U. Keilholz

Evaluation of the interferon-γ ELISPOT-assay for quantification of peptide specific T lymphocytes from peripheral blood

ELISPOT assays for detection of antigen specific HLA class I restricted T lymphocytes have recently been developed. Reliable quantitation of T cell frequencies is crucial for the monitoring of specific cancer vaccines. We have evaluated the accuracy of quantitative results obtained with the IFN-gamma ELISPOT assay and both sensitivity and specificity obtained with several pairs of antibodies was compared. The detection system was tested with IFN-gamma coupled latex beads and the quantitation of influenza specific T cells with several sets of dilution experiments. The reproducibility of the quantitative results was established. Only one pair of monoclonal antibodies had an acceptable specificity. In a serial dilution, almost 100% of IFN-gamma-coated beads could be detected. Furthermore, the number of influenza-peptide specific spots correlated closely with (a) the number of antigen specific T cells derived from an influenza-peptide specific T cell line diluted in PBMC, and (b) the number of CD8+ T cells diluted in autologous CD8-depleted PBMC. In three healthy individuals and three cohorts of healthy volunteers, the number of influenza-peptide specific spots was highly reproducible. The data presented here demonstrate that the frequency of peptide specific CD8+ T cells can be reliably determined from peripheral blood with the IFN-gamma ELISPOT assay.

Analysis of the T cell response to tumor and viral peptide antigens by an IFNγ-ELISPOT assay

We have established a sensitive ELISPOT assay measuring interferon γ (IFN γ) release on a single‐cell basis to detect influenza peptide‐specific CD8+ T cells in uncultured peripheral blood mononuclear cells (PBMC). Using this method, we studied the T cell response to HLA‐A1 and HLA‐A2.1 binding peptide epitopes derived from the MAGE‐1 and MAGE‐3 proteins, from the melanoma‐associated antigens tyrosinase, Melan‐A/MART‐1 and gp100, and from influenza proteins in stage IV melanoma patients and healthy controls. In 18 of 24 HLA‐A2‐positive donors (75%), but only in 9 of 25 HLA‐A2‐positive melanoma patients (36%) T cells reactive with the influenza matrix peptide were demonstrated (p = 0.007). T cells responding to one or several of the melanoma‐associated peptides were detected in 5 of 25 HLA‐A2‐positive patients with metastatic melanoma. Four of these 5 patients had been treated with interleukin‐2‐ and IFNα‐containing therapy. Two of the 24 healthy donors had T cells reactive with the MART‐1 27‐35 peptide. No reactivity with the HLA‐A1‐binding peptides from MAGE‐1 or MAGE‐3 was detected in any of the HLA‐A1‐positive healthy controls or melanoma patients. These results show that the IFNγ‐ELISPOT assay is suitable to determine quantitatively T cells reactive with melanoma‐associated and influenza peptide epitopes in uncultured PBMC. The failure to detect T cells responding to influenza in many melanoma patients with progressive disease may indicate an impairment of their T cell function. Int. J. Cancer 71: 932‐936, 1997.

LIPOPOLYSACCHARIDE EFFECTIVELY UP-REGULATES B7-1 (CD80) EXPRESSION AND COSTIMULATORY FUNCTION OF HUMAN MONOCYTES

The influence of lipopolysaccharide (LPS) and various cytokines on the expression of the costimulatory molecule B7‐1 and intercellular adhesion molecule‐1 (ICAM‐1), lymphocyte function associated antigen‐3 (LFA‐3) and human histocompatibility leucocyte antigen‐DR (HLA‐DR) on human monocytes and their effect on the costimulatory function was investigated. Freshly isolated human monocytes constitutively express ICAM‐1, LFA‐3 and HLA‐DR, but no B7‐1. B7‐1 expression was up‐regulated by LPS and, to a lesser extent, by interferon‐γ (IFN‐γ). The other stimuli tested, including IFN‐α, granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), tumour necrosis factor‐α (TNF‐α) and GM‐CSF + TNF‐α, did not influence expression of B7‐1 on monocytes. ICAM‐1 and HLA‐DR were up‐regulated by IFN‐γ and LPS; LFA‐3 expression was not influenced. LPS also effectively enhanced costimulatory function of monocytes as determined in the tetanustoxoid (TT) assay. Blocking of B7 by CTLA‐4Ig inhibited the LPS‐induced enhancement of costimulatory function almost completely. Our results indicate that the LPS‐mediated up‐regulation of the costimulatory function of human monocytes is mediated by B7. This mechanism may be important for host defence against Gram‐negative bacteria.

Regional adoptive immunotherapy with interleukin-2 and lymphokine-activated killer (LAK) cells for liver metastases

A phase lb trial of a novel regional approach to adoptive immunotherapy is reported. Patients with liver metastases received continuous high-dose infusion of interleukin-2 (IL-2) into the splenic artery or intravenous infusion with subsequent transfer of lymphokine-activated killer (LAK) cells into the portal vein or the hepatic artery. Trafficking studies revealed homogeneous distribution of the LAK cells within the liver. The usual side-effects of IL-2 and LAK cells occurred without limiting liver toxicity. One partial (7+ months) and two complete responses (36 and 26+ months) were observed in 9 patients with metastases from cutaneous melanoma. None of 6 patients with metastases from ocular melanoma responded.

Long‐term endocrine toxicity of myeloablative treatment followed by autologous bone marrow/blood derived stem cell transplantation in patients with malignant lymphohematopoietic disorders

The effect of myeloablative treatment with autologous bone marrow transplantation (ABMT)/autologous blood derived stem cell transplantation (ABSCT) in patients with acute leukemias or lymphomas was studied in 32 adult patients with a mean observation time of 15.8 months after transplantation. The conditioning regimen consisted of hyperfractionated total‐body irradiation (TBI) and high‐dose cyclophosphamide or the cyclophosphamide, carmustine, and etoposid (CBV) regimen. In all of the female patients, we observed primary ovarian failure requiring estradiol replacement therapy. In all of the male patients, testosterone levels were normal but follicle stimulating hormone (FSH) levels were increased, suggestive of germinal aplasia which was proved by semen analysis in several patients. In contrast to the reports of other groups, we did not find any abnormalities in thyroid function, most likely because TBI was hyperfractionated. Moderate toxicity to the adrenal cortex was noticed and was more pronounced in women than in men. Our results are similar to findings reported after allogeneic bone marrow transplantation, with the exception of normal thyroid function in our patients. These results should be taken into consideration when counseling patients about the long‐term consequences of myeloablative treatment. Cryopreservation of semen should be offered to men before myeloablative treatment. Estrogen replacement should be initiated after transplantation in women to prevent adverse effects of long‐term ovarian failure.

Detection of clonally rearranged T-cell-receptor gamma chain genes from T-cell malignancies and acute inflammatory rheumatic disease using PCR amplification, PAGE, and automated analysis

Abstract Clonal expansions of T cells carrying identical T-cell-receptor (TCR) genes are the hallmark of T-cell malignancies, but they can also result from a strong immune reaction to a dominant epitope. The basis for the molecular detection of clonal T cells is amplification of the V-(D)-N-J region of the TCR gene. We evaluated PCR amplification of the rearranged gamma TCR from genomic DNA extracted from peripheral blood and subsequent polyacrylamide gel electrophoresis (PAGE) in an automated DNA sequencer. We determined the sensitivity for the detection of clonal T cells and propose a standardized evaluation procedure for the electrophoretic profiles generated by the DNA sequencer. The sensitivity of our method was 0.6–1.25% of clonal T cells within a polyclonal background. Sixteen patients with T-cell malignancies, ten with acute inflammatory rheumatic diseases, and twelve healthy controls were examined. Among the systemic T-cell malignancies, all but one patient with T-PLL (8/9) revealed a clonal PCR signal. No clonal signal was detectable in any patient in clinical complete remission (5/5) or in either of the two patients with lymphomas limited to cutaneous sites. However, clonal T cells were detected in one patient with polymyalgia rheumatica and in one with reactive arthritis. A polyclonal signal was found in the remaining eight patients with acute inflammatory rheumatic diseases and in 12 healthy controls. Taking our results together, the PCR/PAGE assay is able to reliably distinguish clonal from polyclonal T-cell populations. However, although the sensitivity is limited to approximately 1%, clonal T cells can be found in the peripheral blood of some patients with autoimmune diseases and not only in T-cell malignancies.

Immunotherapy of metastatic melanoma with interferon-α and interleukin-2: Pattern of progression in responders and patients with stable disease with or without resection of residual lesions

This evaluation was performed in melanoma patients after successful immunotherapy to describe the pattern of relapse. 63 patients received interferon (IFN)-alpha and high-dose interleukin (IL)-2, resulting in three complete responses (CR), 13 partial responses (PR), three mixed responses (MR) and 17 stable diseases (SD). Median duration of response was 7 months (range 3-28) without surgery. Most relapses occurred at pre-existing sites. Duration of CR was 14-37+ months. In 11 patients, residual tumour lesions were resected. Interestingly, histology revealed almost complete tumour regression in 6 patients, including 2 of 4 with SD. 5 of these 11 patients have relapsed so far, 6 patients are still free of disease with a median of 17 months (range 8-34). Following relapse, 4 of 6 patients responded to retreatment with the identical IFN alpha/IL-2 protocol. The authors conclude that initial disease progression is mostly at previous sites of disease. Resection of residual lesions may offer a chance for extended disease-free survival similar to patients with CR to immunotherapy. Retreatment of relapsing patients is favourable.

Regional administration of lymphokine-activated killer cells can be superior to intravenous application

A patient with liver metastases of human lymphocyte antigen (HLA) class II‐negative malignant melanoma was treated with several cycles of adoptive immunotherapy with interleukin‐2 and lymphokine‐activated killer (LAK) cells. The authors evaluated the efficacy of regional transfer of LAK cells versus systemic intravenous administration. Initially, the patient was treated according to a regional treatment protocol, consisting of perfusion of the spleen with interleukin‐2 and transfer of LAK cells into the portal vein; a partial remission was observed. Because of technical problems, interleukin‐2 and LAK cells were administered intravenously in a second treatment cycle. This systemic treatment course resulted only in a minor mixed response of the hepatic metastases. A third treatment course was administered with the use of intravenous interleukin‐2 infusion and arterial perfusion of the liver with LAK cells. The patient had separate hepatic arteries to both lobes of the liver as an anatomic variation. Because most of the tumor mass was present in the right lobe of the liver, a third of the LAK cells were injected into the right hepatic artery and the remaining cells were administered intravenously. The lesions in the right lobe of the liver regressed, but disease progression occurred in the left lobe. A fourth treatment cycle, consisting of intravenous infusion of interleukin‐2 and arterial perfusion of both lobes of the liver with LAK cells, resulted in a complete response of all hepatic lesions, which has lasted 18 months to date. Because, in this patient, tumor regression was observed only in anatomic areas of the liver, which were perfused with LAK cells, it is suggested that the regional administration of LAK cells was essential for successful treatment.

A phase II study of dacarbazine, cisplatin, interferon-α and high-dose interleukin-2 in ‘poor-risk’ metastatic melanoma

Melanoma patients with very advanced disease have usually not been included in chemo-immunotherapy trials. We report on 22 melanoma patients, including 5 with reduced performance status (Karnofsky PS < 70), 8 with metastatic ocular melanoma, 6 with brain metastases, and 4 who had pretreatment with interleukin-2. These were treated with a combination regimen of dacarbazine (250 mg/m2, days 1-3), cisplatin (30 mg/m2, days 1-3), interferon-alpha 2a (IFN-alpha, 10 Mio IU/m2 s.c., days 1-5) and IL-2 (i.v., 18 Mio IU/m2 for 6, 12, 24 h, followed by 13.5 Mio IU/m2 in 72 h). In the case of brain metastases radiotherapy was added. No grade IV toxicity occurred and no dose reductions were necessary. 21 patients were evaluable for response. 6 (29%) had disease progression, 5 (24%) had partial response and 10 (48%), had stable disease. Sites of response included skin, lymph nodes, muscle, lung, pleura, liver, pancreas, adrenal gland and brain. The described treatment schedule is safe and active even in patients with metastatic melanoma and poor prognosis.

Interleukin-2 in Combination with Interferon-Alpha in Disseminated Malignant Melanoma and Advanced Renal Cell Carcinoma

In vitro, the combination of interleukin-2 (Il-2) with interferon-alpha (IFN-alpha) seems to act synergistically on the generation of lymphokine activated killer (LAK) cells. Due to this fact two clinical trials with the combination of Il-2 and IFN-alpha were initiated in malignant melanoma (MM) and renal cell cancer (RCC). Patients with disseminated MM were treated by a sequential application of 10 x 10(6) U/m2 rIFN-alpha 2b s.c. on days 1-7 followed by continuous intravenous infusion of 3 x 10(6) U/m2 rIl-2 on days 8-13 and 15-20. After a pause of 4 weeks the cycle was repeated. In advanced or disseminated RCC, the patients were treated with a daily alternating scheme of 10 x 10(6) U/m2 rIFN-alpha and rIl-2 as 1 h infusion 1 x /day for 14 days. The rIl-2 escalates intra- and interindividually beginning with a dose of 3 x 10(6) U/m2. The cycles were repeated after a pause of 3 and 4 weeks, respectively. The preliminary results show that the schedules are practicable and that the toxicity of the combination of rIl-2 and IFN-alpha does not accumulate. Within the MM group 3/11 evaluable patients achieved partial remission and 2/11 stable disease. In the RCC-group 2/5 evaluable patients achieved partial remission and 2/5 had stable disease so far.