Oligoclonal T cells are infiltrating the brains of children with AIDS: sequence analysis reveals high proportions of identical beta-chain T-cell receptor transcripts.

We have recently described the presence of perivascular CD3+ CD45RO+ T cells infiltrating the brains of children with AIDS. To determine whether these infiltrates contain oligoclonal populations of T cells, we amplified by PCR beta-chain T-cell receptor (TCR) transcripts from autopsy brains of four paediatric patients with AIDS. The amplified transcripts were cloned and sequenced. Sequence analysis of the beta-chain TCR transcripts from all four patients revealed multiple identical copies of TCR beta-chain transcripts, suggesting the presence of oligoclonal populations of T-cells. These TCR transcripts were novel. The presence of oligoclonal populations of T cells in the brains of these four paediatric patients with AIDS suggests that these T cells have undergone antigen-driven proliferation and clonal expansion very likely in situ, in the brains of these AIDS patients, in response to viral or self-antigens. Although the specificity of the clonally expanded beta-chain TCR transcripts remains to be elucidated, none of the beta-chain TCR transcripts identified in this study were identical to those specific for HIV-1 antigens that are currently reported in the GENBANK/EMBL databases. Certain common CDR3 motifs were observed in brain-infiltrating T cells within and between certain patients. Large proportions (24 of 61; 39%) of beta-chain TCR clones from one patient (NP95-73) and 2 of 27 (7%) of another patient (NP95-184-O) exhibited substantial CDR3 homology to myelin basic protein (MBP)-specific TCR derived from normal donors or TCR expressed in the brain of patients with multiple sclerosis (MS) or with viral encephalitis. These two patients (NP95-73 and NP95-184-O) also shared HLA class II with the normal donors and the MS patients who expressed these homologous TCR. Pathologic examination at autopsy of the brains revealed the presence of myelin pallor only in patient NP95-73. T-cell clones identified in the brain of patients NP95-73 and NP95-184-O may recognize MBP or another CNS self antigen and this recognition may be restricted by either DRB1*15 or DQB1*0602 specificities.

Presence of oligoclonal T cells in cerebrospinal fluid of a child with multiphasic disseminated encephalomyelitis following hepatitis A virus infection.

We have investigated the clonality of beta-chain T-cell receptor (TCR) transcripts from the cerebrospinal fluid (CSF) and peripheral blood from a 7-year old child who developed a multiphasic disseminated encephalomyelitis following an infection with hepatitis A virus. We amplified beta-chain TCR transcripts by nonpalindromic adaptor (NPA)-PCR-Vbeta-specific PCR. TCR transcripts from only five Vbeta families (Vbeta13, Vbeta3, Vbeta17, Vbeta8, and Vbeta20) were detected in CSF. The amplified products were combined, cloned, and sequenced. Sequence analysis revealed in the CSF substantial proportions of identical beta-chain of TCR transcripts, demonstrating oligoclonal populations of T cells. Seventeen of 35 (48%) transcripts were 100% identical, demonstrating a major Vbeta13.3 Dbeta2.1 Jbeta1.3 clonal expansion. Six of 35 (17%) transcripts were also 100% identical, revealing a second Vbeta13 clonal expansion (Vbeta13.1 Dbeta2.1 Jbeta1.2). Clonal expansions were also found within the Vbeta3 family (transcript Vbeta3.1 Dbeta2.1 Jbeta1.5 accounted for 5 of 35 transcripts [14%]) and within the Vbeta20 family (transcript Vbeta20.1 Dbeta1.1 Jbeta2.4 accounted for 3 of 35 transcripts [8%]). These results demonstrate the presence of T-cell oligoclonal expansions in the CSF of this patient following infection with hepatitis A virus. Analysis of the CDR3 motifs revealed that two of the clonally expanded T-cell clones exhibited substantial homology to myelin basic protein-reactive T-cell clones. In contrast, all Vbeta TCR families were expressed in peripheral blood lymphocytes. Oligoclonal expansions of T cells were not detected in the peripheral blood of this patient. It remains to be determined whether these clonally expanded T cells are specific for hepatitis A viral antigen(s) or host central nervous system antigen(s) and whether molecular mimicry between hepatitis A viral protein and a host protein is responsible for demyelinating disease in this patient.

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.

Differential expression of TGF-beta, IL-2, and other cytokines in the CNS of Theiler's murine encephalomyelitis virus-infected susceptible and resistant strains of mice.

Intracranial inoculation of susceptible SJL mice with Theiler's murine encephalomyelitis virus (TMEV) results in biphasic disease consisting of early acute disease, followed by late chronic demyelinating disease, associated with mononuclear infiltrates and demyelinating lesions. In contrast, resistant C57BL/6 (B6) mice develop only early acute disease. We employed cytokine-specific RT-PCR to determine the expression of cytokine transcripts in the CNS of TMEV-infected SJL and B6 mice. During early acute disease, we have found a strong proinflammatory (Th1) cytokine response in the CNS of both TMEV-infected SJL and B6 mice, demonstrated by the expression of transcripts for IFN-gamma, IL-1, IL-6, IL-12p40, and TNF-alpha. At 8 days postinfection (p.i.), TGF-beta1 and TNF-alpha transcripts were present at significantly higher levels (P < 0.01) in the CNS of SJL susceptible mice in comparison to those found in the CNS of B6 mice. Immunohistochemical staining revealed that TGF-beta protein was expressed in leptomeningeal mononuclear inflammatory cell infiltrates in the brain of SJL mice but not in B6 mice, at 8 days p.i. TGF-beta may be responsible for the failure of SJL mice to develop an effective anti-TMEV CTL response. During late chronic demyelinating disease, high levels of proinflammatory Th1 cytokines were found in the CNS of SJL mice, but not B6 mice. Significantly higher levels (P < 0.01) of anti-inflammatory cytokine transcripts (IL-4, IL-5, and IL-10 (Th2 cytokines) and TGF-beta) were found in the spinal cord of TMEV-infected SJL mice with chronic demyelinating disease than in the spinal cord of B6 mice during the same time period (39 or 60 days p.i.). These anti-inflammatory cytokines may contribute to the downregulation of the proinflammatory response in SJL mice. High levels of IL-2 transcripts and protein appeared transiently in the spinal cord of TMEV-infected SJL mice before the onset of demyelinating disease and coincided with an influx of new T cells into the CNS and/or expansion of remaining T cells that have not been eliminated after viral clearance.

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.

Increased levels of alternatively spliced interleukin 4 (IL-4delta2) transcripts in peripheral blood mononuclear cells from patients with systemic sclerosis.

Recent in vitro studies have shown that interleukin 4 (IL-4) induces and gamma interferon (IFN-gamma) inhibits collagen production. To define the TH1(IFN-gamma) and TH2(IL-4) cytokine profiles in systemic sclerosis (Sscl), a disease characterized by widespread fibrosis, we investigated IL-4 and IFN-gamma transcripts in peripheral blood mononuclear cells and plasma protein levels in 13 patients with Sscl. Two previously identified IL-4 transcripts, a full-length transcript and an alternatively spliced (truncated) transcript (designated IL-4delta2), were identified in patients and normal controls. Significantly increased levels of total IL-4 transcripts (full-length plus IL-4delta2 transcripts) were found in patients with Sscl in comparison to those found in healthy controls (P = 0.003), and this increase was primarily due to an increase in the level of the alternatively spliced IL-4delta2 form. The IL-4delta2/full-length-IL-4 transcript ratio was significantly increased in Sscl patients (P < 0.0001, versus healthy controls). Sequencing analysis revealed that the frequency of IL-4 clones carrying the IL-4delta2 transcript was also substantially increased in patients with Sscl. Plasma IL-4 protein levels were increased in Sscl patients compared to those in healthy controls (P = 0.001) and correlated with total IL-4 transcript levels. The up-regulation of the fibrogenic IL-4 (a TH2 cytokine) in Sscl suggests a pathogenic role for IL-4 in this disease.

Angiocentric CD3(+) T-cell infiltrates in human immunodeficiency virus type 1-associated central nervous system disease in children.

A significant proportion of brain tissue specimens from children with AIDS show evidence of vascular inflammation in the form of transmural and/or perivascular mononuclear-cell infiltrates at autopsy. Previous studies have shown that in contrast to inflammatory lesions observed in human immunodeficiency virus type 1 (HIV-1) encephalitis, in which monocytes/macrophages are the prevailing mononuclear cells, these infiltrates consist mostly of lymphocytes. Perivascular mononuclear-cell infiltrates were found in brain tissue specimens collected at autopsy from five of six children with AIDS and consisted of CD3(+) T cells and equal or greater proportions of CD68(+) monocytes/macrophages. Transmural (including endothelial) mononuclear-cell infiltrates were evident in one patient and comprised predominantly CD3(+) T cells and small or, in certain vessels, approximately equal proportions of CD68(+) monocytes/macrophages. There was a clear preponderance of CD3(+) CD8(+) T cells on the endothelial side of transmural infiltrates. In active lesions of transmural vasculitis, CD3(+) T-cell infiltrates exhibited a distinctive zonal distribution. The majority of CD3(+) cells were also CD8(+) and CD45RO+. Scattered perivascular monocytes/macrophages in foci of florid vasculitis were immunoreactive for the p24 core protein. In contrast to the perivascular space, the intervening brain neuropil was dominated by monocytes/macrophages, microglia, and reactive astrocytes, containing only scant CD3(+) CD8(+) cells. Five of six patients showed evidence of calcific vasculopathy, but only two exhibited HIV-1 encephalitis. One patient had multiple subacute cerebral and brainstem infarcts associated with a widespread, fulminant mononuclear-cell vasculitis. A second patient had an old brain infarct associated with fibrointimal thickening of large leptomeningeal vessels. These infiltrating CD3(+) T cells may be responsible for HIV-1-associated CNS vasculitis and vasculopathy and for endothelial-cell injury and the opening of the blood-brain barrier in children with AIDS.

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.

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.

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.

T cells and T-cell cytokine transcripts in the synovial membrane in patients with osteoarthritis.

The synovial membrane in osteoarthritis (OA) often exhibits inflammatory infiltrates, but the role of T cells in these infiltrates is not known. T-cell activation antigens were analyzed by immunohistochemistry, and T-cell cytokine transcripts were measured by competitive PCR in synovial membranes from patients with OA and rheumatoid arthritis (RA). Lymphoid cell aggregates, containing primarily CD3+ T lymphocytes, were found in 65% of patients with OA. Mononuclear cells expressing the activation antigens CD69, CD25, CD38, CD43, CD45RO, and HLA class II were present in both patient groups, although in higher numbers in patients with RA. Interleukin 2 (IL-2) transcripts were found in 10 of 18 patients with OA versus 12 of 13 patients with RA (P = 0.03). Gamma interferon (IFN-gamma) transcripts were detected in 9 of 18 patients with OA versus 10 of 13 patients with RA (not significant), whereas IL-10 transcripts were found in nearly all patients. IL-4 and IL-5 were not detected in any patients. The levels of IFN-gamma and IL-2 transcripts, normalized for T-cell number equivalents, were not statistically different between OA and RA, but the levels of IFN-gamma, normalized for total cell number equivalents, were lower in OA than in RA (P = 0.01). Synovial membranes that expressed IL-2 and IFN-gamma transcripts were more likely to have heavier infiltrations of T cells and cells bearing activation markers than synovial membranes that did not express these cytokines. The presence of activated T cells and TH1 cytokine transcripts in chronic joint lesions of patients with OA suggests that T cells contribute to chronic inflammation in a large proportion of these patients.

Amplification of T-cell receptor alpha- and beta-chain transcripts from mouse spleen lymphocytes by the nonpalindromic adaptor-polymerase chain reaction.

We employed the nonpalindromic adaptor-PCR (NPA-PCR) method to amplify T-cell receptor (TCR) alpha- and beta-chain transcripts from the spleen of normal SJL mice. The NPA-PCR method has been specifically designed for the amplification of transcripts with variable or unknown 5' ends, such as TCRs and immunoglobulins (Ig). This method has certain distinct advantages over existing two-sided PCR methods for the amplification of TCR transcripts. Two NPA-PCR amplifications are sufficient to amplify all the TCR transcripts (one for the alpha-chain and another one for the beta-chain). Amplification of TCR transcripts by classical two-sided PCR requires a minimum of 45 amplification reactions for the murine TCR (20 for the V alpha families and 25 for the V beta families), using 45 different V-family-specific amplification primers. cDNA was synthesized from spleen RNA, using oligonucleotides complementary to sequences of either the murine TCR C alpha or C beta regions. The NotI restriction site was conjugated to these primers and therefore, a NotI restriction site was incorporated at the 3' end of the cDNA. A double-stranded nonpalindromic adaptor (EcoRI-XmnI strand and XmnI G strand, which are complementary to each other) was ligated onto both ends of the double-stranded cDNA. The adaptor was removed from the 3' end by NotI nuclease digestion whereas the adaptor was retained at the 5' end. Two rounds of PCR amplification were carried out. In the first, the EcoRI-XmnI adaptor was used as 5' end amplification primer; an antisense C region primer, designated mC alpha 2 or mC beta 2 (for the alpha- and beta-chain, respectively), was used as 3' amplification primer. In the second round of PCR amplification the same 5' end primer and a 3' end antisense primer, designated mC alpha 1 or mC beta 1, were used. These mC alpha 1 and mC beta 1 primers are located 5' to the mC alpha 2/mC beta 2 primers that were used for the first amplification. The amplified transcripts were cloned. Colonies were screened using a 32P-labeled probe, either C alpha or C beta, located 5' to those used for the last amplification and many positive clones were isolated and sequenced. All clones were unique when compared to each other, as anticipated for polyclonal T-cell populations. Comparison of the sequences obtained to those in the GENBANK/EMBL database revealed that they were typical of mouse alpha- or beta-chain TCR. With the exception of two beta-chain TCR transcripts, all the sequences shown here (36 alpha-chain and 20 beta-beta chain) have not been previously reported to the GENBANK/EMBL database.