Treatment of advanced metastasized breast cancer with bone marrow-derived tumour-reactive memory T cells: a pilot clinical study

Background  Breast cancer patients frequently harbour tumour-reactive memory T cells in their bone marrow (BM) but not in the blood. After reactivation ex-vivo these cells rejected autologous breast tumours in xenotransplanted mice demonstrating therapeutic potential upon reactivation and mobilization into the blood. We conducted a clinical pilot study on metastasized breast cancer patients to investigate if ex-vivo reactivation of tumour-reactive BM memory T cells and their adoptive transfer is feasible and increases the frequencies of tumour-reactive T cells in the blood. Methods  The study protocol involved one transfusion of T cells which were reactivated in vitro with autologous dendritic cells pulsed with lysate of MCF7 breast cancer cells as source of tumour antigens. Immunomonitoring included characterization of T cell activation in vitro and of tumour-specific T cells in the blood by interferon (IFN)-γ ELISPOT assay, HLA-tetramers and antigen-induced interleukin (IL)-4 secretion. Results  Twelve patients with pre-existing tumour-reactive BM memory T cells were included into the study. In all cases, the treatment was feasible and well tolerated. Six patients (responders) showed by ELISPOT assay de-novo tumour antigen-specific, IFN-γ-secreting T cells in the blood after 7 days. In contrast, non responders showed in the blood tumour antigen-induced IL-4 responses. All responders received more than 6.5 × 10³ tumour-reactive T cells. In contrast, all non responders received lower numbers of tumour antigen-reactive T cells. This was associated with reduced activation of memory T cells in activation cultures, increased amounts of CD4⁺ CD25^high regulatory T cells in the BM and increased tumour antigen-dependent IL-10 secretion. The latter was prevented by preceding depletion of regulatory T cells suggesting that regulatory T cells in the BM can inhibit reactivation of tumour-specific T cells. Conclusion  Taken together, adoptive transfer of ex-vivo re-stimulated tumour-reactive memory T cells from BM of metastasized breast cancer patients can induce the presence of tumour antigen-reactive type-1 T cells in the peripheral blood. Florian Schuetz and Katrin Ehlert contributed equally to the study.     

A Th1 cytokine–enriched microenvironment enhances tumor killing by activated T cells armed with bispecific antibodies and inhibits the development of myeloid-derived suppressor cells

In this study, we investigated whether activated T cells (ATC) armed with bispecific antibodies (aATC) can inhibits tumor growth and MDSC development in a Th₁ cytokine–enriched (IL-2 and IFN-γ) microenvironment. Cytotoxicity mediated by aATC was significantly higher (P < 0.001) against breast cancer cell lines in the presence of Th₁ cytokines as compared with control co-cultures. In the presence of aATC, CD33⁺/CD11b⁺/CD14⁻/HLA-DR⁻ MDSC population was reduced significantly under both control (P < 0.03) and Th₁-enriched (P < 0.036) culture conditions. Cytokine analysis in the culture supernatants showed high levels of MDSC suppressive chemokines CXCL9 and CXCL10 in Th₁-enriched culture supernatants with highly significant increase (P < 0.001) in the presence of aATC. Interestingly, MDSC recovered from co-cultures without aATC showed potent ability to suppress activated T-cell-mediated cytotoxicity (P < 0.001), IFN-γ production (P < 0.01) and T-cell proliferation (P < 0.05) compared to those recovered from aATC-containing co-cultures. These data suggest that aATC can mediate enhanced killing of tumor cells and may suppress MDSC and T_reg differentiation, and presence of Th₁ cytokines potentiates aATC-induced suppression of MDSC, suggesting that Th₁-enriching immunotherapy may be beneficial in cancer treatment.     

Resistance to the proapoptotic effects of interferon-gamma on melanoma cells used in patient-specific dendritic cell immunotherapy is associated with improved overall survival

The use of whole cell tumor vaccines and various means of loading antigen onto dendritic cells have been under investigation for over a decade. Induction of apoptosis and the exposure of immune-stimulating proteins are thought to be beneficial for the use in immunotherapy protocols, but conclusive evidence in the clinical setting has been lacking. Incubation of melanoma cell lines with interferon-gamma (IFN-γ) increased phosphatidylserine and calreticulin exposure, but not in the IFN-γ-resistant cell line Lu-1205. Short-term autologous melanoma cell lines used for loading dendritic cells for immunotherapy showed differential response to the pro-apoptotic effects of IFN-γ. These IFN-γ-treated tumor cells (TCs) were irradiated and used for loading antigen for dendritic cell therapy. A log-rank comparison of survival for patients whose TCs were found to be either sensitive (upregulated phosphatidylserine and calreticulin) or insensitive to IFN-γ revealed a strongly significant correlation to progression-free (p = 0.003) and overall survival (p = 0.002) favorably in those patients whose cell lines were resistant to the proapoptotic effect of IFN-γ. These results suggest that the use of IFN-γ in anti-melanoma dendritic cell-based immunotherapy may only be beneficial when the cells do not undergo apoptosis in response to IFN-γ and support the contention that the use of some apoptotic cells in vaccines may be detrimental.     

Long-term survival after adoptive bone marrow T cell therapy of advanced metastasized breast cancer: follow-up analysis of a clinical pilot trial

Background

The bone marrow (BM) of breast cancer patients harbors tumor-reactive memory T cells (TCs) with therapeutic potential. We recently described the immunologic effects of adoptive transfer of ex vivo restimulated tumor-reactive memory TCs from the BM of 12 metastasized breast cancer patients in a clinical phase-I study. In this trial, adoptive T cell transfer resulted in the occurrence of circulating tumor antigen-reactive type-1 TCs. We here describe the long-term clinical outcome and its correlation with tumor-specific cellular immune response in 16 metastasized breast cancer patients, including 12 included in the original study.

Methods

Sixteen metastatic breast cancer patients with preexisting tumor-reactive BM memory TCs were included into the study. The study protocol involved one transfusion of TCs which were reactivated in vitro with autologous dendritic cells pulsed with lysates of MCF-7 breast cancer cells as source of tumor antigens. The presence of tumor-reactive memory TCs was analyzed by IFN-γ ELISpot assays.

Results

Tumor-reactive memory TCs in the peripheral blood were induced de novo in 7/16 patients (44 %) after adoptive TC transfer. These patients were considered immunologic responders to the therapy. Positive adoptive immunotherapy (ADI) response was observed significantly more often in patients without bone metastases (p = 0.0051), in patients with high levels of tumor-reactive BM TCs prior to therapy (p = 0.036) and correlated significantly with the estimated numbers of transferred tumor-reactive TCs (p = 0.0021). After the treatment, we observed an overall median survival of 33.8 months in the total cohort with three patients alive at last follow-up and more than 7 years after ADI. Numbers of transferred tumor-reactive TCs correlated significantly with the overall survival of patients (p = 0.017). Patients with an immunologic response to ADI in the peripheral blood had a significantly longer median survival than nonresponders (median survival 58.6 vs. 13.6 months; p = 0.009).

Conclusion

In metastasized breast cancer patients, adoptive transfer of BM TCs can induce the presence of tumor antigen-reactive type-1 TCs in the peripheral blood. Patients with immunologic response after ADI show a significantly longer overall survival. Patients with bone metastases significantly less frequently respond to the treatment and, therefore, might not be optimal candidates for ADI. Although the present study does not yet prove the therapeutic effect of ADI, these findings shed light on the relation between immune response and cancer prognosis and suggest that transfer of reactivated BM TCs might bear therapeutic potential.     

Phenotypic and functional alterations of Vγ2Vδ2 T cell subsets in patients with active nasopharyngeal carcinoma

Introduction  Human Vγ2Vδ2 T cells play important role in immunity to infection and cancer by monitoring self and foreign isoprenoid metabolites with their γδ T cell antigen receptors. Like CD4 and CD8 αβ T cells, adult peripheral Vγ2Vδ2 T cells represent a pool of heterogeneous cells with distinct functional capabilities. Purpose  The aim of this study was to characterize the phenotypes and functions of various Vγ2Vδ2 T cell subsets in patients with nasopharyngeal carcinoma (NPC). We sought to develop a better understanding of the role of these cells during the course of disease and to facilitate the development of immunotherapeutic strategies against NPC. Results  Although similar total percentages of peripheral blood Vγ2Vδ2 T cells were found in both NPC patients and normal donors, Vγ2Vδ2 T cells from NPC patients showed decreased cytotoxicity against tumor cells whereas Vγ2Vδ2 T cells from normal donors showed potent cytotoxicity. To investigate further, we compared the phenotypic characteristics of Vγ2Vδ2 T cells from 96 patients with NPC and 54 healthy controls. The fraction of late effector memory Vγ2Vδ2 T cells (T_{EM RA}) was significantly increased in NPC patients with corresponding decreases in the fraction of early memory Vγ2Vδ2 T cells (T_CM) compared with those in healthy controls. Moreover, T_{EM RA} and T_CM Vγ2Vδ2 cells from NPC patients produced significantly less IFN-γ and TNF-α, potentially contributing to their impaired cytotoxicity. Radiotherapy or concurrent chemo-radiotherapy further increased the T_{EM RA} Vγ2Vδ2 T cell population but did not correct the impaired production of IFN-γ and TNF-α observed for T_{EM RA} Vγ2Vδ2 T cells. Conclusion  We have identified distinct alterations in the Vγ2Vδ2 T cell subsets of patients with NPC. Moreover, the overall cellular effector function of γδ T cells is compromised in these patients. Our data suggest that the contribution of Vγ2Vδ2 T cells to control NPC may depend on the activation state and differentiation of these cells. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Using IFN-γ as a Biomarker for Detecting Exposure to Viral Pathogens

To determine whether interferon gamma (IFN-γ) can be used as a biomarker of exposure to viral pathogens, 12-week-old BALB/c mice were injected intraperitoneally with coxsackievirus B3 (CVB3) or coxsackievirus B4 (CVB4) diluted in sterilized phosphate-buffered saline (PBS). Control mice were injected with PBS only. Four months after viral infection, mouse spleen cells were harvested and assayed for the release of IFN-γ by memory T cells after in vitro stimulation with viral antigens, phytohemagglutinin (PHA), and PBS, respectively. The level of IFN-γ was examined by an antibody-capture enzyme-linked immunosorbent assay (ELISA). A marked increase in the level of IFN-γ was observed when memory T cells from CVB3-infected mice were incubated with CVB3 virus, but not with CVB4 or PBS. Conversely, memory T cells from mice infected by CVB4 were not stimulated to produce IFN-γ when they were incubated with CVB3 and PBS, but did significantly produce IFN-γ when stimulated with CVB4. T cells from mice injected with PBS did not release IFN-γ after stimulation with CVB3 or CVB4. However, these T cells did release IFN-γ after stimulation with PHA. Our results demonstrated that IFN-γ produced by memory T cells is virus-specific and may have use as a biomarker in viral exposure studies. The results of this study may be extended to the study of infection by pathogens that are capable of inducing cell-mediated immune response in humans. Disclaimer: The United States Environmental Protection Agency through its Office of Research and Development funded and managed the research described here. It has been subjected to Agency’s administrative review and approved for publication.     

Cytokine gene polymorphism and asthma susceptibility, progress and control level

Asthma is a multifactor inflammatory disorder, and its management requires understanding of its various pathogenesis and control mechanisms. Cytokines and other inflammatory mediators are important factors in asthma pathophysiology. In this study, we evaluated the role of cytokine polymorphisms in the asthma susceptibility, progress, control, and lung functions. IL-4-C590T polymorphism by PCR-RFLP method, IFN-γ T+874A, TNF-α-A308G, IL-6 G−174C and TGF-β T+869C variants by ARMS-PCR method and IgE serum level by ELISA technique were determined in 81 asthmatic patients and 124 normal subjects. Asthma diagnosis, treatment and control levels were considered using standard schemes and criteria. TNF-α−308GA genotype was more frequent in asthmatics (P = 0.025, OR 3.352), and polymorphisms between different asthma control levels (P > 0.05) were not different. IFN-γ+874AT genotype had a positive correlation with the familial history of asthma (P = 0.034, OR 2.688). IL-6−174C allele (P = 0.045), TNF-α−308GG genotype (P = 0.002) and TNF-α−308G allele (P = 0.004) showed reduced values, and TNF-α−308GA genotype (P = 0.002) increased FEF25-75 value in asthmatics. IFN-γ+874AA genotype caused a decrease in FVC factor (P = 0.045). This study showed that TNF-α−308GA is a risk factor for asthma, but cytokine gene variants do not affect asthma control and IgE serum levels. Variants producing lower levels of IL-6, TNF-α and IFN-γ are associated with reduced pulmonary capacities. To achieve an appropriate schema for asthma management, further studies with consideration of different aspects in a larger group of patients would be more elucidative.     

Human CD80/IL2 lentivirus transduced acute myeloid leukaemia cells enhance cytolytic activity in vitro in spite of an increase in regulatory CD4<Superscript>+</Superscript> T cells in a subset of cultures

Immunotherapeutic strategies are increasingly being explored as a method of enhancing anti-tumour immune responses in patients with acute myeloid leukaemia (AML). Regulatory CD4⁺ T cells (Tregs) suppress effector T and natural killer (NK) cells and therefore pose a potential challenge to the efficacy of immunotherapy. AML cells transduced with a lentivirus expressing CD80 (B7.1) and IL2 (LV-CD80/IL2) are capable of stimulating T and NK cell cytotoxicity in vitro. This study examines the effect of CD80/IL2 modified AML cells on Treg number and function. We report a significant increase in the number of CD8⁺ T cells (P = 0.046) CD3⁻CD56⁺ NK cells (P = 0.028) and CD3⁺CD4⁺CD25^highFoxp3⁺ Tregs (P = 0.043) following stimulation for 7 days with allogeneic LV-CD80/IL2 AMLs. In contrast, autologous LV-CD80/IL2 AML cell cultures provide a weaker stimulation with a lower number of CD8⁺ T cells (P = 0.011) and no change in NK cell or Treg numbers. However, an increase in cytotoxic CD8⁺ T cells and NK cells are detected following both allogeneic and autologous LV-CD80/IL2 stimulation as demonstrated by an increase in IFN-γ and CD107a expression. Despite the presence of increased numbers of Tregs with suppressive activity in a subset of cultures, increased lysis of unmodified AMLs was still achieved following allogeneic (day 0, 2.2%; day 7, 20.4%) and more importantly, autologous LV-CD80/IL2 culture in which AML patients had recently received intensive chemotherapy (day 0, 0%; day 7, 16%). Vaccination with LV-CD80/IL2 therefore provides a potential strategy to enhance anti-leukaemia immune responses without a concomitant stimulation of Treg-mediated inhibition of cytotoxic immunological responses.     

Role of IFN-γ +874 T/A single nucleotide polymorphism in the tuberculosis outcome among Brazilians subjects

Several genetic cytokine gene variants have been associated with host susceptibility to infectious diseases, including tuberculosis. Based upon the importance of IFN-γ in protective immunity against Mycobacterium tuberculosis, and the functional role of the IFN-γ + 874T/A single nucleotide polymorphism in IFN-γ production, we genotyped 93 Brazilian tuberculosis patients and 266 asymptomatic health care workers, including 150 individuals with a positive tuberculin skin test, and analyzed the possible association of the +874A low IFN-γ producer allele with tuberculosis occurrence. Using multivariable logistic regression models, genotype and allele frequencies of the mutant + 874A (low IFN-γ producer) allele were significantly associated with tuberculosis disease. Heterozygous carriers had a 25% increased chance, while individuals presenting the A/A homozygous genotype had an over two-fold risk of having active tuberculosis (95% CI, 1.16–5.91, P = 0.03). Despite the mixed ethnicity observed in Brazilian populations, the present data agree with observations reported in other populations and thus demonstrate that the functional +874T/A IFN-γ gene polymorphism is associated with tuberculosis in different populations.     

Association of IL-6 promoter and IFN-γ gene polymorphisms with acute rejection of liver transplantation

Liver transplantation is one of the most important therapies for end-stage liver diseases and is associated with major problems including infections and acute rejection. The outcome of transplantation can be determined by immune responses as a key role in response to the graft. Inflammatory and anti-inflammatory mediators especially cytokines influence the graft microenvironment. Th1 and Th2 immune responses in contrast to regulatory responses cause acute rejection or help graft survival. In this study, we evaluated the gene polymorphisms of IL-6 G-174C, TGF-β T + 869C, IL-4 C-590T, and IFN-γ T + 874A cytokines in liver transplant patients. ARMS-PCR method was used to characterize IL-6 G-174C, TGF-β T + 869C and IFN-γ T + 874A polymorphisms and PCR-RFLP using AvaII restriction enzyme was done for IL-4 C-590T characterization in 70 liver transplant patients. Acute rejection episodes were diagnosed according to standard criteria. The analysis of the results showed that IL-6-174 GG genotype ( P = 0.009, OR = 4.333, 95% CI = 1.043–18.000), IL-6-174G allele (P = 0.011, OR = 5.273, 95% CI = 1.454–19.127) was more frequent and IFN-γ +874 TT genotype was less frequent (P = 0.043, OR = 0.143, 95% CI = 0.0118–1.190) in acute rejection than in non-rejection patients. TGF-β T + 869C and IL-4 C-590T frequencies were not significantly different (P > 0.05). According to the results, it can be conclude that IL-6 G-174C and IFN-γ T + 874A gene polymorphisms have predictive values for acute rejection after liver transplantation. High producer genotype of IL-6 is a genetic risk factor and IFN-γ is a protective factor for acute rejection development.     

In vivo induction of necrosis in mice fibrosarcoma via intravenous injection of type B staphylococcal enterotoxin

The bacterial superantigen staphylococcal enterotoxin B (SEB) is a potent inducer of cytotoxic T-cell activity and cytokine production in vivo. We investigated the possibility of the therapeutic application of SEB in patients with fibrosarcoma. The anti-tumor effect of SEB in mice with inoculated fibrosarcoma (WEHI-164) was examined by intravenous (IV) and intratumoral (IT) injection and the sizes of the inoculated tumors, IFN-γ production, and CD4+/CD8+ T cell infiltration were determined. The inoculated tumors were also examined histologically. In the mice in the IV-injected group, a significant reduction (P < 0.02) of tumor size was observed in comparison with mice in the IT-injected and control groups. Furthermore, the mice in the IV-injected group showed significantly higher levels of IFN-γ (P < 0.009) and CD4+/CD8+ T cell infiltration when compared with the other groups (P < 0.02). A significantly higher frequency of necrosis in tumor tissues was also observed in mice in the IV-injected group (P < 0.05). Our present findings suggest that tumor cell death is caused by increased cytotoxic T-cell activity and cytokine levels in response to the IV injection of SEB and that SEB may be a good option for use as a novel therapy in patients with fibrosarcoma. An erratum to this article can be found at     

Rheumatoid synovial CD4+ T cells exhibit a reduced capacity to differentiate into IL-4-producing T-helper-2 effector cells

CD4⁺ memory T cells (Tm) from rheumatoid arthritis peripheral blood (RAPB) or peripheral blood from normal donors produced IL-2, whereas fewer cells secreted IFN-γ or IL-4 after a brief stimulation. RAPB Tm contained significantly more IFN-γ producers than normal cells. Many rheumatoid arthritis (RA) synovial Tm produced IFN-γ alone (40%) and fewer cells produced IL-2 or IL-4. An in vitro model was employed to generate polarized T-helper (Th) effectors. Normal and RAPB Tm differentiated into both IFN-γ- and IL-4-producing effectors. RA synovial fluid (RASF) Tm demonstrated defective responsiveness, exhibiting diminished differentiation of IL-4 effectors, whereas RA synovial tissue (RAST) Tm exhibited defective generation of IFN-γ and IL-4 producers.     

NMR Based Metabonomics Study of DAG Treatment in a C2C12 Mouse Skeletal Muscle Cell Line Myotube Model of Burn-Injury

After severe burn injury, proinflammatory cytokine levels are elevated in serum and skeletal muscle, which in turn increases protein breakdown and decreases protein synthesis. In this study, C2C12 mouse skeletal muscle cell line myotubes were exposed to proinflammatory cytokines tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) as an in vitro cell-line model of catabolic response to burn injury and then treated with des-acyl ghrelin (DAG), a 28 amino acid polypeptide hormone thought to inhibit protein breakdown and increase protein synthesis, to assess its therapeutic potential. Nuclear magnetic resonance-based metabonomics was used to monitor metabolic activity of C2C12 myotubes under four treatment conditions: (1) control, (2) TNF-α/IFN-γ (TI), (3) DAG (DA), and (4) TNF-α/IFN-γ followed by DAG (TIDA) to assess the effect of DAG treatment on cellular metabolic response during basal or catabolic conditions. Twelve metabolites showed significant changes in concentrations following treatments in the hydrophilic cell extracts. Lactate (P < 10⁻⁴) and citrulline (P < 10⁻⁹) increased with TNF-α/IFN-γ treatment, indicating increased protein degradation, and returned to control levels in the TIDA group. Adenosine nucleotide levels had decreased trends in TI myotubes that returned to baseline levels after DAG treatment (P < 10⁻⁴). Guanidinoacetate and pantothenate, metabolites involved in protein synthesis and cell proliferation, had increased concentration trends following DAG treatment in both the DA and TIDA groups. Our metabonomics analysis provides further evidence that DAG counteracts the catabolic response caused by elevated muscle TNF-α/IFN-γ cytokine levels following severe burns and can play a potential therapeutic role in treatment of burn injury.     

Three novel SNPs in the coding region of <Emphasis Type="Italic">PPARγ</Emphasis> gene and their associations with meat quality traits in cattle

The peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear hormone receptor that regulates adipogenesis and many other biological processes. In the present study, we carried out PCR–SSCP and DNA sequencing analyses to examine SNPs in coding region of the PPARγ gene. A total of 660 individuals from five Chinese cattle breeds were genotyped. We identified three SNPs and their associations with meat quality traits were analyzed in 108 Qinchuan cattle. Two missense mutations and one synonymous mutation were found: 200 A > G (genotypes AA, AB and BB) resulting in D7G change, the silent substitution 42895 C > T (genotypes JJ and JI) and 72472 G > T (genotypes CC, DC and DD) producing Q448H change, respectively. The frequencies of PPARγ-A allele were 0.86, 0.83, 0.80, 0.72 and 0.87 for Qinchuan, Nanyang, Jiaxian, Luxi and Xianan populations, respectively. The frequencies of PPARγ-J allele varied from 0.87 to 0.96 in the five populations. In the 72472 G > T locus, the frequencies of PPARγ-C allele were higher than PPARγ-D allele in the five populations, and ranged from 0.58 to 0.82. Least squares analysis revealed that in 42895 C > T locus, there was a significant effect on tenderness in 18-20 months Qinchuan cattle (P < 0.01), and in the 72472 G > T locus, animals with the genotype DC had lower mean values than these with genotype CC (P < 0.01) for back fat thickness in 18–20 months, and animals with the genotype DD had lower mean values than these with genotypes CC and DC (P < 0.01) for water holding capacity in 21–24 months (P < 0.01). The SNPs we have identified may contribute to establishing a more efficient selection program for improving of genetic characteristics in indigenous Chinese cattle     

Vaccine-specific local T cell reactivity in immunotherapy-associated vitiligo in melanoma patients

The occurrence of vitiligo in patients with melanoma is especially reported for patients undergoing immunotherapy. While vitiligo in these patients is thought to be related to an immune response directed against melanoma cells, solid evidence is lacking. Here we report local cytotoxic T cell reactivity in three melanoma patients who developed vitiligo, after experimental immunotherapy using dendritic cell vaccinations. Tetramer analysis showed that vaccine-induced T cells recognizing gp100 and tyrosinase are present at the vitiligo lesions. These T cells secrete IFN-γ and IL-2 upon peptide specific stimulation as well as upon recognition of the autologous tumor. We show that functional CD8⁺ T cells specific for melanoma differentiation antigens used in a melanoma immunotherapy trial, do not only invade the tumor, but also the vitiligo lesions. This directly links vitiligo to the immuno-therapeutic intervention and supports the hypothesis that vitiligo is a marker of immunity against melanoma cells. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Enhanced anti-tumor activity of interferon-alpha in SOCS1-deficient mice is mediated by CD4<Superscript>+</Superscript> and CD8<Superscript>+</Superscript> T cells

Interferon-alpha (IFN-α) is an immunomodulatory cytokine that is used clinically for the treatment of melanoma in the adjuvant setting. The cellular actions of IFN-α are regulated by the suppressors of cytokine signaling (SOCS) family of proteins. We hypothesized that the anti-tumor activity of exogenous IFN-α would be enhanced in SOCS1-deficient mice. SOCS1-deficient (SOCS1^−/−) or control (SOCS1^+/+) mice on an IFN-γ^−/− C57BL/6 background bearing intraperitoneal (i.p.) JB/MS murine melanoma cells were treated for 30 days with i.p. injections of IFN-A/D or PBS (vehicle). Log-rank Kaplan-Meier survival curves were used to evaluate survival. Tumor-bearing control SOCS1^+/+ mice receiving IFN-A/D had significantly enhanced survival versus PBS–treated mice (P = 0.0048). The anti-tumor effects of IFN-A/D therapy were significantly enhanced in tumor-bearing SOCS1^−/− mice; 75% of these mice survived tumor challenge, whereas PBS-treated SOCS1^−/− mice all died at 13-16 days (P = 0.00038). Antibody (Ab) depletion of CD8⁺ T cells abrogated the anti-tumor effects of IFN-A/D in SOCS1^−/− mice as compared with mice receiving a control antibody (P = 0.0021). CD4⁺ T-cell depletion from SOCS1^−/− mice also inhibited the effects of IFN-A/D (P = 0.0003). IFN-A/D did not alter expression of CD80 or CD86 on splenocytes of SOCS1^+/+ or SOCS1^−/− mice, or the proportion of T regulatory cells or myeloid-derived suppressor cells in SOCS1^+/+ or SOCS1^−/− mice. An analysis of T-cell function did reveal increased proliferation of SOCS1-deficient splenocytes at baseline and in response to mitogenic stimuli. These data suggest that modulation of SOCS1 function in T-cell subsets could enhance the anti-tumor effects of IFN-α in the setting of melanoma.     

Immune Response to Nocardia brasiliensis Extracellular Antigens in Patients with Mycetoma

The ability of culture-filtrate proteins to induce a cellular immune response in infected mice and humans was investigated. A crude extract culture filtrate of Nocardia brasiliensis (CFA) and five semi-purified CFA fractions (P1, P2, P3, P4, P5) were used to stimulate BALB/c mice spleen-cell cultures. The animals were divided into three groups: the first group was infected with 1 × 10⁷ CFU of N. brasiliensis in the footpad, the second group was immunized with heat-killed bacteria, and the third was injected with sterile saline. IFN-γ, IL-1α, and IL-4 concentrations were determined in culture supernatants. Protein fractions eliciting IFN-γ production in mice, as well as the CFA, were used to stimulate IFN-γ production and in vitro cell proliferation assays with peripheral blood mononuclear cells of patients with actinomycetoma by N. brasiliensis, individuals with pulmonary tuberculosis, and healthy controls. In mice, CFA and three of the protein fractions (P3, P4 and P5) induced significant IFN-γ production in the infected group. In humans, only the CFA-induced IFN-γ production and cell proliferation in the group of patients with actinomycetoma. There was no stimulation in tuberculosis patients nor healthy controls. These results suggest that some culture-filtrate antigens are recognized by patients with active actinomycetoma and do not cross-react with M. tuberculosis antigens, being therefore potential candidates to develop a diagnostic test.     

Distinct myeloid suppressor cell subsets correlate with plasma IL-6 and IL-10 and reduced interferon-alpha signaling in CD4<Superscript>+</Superscript> T cells from patients with GI malignancy

Interferon-alpha (IFN-α) promotes anti-tumor immunity through its actions on immune cells. We hypothesized that elevated percentages of myeloid-derived suppressor cells (MDSC) and increased pro-inflammatory cytokines in peripheral blood would be associated with impaired response to IFN-α in patients with gastrointestinal (GI) malignancies. This study evaluated relationships between plasma IL-6, IL-10, circulating MDSC subsets, and IFN-α-induced signal transduction in 40 patients with GI malignancies. Plasma IL-6 and IL-10 were significantly higher in patients versus normal donors. CD33⁺HLADR⁻CD11b⁺CD15⁺ and CD33⁺HLADR^−/lowCD14⁺ MDSC subsets were also elevated in patients versus normal donors (P < 0.0001). Plasma IL-6 was correlated with CD33⁺HLADR⁻CD15⁺ MDSC (P = 0.008) and IL-10 with CD33⁺HLADR⁻CD15⁻ MDSC (P = 0.002). The percentage of CD15⁺ and CD15⁻ but not CD14⁺ MDSC subsets were inversely correlated with IFN-α-induced STAT1 phosphorylation in CD4⁺ T cells, while co-culture with in vitro generated MDSC led to reduced IFN-α responsiveness in both PBMC and the CD4⁺ subset of T cells from normal donors. Exploratory multivariable Cox proportional hazards models revealed that an increased percentage of the CD33⁺HLADR⁻CD15⁻ MDSC subset was associated with reduced overall survival (P = 0.049), while an increased percentage of the CD33⁺HLADR^−/lowCD14⁺ subset was associated with greater overall survival (P = 0.033). These data provide evidence for a unique relationship between specific cytokines, MDSC subsets, and IFN-α responsiveness in patients with GI malignancies.     

Differential modulation by interferon γ of the sensitivity of human melanoma cells to cytolytic T cell clones that recognize differentiation or progression antigens

 Human melanoma is a highly immunogenic tumor capable of inducing a specific immune response. A number of melanoma-associated antigens have been characterized during the past several years and can be classified into two groups: differentiation antigens  –  present also in normal melanocytes  –  and tumor-specific antigens, which, with the exception of testis, are present only in tumor cells. In a previous publication [Kirkin A. F., Petersen T. R., Olsen A. C., Li L., thor Straten P., Zeuthen J. (1995) Cancer Immunol Immunother 41:71] we have described the production of clones of cytotoxic T lymphocytes (CTL) against the highly immunogenic human melanoma cell line FM3. Using these clones we have defined four previously unknown melanoma-associated antigens, which could be subdivided into differentiation and progression antigens. In the experiments reported in this paper, we have further compared CTL clones from different groups and shown that the sensitivity of melanoma cells to CTL that recognize differentiation or progression antigens is differentially modulated during tumor progression as well as by the lymphokines interferon γ (IFNγ) and interleukin-10 (IL-10). The interaction of CTL clones recognizing progression antigens was strongly increased after treatment of melanoma cells with IFNγ, while the recognition by CTL clones specific for differentiation antigens either was unchanged or significantly decreased. IL-10 treatment of melanoma cells induced up-regulation with respect to recognition by CTL clones specific for differentiation antigens without affecting the recognition of melanoma cells by CTL clones specific for progression antigens. Using cellular systems at different stages of tumor progression, we demonstrated that the progressed state of melanoma cells is associated with increased sensitivity to recognition by CTL clones detecting progression antigens, and with decreased sensitivity to CTL clones recognizing differentiation antigens. Mimicking tumor progression, treatment with IFN-γ induced apparent down-regulation of differentiation antigens. A hypothesis is suggested in which IFN-γ plays different roles in the immune response against poorly immunogenic and highly immunogenic melanoma cells, increasing the progression of poorly immunogenic tumor cells or promoting a strong immune response and regression of highly immunogenic melanoma cells. Received: 23 November 1995 / Accepted: 7 March 1996