Pooled peptides from HER-2/neu-overexpressing primary ovarian tumours induce CTL with potent antitumour responses in vitro and in vivo.

Unfractionated peptides (MW: up to 10 kDa), derived from HLA-A2.1 positive (+) HER-2/neu-overexpressing primary tumour cell acid cell extracts (ACE), were successfully used to generate in vitro cytotoxic T lymphocytes (CTL). Primary tumour cells were collected from peritoneal malignant effusions of patients with ovarian cancer. Acid cell extracts-induced CTL specifically lysed in an HLA-A2-restricted manner HER-2/neu+ autologous primary tumour cells as well as HER-2/neu+ tumour cell lines. In addition, adoptive transfer of such CTL significantly prolonged the survival of SCID mice xenografted with HLA-A2.1+, HER-2/neu+ human breast and ovarian tumour cell lines. Acid cell extracts collected from HLA-A2.1+ HER-2/neu negative (-) primary ovarian tumours induced HLA-A2.1-restricted CTL with weak in vitro and in vivo antitumour capacity, suggesting that HER-2/neu peptides within ACE from HER-2/neu-overexpressing primary ovarian tumour cells are immunodominant. The results presented herein serve as a rationale for the initiation of vaccination studies in patients with HER-2/neu-overexpressing ovarian tumours utilising autologous tumour-derived ACE.

Redirecting mouse T hybridoma against human breast and ovarian carcinomas: in vivo activity against HER-2/neu expressing cancer cells.

Chimeric receptors comprising of the T-cell receptor-zeta cytoplasmic signalling chain fused to an extracellular ligand-binding domain of a single-chain antibody (scFv) have served as effective tools for redirecting cytotoxic T lymphocytes (CTL) against tumour cells. In this report, we constructed a chimeric scFv/zeta gene composed of the variable regions of an HER-2/neu-specific monoclonal antibody (MAb) joined to the TCR-zeta chain. The scFv(anti-HER-2/neu)/zeta chimeric gene was successfully expressed as a functional surface receptor in the MD.45 CTL hybridoma (MD.45-HER/zeta). More importantly, the scFv(anti-HER-2/neu)/zeta receptor was functionally active, since it triggered cytokine secretion by the MD.45-HER/zeta cells upon recognition of HER-2/neu-positive (+) tumour cell lines, or primary tumour cells from patients with HER-2/neu(+) cancers. The MD.45-HER/zeta-transduced cells also lysed HER-2/neu(+) target cells in vitro with high specificity. We tested the antitumour efficacy of scFv(anti-HER-2/neu)/zeta expressing MD.45 cells in severe combined immunodeficiency disease mice/human and murine tumour models. The adoptively transferred MD.45-HER/zeta cells both slowed significantly the growth of human FM3 melanoma or murine ALC leukaemic cells both transfected to express HER-2/neu. Our data demonstrate the feasibility of redirecting MD.45 CTL with the scFv(anti-HER-2/neu)/zeta chimeric receptor to respond specifically against HER-2/neu expressing tumour cells in vitro and in vivo. Moreover, they make it likely that T cells transduced with the same chimeric gene might be utilised in the treatment of patients with HER-2/neu(+) tumours.

Large-scale expansion of CD3(+)CD56(+) lymphocytes capable of lysing autologous tumor cells with cytokine-rich supernatants.

We have developed culture conditions for the efficient expansion of cytotoxic effector cells from peripheral blood mononuclear cells (PBMC) by the timed addition of cytokine-rich supernatants collected from allogeneic PBMC cultures stimulated with anti-CD3 monoclonal antibody (mAb) (allogeneic CD3 supernatants; ACD3S). These cytotoxic effectors belonged primarily to CD56(+) natural killer (NK) cells, and the cell subset with the greatest cytotoxic activity was an otherwise rare population of CD3(+)CD56(+) cells (NKT cells) that expand dramatically under these conditions. CD3(+)CD56(+) cytotoxic effectors were generated from the PBMC of 16 patients with several types of cancer. The CD3(+)CD56(+) cell subset expanded significantly and efficiently lysed NK- as well as lymphokine-activated killer (LAK)-sensitive targets. More importantly, ACD3S-activated CD3(+)CD56(+) cells were capable of efficiently lysing autologous tumor cells including metastatic colorectal, ovarian, breast, lung and pancreatic tumor cells as well as melanoma cells. ACD3S-expanded CD3(+)CD56(+) cells exhibited increased levels of cytoplasmic interleukin-2 (IL-2), tumor necrosis factor-alpha (TNF-alpha), gamma-interferon (IFN-gamma) and perforin. CD3(+)CD56(+) cell-mediated cytotoxicity was not restricted by major histocompatibility complex (MHC) gene products, since it was not blocked by anti-MHC class I mAb but was highly inhibited in the presence of CD2- and CD18-specific mAb. These data suggest that CD3(+)CD56(+) cells expanded under the presence of ACD3S may be utilized in clinical protocols for cancer immunotherapy.

Peptide HER2(776-788) represents a naturally processed broad MHC class II-restricted T cell epitope.

HER2/neu-derived peptides inducing MHC class II-restricted CD4+ T helper lymphocyte (Th) responses, although critical for tumour rejection, are not thoroughly characterized. Here, we report the generation and characterization of CD4+ T cell clones specifically recognizing a HER-2/neu-derived peptide (776-788) [designated HER2(776-788)]. Such clones yielded specific proliferative and cytokine [gamma-interferon(IFN)-gamma] responses when challenged with autologous dendritic cells (DCs) loaded with HER2(776-788). By performing blocking studies with monoclonal antibodies (MAbs) and by using DCs from allogeneic donors sharing certain HLA-DR alleles, we found that HER2(776-788) is a promiscuous peptide presented, at least, by DRB5*0101, DRB1*0701 and DRB1*0405 alleles. One TCRV beta 6.7+ clone recognized the HLA-DRB5*0101+ FM3 melanoma cell line transfected with a full length HER-2/neu cDNA. Moreover, this clone recognized the HER-2/neu+ SKBR3 breast cancer cell line induced to express HLA-DR, thus demonstrating that HER2(776-788) represents a naturally processed and presented epitope. Our data demonstrate that helper peptide HER2(776-788) represents a promiscuous epitope binding to at least three HLA-DR alleles, thus offering a broad population coverage. The use of antigenic peptides presented by major histocompatibility complex (MHC) class II in addition to those presented by class I may improve the therapeutic efficacy of active immunization.

A novel culture environment for generating mature human dentritic cells from peripheral blood CD14+ cells.

BACKGROUND: We recently demonstrated that supernatants from cultures of peripheral blood mononuclear cells (PBMC) activated with anti-CD3-specific antibody (ACD3S) can induce, upon brief exposure, tumor-reactive lymphocytes in cancer patients. Here, we report that ACD3S can also induce rapid and stable maturation of dendritic cells (DC) which can be used as antigen presenting cells in in vitro protocols and for cancer immunotherapy in vivo. MATERIALS AND METHODS: A short (4-day) priming of CD14+ monocytes with granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) followed by only a 24 hour-incubation in ACD3S, is sufficient to generate fully mature and stable DC. RESULTS: These DC (i) stimulated strong T cell proliferative responses in the mixed lymphocyte reaction, (ii) when pulsed with unfractionated peptides from autologous tumor membrane extracts activated CD4+ T cells which proliferated in response to the autologous tumor and CD8+ cytotoxic T cells (CTL) which specifically lyse autologous tumor targets and (iii) produced high levels of IL-12. CONCLUSION: ACD3S-treated DC are functionally superior to monocyte-conditioned medium (MCM)-treated DC generated under the same short-term protocol and as efficient as DC induced by the standard 10-day protocol. Our data present an efficient and effective method for generating in a very short period of time, highly mature and functionally competent DC.

Cytokine serum levels, autologous mixed lymphocyte reaction and surface marker analysis in never medicated and chronically medicated schizophrenic patients.

A number of immunological parameters were studied in 82 DSM-III-R diagnosed schizophrenic patients (53 first drug-naive and 29 medicated chronic patients) as well as 62 healthy blood donors. The serum levels of interleukin-2 (IL-2), interleukin-1 beta (IL-1beta) and tumour necrosis factor-alpha (TNF-alpha) were measured and correlated with cellular immunity, as assessed by the autologous mixed lymphocyte reaction (AMLR). T lymphocyte subsets were also examined. The above immune parameters were reassessed in a subgroup of 11 first-episode, drug-naive patients 1month after neuroleptic medication. IL-2 serum levels were significantly lower, and IL-1beta and TNF-alpha were significantly higher in schizophrenic patients compared with healthy donors (P<0.001); no significant difference was observed between the two patient groups (medicated and not medicated). Abnormal cytokine serum levels were associated with decreased AMLR responses in vitro. Increased percentages of activated CD4+ and CD16+ natural killer cells, as well as cells expressing ICAM-1 adhesion molecules and IL-2 specific receptors, were detected in the patients. Immunophenotype studies revealed a higher percentage of cells expressing IL-2 receptors in medicated chronic schizophrenic patients compared with drug-naive patients. The abnormal cytokine production in vivo, along with the low AMLR responses in vitro, and the high percentage of activated CD4+ lymphocytes presented in this study suggest alterations in the immune system of schizophrenic patients (medicated or not medicated) consistent with immune activation.

Synergy between interleukin-2 and prothymosin alpha for the increased generation of cytotoxic T lymphocytes against autologous human carcinomas.

Peripheral blood mononuclear cells (PBMC) from cancer patients were cultured in vitro with irradiated autologous tumor cells isolated from malignant effusions (mixed lymphocyte tumor cultures, MLTC) and low-dose (50 IU/ml) recombinant interleukin-2 (IL-2). The combination of IL-2 and prothymosin alpha (ProTalpha) resulted in a greater PBMC-induced response to the autologous tumor than that brought about by IL-2 alone. In particular, ProTalpha specifically enhanced the CD4+ T-cell-mediated proliferation against the autologous tumor. CD4+ T cells seemed to recognize tumor antigens presented by HLA-DR molecules expressed on the autologous monocytes, since preincubation of the latter with an anti-HLA-DR monoclonal antibody (mAb) abrogated the response. In addition, MLTC set up with IL-2 and ProTalpha also generated more MHC-class-I-restricted cytotoxic T lymphocytes (CTL) against the autologous tumor than did MLTC set up with IL-2 alone. The MLTC-induced CTL contained high levels of cytoplasmic perforin and their development was strictly dependent on the presence of both autologous CD4+ T cells and monocytes. In the absence of either population there was a strong impairment of both proliferative and cytotoxic responses which was not restored by the presence of ProTalpha. In contrast, when both cell populations were present, ProTalpha exerted optimal enhancement of CD4+ T cell proliferation, which was associated with potentiated CTL responses. Our data emphasize the role of ProTalpha for the enhancement of IL-2-induced CTL responses against autologous tumor cells. Such responses require collaborative interactions between CD4+, CD8+ T cells and monocytes as antigen-presenting cells. Our data are relevant for adoptive immunotherapeutic settings utilizing IL-2 and ProTalpha-induced autologous-tumor-specific CTL.

Compromised anti-tumor responses in tumor necrosis factor-alpha knockout mice.

The response of lymphokine-activated killer (LAK) and natural killer (NK) cells from mice lacking tumor necrosis factor-alpha (TNF-alpha-/- mice) was impaired in cytotoxicity assays against various tumor cell targets. Furthermore, allogeneic cytotoxic T lymphocyte (CTL) responses were also impaired as compared to TNF-alpha+/+ littermates (control mice). Cytotoxicity was restored both upon in vitro incubation of TNF-alpha-/- lymphocytes with recombinant TNF-alpha (rTNF-alpha) or upon in vivo treatment of TNF-alpha-/- mice with rTNF-alpha. Using combinations of monoclonal antibodies we were able to show that TNF-alpha-/- effector lymphocytes exhibit both perforin- and Fas ligand-based cytotoxicity. Furthermore, upon in vivo administration of rTNF-alpha these effectors, in addition to perforin and Fas ligand, are also armed with TNF-alpha cytotoxic molecules, thus resembling to the cytotoxic effectors from control mice. In a tumor model, immunized TNF-alpha-/- mice failed to reject the syngeneic fibrosarcoma MC57X, but did so when injected with rTNF-alpha. In vivo administration of anti-TNF-alpha mAb neutralized the effect of rTNF-alpha supporting the growth of MC57X cells. Our data provide novel evidence for TNF-alpha as an essential factor in (i) controlling cytotoxicity in vitro and in vivo and (ii) promoting tumor rejection in vivo.

Tumor-specific CD4+ T lymphocytes from cancer patients are required for optimal induction of cytotoxic T cells against the autologous tumor.

This study focuses on the specific CD4+ T cell requirement for optimal induction of cytotoxicity against MHC class II negative autologous tumors (AuTu) collected from patients with various types of cancer at advanced stages. CD4+ T cells were induced in cultures of cancer patients' malignant effusion-associated mononuclear cells with irradiated AuTu (mixed lymphocyte tumor cultures (MLTC)) in the presence of recombinant IL-2 and recombinant IL-7. Tumor-specific CD4+ T cells did not directly recognize the AuTu cells, but there was an MHC class II-restricted cross-priming by autologous dendritic cells (DCs), used as APC. CD8+ CTL, also induced during the MLTC, lysed specifically AuTu cells or DCs pulsed with AuTu peptide extracts (acid wash extracts (AWE)) in an MHC class I-restricted manner. Removal of CD4+ T cells or DCs from the MLTC drastically reduced the CD8+ CTL-mediated cytotoxic response against the AuTu. AWE-pulsed DCs preincubated with autologous CD4+ T cells were able, in the absence of CD4+ T cells, to stimulate CD8+ T cells to lyse autologous tumor targets. Such activated CD8+ T cells produced IL-2, IFN-gamma, TNF-alpha, and GM-CSF. The process of the activation of AWE-pulsed DCs by CD4+ T cells could be inhibited with anti-CD40 ligand mAb. Moreover, the role of CD4+ T cells in activating AWE-pulsed DCs was undertaken by anti-CD40 mAb. Our data demonstrate for the first time in patients with metastatic cancer the essential role of CD4+ Th cell-activated DCs for optimal CD8+ T cell-mediated killing of autologous tumors and provide the basis for the design of novel protocols in cellular adoptive immunotherapy of cancer, utilizing synthetic peptides capable of inducing T cell help in vivo.

Increased generation of autologous tumor-reactive lymphocytes by anti-CD3 monoclonal antibody and prothymosin alpha.

Anti-CD3 monoclonal antibody (mAb) activates in vitro peripheral blood mononuclear cells (PBMC) to lyse a variety of tumor cell lines in a non-major histocompatibility-complex(MHC)-restricted manner [subsequently referred to as anti-CD3-activated killer (AAK) cytotoxicity]. Prothymosin alpha (ProTalpha) is a biological response modifier that exerts its effects primarily on mononuclear cells, especially when these cells' effector functions are impaired. In this study, we report that ProTalpha enhances the AAK cytotoxicity in PBMC from healthy donors. This effect was more profound with cancer patients' PBMC, which were deficient in their ability to respond with enhanced AAK cytotoxicity upon in vitro stimulation with anti-CD3. Thus, cancer patients' PBMC, activated with a combination of anti-CD3 and ProTalpha, exhibited increased AAK activity and efficiently lysed both autologous tumor and Daudi targets. The ProTalpha effect on PBMC was demonstrated to involve stimulation of adhesion molecules (CD2, CD18, CD54, CD49f) and CD25 expression, up-regulation of perforin mRNA transcription, increased numbers of perforin-positive (+) cells and elevated production of interleukin-2 (IL-2), interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha). Moreover, effector CD8+ and CD56+ cells pretreated with anti-CD3 and ProTalpha contained high cytoplasmic perforin levels and increased expression of IL-1beta- and TNFalpha-specific receptors. The induction of autologous-tumor-reactive CD8+ and CD56+ lymphocytes in anti-CD3-activated PBMC by ProTalpha provides an alternative protocol aimed at the improvement of clinical results in cellular adoptive immunotherapy of cancer.

Mistletoe lectin I-induced effects on human cytotoxic lymphocytes. I. Synergism with IL-2 in the induction of enhanced LAK cytotoxicity.

This report demonstrates that in vitro activation of human cells with the beta-galactoside-specific lectin from mistletoe (ML-I) or interleukin-2 (IL-2) results in different patterns of activation and function of cytotoxic cells. It is now well established that natural killer (NK) and lymphokine-activated killer (LAK) cytotoxicity is mainly mediated by resting (NK) and IL-2-activated (LAK) CD56-positive (+) cells respectively. Culture of peripheral blood lymphocytes (PBL) for 3 days with ML-I led to expansion and activation of T cells which demonstrated NK- and LAK-like cytotoxicity. T lymphocyte subset analysis revealed that in total PBL, ML-I preferentially stimulated and expanded CD8+ T cells which mediated the cytotoxic effect. Incubation of highly purified CD8+ T cells alone with ML-I did not lead to induction of cytotoxicity, which required the presence of both CD4+ and CD14+ (monocytes) cells, suggesting that ML-I does not exert a direct effect on CD8+ T cells. Activation of PBL with both ML-I and IL-2 resulted in simultaneous induction of T and CD56+ cell-mediated NK and LAK cytotoxicity. These data suggest that treatment with ML-I and IL-2 might provide an approach to induce maximum cytotoxicity against tumors and to recruit both T and NK cells for tumor therapy.

Production and characterization of a monoclonal antibody against epirubicin.

Epirubicin is an anthracyclinic antibiotic that has been increasingly used in the treatment of a variety of malignancies. A hybridoma producing monoclonal antibody (MAb) against the drug was obtained by cell fusion. The MAb is of the IgM isotype and has an affinity constant of 1.4 x 10(-7) M. Inhibition analysis showed that the antibody recognizes an epitope related to the C 4'-hydroxyl group in the amino sugar moiety, distinguishing epirubicin from the closely related doxorubicin. Since the precise mechanism of anthracycline action as well as its immunomodulating effects are still under scrutiny, powerful tools for their study are clearly needed. Moreover, this MAb can be useful in monitoring the levels of epirubicin in treated patients, as well as for the construction of bispecific antibodies in tumor-targeting immunotherapy.

Induction of tumor-specific T lymphocyte responses in vivo by prothymosin alpha.

We have recently reported that administration of ProT alpha to DBA/2 mice before the inoculation of syngeneic L1210 leukemic cells prolonged the survival of these animals by (a) inducing tumoricidal peritoneal macrophages, (b) enhancing natural killer (NK) and inducing lymphokine-activated killer (LAK) activities in splenocytes and (c) inducing the production of interleukin-2 and tumor necrosis factor alpha [Papanastasiou et al. (1992) Cancer Immunol Immunother 35:145; Baxevanis et al. (1994) Cancer Immunol Immunothera 38:281]. In this report we demonstrated that ProT alpha, when administered simultaneously with L1210 tumor cells, is capable of generating in DBA/2 animals tumor-specific CD8+ cytotoxic T lymphocytes (CTL). The ProT alpha-induced CD8+ CTL lysed their syngeneic L1210 targets in a major histocompatibility complex (MHC)-restricted fashion since monoclonal antibodies (mAb) against the H-2Kd allelic product could inhibit the cytotoxic response. Mice receiving only ProT alpha developed non-MHC-restricted cytotoxic activity (NK, and LAK activities) whereas those receiving ProT alpha and L1210 tumor cells developed both MHC-restricted (CTL) and non-MHC-restricted cytotoxic activities and survived longer. The ProT alpha-induced CD8+ CTL activity was regulated by ProT alpha-induced L1210-specific syngeneic CD4+ cells. This was shown in two different ways: first, CD8(+)-cell-mediated cytotoxic responses against L1210 targets were associated with L1210-specific and MHC-restricted proliferative responses of syngeneic CD4+ cells and, second, CD4+ cells from mice that had received both ProT alpha and L1210 tumor cells could enhance in vitro the otherwise weak, MHC-restricted and L1210-specific cytotoxicity of syngeneic CD8+ cells from mice that had received only L1210 cells. Our data suggest that ProT alpha is capable of inducing nonspecific, as well as tumor-specific CTL responses in vivo. This is of importance since ProT alpha may prove to be useful in clinical protocols aimed at cancer immunotherapy.

An improved fluorescence assay for the determination of lymphocyte-mediated cytotoxicity using flow cytometry.

The use of the chromium-release assay to determine cytotoxicity of effector against target cells has various limitations mostly due to the inherent properties of the radioactive substance. We have developed an improved flow cytometric method that is able to measure cytotoxicity, based on two fluorescent dyes. Calcein-AM, a non-fluorescent substance which is intracellularly converted to the green fluorescent calcein by esterase activity in viable cells, is initially used to stain target cells. After incubating targets with effectors for 2 h, ethidium homodimer-1, a red DNA stain non-permeable to viable cells, is added. Dead target cells are distinguished by their double (green-red) staining. Data analysis is performed by gating the regions of living target, dead target and living effector cells, based on appropriate controls. Non-specific events are subtracted from the dead target region and the ratio of specific dead target events to total target events is expressed as percent cytotoxicity. The method is used to quantify natural killer (NK) and lymphokine-activated killer (LAK) activities against the human K562 and Daudi cell lines and the murine YAC-1 and L1210 cell lines respectively, as well as cell-mediated lympholysis (CML) exerted by tumor-infiltrating lymphocytes (TIL) against autologous and allogeneic human breast cancer tumor cells. The method is fast, reliable and correlates well with the standard 51Cr-release assay.

Interleukin 1 beta synergises with interleukin 2 in the outgrowth of autologous tumour-reactive CD8+ effectors.

Using peritoneal fluid or pleural effusion obtained from 20 patients with lung, ovarian or metastatic breast cancer, we separated tumour cells from malignant effusion-associated mononuclear cells (MEMNCs) using discontinuous Ficoll-Hypaque density gradients. CD3+ T lymphocytes represented the main population of MEMNCs. The mean +/- s.d. CD4/CD8 ratio of MEMNC suspensions was 1.18 +/- 0.40. MEMNCs proliferated and expanded in vitro with human interleukin 2 (IL-2) either as CD3+ CD8+ cells or as CD3+ CD4+ cells or as mixed populations of CD8+ and CD4+ cells. Preferential cytolytic activity against autologous tumour cells was demonstrated in IL-2-activated MEMNC cultures with excess CD3+ CD8+ cells. In contrast, effectors derived from IL-2-activated cultures with excess CD3+ CD4+ cells lysed both autologous and allogeneic tumour target cells. The addition on day 0 of interleukin 1 beta (IL-1 beta) to MEMNCs cultured in the presence of IL-2 was effective in promoting the growth of CD3+ CD8+ cells and augmenting the cytotoxicity against autologous tumour. Simultaneously, the production of gamma-interferon (IFN-gamma) was increased in these cultures. This is the first report suggesting that IL-1 beta synergises with IL-2 to induce autologous tumour-specific CD8+ cytotoxic T lymphocytes (CTLs) within the MEMNC population. Selective enrichment in T-cell subsets by IL-1 beta may be useful in cellular adoptive immunotherapy using cells isolated from malignant effusions.

On the role of monokines in the generation of nonspecific suppressor T cell activity in vitro.

We examined the role of endogenously produced interleukin 1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) in lectin-induced nonspecific suppressor activity in vitro. The cultures consisted of highly purified T lymphocytes, autologous monocytes and phytohemagglutinin (PHA). Kinetic studies revealed peak levels for both TNF-alpha and IL-1 beta production 4 hr after initiation of cultures which then declined and reached minimal levels on day 3. At this time point maximal levels of interleukin-2 (IL-2) were detected which declined sharply 24 hr later. The decline in cytokine levels in culture supernatants was most probably due to their consumption by the mononuclear cells which were found to express specific receptors for IL-1 beta, (IL-1 beta R), TNF-alpha (TNF-alpha R) and IL-2 (IL-2R) after 3- and 6-days of culture. After their first cycle of production and consumption both monokines were reproduced and the events followed the same patterns as for the first cycle: both monokines were first produced and at the time point of their consumption, IL-2 production reached maximal levels. The requirement for IL-1 beta and TNF-alpha in both IL-2 production and generation of suppressor activity was shown by three different approaches which included (a) blocking of HLA-DR molecules on monocytes which prevented monokine consumption during the early stages of culture, (b) blocking of HLA-A,B,C molecules on monocytes which prevented monokine consumption and IL-2 production late in culture, and (c) neutralization of monokine activity late in culture which resulted in highly reduced IL-2 production. T lymphocytes harvested from such cultures exhibited diminished suppressor activity. Our data suggest that the generation of nonspecific suppressor cell activity in vitro represents a complex system that requires cell interactions via self-major histocompatibility complex (MHC) antigen recognition and two cycles of cytokine production, where IL-1 beta and TNF-alpha production and consumption is a prerequisite for IL-2 production. Since lectin-induced nonspecific suppressor activity in vitro is deficient in certain autoimmune disorders the data presented herein might help in understanding the cellular basis for this immunodeficiency.

Induction of lymphokine-activated killer activity in mice by prothymosin alpha.

We have recently demonstrated that prothymosin alpha (ProT alpha) when administered intraperitoneally (i.p.) protects DBA/2 mice against the growth of syngeneic leukemic L1210 cells through the induction of tumoricidal peritoneal cells producing high levels of tumor necrosis factor alpha (TNF alpha) [Papanastasiou et al. (1992) Cancer Immunol Immunother 35: 145]. In this report we tested further immunological alterations that may be caused by the administration of ProT alpha in vivo. We demonstrate that i.p. injections of ProT alpha enhance natural killer (NK) cell activity and induce lymphokine-activated (LAK) activity in vivo. Thus, splenocytes from ProT alpha-treated DBA/2 animals exhibited significantly higher cytotoxic activity (up to threefold) against the NK-sensitive YAC cell line and the NK-resistant P815 and L1210 syngeneic tumor cells, as compared to splenocytes from syngeneic control mice. The enhancement of the cytotoxic profile of DBA/2 splenocytes was associated with increased percentages of CD8+ cells, NK cells and activated CD3+ cells. The ProT alpha-induced effect persisted for 30 days after the end of the ProT alpha treatment period and returned to normal levels 20 days later. Splenocytes from non-treated DBA/2 animals generated high NK and LAK activities in response to ProT alpha in vitro. The ProT alpha-induced NK and LAK activities reached 84% and 75% respectively of what was obtained with interleukin-2 (IL-2). High concentrations of TNF alpha and IL-2 were generated in response to ProT alpha in LAK cultures. These findings suggest that ProT alpha may provide an overall protective effect against tumor growth in vivo through induction of NK and LAK activities possibly indirectly via the production of IL-2 and TNF alpha in the spleen, peritoneal cavity and probably other lymphoid organs.

Elevated prostaglandin E2 production by monocytes is responsible for the depressed levels of natural killer and lymphokine-activated killer cell function in patients with breast cancer.

BACKGROUND: Human natural killer (NK) cells mediate spontaneous cytotoxicity against tumor cells and represent the main precursors of lymphokine-activated killer (LAK) cell activity. A comparison of some aspects of NK and LAK cell activity was undertaken in 85 preoperative patients with breast cancer and 75 healthy donors. METHODS: NK cell activity (tested in 18-hour cultures of effector peripheral blood mononuclear cells [PBMC] with K562 or MOLT-4 tumor target cells) was significantly diminished in these patients as it was the fully mature LAK cell activity (i.e., interleukin-2 (IL-2)-induced cytotoxicity in PBMC) against NK resistant target cells. Using immunoenzymatic methods we showed that the reduced NK cell activity was due to abnormally high levels of prostaglandin E2 (PGE2) produced by monocytes in culture. RESULTS: PGE2 was found to suppress the production of IL-2 in these cultures. Removal of monocytes from PBMC restored to almost normal levels the deficient NK and LAK cell activity in patients with breast cancer and was also associated with a normalization in the levels of PGE2 and IL-2. Indomethacin and gamma-interferon (IFN-gamma) increased the NK and LAK cell activity in these patients up to the levels of healthy donors. When highly purified CD56+ cells (obtained by an immunomagnetic isolation technique) were used as effector cells, no differences in LAK cell activity could be noticed between healthy donors and patients with cancer. FACS and northern blot analyses demonstrated a PGE2-mediated down-regulation of IL-2 receptor (IL-2R) expression on CD56+ cells that correlated with reduced LAK cell activity. This inhibitory effect of PGE2 was noticeable in long-term LAK cultures and was abrogated in the presence of IFN-gamma or indomethacin. CONCLUSION: This study may have important implications in the potentiation of NK and LAK cell activity for immunotherapeutic protocols in patients with breast cancer.

Comparison of immune parameters in patients with one or two primary malignant neoplasms.

The purpose of this study was to correlate cellular immune responses and cytokine production in vitro between patients with one or two primary malignant neoplasms. One hundred and ninety-three patients (110 patients with one primary malignant neoplasm (group I), and 83 patients with two primary tumors (group II), entered this study. Mononuclear cells isolated from peripheral blood were tested in the following tests: (a) proliferative responses in the autologous and allogeneic mixed lymphocyte reaction (auto- and allo-MLR respectively): (b) natural killer cell activity; (c) production of interleukin-2 during the allo-MLR, and (d) interleukin-1 beta production by lipopolysaccharide-stimulated monocytes. All these parameters were found to be decreased in cancer patients as compared to normal donors (p < 10(-3)). In addition, we were able to detect significant differences between the values obtained from patients in groups I and II (p < 10(-2)). These data suggest a further impairment in cancer patients' immune status after the diagnosis of a second malignancy.

Monocyte disorders associated with T cell defects in patients with solid tumors.

T cells proliferate in response to autologous monocytes in the autologous mixed lymphocyte reaction (AMLR). AMLR was found to be impaired in patients with advanced cancer (stages III and IV), whereas normal values were found in the early stages of the disease (stages I and II). Peripheral T lymphocytes from patients with advanced stages also exhibited a decreased ability to produce Interleukin-2 (IL-2) during an AMLR response, whereas production of IL-2 by T cells in stages I and II was comparable to that of normal donors. The impaired IL-2 production by T lymphocytes in the AMLR was associated with high concentrations of soluble interleukin-2 receptor (sIL-2R) in culture supernatants and reduced expression of membrane-bound interleukin-2 receptors (IL-2R) on the same AMLR-activated T lymphocytes. These abnormalities in T cells from cancer patients were demonstrated to be associated with dysfunctions of autologous monocytes. Thus monocytes from patients with advanced cancer exhibited diminished expression of HLA-DR antigens and produced low levels of Interleukin-1 beta (IL-1 beta) and Tumor Necrosis Factor a (TNFa). No changes were detected in the expression of HLA-A, -B, -C antigens. The results presented here demonstrate that decreased in vitro T cell responses may be attributed to monocyte dysfunctions in these patients and provide new information for a better understanding of the impaired T cell function in cancer patients.