Vaccination of melanoma patients using dendritic cells loaded with an allogeneic tumor cell lysate

The aim of the present phase I/II study was to evaluate the safety, immune responses and clinical activity of a vaccine based on autologous dendritic cells (DC) loaded with an allogeneic tumor cell lysate in advanced melanoma patients. DC derived from monocytes were generated in serum-free medium containing GM-CSF and IL-13 according to Good Manufacturing Practices. Fifteen patients with metastatic melanoma (stage III or IV) received four subcutaneous, intradermal, and intranodal vaccinations of both DC loaded with tumor cell lysate and DC loaded with hepatitis B surface protein (HBs) and/or tetanus toxoid (TT). No grade 3 or 4 adverse events related to the vaccination were observed. Enhanced immunity to the allogeneic tumor cell lysate and to TAA-derived peptides were documented, as well as immune responses to HBs/TT antigens. Four out of nine patients who received the full treatment survived for more than 20 months. Two patients showed signs of clinical response and received 3 additional doses of vaccine: one patient showed regression of in-transit metastases leading to complete remission. Eighteen months later, the patient was still free of disease. The second patient experienced stabilization of lung metastases for approximately 10 months. Overall, our results show that vaccination with DC loaded with an allogeneic melanoma cell lysate was feasible in large-scale and well-tolerated in this group of advanced melanoma patients. Immune responses to tumor-related antigens documented in some treated patients support further investigations to optimize the vaccine formulation. Margarita Salcedo and Nadège Bercovici both contributed equally to this work     

Pilot study of treatment of biochemotherapy-refractory stage IV melanoma patients with autologous dendritic cells pulsed with a heterologous melanoma cell line lysate

Eleven AJCC stage IV melanoma patients with progressive disease after treatment with biochemotherapy were treated with autologous dendritic cells pulsed with heterologous tumor cell lysates. The vaccine used mature DCs (CD1a⁺⁺⁺, CD40⁺⁺, CD80⁺⁺, CD83⁺, and CD86⁺⁺⁺) generated from peripheral blood monocytes in the presence of GM-CSF and IL-4. After 7 days, DCs were matured with a defined cocktail of cytokines (IL-1+IL-6+TNF-+PGE₂) and simultaneously pulsed with lysates of heterologous melanoma cell lines, for 2 days. A total of 4×10⁶ DCs was injected monthly under ultrasound control in an inguinal lymph node of normal appearance. The study was closed when all patients died as a consequence of tumor progression. No sign of toxicity was observed during the study. One patient experienced a partial response lasting 5 months, and two patients showed a mixed response which lasted 3 months. The median survival of the whole group was 7.3 months (range 3–14 months). This vaccination program had specific antitumoral activity in highly pretreated and large tumor burden stage IV melanoma patients and was well tolerated. The clinical responses and the median survival of the group of patients, together with the low toxicity of our DC vaccine, suggest that this approach could be applied to earlier AJCC stage IV melanoma patients.     

Expression of interleukin-2 receptor on blood lymphocytes stimulated with allogeneic lymphocytes or autologous tumor cells

Summary The kinetics of interleukin-2 receptor (IL-2R) expression and the [³H]dT incorporation of blood lymphocytes after the first and the second stimulation with allogeneic leukocytes (primary and secondary MLC) or with the autologous tumor cells (primary and secondary MLTC) were compared. The expression of IL-2R paralleled the induction of DNA synthesis. The proportion of IL-2R⁺ cells of the unprimed donors peaked earlier in the secondary MLC as compared to the primary MLC (on days 3 and 5 respectively). In MLC of alloimmunized healthy individuals and in the MLTC of cancer patients the highest proportions of IL-2R⁺ cells were detected between days 2 and 3 after both the first and second stimulations. Thus the first in vitro stimulation in the MLTC showed similar kinetics to those of the secondary MLC of unprimed individuals and to the primary MLC response of the allo-immunized individuals. The findings in the MLTC substantiate the hypothesis that cancer patients can be sensitized to their own tumors. The kinetics of the appearance of the IL-2R together with the characteristics of the IL-2-propagated cultures provide useful information for the strategy of expansion of auto-tumor reactive lymphocyte populations.     

Simultaneous transduction of B7-1 and IL-2 genes into human melanoma cells to be used as vaccine: enhancement of stimulatory activity for autologous and allogeneic lymphocytes

 In order to construct an immunogenic cellular vaccine, we transduced three HLA-A*0201 human melanoma lines, selected for expression of classes I and II HLA, adhesion molecules and the T cell-defined melanoma antigens Melan/MART-1, gp100 and tyrosinase, with both interleukin-2 (IL-2) and B7-1 genes by the use of a polycistronic retroviral vector. The lines were selected to share only the HLA-A*0201 allele to avoid generation of strong alloreactivity in case of their multiple in vivo use in HLA-A*0201 + patients. Phenotypic and functional analysis of B7-1-IL2 transduced melanoma lines in comparison with B7-1 transduced and/or parental untransduced counterparts were then carried out. Tumor cells expressing either B7-1 or both genes did not change their original antigenic profile. From a functional point of view, expression of both genes in melanoma lines: (1) improved the response of anti-melanoma cytotoxic T lymphocytes (CTL) over singly transduced or untransduced melanoma cells when subthreshold levels of MHC-peptide complexes were expressed by melanoma cells; (2) conferred a distinct advantage in the ability to stimulate cytotoxicity and interferon-γ release by autologous and/or HLA-A*0201-compatible allogeneic lymphocytes; (3) allowed the generation of a high number of specific CTL by in vitro stimulation of lymphocytes of HLA-A*0201-melanoma patients. Thus, B7-IL2 gene-transduced melanoma lines appear to display a high immunogenicity and could be used as vaccine in melanoma patients. Received: 17 August 2000 / Accepted: 1 February 2001     

Active immunization of metastatic melanoma patients with interleukin-2-transduced allogeneic melanoma cells: evaluation of efficacy and tolerability

 From January 1994 to July 1996 we immunized metastatic melanoma patients with HLA-A2-compatible, interleukin-2 (IL-2)-secreting, immunogenic melanoma lines in an attempt to induce a systemic reaction that might also affect distant melanoma lesions. Twelve patients (6 male and 6 female) aged from 28 to 72 years, affected with visceral and/or subcutaneous (s.c.) melanoma metastases, were treated. Two different HLA-A2⁺ melanoma lines were transduced with the human IL-2 gene (14932/IL-2 and 1B6/IL-2) and used as vaccine. Two groups of 4 patients each were injected s.c. with 5×10⁷ and 15×10⁷ irradiated 14932/IL-2 melanoma cells respectively, whereas a third group received 5×10⁷ cells of the second line (1B6/IL-2). All patients received the vaccine on days 1, 13, 26; if no progression was evident, further immunizations were administered at monthly intervals. All patients were assessable for clinical response after at least three injections of the vaccine. In 4 cases a stabilization of disease lasting from 2 to 6 months was observed; in 2 of them a mixed type of response to treatment was noted with simultaneous evidence of regressing and non-responding lesions in the same patients. No signs of clinical response were found in the remaining patients. Nine patients died of disease between 3 and 24 months after the onset of therapy, whereas 3 were alive 3 months after the end of therapy. The local and systemic side-effects of treatment were mild. These results indicate that vaccination with cells bearing the appropriate antigens and releasing IL-2 locally can produce weak clinical responses, but also indicate that better results may be achieved through modifications of the vaccine, the schedule of immunization and/or a more appropriate selection of patients. Received: 20 December 1996 / Accepted: 27 February 1997     

Fusions of human ovarian carcinoma cells with autologous or allogeneic dendritic cells induce antitumor immunity

Human ovarian carcinomas express the CA-125, HER2/neu, and MUC1 tumor-associated Ags as potential targets for the induction of active specific immunotherapy. In the present studies, human ovarian cancer cells were fused to human dendritic cells (DC) as an alternative strategy to induce immunity against known and unidentified tumor Ags. Fusions of ovarian cancer cells to autologous DC resulted in the formation of heterokaryons that express the CA-125 Ag and DC-derived costimulatory and adhesion molecules. Similar findings were obtained with ovarian cancer cells fused to allogeneic DC. The fusion cells were functional in stimulating the proliferation of autologous T cells. The results also demonstrate that fusions of ovarian cancer cells to autologous or allogeneic DC induce cytolytic T cell activity and lysis of autologous tumor cells by a MHC class I-restricted mechanism. These findings demonstrate that fusions of ovarian carcinoma cells and DC activate T cell responses against autologous tumor and that the fusions are functional when generated with either autologous or allogeneic DC.     

Analysis of in vitro immunization: generation of cytotoxic T-lymphocytes against allogeneic melanoma cells with tumor lysate-loaded or tumor RNA-transfected antigen-presenting cells

In this study we compared several protocols for in vitro induction of cytotoxic T lymphocytes (CTL) from na?ve HLA-A*0201(+) peripheral blood mononuclear cells (PBMC) against allogeneic melanoma cells. As immunization material we compared: (1) the lysate of apoptotic or live melanoma tumor cells [MSM-M14 (M14) and MSM-M3 (M3)]; and (2) total RNA extracted from the same melanoma cell lines, either unconjugated or conjugated with a charged carrier (DMRIE-C). Overall killing activity was very similar in CTL induced by tumor lysate or RNA. CTL induced by both methods preferentially killed an HLA class I-matched M14 melanoma cell line rather than HLA class I-unmatched M3 cells. Cytotoxicity could be partially blocked by anti-HLA class I antibodies. There were no significant differences in cytotoxicity and in other clonal characteristics in CTL lines induced by a lysate of apoptotic bodies as compared to lines induced by lysate of viable cells. However, CTL induced by DMRIE-C-bound total RNA demonstrated superior cytotoxicity when compared with CTL induced by unconjugated total RNA. Polyclonal CTL induced by tumor lysate contained a substantial percentage of tyrosinase(368-376 370N) tetramer-positive cells and demonstrated specific killing activity against tyrosinase(368-376 370N) peptide-labeled T2 cells, comparable to cytotoxicity of the CTL developed against this peptide alone. In contrast, there were no detectable tyrosinase(368-376 370N)-tetramer positive cells and no specific anti-tyrosinase peptide(368-376 370N) response in polyclonal CTL induced by immunization with tumor RNA. These data demonstrate that both total tumor RNA and tumor lysate are effective for inducing of cytotoxic anti-melanoma CTL, but tyrosinase(368-376 370N) specific cells were detected only in lysate-induced CTL cultures. This suggests that nature of the antigens present in tumor lysate might be different from those in tumor RNA.     

Cellular interaction against autologous tumor cells between IL-2-cultured lymphocytes and fresh peripheral blood lymphocytes in patients with breast cancer given immuno-chemotherapy

In patients with Stage II or III breast cancer and in patients with liver metastases from breast cancer, we examined cellular interaction in the cytotoxicity against autologous tumor cells by interleukin-2(IL-2)-cultured lymphocytes (CL) and fresh peripheral blood lymphocytes (FPBL) treated with immunochemotherapy including OK-432 and cyclophosphamide. In flow cytometric analysis, CD8 + CD11b+ and CD16+ cells significantly decreased after immuno-chemotherapy in both groups of patients. A protocol study in Stage II or III breast cancer patients showed suppressive activity of FPBL on the cytotoxic activity of CL in 3/9 of the non-treatment group but no suppressive activity and enhancing activity in 3/7 in the immuno-chemotherapy group. Moreover, in 19 patients with liver metastases from breast cancer treated with immuno-chemotherapy including adoptive immunotherapy, FPBL in 6/19 showed enhancing activity, and in 8/19 suppressive activity in the lysis of autologous tumor cells. In assaysin vitro using autologous and allogeneic tumor cells, FPBL showed a partial specificity in cellular interaction against autologous tumor cells. CD4-depleted FPBL inhibited cytotoxicity of CL, while CD8-depleted FPBL enhanced cytotoxicity of CL in patients with liver metastases. These results suggest that immuno-chemotherapy eliminates the suppressive population in FPBL and may induce tumor regression if combined with adoptive immunotherapy using CL.Abbreviations IL-2 interleukin-2 - CL IL-2-cultured lymphocytes - FPBL fresh peripheral blood lymphocytes - AIT adoptive immunotherapy     

Phase I/II study of treatment with matured dendritic cells with or without low dose IL-2 in patients with disseminated melanoma

Background In the present study, we have examined whether treatment of patients with metastatic melanoma with matured dendritic cell (DC) vaccines with or without low dose IL-2 may improve treatment outcomes. Methods Sixteen patients received DC vaccines (DCs) sensitized with autologous melanoma lysates and 18 patients received DCs sensitized with peptides from gp100, MART-1, tyrosinase, MAGE-3.A2, MAGE-A10 and NA17. IL-2 was given subcutaneously (sc) at 1 MU/m² on the second day after each injection for 5–14 days in half of each group. DCs were given by intranodal injection. Results There were 2 partial responses (PR) and 3 with stable disease (SD) in the nine patients receiving DCs + peptides + IL-2, and 1 PR and 1 SD in nine patients treated with DCs + peptides without IL-2. There were only two patients with SD in the group receiving DCs + autologous lysates and no IL-2. Median overall survival for all patients was very good at 18.5 months but this was most probably due to selection of a favourable group of patients for the study. There was no significant difference in survival between the groups by log rank analysis. Treatment was not associated with significant side effects. The quality and yield of the DCs in the preparations were generally good. Conclusions We conclude that mature DC preparations may be superior to immature DC preparations for presentation of melanoma peptides and that IL-2 may increase clinical responses to the DCs plus peptides. However, in our view the low response rates do not justify the cost and complexity of this treatment approach.     

Suppression of human monocyte function against Candida albicans by autologous IL-2-induced lymphokine-activated killer cells

We have previously reported that IL-2-induced lymphokine-activated killer (LAK) cells have the capacity to lyse autologous and allogeneic monocytes. To understand the biologic significance of this interaction, we investigated the function of human monocytes against the opportunistic pathogen, Candida albicans, subsequent to a short exposure to autologous LAK cells. A highly sensitive radiolabel assay, which makes use of the incorporation of [3H]glucose into residual Candida after their incubation with monocytes, was developed to measure antifungal activity. Cultured monocytes, after 2 to 6 h exposure to LAK cells, were found to be substantially suppressed in their ability to control fungal growth. Moreover, monocytes cultured in the presence of granulocyte/macrophage (GM)-CSF or IL-3, were even more suppressed in function after a short incubation with LAK cells. The effect of GM-CSF was both time and dose dependent, with peak susceptibility induced after 4 days of culture with as little as 10 U/ml of the cytokine. These GM-CSF-cultured monocytes, however, were relatively resistant to inhibition by freshly isolated large granular lymphocytic NK cells. Therefore, IL-2 induces in large granular lymphocytic cells the capacity to inhibit monocyte function. In contrast to GM-CSF and IL-3, IFN-gamma was found to have a protective effect on monocytes, because monocytes cultured 4 days in IFN-gamma were not significantly inhibited by LAK cells. These results indicate that LAK cells may be involved in regulation of monocyte function and suggest that the state of differentiation induced by different cytokines may dictate the level of control of the monocytes by LAK cells.     

IL-4 and T cells are required for the generation of IgG1 isotype antibodies against cardiolipin

Infection with Mycobacterium tuberculosis induces Abs against a vast array of mycobacterial lipids and glycolipids. One of the most prominent lipid Ags recognized is cardiolipin (CL). The kinetics of the generation of anti-CL Abs during infection reveals that IgM titers to CL increase over time. Interestingly, at day 30 postinfection CL-specific IgG1 appears, an isotype usually dependent on T cell help. Using an immunization schedule with CL/anti-CL Ab complexes, which induces antiphospholipid syndrome in mice, we show that the generation of IgG1 to CL requires IL-4 and that optimal production is T cell dependent. IgG1 production to CL was impaired in nude (nu/nu) mice devoid in conventional T cells, but was not affected in mice deficient for either alphabeta TCR(+), gammadelta TCR(+), CD4(+), CD8(+), or NK1.1(+) T cells. We conclude that IgG1 production to CL depends on T cell help and IL-4, which can be provided by different T cell populations. This is the first report that IL-4 is indispensable for the induction of IgG1 Abs to lipid Ags.     

Vaccination with autologous dendritic cells pulsed with multiple tumor antigens for treatment of patients with malignant melanoma: results from a phase I/II trial

Background aimsDendritic cells are regarded as the most effective antigen presenting cells and coordinators of the immune response and therefore suitable as vaccine basis. Here we present results from a clinical study in which patients with malignant melanoma (MM) with verified progressive disease received vaccination with autologous monocyte-derived mature dendritic cells (DC) pulsed with p53, survivin and telomerase-derived peptides (HLA-A2⁺ patients) or with autologous/allogeneic tumor lysate (HLA-A2^? patients) in combination with low-dose interleukin (IL)-2 and interferon (IFN)-α2b.ResultsOf 46 patients who initiated treatment, 10 stopped treatment within 1–4 weeks because of rapid disease progression and deterioration. After 8 weeks, 36 patients were evaluable: no patient had an objective response, 11 patients had stable disease (SD); six had continued SD after 4 months, and three patients had prolonged SD for more than 6 months. The mean overall survival time was 9 months, with a significantly longer survival (18.4 months) of patients who attained SD compared with patients with progressive disease (PD) (5 months). Induction of antigen-specific T-cell responses was analyzed by multidimensional encoding of T cells using HLA-A2 major histocompatibility complex (MHC) multimers. Immune responses against five high-affinity vaccine peptides were detectable in the peripheral blood of six out of 10 analyzed HLA-A2⁺ patients. There was no observed correlation between the induction of immune responses and disease stabilization. A significant lower blood level of regulatory T cells (CD25^high CD4 T cells) was demonstrable after six vaccinations in patients with SD compared with PD.ConclusionsVaccination was feasible and safe. Treatment-associated SD was observed in 24% of the patients. SD correlated with prolonged survival suggesting a clinical benefit. Differences in the level of regulatory T cells among SD and PD patients could indicate a significant role of these immune suppressive cells.     

Frequencies of IL-2- and IL-4-secreting T cells in naive and antigen-stimulated lymphocyte populations

The relative frequencies of IL-2-and IL-4-secreting precursors in naive and Ag-primed populations were investigated by using limiting dilution analysis. Cells capable of IL-4 production in lymphoid populations freshly isolated from mice were rare in comparison with those producing IL-2 when the cells were stimulated by nominal Ag, by alloantigens, or by mitogens. One cycle of in vitro Ag restimulation and rest, however, enabled us to detect high proportions of IL-4-secreting cells among keyhole limpet hemocyanin-primed lymph node cells. With cultures set up at monoclonal cell doses, it was shown that IL-2 and IL-4 are secreted by separate precursor populations at this stage of their development. The IL-4-secreting cells were further shown to be dependent upon the presence of IL-2, either secreted by separate precursors or exogenously added, for the production of detectable amounts of IL-4. Analysis of the frequencies of helper cells producing both IL-2 and IL-4 at various stages of the in vivo immune response and the requirements for their growth and differentiation should give a better understanding of the relative contributions of each cell type.     

Comparative analysis of DC fused with allogeneic hepatocellular carcinoma cell line HepG2 and autologous tumor cells as potential cancer vaccines against hepatocellular carcinoma

Fusions of patient-derived dendritic cells (DCs) and autologous tumor cells induce T-cell responses against autologous tumors in animal models and human clinical trials. These fusion cells require patient-derived tumor cells, which are not, however, always available. Here we fused autologous DCs from patients with hepatocellular carcinoma (HCC) to an allogeneic HCC cell line (HepG2). These fusion cells co-expressed tumor-associated antigens (TAAs) and DC-derived costimulatory and MHC molecules. Both CD4⁺ and CD8⁺ T cells were activated by the fusion cells. Cytotoxic T lymphocytes (CTLs) induced by the fusion cells were able to kill autologous HCC by HLA-A2- and/or HLA-A24-restricted mechanisms. CTL activity against shared TAAs indicates that the presence of alloantigens does not prevent the development of CTLs with activity against autologous HCC cells. These fusion cells may have applications in anti-tumor immunotherapy through cross-priming against shared tumor antigens and may provide a platform for adoptive immunotherapy.     

Lysis by interleukin 2-stimulated tumor-infiltrating lymphocytes of autologous and allogeneic tumor target cells

Summary Stepwise counterflow centrifugal elutriation of leukapheresed human mononuclear cells (MNC) in a Beckman JE-6B rotor and J6-M/E centrifuge yielded a population highly enriched in natural killer (NK) cells (70–75% large granular lymphocytes with 10–13 times greater NK activity) at a flow rate of 38–44 ml/min using a fixed rotor speed of 3000 rpm at 27° C. However, the mean cell recovery was <1%. To obtain sufficient numbers of purified NK cells for adoptive immunotherapy, a strategy combining counterflow centrifugal elutriation with adherence of recombinant interleukin-2(rIL-2)-activated NK cells to plastic was developed. First, MNC were elutriated to give a twofold enrichment in NK cells, containing 22% Leu19⁺ cells, 18% large granular lymphocytes and 51 lytic units of activity against K562 targets as opposed to the unfractionated MNC containing 10% Leu19⁺ cells, 7% large granular lymphocytes and 26 lytic units of activity. The mean recovery was 80±15% (n=10). Further enrichment was obtained by isolation of the elutriated cells that adhered to plastic after culture for 24 h in the presence of 1000 U/ml rIL-2. The initial adherent lymphokine-activated killer (A-LAK) cells represented 1–4% of total MNC, but their subsequent expansion was at least 10–22-fold during 8–14 days in culture with 1000 U/ml rIL-2. Using this strategy, 2 × 10⁹ normal MNC, obtained by leukapheresis, yielded 5 × 10⁸ A-LAK cells with a total of 5.7 × 10⁵ lytic units of cytotoxicity against K562 and a total of 3.3 × 10⁵ lytic units against Daudi targets. This enrichment method has yielded sufficient numbers of A-LAK cells to form the basis for a phase I clinical trial of adoptive immunotherapy in patients with advanced cancer.     

Human autologous rosettes. IV. Their relation with interleukin 2 activity production and natural killer cells in cancer patients

Peripheral blood lymphocytes (PBL) of solid-tumor-bearing cancer patients produced a lower interleukin 2 (IL-2) activity after lectin stimulation than did those from normal subjects. Moreover natural killer (NK) cell activity and autologous rosette forming (ARF) cell rate are found significantly correlated with IL-2 production in these patients. No direct relation is observed between ARF cell ratio and NK cell activity in a given patient. A central role for IL-2 in cancer patient immune dysfunctions is suggested. Two lines of pathogenetic mechanisms are documented. First, PBL exhibited cellular function defects, namely, autologous receptor expression, IL-2 production, and NK activity. Second, these dysfunctions involved, at least partly, plasma factors. The possibility of specific deficiency, (e.g., thymic factors) is not documented. Conversely it is demonstrated that patient plasma contain immunosuppressive factor(s) that block(s) IL-2 production and ARF cell expression. Involvement of ARF cell receptor in T-cell activation is discussed.     

IL-4 inhibits IL-2 receptor expression and IL-2-dependent proliferation of human T cells

Recent studies have shown that IL-4 can affect lymphocyte responses to IL-2. To evaluate the effects of IL-4 on T cell responses to physiologically relevant stimuli, we studied normal human T cells cultured with a low concentration of anti-CD3 mAb and IL-2 in the presence and absence of added IL-4. The addition of IL-4 to cultures of T cells stimulated with anti-CD3 mAb and IL-2 reduced the proliferative response by 49 to 59%. The inhibitory effect was observed in 3-, 5-, and 7-day cultures. Inhibition was dose-dependent with maximal inhibition at concentrations greater than or equal to 5 to 10 U/ml IL-4. IL-4-mediated inhibition occurred early during the T cell response, inasmuch as addition of IL-4 after stimulation for 24 h did not result in significant inhibition. Phenotypic analyses of cells cultured in the presence of anti-CD3 mAb, IL-2, and IL-4 suggested that the mechanism of regulation by IL-4 involves the inhibition of IL-2R expression. The proportion of both CD4+ and CD8+ cells that expressed IL-2R in response to IL-2 was diminished in the presence of IL-4, although HLA-DR levels were unaffected. Soluble IL-2R was also reduced in supernatants of cultures stimulated with anti-CD3 mAb, IL-2, and IL-4 as compared to cultures stimulated with anti-CD3 mAb and IL-2. These findings indicate that when normal human T cells are stimulated in vitro in a manner that approximates a physiologic interaction with Ag in vivo, rIL-4 provides a potent inhibitory signal to IL-2 responsive cells that is likely mediated by IL-4-induced inhibition of IL-2R expression.     

Brief antigenic stimulation generates effector CD8 T cells with low cytotoxic activity and high IL-2 production

It is currently believed that a brief antigenic stimulation is sufficient to induce CD8 T cells to complete their differentiation program, become effector T cells, and subsequently generate memory. Because this concept was derived from studies in which only a single effector function was analyzed (either IFN-gamma production or target cell lysis), we wondered whether monitoring for multiple effector functions might reveal novel characteristics of effector CD8 T cells elicited by brief or prolonged Ag exposure. Using an in vitro system to generate effector T cells and an in vivo adoptive transfer model to track donor CD8 T cells, we found that the differentiation programs acquired by CD8 T cells after brief or prolonged antigenic stimulation were different. Although the frequencies of IFN-gamma and TNF-alpha producers were comparable for both effector CD8 T cell populations, there were major differences in cytotoxic potential and IL-2 production. Whereas prolonged (>24 h) Ag exposure stimulated effector CD8 T cells with high cytotoxic activity and low IL-2 production, brief (<24 h) stimulation generated effector CD8 T cells with low cytotoxic activity and high IL-2 production. The latter effector T cells rapidly converted into central memory-like CD8 T cells, exhibited long-term survival in adoptively transferred hosts, and gave robust recall responses upon Ag challenge. These data suggest that not all functions of effector CD8 T cells are equally inherited after brief or prolonged antigenic stimulation.