Lack of T-cell responses following autologous tumour lysate pulsed dendritic cell vaccination, in patients with relapsed osteosarcoma

Background

Immunotherapy using autologous dendritic cell (DC) vaccination has not been systematically evaluated in osteosarcoma. We therefore conducted a phase I trial to assess feasibility, safety and tumour-specific immune responses in patients with relapsed disease.

Patients and methods

Of 13 recruited patients with relapsed osteosarcoma, 12 received 3 weekly vaccines of autologous DCs matured with autologous tumour lysate and keyhole limpet haemocyanin (KLH), to a maximum of 6 vaccinations. An additional 3 paediatric patients afflicted with other tumour types and with relapsed disease received vaccines generated with identical methodology. Immune responses were assessed using an ELISpot assay for the detection of interferon gamma, whilst interleukin-2 and granzyme B were additionally assessed in cases where interferon-?? responses were induced.

Results

In total 61 vaccines, of homogeneous maturation phenotype and viability, were administered with no significant toxicity. Only in 2 out of 12 treated osteosarcoma cases was there an induction of specific T-cell immune response to the tumour, whilst a strong but non-specific immune response was induced in 1 further osteosarcoma patient. Immune response against KLH was induced in only 3 out of 12 osteosarcoma patients. In contrast, three additional non-osteosarcoma patients showed significant T-cell responses to vaccine.

Conclusion

We have shown the strategy of DC vaccination in relapsed osteosarcoma is safe and feasible. However, significant anti-tumour responses were induced in only 2 out of 12 vaccinated patients with no evidence of clinical benefit. Comparison of results with identically treated control patients suggests that osteosarcoma patients might be relatively insensitive to DC-based vaccine treatments.     

An inducible transgenic mouse breast cancer model for the analysis of tumor antigen specific CD8+ T-cell responses

In Simian virus 40 (SV40) transgenic BALB/c WAP-T mice tumor development and progression is driven by SV40 tumor antigens encoded by inducible transgenes. WAP-T mice constitute a well characterized mouse model for breast cancer with strong similarities to the corresponding human disease. BALB/c mice mount only a weak cellular immune response against SV40 T-antigen (T-Ag). For studying tumor antigen specific CD8⁺ T-cell responses against transgene expressing cells, we created WAP-T_NP mice, in which the transgene additionally codes for the NP_118–126-epitope contained within the nucleoprotein of lymphocytic choriomeningitis virus (LCMV), the immune-dominant T-cell epitope in BALB/c mice. We then investigated in WAP-T_NP mice the immune responses against SV40 tumor antigens and the NP-epitope within the chimeric T-Ag/NP protein (T-Ag_NP). Analysis of the immune-reactivity against T-Ag in WAP-T and of T-Ag_NP in WAP-T_NP mice revealed that, in contrast to wild type (wt) BALB/c mice, WAP-T and WAP-T_NP mice were non-reactive against T-Ag. However, like wtBALB/c mice, WAP-T as well as WAP-T_NP mice were highly reactive against the immune-dominant LCMV NP-epitope, thereby allowing the analysis of NP-epitope specific cellular immune responses in WAP-T_NP mice. LCMV infection of WAP-T_NP mice induced a strong, LCMV NP-epitope specific CD8⁺ T-cell response, which was able to specifically eliminate T-Ag_NP expressing mammary epithelial cells both prior to tumor formation (i.e. in cells of lactating mammary glands), as well as in invasive tumors. Elimination of tumor cells, however, was only transient, even after repeated LCMV infections. Further studies showed that already non-infected WAP-T_NP tumor mice contained LCMV NP-epitope specific CD8⁺ T-cells, albeit with strongly reduced, though measurable activity. Functional impairment of these ‘endogenous’ NP-epitope specific T-cells seems to be caused by expression of the programmed death-1 protein (PD1), as anti-PD1 treatment of splenocytes from WAP-T_NP tumor mice restored their activity. These characteristics are similar to those found in many tumor patients and render WAP-T_NP mice a suitable model for analyzing parameters to overcome the blockade of immune checkpoints in tumor patients.     

A phase I study of active specific intralymphatic immunotherapy (ASILI).

Twenty-one patients with advanced malignancies who had exhausted or refused conventional modalities of treatment were entered in a Phase I toxicology trial of active specific intralymphatic immunotherapy (ASILI). The patients were immunized with 1 X 10(7) to 1.2 X 10(8) viable autochthonous or allogeneic irradiated tumor cells intralymphatically each month and received no other antineoplastic treatment. To date, 274 intralymphatic injections have been performed and except for one case of bacterial lymphangitis, no adverse side effects have been observed. ASILI did not significantly alter peripheral blood lymphocyte counts, absolute E-rosette forming cell levels, or EA-rosette forming cell levels. PHA reactivity of peripheral blood lymphocytes increased slightly in all but one patient tested. Seven out of nine patients who had not had delayed hypersensitivity to recall antigens developed positive reactions following ASILI. Sixteen out of twenty patients tested also developed reactivity to their immunizing cells after treatment. Objective regression (greater than 50% reduction of tumor mass) was observed in five out of nineteen evaluable patients. Six patient showed stabilization of tumor growth and eight patients continued to progress under treatment.     

105Ad7 cancer vaccine stimulates anti-tumour helper and cytotoxic T-cell responses in colorectal cancer patients but repeated immunisations are required to maintain these responses

105AD7 is a human anti-idiotypic antibody that recognises the binding site of the anti-tumour antibody 791T/36 and can thereby mimic the CD55 antigen. The molecular basis of 105AD7 mimicry has been identified with 3 CDR regions of 105AD7 showing similarity to 3 regions of CD55. These regions have been analysed for potential T-cell epitopes, and sequences that are predicted to bind to HLA/A1, 3,24 and to HLA/DR1,3,7 have been identified within the CDRH3 region of 105AD7. These epitopes should be stimulating CD8 and CD4 responses, respectively. This prediction was tested on 12 colorectal cancer patients receiving 105AD7 therapy. There was good concordance in 10 of 11 patients between accumulation of CD8RO cells or tumour killing and expression of HLA/A1,3,24. The only patient who failed to respond had a non-permissive class II haplotype and failed to show a helper response. Again 10 of 11 patients showing accumulation of CD4RO cells, in vitro blastogenesis responses, enhanced IL-2 or enhanced NK activity expressed one or more of the HLA/DR1,3,7 haplotypes. Although there was a consistent accumulation of CD45RO cells following 14 of 18 immunisations, only one patient showed a sustained memory response. Our results suggest that 105AD7 can stimulate CD4 and CD8 responses in patients with the appropriate haplotype. However, it may be necessary to continue to immunise, since few patients produce a sustained memory response.     

Effects of specific active immunization on tumor recurrence following primary tumor resection in WF rats with 1,2-dimethylhydrazine-induced bowel cancer

Primary gastrointestinal tumors were induced in male WF rats by 16 weekly sc injections of 1,2-dimethylhydrazine [(DMH) CAS: 540-73-8; 20 mg/kg/wk]. Twenty-four to 28 weeks after the start of DMH injections, all rats were surgically explored and gastrointestinal tumors were resected. Rats with no remaining microscopic disease after operation were immunized with one of four tumor isografts. The first isograft, DMH-W163, is a poorly differentiated mucinous adenocarcinoma explanted from a colon cancer in a DMH-treated animal. It has been shown to possess antigens that cross-react with other DMH-induced bowel adenocarcinoma isografts. The second isograft, DMH-W49, is a carcinosarcoma explanted from a DMH-treated primary colon cancer. It has intermediate antigenic cross-reactivity with other colon adenocarcinoma isografts in the WF model. The third isograft, DMH-W15, is a sarcoma explanted from a DMH-induced colon cancer that does not possess antigens cross-reactive with other DMH-induced colon adenocarcinomas. The fourth isograft, SPK, is a spontaneous (non-DMH-induced) renal cell carcinoma that is immunogenic but should not contain tissue-type-specific antigens cross-reacting with the bowel cancers. Immunized rats received three sc weekly injections of 1 X 10(3) irradiated cells. Concomitant control rats received no immunization after resection of the primary tumor. Within 24 weeks of primary tumor resection, 12 of 16 (75%) rats not immunized had tumor recurrence. Only 8 of 24 (34%) rats immunized with DMH-W163 had tumor recurrence (P less than .025 compared to controls). Fifty percent of animals (10/20) immunized with the carcinosarcoma DMH-W49 had a recurrence. Animals immunized with the non-cross-reacting DMH-W15 sarcoma isograft had a recurrence rate similar to that of controls (16/20, 75%). The rats immunized with SPK were not protected from recurrence. Twelve of 19 (63%) had a recurrence at or near the suture line within 24 weeks following primary tumor resection. These results confirm that adjuvant immunotherapy can decrease the rate of recurrence following primary tumor resection in this model. In addition, immunogens that possessed tissue-type-specific antigens were more effective in preventing tumor recurrence than those that did not.     

Western blot analysis of serological responses following immunization with vaccinia viral lysates of melanoma cells

Western blot analysis was used to characterize the antibody response of melanoma patients during immunization with vaccinia-virus-induced melanoma cell lysates (VMCL). Strong antibody responses were detectable within 4 weeks from commencement of immunization against different fractions (from 3 to 10) in blots of VMCL. The approximate MW of the main fractions identified were 100, 91, 85, 77, 64-66, 49, 46, 43 and 34-36 kDa. Comparison of the reactivity of the sera with extracts of vaccinia virus suggested that the reactivity against VMCL was not against viral antigens in the lysates. Studies on autologous extracts from 7 patients immunized with VMCL indicated that the majority of the fractions identified in extracts of VMCL were also identified in autologous extracts of melanoma cells. This indicated vaccine-induced responses against the patients' tumor cells and not merely against alloantigens in the vaccine. Sera from the immunized patients appeared to identify a number of fractions of similar MW in extracts of colon and glioma cells, suggesting that the responses were not specific for melanoma. Possible exceptions were reactivity against melanoma fractions of 85, 64-66 and 36 kDa. Our studies provide evidence for the effectiveness of VMCL in inducing antibody responses against autologous melanoma cells and form a basis for subsequent biochemical and genetic analysis of the antigens identified by antibody responses in immunized patients.     

An improved procedure for autologous gene-modified cancer vaccine preparation for active specific immunotherapy of disseminated solid tumors

Resected material was used from 108 patients with disseminated skin melanoma and 47 patients suffering metastasized renal tumors to test procedures of tumor cell culture preparation and to search the best parameters. Gene tag 7 transfer, liposome delivery and electroporation were employed to stimulate immunogenic tumor cells. The transfer results were evaluated by expression of beta-galactosidase and EGFP genes whose products were detected microscopically. Transfer efficiency was boosted by 30% due to selecting suitable parameters of tumor cell modification. Maximum effectiveness was attained by individualized choice of the parameters. Yet, undoubtedly, the best way of cell isolation was mechanical fragmentation of tumor. To speed up cell production, DMEM/F12 medium should be recommended. It should contain cattle embryonic serum (20%), conditioned medium of cultured fibroblasts of human embryonic lungs (20%), transferin, insulin and selenium (standard dose).     

自体肿瘤疫苗主动特异性免疫治疗对结肠癌病人细胞免疫功能的影响

目的　探讨自体肿瘤疫苗的作用机制。方法　 2 0例结肠癌患者术后 ,以自体肿瘤疫苗行主动免疫治疗。术后第 4周开始免疫接种 (共 4次 ,每次间隔 7～ 10天 ) ;接种前 3天及 4次接种后 1周 ,采集外周血 ,分离单个核细胞 ,测定CD+ 8-IFN -γ+ ,CD+ 8-IL - 10 + ,细胞比例及CD+ 4 -IFN -γ+ 、CD+ 4-IL - 10 + 细胞比例 ;同时采集血清检测血清IFN -γ、IL - 10水平 ;用自体肿瘤抗原做皮肤迟发型过敏反应试验 ,4 8小时后测量红斑、硬结大小 (mm) ;临床随访。结果　自体疫苗治疗后 :血清IFN -γ水平升高 ,由 (6 .2 9± 1.96 )pg/ml升至 (10 .94± 3.2 1) pg/ml;而IL - 10水平由 (2 1.91± 10 .19)pg/ml降至(10 .84± 6 .0 1) pg/ml。有显著性差异 (P <0 .0 5 )。CD+ 8-IFN -γ+ 双阳性细胞比例由 (4.0 2± 1.13) %升至 (8.81± 2 .90 ) % ;CD+ 4 -IFN -γ+ 双阳性细胞比例由 (4.19± 1.10 ) %升至 (6 .99± 1.87) %。有显著性差异 (P <0 .0 5 )。治疗前后患者对自体肿瘤抗原的特异性DTH反应明显增强 (P <0 .0 1)。患者耐受性良好 ,无溃疡等严重副作用发生。 11例无瘤生存时间为 36～ 4 8个月 (仍健在 ) ;6例无瘤生存时间为 11～ 14个月 (仍健在 ) ;3例生存 2 4～ 2 6个月 (死于肝转移 )。结论　自体肿瘤疫苗可     

Effect of active immunization with irradiated tumour cells on specific serum inhibitors of cell-mediated immunity in patients with disseminated cancer

The sera from patients with advanced cancer were tested for their specific inhibitory effects on the cytotoxicity of autologous lymphocytes on tumour cells in a microculture assay. By adding a standard volume of the sera to suspensions of well-washed lymphocytes the inhibitory effect was quantitated by comparison with the effect of normal allogeneic serum. Significant levels of inhibitory activity were detected in 7 patients (one massive primary melanoma, 4 with disseminated melanoma, one with metastatic hypernephroma and one with a recurrent leiomyosarcoma). The patient with a massive primary melanoma was treated by extensive surgical excision. This procedure was associated with the rapid and complete disappearance of the serum inhibitory effect. In the other cases surgical intervention was minimal and the serum inhibitor was unaffected. All 6 of these patients were then immunized with irradiated autologous tumour cells and the serum inhibitory activity assayed. In 5 cases the serum inhibitor rapidly became undetectable after a single immunization. The one patient who failed to respond in this manner had very extensive disease and died within 2 weeks of the study. Repeated monthly immunization in the case of recurrent leiomyosarcoma was associated with the maintenance of the serum inhibitory activity at very low levels and with good clinical progress. The response to a single immunization is transient, the inhibitor becoming detectable again at 14-21 days. The possible role of circulating antigen in this serum inhibitory activity is discussed, as is the potential value of assaying the sera of cancer patients for serum inhibitory activity, as a means of monitoring the effects of treatment.     

Phase I trial of active specific immunotherapy with autologous dendritic cells pulsed with autologous irradiated tumor stem cells in hepatitis B‐positive patients with hepatocellular carcinoma

Background and Objectives

Hepatocellular carcinoma (HCC) is often associated with chronic hepatitis due to hepatitis‐B or ‐C viruses. Active specific immunotherapy (ASI) with autologous dendritic cells (DC) presenting antigens from autologous tumor stem cell (TC) lines is associated with promising long‐term survival in metastatic cancer, but hepatitis patients were excluded. ASI might benefit high‐risk primary HCC patients following surgical resection, but first it is important to show that ASI does not exacerbate hepatitis.

Methods

Previously untreated HCC patients with a solitary lesion > 5 cm, or three lesions with at least one > 3 cm, or more than three lesions, underwent surgical resection from which autologous TC lines were established. Irradiated TC were incubated with autologous DC to create DC‐TC. After one course of trans‐arterial chemoembolization therapy (TACE), three weekly subcutaneous injections of DC‐TC suspended in granulocyte‐macrophage colony stimulating factor were administered. Patients were monitored for eight weeks.

Results

HCC cell lines were established within five weeks for 15/15 patients. Eight patients, all with chronic hepatitis B, were treated. There was no increase in hepatic transaminases, hepatitis B antigens, or viral DNA.

Conclusion

Autologous DC‐TC did not exacerbate HBV in these HCC patients. A phase II efficacy trial is being planned. J. Surg. Oncol. 2015 111:862–867. © 2014 Wiley Periodicals, Inc.     

Dendritic cells pulsed with HER-2/neu-derived peptides can induce specific T-cell responses in patients with gastric cancer.

PURPOSE: We have previously reported (K. Kono et al., Int. J. Cancer, 78: 202-208, 1998) that HER-2/neu-derived peptides are naturally processed as tumor-associated antigens recognized by tumor-specific, human leukocyte antigen (HLA)-A2-restricted CTLs in gastric cancer. In the present study, we described a Phase-1 vaccination trial in gastric cancer patients using dendritic cells (DCs) pulsed with the immunodominant HER-2/neu(p369) peptides. EXPERIMENTAL DESIGN: Nine enrolled patients, who had HER-2/neu-overexpressing tumors and who were HLA-A2 positive, received four vaccinations by DCs pulsed with HER-2(p369) peptide at 2-week intervals intradermally. RESULTS: There were no serious adverse effects noted in the immunized patients. Peripheral blood mononuclear cells, preimmunization and after the fourth immunization, were cultured with autologous, HER-2(p369)-pulsed antigen-presenting cells for 12 days. Thereafter, peptide specificity was evaluated by IFN-gamma secretion assay from cultured T cells against T2 cells pulsed with HER-2(p369) peptide. HER-2/neu peptide-specific recognition could be demonstrated in six of nine patients after immunization, whereas there was no HER-2/neu peptide-specific recognition before immunization. The peptide-specific CTL lines isolated from two of the patients could also lyse a HER2/neu-transfected cell line. Furthermore, a peptide-specific delayed-type hypersensitivity response occurred in three of nine patients. One of the patients underwent a partial clinical response concurrent with a decrease of tumor marker. Another patient demonstrated a stabilization of disease status for a period of 3 months. CONCLUSIONS: Taken together, tumor vaccination therapy with DCs pulsed with HER-2/neu-peptides may be a potential candidate for the novel treatment of gastric cancer patients.     

Role of up-front autologous stem-cell transplantation in peripheral T-cell lymphoma for patients in response after induction: an analysis of patients from LYSA centers

BackgroundPeripheral T-cell lymphoma (PTCL) remains a therapeutic challenge. Due to the rarity and the heterogeneity of PTCL, no consensus has been achieved regarding even the type of first-line treatment. The benefit of autologous stem-cell transplantation (ASCT) is, therefore, still intensely debated.Patients and methodsIn the absence of randomized trials addressing the role of ASCT, we performed a large multicentric retrospective study and used both a multivariate proportional hazard model and a propensity score matching approach to correct for sample selection bias between patients allocated or not to ASCT in intention-to-treat (ITT).ResultsAmong 527 patients screened from 14 centers in France, Belgium and Portugal, a final cohort of 269 patients ≤65 years old with PTCL-not otherwise specified (NOS) (N = 78, 29%), angioimmunoblastic T-cell lymphoma (AITL) (N = 123, 46%) and anaplastic lymphoma kinase-positive anaplastic large cell lymphoma (ALK-ALCL) (N = 68, 25%) with partial (N = 52, 19%) or complete responses (N = 217, 81%) after induction was identified and information about treatment allocation was carefully collected before therapy initiation from medical records. One hundred and thirty-four patients were allocated to ASCT in ITT and 135 were not. Neither the Cox multivariate model (HR = 1.02; 95% CI: 0.69–1.50 for PFS and HR = 1.08; 95% CI: 0.68–1.69 for OS) nor the propensity score analysis after stringent matching for potential confounding factors (logrank P = 0.90 and 0.66 for PFS and OS, respectively) found a survival advantage in favor of ASCT as a consolidation procedure for patients in response after induction. Subgroup analyses did not reveal any further difference for patients according to response status, stage disease or risk category.ConclusionsThe present data do not support the use of ASCT for up-front consolidation for all patients with PTCL-NOS, AITL, or ALK-ALCL with partial or complete response after induction.     

Adjuvant immunization of HLA-A2-positive melanoma patients with a modified gp100 peptide induces peptide-specific CD8+ T-cell responses.

PURPOSE: To measure the CD8+ T-cell response to a melanoma peptide vaccine and to compare an every-2-weeks with an every-3-weeks vaccination schedule. PATIENTS AND METHODS: Thirty HLA-A2-positive patients with resected stage I to III melanoma were randomly assigned to receive vaccinations every 2 weeks (13 vaccines) or every 3 weeks (nine vaccines) for 6 months. The synthetic, modified gp100 peptide, g209-2M, and a control peptide, HPV16 E7, were mixed in incomplete Freund's adjuvant and injected subcutaneously. Peripheral blood mononuclear cells obtained before and after vaccination by leukapheresis were analyzed using a fluorescence-based HLA/peptide-tetramer binding assay and cytokine flow cytometry. RESULTS: Vaccination induced an increase in peptide-specific T cells in 28 of 29 patients. The median frequency of CD8+ T cells specific for the g209-2M peptide increased markedly from 0.02% before to 0.34% after vaccination (P <.0001). Eight patients (28%) exhibited peptide-specific CD8+ T-cell frequencies greater than 1%, including two patients with frequencies of 4.96% and 8.86%, respectively. Interferon alfa-2b-treated patients also had significant increases in tetramer-binding cells (P <.0001). No difference was observed between the every-2-weeks and the every-3-weeks vaccination schedules (P =.59). CONCLUSION: Flow cytometric analysis of HLA/peptide-tetramer binding cells was a reliable means of quantifying the CD8+ T-cell response to peptide immunization. This assay may be suitable for use in future trials to optimize different vaccination strategies. Concurrent interferon treatment did not inhibit the development of a peptide-specific immune response and vaccination every 2 weeks, and every 3 weeks produced similar results.     

Incidence of post transplant myelodysplasia/acute leukemia in non-Hodgkin's lymphoma patients compared with Hodgkin's disease patients undergoing autologous transplantation following cyclophosphamide, carmustine, and etoposide (CBV).

Secondary malignancies, particularly myelodysplasia (MDS), are serious events following high dose therapy with autologous stem cell support. We observed a higher frequency of secondary malignancies in patients with Hodgkin's disease (HD) than in patients with non-Hodgkin's lymphoma (NHL) undergoing high dose therapy with the same non-TBI conditioning regimen. Three hundred patients with Hodgkin's disease (HD) and non-Hodgkin's lymphoma (NHL) were treated with cyclophosphamide, carmustine and etoposide and autologous stem cell support from 1986 through 1994. Median follow up of survivors is 3.9 years. Five-year survival is 51% for HD and 48% for NHL. Eleven patients developed second malignancies (9/150 treated for HD vs. 2/150 treated for NHL) a median of 2.4 years from transplantation and 5.2 years from initial diagnosis. Six patients had myelodysplasia or acute leukemia (MDS/AML) and 5 had lymphomas or solid tumors. Actuarial risk of MDS/AML at five years for patients transplanted for non-Hodgkin's lymphoma is 3% (95% CI 0.6-9.6%). HD patients had significantly different pretreatment characteristics than patients with NHL. A Cox model showed that greater number of prior relapses and prior radiation therapy were significant risk factors for the development of MDS/AML. These data suggest that CBV is associated with a lower risk of secondary MDS/AML than TBI containing regimens and that much of the risk is associated with the pre-transplantation therapy. The use of autotransplantation early in the course of therapy for relapsed lymphoma might prevent some cases of MDS/AML.     

Diverse CD8+ T-cell responses to renal cell carcinoma antigens in patients treated with an autologous granulocyte-macrophage colony-stimulating factor gene-transduced renal tumor cell vaccine

A phase I clinical trial with granulocyte-macrophage colony-stimulating factor tumor cell vaccines in patients with metastatic renal cell carcinoma (RCC) showed immune cell infiltration at vaccine sites and delayed-type hypersensitivity (DTH) responses to autologous tumor cells indicative of T-cell immunity. To further characterize RCC T-cell responses and identify relevant RCC-associated antigens, we did a detailed analysis of CD8+ T-cell responses in two vaccinated RCC patients who generated the greatest magnitude of DTH response and also displayed a strong clinical response to vaccination (>90% reduction in metastatic tumor volume). Three separate CD8+ T-cell lines (and subsequent derived clones) derived from patient 24 recognized distinct RCC-associated antigens. One recognized a shared HLA-A*0201-restricted antigen expressed by both renal cancer cells and normal kidney cells. This recognition pattern correlated with a positive DTH test to normal kidney cells despite no evidence of impairment of renal function by the patient's remaining kidney after vaccination. A second line recognized a shared HLA-C7-restricted antigen that was IFN-gamma inducible. A third line recognized a unique HLA-A*0101-restricted RCC antigen derived from a mutated KIAA1440 gene specific to the tumor. In addition, two independent CTL lines and three clones were also generated from patient 26 and they recognized autologous tumor cells restricted through HLA-A*0205, HLA-A/B/C, and HLA-B/C. These results show that paracrine granulocyte-macrophage colony-stimulating factor tumor vaccines may generate a diverse repertoire of tumor-reactive CD8+ T-cell responses and emphasize the importance of polyvalency in the design of cancer immunotherapies.     

Generation in vitro of EBV-induced specific cytotoxic T cells in autologous serum avoids complications due to self-preferred foetal calf serum-specific T-cell cytotoxicity

A previous report has established that in cultures of human mononuclear leukocytes, foetal calf serum (FCS) is capable of generating high levels of T cells preferentially cytotoxic for the autologous lymphoblastoid cell line (LCL). The present study compared the capacity of Epstein-Barr virus (EBV) to generate cytotoxic T cells in cultures of mononuclear cells grown in FCS in this system. Five EBV-seropositive and three seronegative donors were used and cultures were harvested at 14 days. With cultures from seropositive donors, whether grown in FCS or in autologous serum, EBV infection generated T cells cytotoxic for the autologous LCL; the response in uninfected control cultures was markedly lower. With seronegative donor cultures grown in FCS, there was virtually no difference in the capacity of T cells generated in infected or uninfected cultures to lyse the autologous LCL. Moreover, cells from seronegative donors cultured in human serum gave no detectable lysis of autologous LCL in either infected or uninfected cultures, clearly showing the absence of a response to EBV. This evidence shows that it is possible to distinguish the generation of specific cytotoxic T cells by FCS from generation by EBV, and with certain donors the apparently EBV-induced response may actually include a significant component induced by FCS in the medium. The cytotoxicity patterns of EBV-induced and FCS-induced T cells for autologous and allogeneic LCL targets showed a degree of parallelism, stressing the need for caution in interpretation of data obtained from cultures using FCS.