Autologous tumor-specific cytotoxic T lymphocytes in the infiltrate of human metastatic melanomas. Activation by interleukin 2 and autologous tumor cells, and involvement of the T cell receptor.

TIL from metastatic melanoma proliferated by greater than 1,000-fold (840-3,675, mean 1,543) after 6 wk in culture of mixtures of TIL and tumor cells with rIL-2 alone. Cytolysis was restricted to autologous tumor cells. CD8+ T cells were the predominant population of TIL before and after expansion, and were primarily responsible for autologous tumor-specific CTL activity. No other rIL-2-activated lymphocytes from peripheral blood, lymph nodes with melanoma metastasis, or TIL from sarcoma or renal cell carcinoma had autologous tumor-specific CTL activity. There were few or no CD16+ NK cells in TIL from metastatic melanoma before or after incubation with rIL-2, respectively. However, TIL from sarcoma or renal cell carcinoma contained a substantial proportion of CD3-CD16+ NK cells, which increased in number in culture with rIL-2. Purified CD16+ NK cells as well as CD3+CD16- T cells from rIL-2-activated TIL of renal cell carcinoma displayed MHC-nonrestricted cytotoxicity. At the clonal level as determined by limiting dilution, 8 of 10 clones from melanoma TIL displayed cytotoxicity restricted to autologous tumor cells, while all 13 clones from renal cancer TIL equally lysed autologous and allogeneic tumor cells. Anti-T cell receptor (TCR)-alpha/beta(WT31) mAb as well as anti-CD3 mAb inhibited autologous melanoma cell-specific CTL activity mediated by rIL-2-activated TIL at the effector phase. These two mAbs also inhibited rIL-2-dependent proliferation of these TIL when added to the culture. Pretreatment of fresh melanoma cells with mAb to MHC antigens followed by washing inhibited specific CTL activity. These results suggest that both TCR-alpha/beta on effector TIL and MHC antigens on fresh tumor cells are involved in the specific immune-recognition. After reaching maximum propagation, TIL from metastatic melanoma responded poorly to rIL-2 alone. However, stimulation with fresh autologous melanoma cells restored both CTL activity and proliferation in response to rIL-2. The latter is associated with IL-2 receptor (Tac antigen) expression on the surface. These results indicate that TIL from metastatic melanomas may have unique characteristics different from lymphocytes obtained from the other sources, and may contain precursor CTL sensitized in vivo to autologous tumor cells, and thus can be propagated in larger numbers with rIL-2 alone while retaining autologous tumor-specific CTL activity.

Disulfide-linked gamma delta T cell antigen receptors expressed on T cells derived from patients with primary immunodeficiency disorders.

T cell lines or clones from two patients, one with a partial DiGeorge syndrome and one with severe common variable immunodeficiency expressed disulfide-linked gamma delta T cell antigen receptor (TCR) comprised of a gamma-chain polypeptide of 40-43 kD, and a delta-chain polypeptide of 37-40 kD. This gamma delta TCR appears to be similar to that found on T cell clones, and lines derived from peripheral blood lymphocytes from normal donors. Previous studies have shown that T cell lines derived from the peripheral blood of patients with immunodeficiency disorders express non-disulfide-linked gamma delta TCR. In contrast to the latter and coincident with findings in the present study, the vast majority of T cell lines and clones derived from the peripheral blood of normal donors express disulfide-linked gamma delta TCR.

Adenosine receptors and modulation of natural killer cell activity by purine nucleosides.

Natural killer (NK) cell activity is inhibited by the adenosine analogue tubercidin (Tub) and stimulated by the deoxyadenosine analogue 2-fluoro-1-beta-D-arabinofuranosyladenine 5'-monophosphate (F-ara-AMP) in the spleen lymphocytes from mice treated with the drugs in vivo (T. Priebe et al., Cancer Res., 48:4799, 1988). The present report demonstrates that the inhibition by Tub and stimulation by F-ara-AMP of NK cell activity are readily demonstrable in murine and human lymphocytes exposed to the drugs in vitro. In mouse spleen lymphocytes, NK cell activity is also inhibited by adenosine receptor A2 agonists, whereas potent A1 receptor agonists are more effective stimulators. Inhibition produced by adenosine, deoxyadenosine, and adenosine receptor agonists, but not by Tub, is partially prevented by the adenosine receptor antagonist 1,3-dipropyl-8-phenylxanthine amine congener. Agents that stimulate NK cell activity (deoxyadenosine, A1 receptor agonists, F-ara-AMP) do not increase further the 1.5-fold enhancement produced by a 10(-6) M concentration of 1,3-dipropyl-8-phenylxanthine amine congener. The nucleoside transport inhibitor, p-nitrobenzylthioinosine 5'-monophosphate, has no effect on NK cell activity or intracellular ribonucleotide pools; however, it partially prevents Tub 5'-triphosphate formation, ATP depletion, and NK cell inhibition in mouse spleen cells treated with Tub. p-Nitrobenzylthioinosine 5'-monophosphate also partially prevents the F-ara-AMP stimulation of NK cell activity, but it does not influence the effects of adenosine or deoxyadenosine. The results obtained with the adenosine receptor agonists suggest roles for both A1 and A2 receptors in regulating murine NK cell activity. Tub inhibition of NK cell activity does not appear to involve adenosine receptors; however, inhibition by the other agents may be mediated via an A2 receptor (stimulatory for adenylyl cyclase). Since p-nitrobenzylthioinosine 5'-monophosphate inhibited the stimulation of NK cell activity by F-ara-AMP, this stimulation may occur via an intracellular "P" site (inhibitory to adenylyl cyclase).

Tumor cytolysis by lymphocytes infiltrating ovarian malignant ascites.

Tumor-associated lymphocytes (TAL) were isolated from the ascitic fluid of patients with adenocarcinoma of the ovary. These cells proliferated and expanded by 100-600-fold as either CD3+ CD4+ or CD3+ CD8+ cultures in the presence of moderate concentrations (50-200 cetus units/ml) of recombinant interleukin 2 and reached high numbers (5 x 10(8)-1 x 10(9)). After expansion of 16 TAL samples from 15 patients, 5 of the 7 isolated ovarian cytotoxic T-lymphocyte cell lines of T-cell receptor (TCR) (alpha beta)+ CD3+ CD8+ CD4- phenotype exhibited preferential cytolytic activity against autologous tumor targets and significantly lower cytolytic activity against allogeneic tumor targets and the natural killer-sensitive cell line K562. The cytolytic activity of the CD8+ TAL was inhibited by operationally anti-TCR (alpha beta) monoclonal antibody and monoclonal antibody specific for the CD3 differentiation antigen, indicating that the TCR and CD3 are involved in the cytolytic process. The other TAL cultures demonstrated similar cytolytic activity against both autologous and allogeneic tumors. The phenotype of these TAL was predominantly TCR (alpha beta)+ CD3+ CD4+ CD8-. Certain CD3+ CD8+ T-cell clones isolated from representative TAL exhibited preferential autologous tumor-specific cytotoxicity that may be major histocompatibility complex restricted. Other CD3+ CD8+ and CD3+ CD4+ clones exhibited nonmajor histocompatibility complex restricted cytotoxicity. These results demonstrate that CD3+ CD4+ and CD3+ CD8+ T-cells present in the ovarian malignant ascites can be propagated in large numbers and for long time intervals as T-cell lines in vitro. This finding may be significant for further investigation of ovarian tumor-specific cytotoxic T-lymphocytes and future adoptive specific immunotherapy studies.

Involvement of both Tac and non-Tac interleukin 2-binding peptides in the interleukin 2-dependent proliferation of human tumor-infiltrating lymphocytes.

Interleukin 2 (IL-2) receptor expression was examined on recombinant IL-2 (rIL-2)-propagated tumor-infiltrating lymphocytes (TIL) from eight metastatic melanoma and three sarcoma samples. All 11 TIL expanded with similar growth rates. rIL-2 propagated TIL from five of eight metastatic melanoma specimens contained no Tac antigen-positive lymphocytes as determined by immunofluorescence and flow cytometry performed multiple times during the 4 to 8 week culture period. However, "Tac-negative" TIL did express the non-Tac IL-2-binding peptide, p70-75 as determined by [125I]IL-2 cross-linking and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. IL-2-binding assays revealed that these "Tac-negative" TIL expressed only an intermediate affinity IL-2 receptor. In contrast, TIL from the other three of eight melanoma and all three sarcoma contained one-third Tac-positive cells as assessed by flow cytometry analysis, and expressed surface non-Tac (p70-75) and Tac (p55) peptides by [125I]IL-2 cross-linking. These "Tac-positive" TIL displayed both the high and intermediate affinity IL-2 receptors. However, rIL-2-dependent growth of both "Tac-negative" and "Tac-positive" TIL was significantly inhibited by anti-Tac mAb, suggesting a transient Tac expression on the "Tac-negative" TIL. Additionally, due to the limits of our methodology, we cannot rule out the possibility of a constitutive expression of a low level of Tac, with an indicible expression of higher levels. Addition of culture supernatants from phytohemagglutinin- and phorbol myristate acetate-stimulated peripheral blood mononuclear cells to the "Tac-negative" TIL-induced detectable Tac expression within 48 h. These results indicate that both non-Tac and Tac IL-2 receptors play important roles during IL-2-dependent proliferation of TIL.

Lineage-negative human leukocyte antigen-DR+ cells with the phenotype of undifferentiated dendritic cells in patients with carcinoma of the abdomen and pelvis.

The characteristics of antigen-presenting cells in carcinomas that involve the abdominopelvic cavity are unknown. Dendritic cells, a population of antigen-presenting cells, have been identified as lineage-negative human leukocyte antigen (HLA)-DR+ cells by two-color flow cytometry. We used this criterion to study the putative dendritic cells in ascites from 25 patients with peritoneal carcinomatosis. The mean proportion +/- SD of lineage-negative HLA-DR+ cells in ascites was 3.1 +/- 4.6% (range, 0.05-17.3%). Most lineage-negative HLA-DR+ cells expressed CD45RA or CD4 antigens. Dendritic cells had low proportions of CD80, CD11c, CD45RO, and CD58, suggesting that they were of low maturity. The proportion of lineage-negative HLA-DR+ cells in ascites of seven patients was significantly higher than the proportion in peripheral blood from the identical patients (4.5 +/- 5.7 versus 0.5 +/- 0.4; P < 0.05). In paired specimens of ascites and peripheral blood, the proportion of lineage-negative HLA-DR+ cells that coexpressed CD86 or CD58 was significantly lower in ascites than in peripheral blood, whereas a higher proportion of lineage-negative HLA-DR+ cells in ascites expressed CD4. Relative fluorescence intensity of HLA-DR+ was also lower in dendritic cells from ascites and blood from patients with carcinomatosis than it was in blood from normal donors. As an indicator of macrophage activation, the concentration of neopterin in ascitic fluid correlated negatively with the numbers of lineage-negative HLA-DR+ cells in ascites (Spearman correlation coefficient, -0.44; P = 0.05) correlated positively with the concentration of interleukin 10 in ascitic fluid (Spearman correlation coefficient, -0.40; P = 0.05). IFN-gamma and tumor necrosis factor alpha were also not detected. These findings suggest that certain factors associated with the tumor microenvironment might influence the number of these dendritic cells and their expression of function-associated markers.

Quantitative analysis of transforming growth factor beta 1 and 2 in ovarian carcinoma.

Transforming growth factor beta (TGF-beta) is an important family of cytokines that may promote tumor growth in vivo through several mechanisms including interference with antitumor T-cell immune responses, alteration of factors in the stroma and matrix, and the promotion of angiogenesis. TGF-beta isotypes have been detected in malignant and normal ovarian tissues. We have determined by quantitative immunohistochemistry the density of TGF-beta1, TGF-beta2, and human leukocyte antigen (HLA) Class I and Class II antigens on malignant cells in paired primary and metastatic specimens from 10 patients with ovarian carcinoma. Cryostat sections of specimens from the carcinomas and from normal ovaries of three women of similar age without ovarian cancer were stained respectively with specific antibodies to TGF-beta1, TGF-beta2, and HLA Class I and II antigens, and with isotype-matched control antibodies. Antigen density was quantitated blindly as mean absorbance on a SAMBA 4000 image analyzer. TGF-beta1 and TGF-beta2 were overexpressed in both primary and metastatic tumor specimens in comparison with normal ovarian tissue. No statistical correlation was found between the expression of TGF-beta1 or TGF-beta2 and HLA class I or HLA class II, which suggests that TGF-beta isotypes could have effects on the immune system other than down-modulation of these HLA molecules. Furthermore, the lack of association between levels of TGF-beta expression and the reduced expression of HLA molecules could suggest that tumor cells expressing both HLA and TGF-beta may be suitable targets for adaptive immunotherapy. Additional studies are necessary to determine whether TGF-beta expressed by ovarian cancer cells merits evaluation as a therapeutic target.

Clinical and biological effects of intraperitoneal injections of recombinant interferon-gamma and recombinant interleukin 2 with or without tumor-infiltrating lymphocytes in patients with ovarian or peritoneal carcinoma.

To identify strategies that enhance tumor-specific immunity in patients with ovarian carcinoma, 22 patients received four to six doses of i.p. recombinant IFN-gamma (rIFN-gamma), 200 microg/m2 on days 1, 3, 5, 8, 10, and 12, and i.p. recombinant interleukin 2 (rIL-2), either 6.0 x 10(5) IU/m2 (group A) or 1.0 x 10(5) IU/m2 (group B), on days 9, 10, and 11. Two patients in group A also received T-cell lines expanded from peritoneal tumor-infiltrating lymphocytes (TILs) obtained after i.p. rIFN-gamma/rIL-2 administration. Toxicity was manageable and included five nonhematological grade 3 or 4 events in 22 patients (23%). A patient had normalization of CA-125 values and a progression-free interval of 18 months, after receiving i.p. rIFN-gamma/rIL-2 without TILs. Another patient who received i.p. rIFN-gamma/rIL-2 plus TILs had stabilization of ascites and intra-abdominal tumors and >50% reduction in serum CA-125 values over 6 months. A third patient who received i.p. rIFN-gamma/rIL-2 had stabilization of intra-abdominal tumors and ascites accompanied by CA-125 values of 50 to 100 units over 6 months. T-cell lines for adoptive immunotherapy were developed for only 3 of 20 patients who were treated with rIFN-gamma/rIL-2. Large numbers of CD3- CD56+ adherent cells were expanded in rIL-2 in the remaining patients, precluding the development of T-cell lines. i.p. rIFN-gamma, either alone or followed by rIL-2, increased proportions of human leukocyte antigen (HLA) class I+ and class II+ tumor cells and increased HLA class I staining intensity on peritoneal carcinoma cells. i.p. rIFN-gamma plus rIL-2 also enhanced cytotoxic activity against Daudi and K562 cells and against allogeneic ovarian tumor cells. Increased cytotoxic activity was associated with an increase in the proportion of CD56+ cells. IFN-gamma and IL-2 transcripts were expressed more frequently after rIFN-gamma and rIL-2 treatment. In addition, the proportions of CD45RA+ (naive lymphocytes) were increased, and CD8+ DR+ lymphocytes were increased relative to CD8+ CD69+ cells, which were decreased. IL-10 concentrations in peritoneal fluids were increased after treatment with rIFN-gamma and the higher rIL-2 dosing (group A) but not in those treated with rIFN-gamma and the lower rIL-2 dosing (group B). These results demonstrated that patients with ovarian carcinoma can tolerate treatment with rIFN-gamma and rIL-2 and that rIFN-gamma alone or rIFN-gamma combined with rIL-2 enhances the expression of HLA class I and class II antigens on ovarian tumor cells, although immunosuppressive cytokines, such as transforming growth factor-beta and IL-10, may persist. Treatment with rIFN-gamma/rIL-2 i.p. did not facilitate the production of TIL-derived T-cell lines ex vivo.

Transduction of rIL-2 expanded CD4+ and CD8+ ovarian TIL-derived T cell lines with the G1Na (neor) replication-deficient retroviral vector.

We have expanded ovarian tumor-infiltrating lymphocytes (TIL) in low concentrations of recombinant interleukin-2 (rIL-2) to conduct intraperitoneal adoptive immunotherapy trials in patients with ovarian cancer. We have previously demonstrated that certain T cell lines and clones derived from ovarian TIL exhibit in vitro autologous tumor-specific cytotoxicity and/or cytokine production (interferon-gamma, tumor necrosis factor-alpha) preferentially in response to autologous tumor cells. Studies that utilize a marker gene introduced into the DNA of TIL can provide useful information on specific uptake or localization of TIL at tumor sites and on the survival of TIL in vivo. We have conducted a series of preclinical experiments in which we have successfully transfected TIL with G1Na, which encodes the gene for neomycin phosphotransferase (neoR). NeoR was detected in at least 10% of CD8+ cells (mean = 10.4%) and between 2.5 and 20% of CD4+ TIL (mean = 8.5%). Transduction of ovarian TIL with G1Na caused no substantial changes to the T cell phenotypes or in vitro cytotoxicities against ovarian and hematogenous tumor cell targets, or on the rIL-2 requirements of TIL for growth and proliferation. In addition, the intact G1Na provirus in transduced TIL cells was rescuable by replication-competent retrovirus and was transferred into the genome of NIH-3T3 fibroblasts, which were rendered resistant to G418. An enhanced polymerase chain reaction (PCR) procedure utilizing detection by ethidium bromide staining was developed. The enhanced PCR detected 1 in 100,000 neoR-labeled cells. Furthermore, detection of the G1Na genome in transduced TIL by in situ hybridization with an RNA probe provided evidence for expression of the neoR gene in transduced TIL. Results obtained from these studies suggest that ovarian TIL-derived T cell lines transduced with the neoR gene post infection with the G1Na retroviral vector can be utilized to examine the in vivo trafficking pattern of ovarian TIL-derived T cell lines expanded in low concentrations of rIL-2 and their survival.

Immunopathogenesis of juvenile rheumatoid arthritis: role of T cells and MHC.

Juvenile rheumatoid arthritis (JRA) is defined as chronic arthritis of unknown etiology appearing in patients less than 16 years of age. The disease is heterogeneous and is classified as pauciarticular, polyarticular, or systemic-onset disease. A few lines of evidence suggest that T cells are involved in the pathogenesis of the disease. T cells infiltrating the synovial membrane bear markers of activation and produce cytokines. The association of particular subtypes of JRA with certain HLA class II alleles provides strong evidence in favor of T cell involvement through an HLA-peptide-T cell receptor complex. Limited data from a few patients with JRA on T cell receptor transcripts from synovial membrane or synovial fluid cells point towards oligoclonality. This further supports the concept that T cells infiltrating the synovial membrane or extravasating into synovial fluid in patients with JRA reflect antigen-driven T cell proliferation.

Immunology of Theiler's murine encephalomyelitis virus infection.

Theiler's murine encephalomyelitis virus (TMEV) is a single-stranded RNA virus that belongs to the family of picornaviruses. Intracranial inoculation of susceptible mouse strains with TMEV results in biphasic disease, consisting of early acute disease that resembles poliomyelitis, followed by late chronic demyelinating disease that is characterized by the appearance of chronic inflammatory demyelinating lesions. Susceptibility to TMEV infection is genetically controlled by three loci: one that maps to the H-2D region of the major histocompatibility complex, one to the beta-chain constant region of the T-cell antigen receptor, and one located on chromosome 3. Both early acute and chronic late demyelinating diseases are immunologically mediated. T cells appear to play an important role in the pathogenesis of the disease. TMEV-induced demyelinating disease in mice has extensive similarities with multiple sclerosis, and it is considered one of the best experimental animal models for multiple sclerosis.

T-cell antigen receptors in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a systemic disease of unknown etiology characterized by chronic inflammation mainly in the joints. Several lines of evidence suggest that T cells are involved in the pathogenesis of the disease. RA is associated with certain HLA-DR alleles. Studies analyzing T-cell receptor transcripts in RA have found biased or preferential usage of certain V alpha and/or V beta gene segments by T cells infiltrating the synovial membrane or extravasating into the synovial fluid compared to peripheral blood. In certain patients few T-cell antigen receptor (TCR) clones dominated the infiltrating T cells, suggesting that T cells from the synovial membrane or the synovial fluid comprise oligoclonal populations of T cells. However, other studies have found a polyclonal population of T cells. In interpreting these results the phase of the disease (early vs. late RA), the source of T cells and the limitations of the methods used in these studies should be taken into consideration. However, it appears that synovial T cells comprise oligoclonal populations of T cells and that there is a bias towards particular TCR gene segments, although a specific TCR gene segment in RA has not emerged.

Separation of rat T and B lymphocytes by density gradient electrophoresis.

Density gradient electrophoresis has been employed for the preparative separation of T and B lymphocytes from rat spleen and peripheral blood. The high mobility cells were found to be predominantly T lymphocytes, as determined by their response to phytohemagglutinin and the relative absence of immunoglobulin-positive cells. The low mobility cells were predominantly B cells, as determined by the high percentage of immunoglobulin-positive cells and the total lack of response to PHA, an exclusive T cell mitogen. A better separation of peripheral blood T and B lymphocytes was achieved than with spleen T and B cells.

Density gradient electrophoresis of mouse spleen lymphocytes: age-related differences. A critical thymus-dependent event during development in the young adult mouse.

T and B BALB/c mouse spleen lymphocytes have been separated by preparative density gradient electrophoresis from animals of different ages. Significant age-related differences in the frequency of occurrence of cells exhibiting different mobility were observed in the young adult mouse. In the 6.5-week-old animals, the frequency of occurrence of the high (T lymphocytes) and low (B lymphocytes) mobility cells was changed, so that these lymphocytes exhibit an electrophoretic distribution profile different (essentially unimodal) from younger (3.5--5.5 weeks) or older (7.5--17 weeks) animals. In the latter two, bimodal electrophoretic distributions were observed. However, differences were also found in the frequency of occurrence of high and low mobility cells. The mobility distributions, representing individual cell types, were reproducible. Furthermore, the age-related changes were independent of the method of cell preparation and appeared in all mouse strains examined. Lymphocytes from animals thymectomized at the 5th week of age did not exhibit these changes (i.e. unimodal distribution) by the 6th week of age. Their electrophoretic distributions at the 6th week and thereafter were similar to those obtained from younger (3.5--5.5 weeks old) animals. It is concluded that the observed changes in the electrophoretic distributions of mouse spleen lymphocytes during development are thymus dependent and may be related to thymus involution.

Density gradient electrophoresis of mouse spleen lymphocytes: separation of T and B cell fractions.

Preparative electrophoresis in an isotonic Ficoll--sucrose density gradient has been employed for the separation of mouse (C57Bl/6J) spleen lymphocyte subpopulations. The separated cells were pooled into six fractions according to their relative position (Rp) within the total cell distribution. In general, the high mobility cells were identified as T lymphocytes. These cells exhibited immunofluorescence upon reaction with fluorescein isothiocyanate-conjugated mouse anti-theta globulin and responded in vitro to phytohemagglutinin stimulation. The low mobility cells were activated in vitro by E. coli lipopolysaccharide and showed immunofluorescence upon reaction with fluorescein isothiocyanate-conjugated anti-mouse Ig which is typical of mouse B lymphocytes. Both T and B cells were completely isolated from each other in certain fractions of very high and very low mobility, respectively. Overlapping of the two distributions was observed in the intermediate mobility fractions. The method which utilizes an inexpensive commercially available apparatus should be useful for the preparation of other lymphocyte subpopulations differing in surface charge.

RT-PCR quantitation of cytokine responses in vivo from specimens containing small numbers of cells during bioimmunotherapy.

A Standard Template method has been developed to carry out semiquantitative RT-PCR analysis on mRNA extracted from small specimens that contain low yields of RNA. Using easily prepared templates (made from previously tested reference specimens), standard calibration curves were generated for each of two cytokine products of interest, specifically IL-10 and IFN-gamma. Also, internal standardization was accomplished by quantitating a well-characterized housekeeping gene (GAPDH). Simple densitometry of the RT-PCR product did not demonstrate sufficient reliability for quantitation since a logarithmic relationship was shown between product and template input. Peritoneal exudate cell specimens that were obtained from ovarian cancer patients during intraperitoneal immunotherapy were utilized for the demonstration of IL-10 and IFN-gamma transcript in vivo. Briefly, this method consists of: (1) template preparation: a PCR product for the cytokine of interest is generated from a previously identified positive specimen, purified and stored at -20 degrees C. (2) RT-PCR: cDNAs are produced from RNA extracted from patient specimens. Replicates of each cDNA and serial dilutions of the corresponding template are amplified with primers specific for each cytokine of interest. (3) Quantitation: photographs are made of the PCR products displayed on an agarose gel and are analyzed by densitometry. Determinations of fold-differences in cytokine transcript expression are normalized to the mRNA content of each specimen (based on the expression of GAPDH).

Large-scale expansion in interleukin-2 of tumor-infiltrating lymphocytes from patients with ovarian carcinoma for adoptive immunotherapy.

Tumor infiltrating lymphocytes (TIL) from malignant ascites or solid tumor specimens obtained from patients with ovarian carcinoma were expanded to large numbers in vitro (10(10)-10(11)) by a four-step method using AIM V medium and low concentrations of recombinant interleukin-2 (rIL-2). The expansion procedure employed 24-well culture plates, T-flasks, polyolefin gas-permeable bags (PGPB), and an artificial capillary culture system (ACCS). The mean number of mononuclear leukocytes introduced into the 24-well plates was 16.5 +/- 4.2 x 10(6) cells. TIL from a total of 16 patients were expanded only through the first three steps of the process (24-well-plates, T-flasks, and PGPB) with an overall expansion of 255 +/- 99 fold and mean duration of 27.4 +/- 2.2 days. TIL from 9 of 16 patients were expanded further through the fourth step (ACCS) of the expansion method. The cumulative fold-expansion in nine patients was 8044 +/- 4807 (mean +/- SEM), the median was 2876 and the mean expansion time was 47.1 +/- 4.7 days. TIL from seven additional patients did not grow in rIL-2. Six of these 7 patients received chemotherapy at least four weeks before the specimens were collected. Two ACCS were used in parallel to facilitate expansion of TIL. Viable rIL-2-expanded TIL in the range of 1 x 10(10)-1 x 10(11) were recovered from the two ACCS, a number sufficient for adoptive immunotherapy of patients with ovarian carcinoma. The rIL-2-expanded TIL were predominantly CD3+ CD4+ CD8- alpha beta TCR+, although CD3+ CD4- CD8+ alpha beta TCR+ T cell lines were obtained from certain patients. An increase (43 +/- 8 vs 75 +/- 13; P = 0.05) in the proportion of CD4+ cells was observed over the duration of the four expansion steps. However, CD8+ TIL-derived T cells lines were also expanded in the ACCS. The four-step expansion method described here has several significant advantages over existing techniques. It requires substantially less personnel, equipment and space and the risk of contamination during expansion of the cultures is decreased. These results demonstrate that the four-step method described here can be effectively used for the large-scale expansion of ovarian TIL for the treatment of patients with ovarian carcinoma by adoptive immunotherapy.

Apoptosis in chronic rejection of human cardiac allografts.

BACKGROUND: We investigated the role of apoptosis (programed cell death) in the pathogenesis of chronic rejection. METHODS: Epicardial coronary arteries from cardiac allografts with chronic rejection were examined for apoptosis by the TUNEL assay. Double labeling was carried out using anti-CD3, anti-CD68, and anti-von Willenbrand factor (vWF) monoclonal antibodies. Additional immunostaining was carried using anti-Fas, anti-Fas-L, and anti-Bcl-2 monoclonal antibodies. Apoptosis-associated oligonucleosomal DNA degradation was assessed by DNA agarose gel electrophoresis. The transcription level of apoptosis-related caspase genes were determined using microarrays. RESULTS: Apoptotic cells (TUNEL+) were detected within the arterial wall and in perivascular areas. Double labeling demonstrated that apoptotic cells included T cells (CD3+), monocyte/macrophages (CD68+), and vascular endothelial cells (VWF+). Numbers and densities of TUNEL+ cells did not correlate with the degree of arterial stenosis. Apoptosis-associated oligonucleosomal DNA degradation was assessed by agarose gel electrophoresis of DNA, which showed DNA fragments of approximately 180 bp and multimers thereof (DNA laddering gel), which are characteristic for DNA fragmentation in apoptotic cells. Microarray analysis demonstrated that the apoptosis related caspases 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, were all transcribed (caspases 8, 9, and 10 were highly up-regulated). These results are consistent with the involvement of apoptosis in chronic rejection. Immunoreactivity for Fas/Fas-L was present at the sites of apoptotic cells. Immunoreactivity for Bcl-2 was present in areas with very few apoptotic cells. CONCLUSIONS: Apoptotic cells include T cells, monocyte/macrophages, and endothelial cells. Apoptosis, likely through the Fas/Fas-L system, is involved in the pathogenesis of chronic rejection in cardiac allografts.

Immunoglobulin synthesis and secretion by leukemic B cells from patients with chronic lymphocytic leukemia.

We investigated the ability of purified E-rosette negative largely leukemic B cells from 15 patients with B-cell chronic lymphocytic leukemia (CLL) to synthesize and secrete IgM, IgA and IgG spontaneously or in the presence of purified autologous or allogeneic T4 cells from normal donors, in PWM-induced differentiation system. We observed moderate but significant IgM synthesis and secretion (19.7 +/- 8.9 micrograms/dl, n = 5) by leukemic B cells alone in 5 of 15 patients examined. These IgM concentrations were significantly higher (p less than 0.005) than those produced by purified E-rosette negative cells from normal donors (4.3 +/- 4.5 micrograms/dl; n = 6) in the absence of T cells. Purified E-rosette negative leukemic B cells alone from patients with CLL did not produce IgA or IgG. Addition of purified autologous or allogeneic T4 cells from normal donors resulted in significant increase of IgM production by leukemic B cells from certain patients or initiated IgM secretion in others. However, these IgM levels (73.9 +/- 56.6 micrograms/dl) were significantly lower (p less than 0.003) to those produced by mixtures of T4 cells and B cells form normal donors (211.6 +/- 58.0 micrograms/dl, n = 6). Addition of purified autologous or allogeneic T4 cells from normal donors to purified largely leukemic B cells from patients with CLL resulted in production of very small amounts of IgA in 4 of 15 patients (10.6 +/- 6.3 micrograms/dl vs 154.7 +/- 35.8 micrograms/dl produced by T4 and B cells from normal donors; n = 6), but did not support IgG synthesis and secretion. Purified T4 cells from certain patients with CLL exhibit defective helper function to immunoglobulin production by E-rosette negative cells from normal donors.

Proliferation of leukemic B cells in response to SAC and anti-mu. Evidence for different modes of action and comparison to proliferation and differentiation induced by conditioned medium.

We investigated the proliferative responses and immunoglobulin production of highly purified E-rosette negative largely leukemic B cells from patients with CLL to Staphylococcus aureus Cowan I (SAC) or to SAC in combination with anti-mu or conditioned medium (CM). The latter was derived by stimulating human peripheral blood mononuclear leukocytes with PHA. We observed: (1) that purified E-rosette negative largely leukemic B cells from 25% (five out of 20) of the patients exhibited proliferative responses to SAC; (2) inhibition of SAC-induced proliferation by anti-mu in certain patients, whereas synergism between SAC and anti-mu in inducing proliferative responses in others; (3) the lack of synergism between SAC and CM in inducing proliferative responses, which is in contrast to the strong synergism that was observed between anti-mu and CM in inducing proliferation; and (4) induction or enhancement by SAC alone of Ig production by largely leukemic B-cell populations from few patients with CLL and purified tonsillar B lymphocytes, but not peripheral blood B cells from normal donors. These results suggest that SAC and anti-mu induce proliferation of B cells by different mechanisms and that B-cell proliferation and differentiation is dependent not only on the mitogen but also on the activation state of the cells.