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.

Coronary arteries from human cardiac allografts with chronic rejection contain oligoclonal T cells: persistence of identical clonally expanded TCR transcripts from the early post-transplantation period (endomyocardial biopsies) to chronic rejection (coronary arteries).

Chronic cardiac allograft rejection presents pathologically as graft arteriosclerosis (GA) characterized by recipient T cell and monocyte infiltration. To determine whether oligoclonal T cells are present in coronary arteries of cardiac allografts from patients with GA, we conducted sequencing analysis of beta-chain TCR transcripts from these explanted coronary arteries using the nonpalindromic adaptor-PCR. Substantial proportions of identical beta-chain TCR transcripts in three of five patients were observed, clearly demonstrating the presence of oligoclonal T cells. TCR transcripts from the arteries of two other patients were relative heterogeneous. High proportions of identical CDR3 beta-chain TCR motifs were found in each patient. GENEBANK/EMBL/SWISS PROT database comparison of all sequences revealed that these beta-chain TCR transcripts were novel. Using Vbeta-specific PCR (independent amplification), we found in patient GA03 that the TCR transcript that was clonally expanded in the left anterior descending artery after nonpalindromic adaptor-PCR was also clonally expanded in the right coronary artery of the same allograft. These results demonstrate that this TCR transcript was clonally expanded at different anatomic sides of the cardiac allograft in a systemic manner. In two patients identical beta-chain TCR transcripts that were found to be clonally expanded in the coronary arteries of their explanted cardiac allografts were also found to be clonally explanted in endomyocardial biopsies collected 17 and 21 mo earlier from each patient. The presence of oligoclonal populations of T cells in the rejected graft suggest that these T cells have undergone specific Ag-driven proliferation and clonal expansion early on within the graft and persist throughout the post-transplantation period.

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.

Expression of costimulatory molecules CD80 and CD86 and their receptors CD28, CTLA-4 on malignant ascites CD3+ tumour-infiltrating lymphocytes (TIL) from patients with ovarian and other types of peritoneal carcinomatosis.

Costimulation of T lymphocytes by the leucocyte surface molecules CD80 and CD86 expressed on antigen-presenting cells (APC) is required for the development of T cell responses. The CD28 and CTLA-4 molecules on T cells serve as receptors for the CD80 and CD86 costimulatory antigens. We have examined the frequency of expression of CD80 (B7.1), CD86 (B7.2), CD28 and CTLA-4 surface antigens on TIL isolated from malignant ascites or peritoneal washings of 26 patients with ovarian carcinoma and five patients with non-ovarian peritoneal carcinomatosis. Expression of CD80 and CD86 antigen was detected by reverse transcription-polymerase chain reaction (RT-PCR), and by FACS analysis. Significantly higher proportions of intraperitoneal CD3+ cells expressed CD86 antigen than the CD80 antigen (14 +/- 9% versus 3 +/- 3%, P < 0.05). Moreover, CD3+CD86+ cells were significantly more frequent in the peritoneal fluid (14 +/- 9%) than in the peripheral blood (3 +/- 0.4%, P < 0.05) of ovarian patients or normal controls (3 +/- 1%). CTLA-4 and CD28 antigen were expressed, respectively, on 9 +/- 4% and 86 +/- 14% of ascitic CD3+ cells of ovarian cancer patients. Both CD80 and CD86 antigens were expressed primarily on HLA-DR+ ascites TIL and were present in a very low proportion of HLA-DR- ascites TIL. These HLA-DR+ cells may represent a population of lymphocytes that have been activated in vivo, and function as APC. An anti-CD86 MoAb or a combination of anti-CD86 and anti-CD80 MoAbs significantly inhibited the proliferation of cultured intraperitoneal TIL. We have shown that in addition to CD28 and CTLA-4, CD3+ intraperitoneal TIL express the costimulatory molecules CD80 and CD86. The expression of these molecules on T cells could be dependent upon certain factors in the tumour microenvironment that could determine the outcome of in vivo immune responses.

Identification of an IL-10-producing HLA-DR-negative monocyte subset in the malignant ascites of patients with ovarian carcinoma that inhibits cytokine protein expression and proliferation of autologous T cells.

A newly described subset of monocytes has been identified in peritoneal exudate cells (PEC) from the malignant ascites from patients with ovarian cancer. These cells were characterized by the production of IL-10 and TGF-beta2, but not IL-12, IL-1alpha, or TNF-alpha, and they expressed CD14, CD16, and CD54, but not HLA-DR, CD80, CD86, CD11a, CD11b, or CD25 cell surface Ags. Since this subset of monocytes could affect the modulation of tumor immune responses in vivo, studies were undertaken to determine their effect on the activation and proliferation of autologous T cells from the peritoneal cavity of patients with ovarian carcinoma. Expression of cytokine-specific transcripts in T cells was determined by RT-PCR. Transcripts for the following cytokines were detected in patient specimens that also contained the IL-10-producing monocytes IL-2 (12 of 17 specimens), GM-CSF (9 of 17 specimens), IFN-gamma (6 of 17 specimens), and TNF-alpha (4 of 17 specimens). Cytokine production by T cells was determined by intracellular flow cytometry and by ELISA. IL-2 and IFN-gamma proteins, unlike their transcripts, were detected only in specimens that lacked IL-10-producing monocytes. IL-10-producing monocytes cocultured with autologous T cells inhibited the proliferation of the T cells in response to PHA. However, T cells cocultured with PEC from which the IL-10-producing monocytes had been removed did not inhibit T cell proliferation. Moreover, the inhibition of T cell proliferation by IL-10-producing monocytes could be reversed by adding neutralizing Abs to both IL-10R and TGF-beta2. These results suggest that this subset of monocytes may modulate immune responses by inhibiting T cell proliferation and cytokine protein production.

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.

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).

Interleukin-12 is expressed by infiltrating macrophages and synovial lining cells in rheumatoid arthritis and osteoarthritis.

TH1 cytokines have recently been detected in rheumatoid arthritis (RA) and osteoarthritis (OA). For this reason we studied the TH-1-promoting cytokine IL-12 in synovial membranes from patients with RA and OA. IL-12 transcripts and protein were analyzed by reverse transcriptase-polymerase chain reaction (PCR) and immunohistochemistry, respectively. In addition, IL-12 transcripts were quantitated by competitive PCR. IL-12 transcripts (p40) were detected in 8 of 13 patients with RA and in 10 of 18 patients with OA. Their levels did not differ significantly between RA and OA. IL-12 heterodimer protein was detected by immunostaining using an anti-IL-12p70 mAb. Double labeling with anti-IL-12p70 and anti-CD68 mAbs showed that synovial lining cells and monocytes/macrophages expressed IL-12 p70 protein. The presence of IL-12 p70 protein in the synovial membranes of patients with RA and OA suggests that IL-12 may play an important immunoregulatory role in these diseases by perpetuating inflammation.

Differential expression of cytokine transcripts in human epithelial ovarian carcinoma by solid tumour specimens, peritoneal exudate cells containing tumour, tumour-infiltrating lymphocyte (TIL)-derived T cell lines and established tumour cell lines.

T cell lines derived in low concentrations of recombinant IL-2 (rIL-2) from TIL of patients with epithelial ovarian carcinoma (EOC) often exhibit specific cytotoxicity against autologous tumour cells. However, the ability of T cells at the tumour site to respond to ovarian carcinoma cells may be affected by the production of cytokines by the various cell types present. Using reverse transcriptase-polymerase chain reaction (RT-PCR) we investigated cytokine transcripts in: (i) established EOC tumour cell lines; (ii) solid tumour specimens or peritoneal exudate cells (PEC) from ascites or peritoneal washings of patients with EOC; and (iii) CD4+ TCRalphabeta+ and CD8+ TCRalphabeta+ TIL-derived T cell lines developed in rIL-2. We have found that (i) established EOC tumour cell lines expressed transcripts for transforming growth factor-beta 2 (TGF-beta2) (7/7), but not IL-10 (0/7) or interferon-gamma (IFN-gamma) (0/7) and rarely IL-2 (1/7); (ii) PEC expressed transcripts for IL-2 (12/13), IL-10 (9/13), and TGF-beta2 (12/13), and less often, IFN-gamma (3/13), whereas solid tumour specimens from eight patients with EOC expressed transcripts for IL-2 (4/8), TGF-beta2 (4/8), and IL-10 (5/8), but not for IFN-gamma (0/8); (iii) CD4+ TCRalphabeta+ T cell lines expressed transcripts for IFN-gamma (4/4), IL-2 (4/4) and IL-10 (3/4), whereas CD8+ TCRalphabeta+ T cell lines expressed transcripts for IFN-gamma (5/5), IL-2 (1/5) and IL-10 (2/5). None of these T cell lines expressed TGF-beta2 transcripts. The frequency of IL-2 and TGF-beta2 transcripts in solid tumours was significantly lower than in the PEC (P = 0.0475). CD4+ or CD8+ T cell lines expressing IFN-gamma, IL-2 and IL-10 transcripts were derived in culture with rIL-2 from the TIL of specimens that did not necessarily express these cytokines in the absence of rIL-2. The frequency of cytokine transcripts in T cell lines compared with these same transcripts in the PEC was significantly higher for IFN-gamma (P = 0.0005) and lower for TGF-beta2 (P = 0.0001). An association was observed between the expression of cytokine transcripts in vivo or by TIL-derived cell lines and functions exhibited by either production of cytokines or in vitro cytotoxicity.

Intraperitoneal immunotherapy of peritoneal carcinomatosis.

Carcinomas that originate in the ovary or from different sites in the gastrointestinal tract frequently develop metastases that involve the peritoneal and serosal surfaces. Carcinomatous involvement of the peritoneum is a significant cause of morbidity and mortality. Advances in our understanding of the immunobiology of the peritoneal cavity and the availability of technically advanced immunotherapeutic agents are providing an important opportunity for the intraperitoneal delivery of these agents. This review describes newer concepts in tumor immunology that have a bearing on the further development of intraperitoneal immunotherapy; delivery systems for and issues to be resolved in intraperitoneal immunotherapy, and results of studies with recombinant interferons alpha and gamma and interleukin (IL)-2, cellular therapies including lymphokine-activated killer cells, tumor-infiltrating lymphocytes, monoclonal antibodies, and intraperitoneal radioimmunotherapy. New trials of intraperitoneal immunotherapy employing novel agents, including IL-12 and genetically modified tumor vaccines, are discussed, as are issues related to the integration of immunotherapy with standard chemotherapy agents. A number of immunotherapy agents have been tested intraperitoneally and have shown promising clinical activity with acceptable toxicity. Complete responses have been documented at surgical restaging, and intraperitoneal treatments with these agents may soon be included in the therapeutic armamentarium for patients with peritoneal carcinomatosis.

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.

All-trans-retinoic acid blocks cell cycle progression of human ovarian adenocarcinoma cells at late G1.

We prepared single cell clones from two ovarian carcinoma cell lines, CA-OV3 and SK-OV3, and analyzed the effect of all-trans-RA treatment on cell division, DNA synthesis, and cell cycle stage distribution of these single cell clones. Our results show that despite the well-known heterogeneous nature of these cell lines, all single cell clones of SK-OV3 cells are resistant to the growth inhibitory effects of all-trans-RA. In contrast, all single cell clones of CA-OV3 cells were growth inhibited by all-trans-RA. However, the extent of growth inhibition did vary somewhat from clone to clone. Additional studies employing flow cytometry showed that all-trans-RA blocked CA-OV3 cell cycle progression in the G1 stage. Finally, all-trans-RA was able to inhibit G1 progression in growth-arrested CA-OV3 cells following stimulation with fetal bovine serum, insulin, IGF-1, or estrogen. Since each of these growth factors is known to act via distinct signal transduction pathways, our results suggest that all-trans-RA blocks G1 progression by targeting a downstream process or event which occurs at a point after the insulin/IGF-1, estrogen, and serum signal transduction pathways converge.

An anti-LFA-1 monoclonal antibody (LDA-8) induces cellular aggregation of human lymphoblastoid cell lines and peripheral blood lymphocytes.

The human leukocyte-function-associated antigen-1 (LFA-1) plays a key role in intercellular adhesion interactions of the immune response. A monoclonal antibody (mab), designated LDA-8, is described that recognizes LFA-1. In contrast to nearly all other anti-LFA-1 mabs, which inhibit cellular aggregation, LDA-8 induces cell-cell aggregation. The LDA-8 mab was generated by immunizing mice with membrane fragments from the Jurkat T-cell line. The LDA-8 mab stained peripheral blood mononuclear cells (PBMC), monocyte-depleted peripheral blood lymphocytes, purified monocytes, and a number of T and B tumor cell lines. The LDA-8 mab induced aggregation of PBMC from normal donors, as well as of cells from T-cell lines (MOLT4 and CEM). Control mabs directed against HLA class 1 or CD4 did not induce aggregation. Aggregation was concentration- and time-dependent. EDTA added to the cultures 1 hour prior to or together with the LDA-8 mab did not inhibit LDA-8-induced aggregate formation. Anti LFA-1 alpha-chain mab added to the cells 1 hour prior to LDA-8 mab, or together with the LDA-8 mab, also did not inhibit LDA-8-induced aggregation. In contrast, anti-LFA-1 beta-chain mab, added to the cells together with or 1 hour prior to the LDA-8 mab, significantly inhibited LDA-8-induced aggregate formation. The LDA-8 mab immunoprecipitated two polypeptide chains of 110 kDa and 160 kDa under non-reducing conditions and of 92 kDa and 162 kDa under reducing conditions, from cells of the MOLT-4 or CEM T-cell lines or phytohemagglutinin (PHA)-stimulated PBMC. The molecular mass of these polypeptides was identical to that of polypeptides immunoprecipitated by the anti-LFA-1 TS1.22 mab, suggesting that the LDA-8 mab and the anti-LFA-1 mab recognize the same molecule. This was confirmed by sequential immunoprecipitation. The LDA-8 mab recognizes a unique epitope on LFA-1 and induces cell aggregation that is blocked by mabs recognizing the beta-chain, but not the alpha-chain of the LDA-1 molecule.

HLA class I expression on human ovarian carcinoma cells correlates with T-cell infiltration in vivo and T-cell expansion in vitro in low concentrations of recombinant interleukin-2.

This study was carried out to determine whether HLA class I or class II expression on ovarian tumor cells and lymphocytic infiltration of the epithelial ovarian carcinoma (EOC) tissues were responsible for the ability to expand tumor-infiltrating lymphocytes (TIL) in vitro in low concentrations of recombinant interleukin-2 (rIL-2). Immunohistochemical analysis was performed using monoclonal antibodies that recognize framework determinants of either HLA class I or HLA class II or leukocyte differentiation antigens (LCA, CD3, CD4, and CD8). Cryostat sections of EOC had HLA class I and HLA class II expression on at least 5% of tumor cells in 18 of 20 specimens (90%). From another portion of the same tumor specimens T-cell lines were developed from TIL in low concentrations of rIL-2 (200-600 IU/ml) in 7 of 17 patients. Tumors from which TIL were expanded in vitro with rIL-2 had significantly higher proportions of HLA class I-positive tumor cells (73 +/- 10%) compared to tumors from which TIL failed to grow (40 +/- 10%) (P = 0.036). However, there was no difference in the proportions of HLA class II-positive tumor cells between the two groups. Tumor specimens of patients whose TIL were expanded in rIL-2 had significantly higher numbers per field (423 +/- 114 vs 154 +/- 20; P = 0.005) and proportions (90 +/- 3% vs 77 +/- 4%; P = 0.023) of infiltrating CD3+ cells, significantly higher numbers per field (115 +/- 44 vs 19 +/- 5; P = 0.003) and proportions (25 +/- 5% vs 11 +/- 2%; P = 0.017) of CD8+ cells and significantly higher numbers per field of CD4+ cells (318 +/- 101 vs 113 +/- 18; P = 0.025), in comparison to tumor specimens from patients whose TIL did not grow in vitro. Significant correlations were observed between the proportions of HLA class I-positive EOC tumor cells and the numbers of infiltrating LCA-positive cells (r = 0.67; P = 0.005) CD3+ cells (r = 0.70; P = 0.002), CD4+ cells (r = 0.69; P = 0.003), and CD8+ cells (r = 0.82; P = 0.001). The proportions of HLA class II-positive tumor cells correlated positively (r = 0.45; P = 0.049) only with the numbers of CD8+-infiltrating cells. In conclusion, we report here that HLA class I expression on EOC cells correlates with T-cell infiltration in vivo and T-cell expansion in vitro, in low concentrations of rIL-2.

Immunopharmacology and cytokine production of a low-dose schedule of intraperitoneally administered human recombinant interleukin-2 in patients with advanced epithelial ovarian carcinoma.

We determined in the peritoneal cavity (p.c.) of epithelial ovarian carcinoma patients during a 4-day treatment cycle of low-dose recombinant human interleukin-2 (rIL-2): (a) pharmacokinetics of IL-2, (b) endogenous cytokine production, and (c) numbers and percentages of peritoneal exudate lymphocytes. We administered 6 x 10(5) IU/m2 of rIL-2 (0.03 mg/m2 Proleukin rIL-2) intraperitoneally (i.p.) over 30 min on each of 4 days. One and one-half liters of D5 0.25 NS was injected i.p. before each rIL-2 infusion. Multiple peritoneal fluid samples were obtained from each of four patients on day 1 and day 4 for detection of IL-2, endogenous cytokines, and soluble IL-2 receptor (IL-2R-alpha). IL-2 concentrations in the peritoneal fluid were determined by bioassay and interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, IL-10, transforming growth factor (TGF)-beta 2, and sIL-2R-alpha by enzyme-linked immunosorbent assay. Numbers of cells per microliter and lymphocyte subpopulation percentages after staining with a panel of monoclonal antibodies were determined on day 1, day 4, and subsequent off-treatment days. IL-2 disappearance in the p.c. was well described by a pharmacokinetic model having constant-rate infusion and biexponential disposition. About 90% of the IL-2 disappearance occurred during the beta-phase, during which IL-2 concentrations were sustained at approximately 10-30 ng/ml (day 1 and day 4) and the median t1/2 beta was 21.5 and 9.2 h on days 1 and 4, respectively. In four of four patients, p.c. production of IL-10 was observed on day 1 and day 4 (maximum 387 pg/ml). Maximum levels of IFN-gamma and sIL-2R-alpha were observed on day 4. (IFN-gamma 217 pg/ml; sIL2-R-alpha: 3486 U/ml). No increases in TNF-alpha or TGF-beta 2 were observed. Large increases in p.c. CD3+, CD4+, CD8+, CD16+, and CD56+ cells were observed. We conclude that biologically active levels of IL-2 are generated in p.c. fluids after i.p. administration of rIL-2 at 0.03 mg/m2.

Selective gamma-chain T-cell receptor gene rearrangements in a patient with Omenn's syndrome: absence of V-II subgroup (V gamma 9) transcripts.

Only gamma-chain T-cell receptor transcripts utilizing V-1 subgroup gene segments were found in peripheral blood lymphocytes from a patient with Omenn's syndrome. gamma-Chain T-cell receptor transcripts utilizing the V gamma 9 (V-II subgroup) gene segment were absent in peripheral blood lymphocytes from this patient. V gamma 9 J gamma 1.2 C gamma 1 rearrangements are those primarily found in peripheral blood lymphocytes (70 to 85%) from normal donors.

Malignant melanoma: relationship of the human leukocyte antigen class II gene DQB1*0301 to disease recurrence in American Joint Committee on Cancer Stage I or II.

BACKGROUND: Melanoma patients who carry the human leukocyte antigen (HLA) Class II allele DQB1*0301 have an increased frequency of metastases at presentation compared with those lacking HLA-DQB1*0301. This study was designed to determine whether HLA-DQB1*0301 is associated with an increased risk of recurrence in melanoma patients presenting with American Joint Committee on Cancer (AJCC) Stage I or II (localized) disease. METHODS: Molecular oligotyping of HLA-DQ genes was performed for 259 patients with AJCC Stage I or II melanoma. Rate of disease recurrence was determined by retrospective review and prospective follow-up. Kaplan-Meier analysis, log rank, and proportional hazard (Cox) comparison were performed. RESULTS: Median follow-up was 24 months. Minimum follow-up was 6 months. Although HLA-DQB1*0301-positive and -negative patients were balanced with regard to standard melanoma prognostic factors (primary tumor thickness, level of invasion, presence of ulceration, anatomic location, and sex), HLA-DQB1*0301-positive patients were more likely to develop locally recurrent, regional, or distant metastatic melanoma during follow-up (actuarial median disease free survival 48 months [DQB1*0301-positive patients] vs. 97 months [DQB1*0301-negative patients]; log rank P = 0.0002). HLA-DQB1*0301 status, in addition to primary tumor thickness, was an independent prognostic indicator in these patients (Cox multivariate P = 0.02). CONCLUSIONS: Patients presenting with localized melanoma who carry HLA-DQB1*0301 are at an increased risk of developing recurrent disease compared with stage-matched patients who lack this allele. HLA-DQB1*0301 is a genomic marker which independently identifies melanoma patients in whom recurrence is more likely, and is potentially useful in selecting those most likely to benefit from adjuvant therapy.