Rea-Min Chu

Tumor-Infiltrating Lymphocyte Secretion of IL-6 Antagonizes Tumor-Derived TGF-β1 and Restores the Lymphokine-Activated Killing Activity

IL-6 is a multifunctional cytokine that regulates cell growth, differentiation, and cell survival. Many tumor cells produce TGF-β1, which allows them to evade CTL-mediated immune responses. IL-6 antagonizes TGF-β1 inhibition of CD3 cell activation. However, whether IL-6 restores NK activity, which also is suppressed by TGF-β1, is not known. We used canine transmissible venereal tumor (CTVT), which produces TGF-β1, as a model to determine whether IL-6 restores lymphokine-activated killer (LAK) activity. During the progression phase, CTVT cells stop expressing MHC molecules. During the regression phase, the number of surface MHC molecules increases dramatically on about one-third of tumor cells. Tumor cells that stop expressing MHC should be targeted by NK cells. In this study, we found that TGF-β1 secreted by CTVT cells suppressed LAK cytotoxicity. Interestingly, tumor-infiltrating lymphocytes (TIL) isolated from regressing CTVT secrete high concentrations of IL-6 and antagonize the anti-LAK activity of tumor cell TGF-β1. TIL also produce IL-6 during progression phase, but the concentration is too low to block the anti-LAK activity of TGF-β1. There is probably a threshold concentration of IL-6 needed to reverse TGF-β1-inhibited LAK activity. In addition, in the absence of TGF-β1, IL-6 derived from TIL does not promote the activity of LAK. This new mechanism, in which TIL manufacture high concentrations of IL-6 to block tumor TGF-β1 anti-LAK activity, has potential applications in cancer immunotherapy and tumor prognosis.

Interactions of host IL-6 and IFN-γ and cancer-derived TGF-β1 on MHC molecule expression during tumor spontaneous regression

Many tumors down-regulate major histocompatibility complex (MHC) antigen expression to evade host immune surveillance. However, there are very few in vivo models to study MHC antigen expression during tumor spontaneous regression. In addition, the roles of transforming growth factor beta1 (TGF-β1), interferon gamma (IFN-γ), and interleukin (IL)-6 in modulating MHC antigen expression are ill understood. We previously reported that tumor infiltrating lymphocyte (TIL)-derived IL-6 inhibits TGF-β1 and restores natural killing (NK) activity. Using an in vivo canine-transmissible venereal tumor (CTVT) tumor model, we presently assessed IL-6 and TGF-β involvement associated with the MHC antigen expression that is commonly suppressed in cancers. IL-6, IFN-γ, and TGF-β1, closely interacted with each other and modulated MHC antigen expression. In the presence of tumor-derived TGF-β1, host IFN-γ from TIL was not active and, therefore, there was low expression of MHC antigen during tumor progression. TGF-β1-neutralizing antibody restored IFN-γ-activated MHC antigen expression on tumor cells. The addition of exogenous IL-6 that has potent anti-TGF-β1 activity restored IFN-γ activity and promoted MHC antigen expression. IFN-γ and IL-6 in combination acted synergistically to enhance the expression of MHC antigen. Thus, the three cytokines, IL-6, TGF-β1, and IFN-γ, closely interacted to modulate the MHC antigen expression. Furthermore, transcription factors, including STAT-1, STAT-3, IRF-1, NF-κB, and CREB, were significantly elevated after IL-6 and IFN-γ treatment. We conclude that the host IL-6 derived from TIL works in combination with host IFN-γ to enhance MHC molecule expression formerly inhibited by TGF-β1, driving the tumor toward regression. It is suggested that the treatment of cancer cells that constitutively secrete TGF-β1 should incorporate anti-TGF-β activity. The findings in this in vivo tumor regression model have potential applications in cancer immunotherapy.

Effect of tumor infiltrating lymphocytes on the expression of MHC molecules in canine transmissible venereal tumor cells

Canine transmissible venereal tumor (CTVT) can be allo-transplanted across major histocompatibility complex barriers. The expression of MHC molecules is usually low in the progression (P) stage and then greatly increases during tumor regression (R). We investigated the effects of tumor infiltrating lymphocytes (TIL) on the expression of MHC molecules of CTVT cells. Isolated, viable CTVT cells were inoculated at each of 12 sites (1 x 10(8) CTVT cells per site) on the back of six, mixed-breed dogs. Tumor masses were collected every 2-3 weeks and prepared for histopathologic, immunocytochemistry, flow cytometry and immunoblotting studies. The level of MHC expression on tumor cells from different stages of growth was measured. Initially, expression of MHC I and II molecules in P phase CTVT was low. Twelve weeks post-inoculation (PI), expression increased dramatically and it continued to increase during R phase. Tumor growth slowed after 12 weeks PI and tumors entered R phase around 17 weeks PI. We hypothesize that CTVT evades host immunosurveillance and grows progressively for 12 weeks, when it becomes vulnerable and subject to the hosts anti-tumor immune responses. We further demonstrated that R phase, but not P phase, TIL were closely associated with the over-expression of MHC I and II molecules by CTVT cells. The number and proportion of TIL were higher in R phase tumors. Supernatants, from R phase co-cultures (CTVT+TIL) and TIL only, promoted MHC I and II expression on P phase CTVT cells. After culturing alone for 1 month, expression of MHC classes I and II molecules in R phase CTVT cells decreased to the level of P phase CTVT cells. However, the above-mentioned supernatants restored their expression of MHC I and II molecules. In contrast, supernatants from P phase TIL or CTVT cells increased expression slightly or had no effect. Therefore, TIL, not CTVT cells, produce the effective substance (s) to promote the expression of MHC molecules by the tumor cells. Heat treated supernatant was unable to promote the expression of MHC I and II molecules by CTVT cells. In conclusion, TIL isolated from R phase CTVT secreted a heat-sensitive, soluble substance(s) that triggered over-expression of MHC I and II after 12 weeks PI. This caused the tumor to enter R phase and helped stop CTVT growth. Our findings will facilitate the understanding and further investigation of the mechanisms that initiate host immune surveillance against tumors.

CD5-low expression lymphocytes in canine peripheral blood show characteristics of natural killer cells

NK cell markers and receptors have been discovered in many mammalian species, such as humans, mice, rats, pigs, and cows. However, there is still a lack of information concerning NK cell markers or receptors in canines. We have discovered that canine CD5‐low density (CD5lo) cells in PBL are closely associated with NK cell characteristics. CD5lo cells comprised 14.9 ± 6.68% of the total PBL. A high proportion of the CD5lo cell population expressed CD3 (96.6%), CD8α (77.7%), CD8β (53%), α/β TCR (83%), and CD11/18 (80%), but the expression of γ/δ TCR (6.5%), CD4 (10.6%), and CD21 (2.4%) was low. CD5lo cells were larger than CD5‐high density (CD5hi) cells. Light and electron microscopy revealed numerous large cytoplasmic granules in CD5lo cells, especially after IL‐2 stimulation, which was in contrast to CD5hi, in which intracytoplasmic granules were not frequently seen. After IL‐2 stimulation, CD5lo cells had significantly stronger NK cytotoxicity than CD5hi cells. CD5lo cells had much higher mRNA levels for NKG2D, CD16, CD94, CD160, perforin, and granzyme than CD5hi. Following IL‐2 stimulation, CD5lo cells had significantly higher mRNA levels of NKp30, NKp44, CD16, and CD94 than CD5hi cells. In addition, IL‐2‐stimulated, CD5lo‐depleted PBL showed a loss of NK cytotoxicity. CD5lo cells also showed significantly lower antigen‐specific cytotoxic T cell activity as compared with CD5hi cells. Taken together, the CD5lo subset in canine PBL is closely related to canine NK cells, and CD5lo can be used as a phenotypic marker for an IL‐2‐dependent canine NK cell enrichment.

Canine transmissible venereal tumor cell depletion of B lymphocytes: molecule(s) specifically toxic for B cells

Canine transmissible venereal tumor (CTVT) is an excellent model for investigating the interaction between host immunity and tumor growth. Although CTVT is an allograft, initially the host immune system is unable to destroy the tumor cells, and the tumor grows progressively for about 4-6 months (P phase). After a short stable phase, the tumor undergoes regression (R phase). In this study, CTVT inoculation significantly reduced the proportion of B lymphocytes among all peripheral blood lymphocytes (PBL), but the proportion of B lymphocytes returned to normal after complete removal of CTVT. Following CTVT inoculation, immunoglobulin concentrations decreased gradually, coincident with B lymphocyte decline. Furthermore, CTVT secreted a soluble, heat- and protease K-sensitive cytotoxic molecule(s) that destroyed peripheral blood B lymphocytes (PBBL) but spared other types of immune cells regardless of whether mitogens, such as IL-2 or Con A, were present. The decrease in the proportion and viability of PBBL was caused by a cytotoxic molecule(s) that induced apoptosis. The molecular weight of the CTVT-derived cytotoxic molecule(s) was 30-100kDa. Human, domestic cat, horse and mouse B cells were also sensitive to the substance.

Combined immunogene therapy of IL-6 and IL-15 enhances anti-tumor activity through augmented NK cytotoxicity

Many tumors evade host immunity by lowering expression of major histocompatibility complex (MHC) molecules. Theoretically, low MHC expression should activate natural killer (NK) cells and in some cases suppress tumor growth; nevertheless, some tumors also produce high concentrations of immunosuppressive cytokines, such as transforming growth factor (TGF)-beta, to inhibit the activity of NK cells. Using a canine transmissible venereal tumor (CTVT) model, we have previously demonstrated that IL-6 is a strong antagonist for TGF-beta. Herein, we found that IL-6 alone was unable to significantly promote TGF-beta-inhibited NK activities. Conversely, IL-15 alone strongly promoted NK activities; however, NK activities were inhibited to baseline levels following the addition of TGF-beta. Therefore, a new strategy using combined immunogene therapy of both IL-6 and IL-15 mediated by electroporation was used in this study. This combined IL-6 and IL-15 treatment effectively relieved the inhibitory effect of TGF-beta and activated NK cell cytotoxicity of lymphokine-activated killer (LAK) cells. Similarly, in isolated DX5+ NK cells, only IL-6 and IL-15 in combination significantly overcame the inhibitory effect of TGF-beta and promoted NK cytotoxicity. The group of BALB/c mice injected with plasmids with IL-6 and IL-15 genes (pIL-6/pIL-15) had the highest percentages of DX5+ NK cells as compared with either the pIL-6 or pIL-15 groups. Further, in SCID mice inoculated with CTVT, electroporation-mediated delivery of pIL-6/pIL-15 was significantly more efficient in suppressing both tumor establishment and tumor growth as compared with pIL-6 or pIL-15 inoculation alone. In addition, the anti-asialo GM-1 antibody abolished NK activities in SCID mice and resulted in outgrowth of the tumors. Together, these results suggest that the TGF-beta-associated inhibition of NK cytotoxicity cannot be adequately restored by simply antagonizing TGF-beta with IL-6: the co-existence of NK activating factors such as IL-15 is also important in restoring TGF-beta-inhibited cytotoxicity. This study highlights the therapeutic potential of the pIL-6/pIL-15 combination by inhibiting TGF-beta activity and enhancing NK cytotoxicity.

Effects of immunotherapy of IL-6 and IL-15 plasmids on transmissible venereal tumor in beagles.

Canine transmissible venereal tumor (CTVT) is a tumor with low MHC antigen expression and is an ideal tumor model for studying the interactions between host immunity and cancer cells. CTVTs produce high concentrations of TGF-beta to hamper the host immune responses and facilitate their growth progression. However, during the later stages of tumor progression, tumor-infiltrating lymphocytes secrete IL-6. This cytokine antagonizes TGF-beta and restores the IFN-gamma activities in promoting MHC antigen expression, and the NK cytotoxicity that has been repressed by TGF-beta is also activated. In this study, we applied combinatory treatment of IL-6 plasmid and IL-15 plasmid (pIL-6/pIL-15) to CTVT-bearing beagles. IL-6 was used as an anti-TGF-beta cytokine; IL-15 was used to promote NK- and CTVT-specific cytotoxicity. After intratumoral pIL-6/pIL-15 delivery mediated by electroporation, MHC antigen expression on CTVT cells was dramatically increased from in less than 5.9% to up to 34% of the tumor cells. The proportion of CD8(+) T cells infiltrating the tumor was also significantly elevated from 6.96+/-0.23% to 21.63+/-5.40%. In addition, the tumor-specific cytotoxicity was enhanced along with a marked increase in tumor-specific IFN-gamma-producing cells. These immune responses are believed to be the important forces driving the tumor towards regression. The results indicate that pIL-6/pIL-15 combinatory immunotherapy may facilitate a promising and effective means of treating tumors.

Stimulation of smooth muscle cell proliferation by ox-LDL- and acetyl LDL-induced macrophage-derived foam cells

To test the hypothesis that LDL lacking of initial oxidation may also anticipate an essential role in the progression for atherosclerotic lesions, we studied the in vitro effect of foam cells induced by low density lipoprotein (LDL), oxidized (ox)-LDL or acetyl-LDL on smooth muscle cell (SMC) proliferation. Intraperitoneal macrophages collected from ICR mice were incubated with buffered saline LDL, ox-LDL or acetyl-LDL to induce foam cell formation. Porcine aortas with atherosclerotic lesions were collected from 5 pigs fed high cholesterol diets. The results indicate that foam cells induced by ox-LDL and acetyl-LDL, but not by LDL, promoted SMC proliferation. SMC proliferation was also increased by ruptured, ox-LDL- and acetyl-LDL- induced foam cells. Immunohistochemically, epitopes of the LDL, ox-LDL, and malondialdelyde (MDA)-LDL were present in atherosclerotic lesions, but the acetyl epitope was not. We suggest that foam cells, whether induced by the oxidized or acetyl or acetyl (unoxidized) form, play an essential role in the pathogenesis of atherosclerosis by stimulating SMC proliferation.

Canine CD8 T cells showing NK cytotoxic activity express mRNAs for NK cell-associated surface molecules

Natural killer (NK) cells have been considered to be a group of lymphocytes lacking clonally distributed receptors for antigens typical of T cells and B cells. In some mammalian species, including humans, a subpopulation of CD8(+) peripheral blood lymphocytes (PBLs) exhibits NK activity. This NK subpopulation has not been well characterized in mammals and its characterization is particularly poor in the dog. In this study, we demonstrated that a subset of canine CD8(+) cells derived from PBLs and lymphokine (IL-2)-activated killers (LAKs) of PBLs that was CD3(+), CD4(-), CD21(-), CD5(lo), alpha/betaTCR(+), and gamma/deltaTCR(-) contained substantially higher levels of mRNAs for NK cell-related receptors (NKp30, NKp44, NKG2D, 2B4, and CD16 for PBL, and NKG2D and CD56 for LAK) than the corresponding CD8(-) cells. This subset of CD8(+) lymphocytes derived from LAKs also displayed significantly higher NK cytotoxic activity than the corresponding CD8(-) cells. In contrast, CD8(+) cells derived from nonstimulated PBLs showed very low levels of NK cytotoxic activity. Our results indicate that, in IL-2-stimulated PBLs, canine CD8(+) cells are an important subset associated with NK cytotoxic activity.

Synergistic anti-tumor effect of combination radio- and immunotherapy by electro-gene therapy plus intra-tumor injection of dendritic cells

Interleukin-2 (IL-2) and granulocyte-macrophage colony stimulating factor (GM-CSF) facilitate the maturation and functioning of injected DC. We developed a method of in situ electroporation using IL-2 and GM-CSF genes (EGT/cytokines), followed by intra-tumoral (i.t.) immature DC to determine the immune response at the tumor site using prostate-specific antigen-transfected CT26 cells. Three cycles of EGT/cytokines and i.t. DC inhibited tumor growth most effectively, but not superior to EGT/cytokines alone. However, the role of i.t. DC became significant when radiation was given after immunotherapy, which may have clinical implications on achieving better local control and prevention of systemic relapse.