surg (1994) in press]. This supernatant was found also to promote the growth of a variety of hematopoietic cell lines, including Jurkat, THP-1, K562, NK-92 or Epstein-Barr-virus-transformed B cell lines. The Jurkat cell line was selected as a reporter cell in an 18-h proliferation assay established to measure the growth-promoting activity of PCI-50 supernatant. The presence of soluble tumor-derived factors able to induce proliferation of Jurkat cells was demonstrated in the supernatant produced by several other SCCHN cell lines but not in that produced by a gastric cancer cell line (HR) or renal cell carcinoma line (5117G8). The growth-promoting PCI-50 supernatant was shown to contain 28 +/- 0.5 pg/ml interleukin-6 (IL-6) in vitro but was negative for interferon gamma, IL-1, IL-2, IL-4, tumor necrosis factor alpha, granulocyte/macrophage-colony-stimulating factor and IL-12. The addition of any of these recombinant cytokines to Jurkat cell cultures did not significantly promote growth, while PCI-50 supernatant was consistently growth-stimulatory. This supernatant neither enhanced intracellular Ca2+ concentration in Jurkat cells nor induced up-regulation of activation antigens on the cell surface, although it supported growth of Jurkat cells in the absence of IL-2. The growth-promoting activity in the PCI-50 supernatant was acid-labile at pH 2 for 4 h, heat-resistant at 96 degrees C for 1 h and sensitive to treatments with trypsin and pepsin. Preincubation of the PCI-50 producer cells with tunicamycin or cyclohexamide reduced the level of growth-promoting activity in the supernatant. A partial purification of this activity was achieved using Amicon filtration, chromatography on concanavalin-A-Sepharose and then a hydroxyapatite column and high-pressure liquid chromatography gel filtration. The partially purified glycoprotein had a molecular mass of 50-70 kDa, as determined by gel filtration.

Signaling via CD7 molecules on human NK cells. Induction of tyrosine phosphorylation and beta 1 integrin-mediated adhesion to fibronectin.

We have previously reported that CD7 expressed on resting human NK cells is a signal-transducing molecule, which upon ligation with mAb induces a rapid increase in cytoplasmic free calcium, secretion of IFN-gamma, and augmented NK activity against K562 targets. We now demonstrate that Ab-mediated clustering of CD7 molecules on NK cells results in enhanced phosphorylation on tyrosine residues of intracellular proteins of 60, 70, 80, 97, and 120 kDa. In the presence of genistein, a specific inhibitor of protein tyrosine kinase, the enhanced level of tyrosine phosphorylation was blocked, indicating that CD7 may induce signaling via activation of tyrosine kinases. Cross-linking of CD7 or CD16 molecules with primary and secondary Abs, as well as stimulation of NK cells with phorbol ester (PMA) or with calcium ionophore A23187 also induced beta 1 integrin-mediated adhesion of these cells to fibronectin (FN)-coated plastic surfaces. In contrast, cross-linking of CD2 expressed on the surface of NK cells had no significant effect on NK cell adhesion to FN. This adhesion was not associated with up-regulation of expression of alpha 4 beta 1 or alpha 5 beta 1 FN receptors on NK cells, but it required an intact cytoskeleton. The CD7-induced adhesion to FN was mediated by alpha 4 beta 1 and alpha 5 beta 1 integrins, as it was partially blocked by FN connective segment-1 peptide (EILDVPST), the alpha 4 beta 1-binding domain, as well as by RGD-containing peptides, the alpha 5 beta 1-binding domain, but not by EILEVPST or RGE control peptides. NK cell binding to FN was also partially inhibited by mAb to alpha 4, alpha 5, and beta 1 integrins. The mechanism by which cross-linking of CD7 or CD16 on NK cells induced adhesion to FN appeared to involve both protein tyrosine kinase and protein kinase C, because this adhesion was blocked in the presence of either genistein or a protein kinase C inhibitor, staurosporin. Our data demonstrate that signals transduced via triggering of either CD7 or CD16 molecules are involved in the regulation of the functional activity of beta 1 integrins on NK cells.

Comparison of in vivo and in vitro prostaglandin E2 production by squamous cell carcinoma of the head and neck.

Prostaglandin E2 has been identified as an immunosuppressive factor in patients with squamous cell carcinoma of the head and neck. Spontaneous prostaglandin E2 production by 21 cancer cell lines, which were obtained from 17 patients with squamous cell carcinoma of the head and neck, was determined by radioimmunoassay. In comparison with normal keratinocyte cultures, prostaglandin E2 production by cancer cell lines was significantly decreased (p < 0.0001). Prostaglandin E2 levels demonstrated no correlation to the site, stage, or histopathologic differentiation of the tumor. In a separate group of 17 patients with squamous cell carcinoma of the head and neck, tumor cells were isolated from fresh tumor specimens, and 24-hour PGE2 production in vitro was assayed. No correlation was found with tumor site, stage, or 2-year disease-free survival. Although prostaglandin E2 may have biologic significance in vivo in patients with squamous cell carcinoma of the head and neck, these findings suggest that measurements of tumor cell-derived prostaglandin E2 are not predictive of biologic behavior.

Expression and function of CD7 molecule on human natural killer cells.

The CD7 molecule, one of the earliest T-lymphocyte Ag expressed during ontogeny, has recently been demonstrated to facilitate activation of T cells and to preferentially activate TCR-gamma/delta + subset of T cells. The CD7 Ag is also expressed on human NK cells, but its function has not been determined. In this study, expression and function of CD7 Ag on highly enriched NK cells (94 +/- 3% mean +/- SD, n = 12) obtained by negative selection from peripheral blood of normal donors were investigated. The CD7 Ag was found to be expressed at a significantly (p < 0.002) higher level on fresh NK cells than on IL-2-activated, NK cells. CD7 on human NK cells was found to be a signal-transducing molecule with a rapid increase in cytoplasmic free calcium observed on binding of anti-CD7 mAb to the surface of NK cells. Cross-linking of CD7 induced expression of surface activation molecules such as CD25, CD71, HLA-DR, CD69, and CD54. Activation by anti-CD7 mAb cross-linked to plastic or through goat anti-mouse Ig also induced a variety of NK cell functions: it stimulated secretion of IFN-gamma, led to proliferation of NK cells, as measured by [3H]thymidine incorporation, and significantly enhanced cytotoxicity of NK cells against K562 targets (p < 0.03). However, CD7 on NK cells did not seem to transduce a lytic signal, because it neither mediated redirected killing of Fc gamma R+ murine mastocytoma P815 cells nor triggered lysis of a hybridoma expressing the antibody in a membrane-bound form. CD7 molecules appeared to have a regulatory role in adhesion of NK cells to fibronectin, because cross-linking of CD7 on resting NK cells significantly augmented their adhesion to fibronectin-coated plastic surfaces. However, this induced adhesion was not associated with increased expression of beta 1-integrins on NK cells. Thus, CD7-mediated signals appear to augment function of adhesion molecules on NK cells, which may be involved in NK cell activation by providing both anchorage and costimulatory triggering.

Transfer of interleukin 2 receptor genes into squamous cell carcinoma. Modification of tumor cell growth.

OBJECTIVE: Human squamous cell carcinomas of the head and neck (SCCHN) have been shown to express interleukin 2 receptor (IL-2R), and binding of the ligand, IL-2, to the receptor results in tumor growth inhibition in vitro or in vivo in an SCCHN xenograft model in nude mice. To optimize growth inhibitory effects of IL-2, expression of the alpha or gamma chains of IL-2R in SCCHN was experimentally modified by transfection of tumor cells with the respective IL-2R genes or the lacZ gene as control. DESIGN: Using plasmid vectors containing the IL-2R alpha chain gene under the control of a cytomegalovirus promoter or the IL-2R gamma chain gene under the control of a Rous sarcoma virus promoter, the IL-2R genes were transferred by lipofection into SCCHN cell lines. Stable transfectants were selected, cloned by limiting dilution, and clones were compared with the parental cell lines for their sensitivity to the growth-inhibitory effect of IL-2. RESULTS: Transfer of the IL-2R alpha chain gene into SCCHN cells resulted in significant upregulation of expression of the IL-2R alpha chain on tumor cell surface but not in increased tumor growth inhibition by IL-2. In contrast, SCCHN IL-2R gamma transfectants, which expressed IL-2R gamma chain transcripts as confirmed in RNase protection assays, were significantly inhibited in growth and were sensitive to lower concentrations of IL-2 than the parental cell lines. CONCLUSIONS: Genetic modification of IL-2R expression on IL-2R-positive tumor cells in culture significantly alters their proliferative response to IL-2. These observations open a way for developing new strategies for therapy of SCCHN based on direct interactions of IL-2 with its receptor on tumor cells.

Effect of rhinovirus 39 infection on cellular immune parameters in allergic and nonallergic subjects.

Patients with allergic rhinitis (AR), compared with nonallergic persons, have been reported to respond differently to a variety of stimuli, some of which are immunologic in nature. This study compared the systemic cellular immune responses to experimental rhinovirus (RV) 39 challenge in RV-39-seronegative AR (n = 20) and nonallergic (n = 18) subjects. Peripheral blood was obtained before, 4 or 7 days after, and 23 days after RV-39 intranasal challenge and assayed for the number and function of various white blood cells. All subjects were infected, as manifested by viral shedding in nasal secretions or seroconversion. RV-39 induced marked changes from baseline values in both immune cell number and functions. Compared with nonallergic subjects, AR subjects manifested different responses for the following parameters: (1) numbers of total white blood cells and lymphocytes (smaller increases on day 4), (2) helper/suppressor T cell ratio (absence of an increase on day 7 and presence of an increase on day 23), (3) number of IL-2 receptor-positive suppressor T cells (presence of a decrease on day 7), (4) natural killer (NK) cell numbers (absence of an increase on day 4 and presence of increases on days 7 and 23), (5) NK/T cell ratio (absence of an increase on day 4 and a decrease on day 7), (6) NK cell activity (a blunted decrease on day 7 and absence of a decrease on day 23), and (7) RV-39-induced lymphocyte proliferation (exaggerated increase on day 4). The results show that intranasal challenge with RV-39 induced RV-39-specific and nonspecific systemic cellular immune responses and a unique immunologic response pattern in AR subjects.

Rapid Il-2-induced adherence of human natural killer cells. Expression of mRNA for cytokines and IL-2 receptors in adherent NK cells.

Natural killer (NK) cells selected by IL-2-induced rapid adherence to plastic and called A-NK cells represent a phenotypically and functionally distinct subset of mature peripheral blood NK cells. To further characterize this subset of NK cells functionally, their potential to express mRNA for the IL-2R and various cytokines after IL-2 activation was examined. Highly purified normal human peripheral blood resting NK (R-NK) cells were obtained by negative immunoselection using OKT3 mAb and magnetic beads coated with goat anti-mouse Ig. By two-color flow cytometry, > 90% of these R-NK cells were either CD3-CD56+CD16+ or - or CD3-CD56-CD16+. R-NK cells were activated in the presence of 6000 IU/ml (22 nM) of IL-2 for different periods of time. After 1, 3, 5, or 24 h, plastic-adherent (A) and nonadherent (NA) NK cells were separated and compared for the expression of the IL-2R or cytokine mRNA by in situ hybridization, using 35[S]-cDNA probes. Only low proportions of R-NK cells expressed genes for IL-2Rp55 (16%) or cytokines IL-2 (20%), IFN-gamma (18%), TNF-alpha (16%), and TGF-beta (7%). Thus, the genes for the IL-2Rp55 and these cytokines were not constitutively expressed by most human R-NK cells, and there was no indication that the NK cells used in these experiments were activated in vivo or during the purification procedure. However, larger proportions of R-NK cells showed expression of mRNA for IL-1-beta (35%) and IL-6 (40%), which indicates that genes for these cytokines may be constitutively expressed in a substantial proportion of normal human circulating NK cells. When R-NK cells were incubated in the presence of 22 nM of IL-2 for 1 to 24 h and separated into A-NK cells and NA-NK cells, a large proportion of A-NK cells became positive for IL-2R and cytokine gene expression. In contrast, the proportion of mRNA-positive NA-NK cells was similar or lower than that observed for R-NK cells, with the exception of an increase in TGF-beta.(ABSTRACT TRUNCATED AT 400 WORDS)

Usage of T-cell receptor V beta chain genes in fresh and cultured tumor-infiltrating lymphocytes from human melanoma.

Tumor-infiltrating lymphocytes (TIL) freshly obtained from human malignant melanomas as well as the same TIL grown in the presence of interleukin 2 (IL2) were studied for gene expression of the T-cell receptor (TCR) variable beta regions (V beta). To perform the TCR-V beta analysis, total RNA was isolated from TIL and reverse-transcribed into cDNA, which was then amplified by PCR using 22 different 5' primers specifically recognizing the sequences of 20 V beta gene families and a 3' primer annealing to the constant region of the beta chain. The TCR-alpha constant region (C alpha) gene was co-amplified as a standard for the calculation of the percentage of each TCR-V beta gene expressed. The frequency of individual V beta regions expressed on TIL was computed from the ratio of cpm V beta to cpm C alpha for each V beta region in relation to the total of all 22 ratios. With fresh TIL obtained from 8 different melanomas, oligoclonal distribution of V beta genes expressed on TIL was observed, in comparison with a broader and unrestricted distribution seen with peripheral-blood T cells of 8 normal individuals. The oligoclonal patterns of V beta-gene expression in fresh melanoma TIL were distinct in every tumor. Several of the V beta-genes usually expressed in normal PBL were not expressed in fresh TIL in melanoma TIL cultured in the presence of IL2 and IL4 and in the absence of autologous tumor (AuTu) or antigen-presenting cells for 23 to 65 days, selection of T-cell lines expressing a restricted number of V beta genes occurred. Although in 4/5 TIL cultures this selection involved the V beta 7 gene, no relationship could be established between V beta gene expression in fresh TIL and that in T-cell lines outgrowing in long-term cultures. Selection in culture of CD3+CD8+ T-cell lines with V beta-gene expression restricted to 1 or 2 V beta families did not correlate with the presence or level of AuTu cytotoxicity mediated by these T cells. The results indicate that in TIL cultures random selection of T-cell lines with reactivity not relevant to AuTu may account for poor expression or loss of AuTu cytotoxicity by most TIL cultured long-term in the presence of cytokines and in the absence of specific antigenic stimulation.

Generation and characterization of ex vivo propagated autologous CD8+ cells used for adoptive immunotherapy of patients infected with human immunodeficiency virus.

Cytolytic T lymphocytes play an important role in host defense against viral infections, including human immunodeficiency virus (HIV). In a phase I clinical trial (protocol 080 of the AIDS Clinical Trials Group), generation of CD8+ effector cells from peripheral blood of patients with acquired immunodeficiency syndrome (AIDS)-related complex (ARC) or AIDS and safety of autologous adoptive transfer of these cells were evaluated. For therapeutic infusions, CD8+ T cells were purified by positive selection on anti-CD8 monoclonal antibody-coated flasks from leukapheresed peripheral blood of seven patients. These CD8+ T cells were cultured in the presence of interleukin-2 and phytohemagglutinin for up to 3 weeks to obtain cells sufficient for therapeutic infusions (10(8) to 10(10)). All 31 cell cultures established from the seven patients and used for therapy were highly enriched in CD8+ (mean, 97%), CD8+HLA-DR+ (50%), cytotoxic CD8+CD11b- (82%), and memory CD29+ (78%) T lymphocytes. In vitro expanded CD8+ cells had excellent cytotoxic function at the time they were used for therapy, including HIV-specific activity against autologous targets infected with vaccinia vectors expressing HIV-IIIb antigens, gag, pol, and env. Anti-HIV activity of cultured CD8+ cells was significantly higher than that of autologous fresh peripheral blood lymphocytes. Our results show that CD8+ T lymphocytes obtained from peripheral blood of symptomatic HIV-infected patients can be purified, cultured to obtain large numbers of cells with enhanced anti-HIV activity, and safely infused into patients with AIDS as a form of immunotherapy.

Expression of mRNA for cytokines in tumor-infiltrating mononuclear cells in ovarian adenocarcinoma and invasive breast cancer.

Cytokine gene expression in tumor-infiltrating lymphocytes (TIL) in frozen-tissue sections of 2 types of human solid tumor--ovarian adenocarcinoma and invasive breast cancer--was examined by in situ hybridization with 35S-labeled cDNA probes for human cytokines. The proportion of cells containing mRNA able to hybridize to the antisense c-DNA probes for interleukin 2 (IL-2), tumor necrosis factor alpha (TNF alpha), interferon gamma (IFN gamma) or receptors for IL-2 (either p55 or p70) was also determined in human normal peripheral lymphoid tissues and inflammatory tissues. Few cells were positive for IL2 and TNF alpha mRNA in reactive human lymph nodes and tonsils. Inflammatory lesions, such as salpingitis or chronic active hepatitis, contained 10-20 times more cells positive for cytokine mRNA than reactive lymphoid tissue. In contrast, tumor-infiltrating lymphocytes (TIL) in the stroma of ovarian carcinomas or most ductal breast tumors only rarely expressed mRNA for TNF alpha, IL2 or IFN gamma. The intensity of mononuclear cell infiltration in these tumors correlated positively with the percentage of cells which expressed mRNA for IL-2, TNF alpha and IL-2R. In those ductal breast carcinomas which contained intracellular or intraductal mucins, up to 30% of lymphoid cells in the tumor stroma were positive for IL-2, TNF alpha, IFN gamma and IL-2R. Thus, strong evidence for local activation of mononuclear cells in situ, exemplified by the expression of genes for cytokines, was obtained only in inflammatory lesions and in mucin-producing breast carcinomas. In most carcinomas studied, few TIL expressed genes for cytokines as measured by in situ hybridization. Thus, human solid tumors appear to differ in their ability to induce gene expression for cytokines in TIL.

Immunologic effector cells in head and neck cancer.

Freshly isolated tumor-infiltrating lymphocytes (TIL) and lymph node lymphocytes (LNL) in patients with head and neck cancer (HNC) often have low or undetectable functional responses. Because impaired ability of these cells to produce cytokines could be responsible for their functional incompetence, spontaneous and in vitro-induced production of interleukin-2 (IL2), interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), and interferon gamma (IFN-gamma) by TIL, LNL from tumor-free as well as tumor-involved lymph nodes (LN), and peripheral blood lymphocytes (PBL) were measured. Although TIL or PBL of patients with HNC produced IL-1 beta and TNF-alpha spontaneously or after in vitro activation, LNL did not produce measurable levels of these cytokines. LNL also produced lower levels of IFN-gamma than PBL. In situ hybridization for cytokine mRNA performed with tumor tissues, and LN of patients with HNC showed that TIL as well as LNL localized in the immediate proximity of the tumor were activated, as evidenced by the expression of mRNA for IL2, IFN-gamma, IL-1 beta, TNF-alpha, and both alpha- and beta-chains of the IL2 receptor. In addition, many LNL located next to the tumor expressed mRNA for transforming growth factor-beta (TGF-beta). In contrast, LNL not adjacent to the tumor in involved LN, as well as those in tumor-uninvolved LN, did not express mRNA for cytokines or IL2 receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Extravasation of antitumor effector cells.

Development of effective local immune responses depends on the ability of lymphocytes to extravasate and migrate into nonlymphoid solid tissues. Different lymphocyte subpopulations seem to vary in their abilities to extravasate. In this review, recent advances made in understanding lymphocyte extravasation, interactions between lymphocytes and vascular endothelial cells, receptors on lymphocytes which are involved in their movement through the extracellular matrix, and cytokines, which regulate lymphocyte mobility through tissue, are described. In pathologic conditions, lymphocyte extravasation may be compromised, and delivery of immune effector cells to the site of injury is an important therapeutic end point. Adoptive therapy of solid tumors with antitumor effector cells depends on their successful delivery to the tumor. Although parameters which determine this delivery are not yet defined, considerable progress has been made in studies of the mechanisms involved in lymphocyte movement through tissues.

Cancer therapy with tumor-infiltrating lymphocytes: evaluation of potential and limitations.

Immunotherapy with tumor-infiltrating lymphocytes (TIL) activated in the presence of recombinant interleukin 2 (IL2) in vitro and adoptively transferred to patients with metastatic melanoma or renal cell carcinoma has resulted in complete or partial responses in some cases. These results have generated a wave of optimism and expectations, which may be premature. Much has been learned about TIL biology and their functional characteristics recently, but only few clinical trials have been completed to date. In this review, therapeutic potential of human TIL is evaluated based on limited knowledge of the antitumor mechanisms involved and imperfect understanding of events which occur during systemic administration of TIL. Limitations and advantages of TIL therapy are discussed and approaches to optimizing this form of therapy which are likely to be implemented in the future are summarized.