Decrease In Granzyme Research Articles

Clinical characteristics and molecular features of non-small cell lung cancers (NSCLCs) following disease progression on immune checkpoint inhibitors (ICIs).

e21178 Background: ICIs are cornerstones of therapy for advanced NSCLC. Despite dramatic and sometimes durable responses to therapy, most patients (pts) either (i) do not respond to therapy (intrinsic resistance), or (ii) subsequently progress after initial clinical benefit (acquired resistance). Currently, insights into the molecular mechanisms of resistance to ICIs in NSCLC are lacking. Methods: To investigate clinical and molecular features of pts progressing on ICIs, we identified pts who underwent repeat tumor biopsies on and/or after disease progression on ICIs and were included in the Stand Up 2 Cancer (SU2C)/Mark Foundation multi-institutional cohort. Biopsy specimens underwent whole-exome sequencing (WES) and/or whole transcriptome sequencing (RNAseq). Results: We identified 37 pts who underwent a total of 47 repeat biopsies on or after ICIs. Six pts underwent multiple post-ICI biopsies (range 2-4). Twenty-five pts (68%) received PD-(L)1 inhibitor monotherapy, 6 (16%) received PD-(L)1 plus CTLA-4 inhibitors, and 6 (16%) received other PD-1 inhibitor-based combinations. Overall, the objective response rate was 46% among pts undergoing repeat biopsies (complete response 2 [5%], partial response 15 [41%], stable disease 14 [38%], progressive disease 5 [14%] and not evaluable 1 [3%]). Median progression-free survival (PFS) was 8.1 months. In total, pre-ICI biopsy specimens were available in 20 pts. WES and RNAseq were performed on 67 and 44 specimens, respectively. Median tumor mutation burden (TMB) in pre-ICI specimens was 5.0 mutations/Mb versus 4.9 mutations/Mb in post-ICI specimens ( p= 0.3, Mann-Whitney U test). Among 20 paired pre/post-ICI specimens, there was no significant difference in TMB (pre-treatment median 3.9 mutations/Mb; post-treatment median 4.3 mutations/Mb; p= 0.7, Wilcoxon signed-rank test). One pt with a complete response acquired a nonsense mutation in B2M, and one pt with a partial response acquired a nonsense mutation in JAK1. Among 10 paired pre/post-ICI specimens that underwent RNAseq, we observed significant decreases in granzyme B and perforin in post-ICI specimens ( p= 4×10-5 and p= 2×10-3, respectively, limma-voom analysis). Conclusions: Genomic alterations impairing antigen presentation (e.g., B2M) or immune activation (e.g., JAK1) may enable resistance to ICIs in a small subset of cases. However, the majority of repeat biopsies obtained from pts progressing on ICIs lacked clear genetic mediators of resistance, suggesting the presence of additional tumor-intrinsic and/or tumor-extrinsic factors underlying resistance to ICIs in NSCLC.

Hobit confers tissue-dependent programs to type 1 innate lymphoid cells

Identification of type 1 innate lymphoid cells (ILC1s) has been problematic. The transcription factor Hobit encoded by Zfp683 has been proposed as a major driver of ILC1 programs. Using Zfp683 reporter mice, we showed that correlation of Hobit expression with ILC1s is tissue- and context-dependent. In liver and intestinal mucosa, Zfp683 expression correlated well with ILC1s; in salivary glands, Zfp683 was coexpressed with the natural killer (NK) master transcription factors Eomes and TCF1 in a unique cell population, which we call ILC1-like NK cells; during viral infection, Zfp683 was induced in conventional NK cells of spleen and liver. The impact of Zfp683 deletion on ILC1s and NK cells was also multifaceted, including a marked decrease in granzyme- and interferon-gamma (IFNγ)-producing ILC1s in the liver, slightly fewer ILC1s and more Eomes+ TCF1+ ILC1-like NK cells in salivary glands, and only reduced production of granzyme B by ILC1 in the intestinal mucosa. NK cell-mediated control of viral infection was unaffected. We conclude that Hobit has two major impacts on ILC1s: It sustains liver ILC1 numbers, while promoting ILC1 functional maturation in other tissues by controlling TCF1, Eomes, and granzyme expression.

HCA587 Protein Vaccine Induces Specific Antitumor Immunity Mediated by CD4+ T-cells Expressing Granzyme B in a Mouse Model of Melanoma.

The antigen HCA587 (also known as MAGE-C2), which is considered a cancer-testis antigen, exhibits upregulated expression in a wide range of malignant tumors with unique immunological properties, and may thus serve as a promising target for tumor immunotherapy. The study aimed to explore the antitumor effect of the HCA587 protein vaccine and the response of humoral and cell-mediated immunity. The HCA587 protein vaccine was formulated with adjuvants CpG and ISCOM. B16 melanoma cells were subcutaneously inoculated to C57BL/6 mice, followed by treatment with HCA587 protein vaccine subcutaneously. Mouse survival was monitored daily, and tumor volume was measured every 2 to 3 days. The tumor sizes, survival time and immune cells in tumor tissues were detected. And the vital immune cell subset and effector molecules were explored. After treatment with HCA587 protein vaccine, the vaccination elicited significant immune responses, which delayed tumor growth and improved animal survival. The vaccination increased the proportion of CD4+ T cells expressing IFN-γ and granzyme B in tumor tissues. The depletion of CD4+T cells resulted in an almost complete abrogation of the antitumor effect of the vaccination, suggesting that the antitumor efficacy was mediated by CD4+ T cells. In addition, knockout of IFN-γ resulted in a decrease in granzyme B levels, which were secreted by CD4+ T cells, and the antitumor effect was also significantly attenuated. The HCA587 protein vaccine may increase the levels of granzyme B expressed by CD4+ T cells, and this increase is dependent on IFN-γ, and the vaccine resulted in a specific tumor immune response and subsequent eradication of the tumor.

The impact of the tumor microenvironment on granzyme production by CD8 T cells

Abstract Granzymes are a class of proteases produced by CD8 T cells to mediate their cytotoxic activity against target cells. The objective of this project is to determine the mechanism through which CD8 T cells are executing their granzyme-mediated cytotoxic function and how the tumor microenvironment (TME) impacts the specific granzymes produced. By micro-array and qPCR we determined that CD8 T cells in the TME have altered expression of specific granzymes. Granzyme F had one of the most altered expressions of all genes examined in antigen-specific CD8 T cells taken from a spleen relative to those taken from a tumor. We further examined the effects of the TME on granzyme expression by sorting tumor infiltrating CD8 T cells on exhaustion markers and found a relationship between exhaustion and granzyme expression, characterized by a decrease in granzyme A and an increase in granzyme B mRNA expression. We will determine the influence different ex vivo culture conditions have on differential granzyme expression and function of CD8 T cells. The expression of different granzymes may have further implications for modulation of the TME through the form of cancer cell death induced, especially the increase in granzyme F which has been suggested to induce a more immunogenic form of cell death. Additionally, the impact of exhaustion on the cytotoxic activity of CD8 T cells from the TME may represent a means for exhausted CD8 T cells to regulate their cytotoxic activity by altering the granzymes they express.

Trophoblast-Derived CXCL16 Decreased Granzyme B Production of Decidual γδ T Cells and Promoted Bcl-xL Expression of Trophoblasts.

Decidual γδ T cells are known to regulate the function of trophoblasts at the maternal-fetal interface; however, little is known about the molecular mechanisms of cross talk between trophoblast cells and decidual γδ T cells. Expression of chemokine C-X-C motif ligand 6 (CXCL16) and its receptor CXCR6 was evaluated in first-trimester human villus and decidual tissues by immunohistochemistry. γδ T cells were isolated from first-trimester human deciduae and cocultured with JEG3 trophoblast cells. Cell proliferation and apoptosis-related molecules, together with cytotoxicity factor and cytokine production, were measured by flow cytometry analysis. Expression of CXCL16 and CXCR6 was reduced at the maternal-fetal interface in patients who experienced unexplained recurrent spontaneous abortion as compared to healthy pregnancy women. With the administration of pregnancy-related hormones or coculture with JEG3 cells, CXCR6 expression was upregulated on decidual γδ T cells. CXCL16 derived from JEG3 cells caused a decrease in granzyme B production of decidual γδ T cells. In addition, decidual γδ T cells educated by JEG3-derived CXCL16 upregulated the expression of Bcl-xL in JEG3 cells. This study suggested that the CXCL16/CXCR6 axis may contribute to maintaining normal pregnancy by reducing the secretion of cytotoxic factor granzyme B of decidual γδ T cells and promoting the expression of antiapoptotic marker Bcl-xL of trophoblasts.

Rituximab impedes natural killer cell function in Chronic Fatigue Syndrome/Myalgic Encephalomyelitis patients: A pilot in vitro investigation

BackgroundA recent in vitro pilot investigation reported Rituximab significantly reduced natural killer (NK) cell cytotoxicity in healthy donors. Chronic fatigue syndrome/Myalgic encephalomyelitis (CFS/ME) is a debilitating disorder of unknown etiology. A consistent finding is a significant reduction in NK cell cytotoxicity. Rituximab has been reported having questionable potential therapeutic benefits for the treatment of CFS/ME, however, the potential effects of Rituximab on NK cell cytotoxicity in CFS/ME patients are yet to be determined.MethodsA total of eight CFS/ME patients (48.63 ± 15.69 years) and nine non-fatigued controls (NFC) (37.56 ± 11.06 years) were included using the Fukuda case definition. Apoptotic function, lytic proteins and degranulation markers were measured on isolated NK cells using flow cytometry following overnight incubation with Rituximab at 10 μg/ml and 100 μg/ml.ResultsThere was a significant reduction in NK cell lysis between CFS/ME patients and NFC following incubation with Rituximab at 100 μg/ml at 12.5:1 and 6.25:1 effecter-target (E:T) ratios (p < 0.05). However, there was no significant difference for NFC following incubation with Rituximab at 10 μg/ml and 100 μg/ml.There was no significant difference between CFS/ME patients and NFC for granzyme A and granzyme B prior to incubation with Rituximab and following overnight incubation with Rituximab at 10 μg/ml. There was a significant decrease in granzyme B in CFS/ME patients compared to NFC with 100 μg/ml of Rituximab prior to K562 cells stimulation (p < 0.05).There was a significant increase in CD107a (p < 0.05) and CD107b expression (p < 0.01) in NFC after stimulation with K562 cells prior to incubation with Rituximab. There was a significant increase in CD107b expression between CFS/ME patients and NFC prior to incubation with Rituximab and without stimulation of K562 cells (p < 0.01). Importantly, there was a significant increase in CD107b following overnight incubation with 100 μg/ml of Rituximab in NFC prior to K562 cells stimulation (p < 0.01).ConclusionThis study reports significant decreases in NK cell lysis and a significant increase in NK cell degranulation following Rituximab incubation in vitro in CFS/ME patients, suggesting Rituximab may be toxic for NK cells. Caution should be observed in clinical trials until further investigations in a safe and controlled in vitro setting are completed.

Notch Ligand Delta-like 4 Promotes Regulatory T Cell Identity in Pulmonary Viral Infection.

Regulatory T (Treg) cells establish tolerance, prevent inflammation at mucosal surfaces, and regulate immunopathology during infectious responses. Recent studies have shown that Delta-like ligand 4 (Dll4) was upregulated on APC after respiratory syncytial virus (RSV) infection, and its inhibition leads to exaggerated immunopathology. In the present study, we outline the role of Dll4 in Treg cell differentiation, stability, and function in RSV infection. We found that Dll4 was expressed on CD11b+ pulmonary dendritic cells in the lung and draining lymph nodes in wild-type BALB/c mice after RSV infection. Dll4 neutralization exacerbated RSV-induced disease pathology, mucus production, group 2 innate lymphoid cell infiltration, IL-5 and IL-13 production, as well as IL-17A+ CD4 T cells. Dll4 inhibition decreased the abundance of CD62LhiCD44loFoxp3+ central Treg cells in draining lymph nodes. The RSV-induced disease was accompanied by an increase in Th17-like effector phenotype in Foxp3+ Treg cells and a decrease in granzyme B expression after Dll4 blockade. Finally, Dll4-exposed induced Treg cells maintained the CD62LhiCD44lo central Treg cell phenotype, had increased Foxp3 expression, became more suppressive, and were resistant to Th17 skewing in vitro. These results suggest that Dll4 activation during differentiation sustained Treg cell phenotype and function to control RSV infection.

Anti-GITR therapy promotes immunity against malignant glioma in a murine model.

Regulatory T cells (Tregs) are potently immunosuppressive cells that accumulate within the glioma microenvironment. The reduction in their function and/or trafficking has been previously shown to enhance survival in preclinical models of glioma. Glucocorticoid-induced TNFR-related protein (GITR) is a tumor necrosis factor superfamily receptor enriched on Tregs that has shown promise as a target for immunotherapy. An agonistic antibody against GITR has been demonstrated to inhibit Tregs in a number of models and has only been recently addressed in glioma. In this study, we examined the modality of the antibody function at the tumor site as opposed to the periphery as the blood-brain barrier prevents efficient antibody delivery to brain tumors. Mice harboring established GL261 tumors were treated with anti-GITR monotherapy and were shown to have a significant increase in overall survival (p<0.01) when antibodies were injected directly into the glioma core, whereas peripheral antibody treatment only had a modest effect. Peripheral treatment resulted in a significant decrease in granzyme B (GrB) expression by Tregs, whereas intratumoral treatment resulted in both a decrease in GrB expression by Tregs and their selective depletion, which was largely mediated by FcγR-mediated destruction. We also discovered that anti-GITR treatment results in the enhanced survival and functionality of dendritic cells (DCs)-a previously unreported effect of this immunotherapy. In effect, this study demonstrates that the targeting of GITR is a feasible and noteworthy treatment option for glioma, but is largely dependent on the anatomical location in which the antibodies are delivered.

Abstract 046: The Role of Tumor Necrosis Factor α and Natural Killer Cells in Uterine Artery Function During Pregnancy in the Stroke Prone Spontaneously Hypertensive Rat

Objective: We have previously characterised the stroke prone spontaneously hypertensive rat (SHRSP) as a model of deficient uterine artery remodelling and identified an increase in pro-inflammatory TNFα relative to the normotensive WKY strain during pregnancy. Method: SHRSP were treated with etanercept (0.8 mg/kg) or vehicle at gestational day (GD) 0, 6, 12 and 18. Animals were sacrificed at GD18. Results: Etanercept reduced systolic blood pressure in the SHRSP after GD12 (ΔSBP GD 10-21 SHRSP 12.0 ± 4.17 vs. ETN 25.8 ± 4.27 mmHg; p&lt;0.05). Uterine arteries from GD18 showed that etanercept reduced uterine artery contraction (SHRSP 57.3 ± 8.75 vs. ETN 35.2 ± 2.19 kPa; p&lt;0.01) and increased carbachol response (SHRSP 13.8 ± 3.8 vs. ETN 40.1 ± 3.25 %; p&lt;0.05). Uteroplacental blood flow analysed using Doppler showed that etanercept reduced uterine artery resistance index in SHRSP (SHRSP 0.79 ± 0.02 vs. ETN 0.61 ± 0.02 UARI; p&lt;0.01). Etanercept increased litter size (SHRSP 7.80 ± 0.44 vs. ETN 12.75 ± 0.94 fetuses), reduced resorption frequency (SHRSP 66.7% vs. ETN 25.0% dams with resorption) and decreased glycogen cell loss from the placenta in SHRSP. We sought to identify the source of excess TNFα in the SHRSP. Natural killer (NK) cells (CD3-CD161+) were increased in the SHRSP relative to the WKY in the maternal circulation (WKY 1.5 ± 0.4 vs. SHRSP 6.06 ± 0.28 %; p&lt;0.01) and placenta (WKY 11.6 ± 2.39 vs. SHRSP 659.8 ± 201.2 cells/mg; p&lt;0.01). These NK cells produced excess TNFα in the SHRSP maternal circulation (SHRSP 6.5 ± 0.4 vs. WKY 2.5 ± 0.4 %; p&lt;0.05) and placenta (SHRSP 65.7 ± 4.2 vs. WKY 16.9 ± 1.7 %; p&lt;0.01) relative to the WKY. In the SHRSP placenta, etanercept treatment reduced the number of cytotoxic NK cells (SHRSP 659.8 ± 201.2 vs. ETN 148.0 ± 12.62 cells/mg; p&lt;0.01) by down-regulating CD161 expression associated with a decrease in granzyme B production (CD161+ 71.47 ± 2.1 vs. CD161 Low 14.32 ± 0.77 % granzyme B+; p&lt;0.01). Conclusions: Excess TNFα plays a causative role in adverse pregnancy outcome in the SHRSP. One source of this TNFα is an increase in NK cells during gestation in the SHRSP. Etanercept targets NK cells in the SHRSP placenta and down-regulates cytotoxic granzyme B production.

Blockade of surface bound TGF-β on regulatory T cells abrogates suppression of effector T cell function within the tumor microenvironment (TUM2P.1015)

Abstract Regulatory T cells (Treg) play a role in suppression of anti-melanoma immunity; however, the exact mechanism is poorly understood. Through intravital two photon microcopy, we found that tumor-specific Pmel-1 effectors engage in cell-cell interactions with tumor resident Tregs. To determine if contact between Tregs and Teff hinders killing of tumor cells in vivo, we utilized ex-vivo three-dimensional collagen-fibrin gel cultures of B16 melanoma cells. Collagen-fibrin gel cultures recapitulated the in vivo suppression, rendering the dissociated tumor resistant to killing by in vitro activated antigen specific T cells. In vivo depletion of Tregs in Foxp3-DTR mice prior to tumor excision reversed the suppression. In vivo modulation of Tregs by GITR ligation also reduced the number of intra-tumor Tregs leading to ex-vivo tumor killing. Using neutralizing antibodies, we found that blocking TGF-β reversed the suppression. In addition, soluble factors from collagen-fibrin gel tumors do not inhibit killing suggesting that suppression is contact or proximity dependent. The CD8 T cells recovered from these gels exhibit a decrease in Granzyme B expression and an increase in expression of T cell exhaustion marker PD-1. These findings support the conclusion that intra-tumor contact with Tregs during the effector phase of the immune response is responsible for inhibiting anti-melanoma immunity in a TGF-β dependent manner shedding light into novel ways to inhibit intratumoral Tregs.

Blockade of surface bound TGF-β abrogates Treg suppression of effector T cell function within the tumor microenvironment

Regulatory T cells (Treg) play a role in suppression of anti-melanoma immunity; however, the exact mechanism is poorly understood. Through intravital two photon microcopy, we found that tumor-specific Pmel-1 effectors engage in cell-cell interactions with tumor resident Tregs. To determine if contact between Tregs and Teff hinders killing of tumor cells in vivo, we utilized ex-vivo three-dimensional collagen-fibrin gel cultures of B16 melanoma cells. Collagen-fibrin gel cultures recapitulated the in vivo suppression, rendering the dissociated tumor resistant to killing by in vitro activated antigen specific T cells. In vivo depletion of Tregs in Foxp3-DTR mice prior to tumor excision reversed the suppression. In vivo modulation of Tregs by GITR ligation had a similar effect, reducing the number of intra-tumor Tregs leading to ex-vivo tumor killing. Using neutralizing antibodies, we found that blocking TGF-β reversed the suppression. In addition, soluble factors from collagen-fibrin gel tumors do not inhibit killing suggesting that suppression is contact or proximity dependent. The CD8 T cells recovered from these gels exhibit a decrease in Granzyme B expression and an increase in expression of T cell exhaustion marker PD-1. These findings support the conclusion that intra-tumor contact with Tregs during the effector phase of the immune response is responsible for inhibiting anti-melanoma immunity in a TGF-β dependent manner shedding light into novel ways to inhibit intratumoral Tregs. This study was supported by Swim Across America; NIH grants R01CA56821, P01CA33049, and P01CA59350 (to J.W. and A.H.); D.S. and S.B. received support from the NIH/NCI Immunology Training GrantT32 CA09149-30.

TGF-β dependent suppression of effector T cell function within the tumor microenvironment (P2033)

Abstract Regulatory T cells (Treg) play a role in suppression of anti-melanoma immunity; however, the exact mechanism is poorly understood. Through intravital two photon microcopy, we found that Pmel-1 effectors engage in cell-cell interactions with tumor resident Tregs. To determine if contact between Tregs and Teff hinders killing of tumor cells in vivo, we utilized ex-vivo three-dimensional collagen-fibrin gel cultures of B16 melanoma cells. Collagen-fibrin gel cultures recapitulated the in vivo suppression, rendering the dissociated tumor resistant to killing by in vitro activated antigen specific T cells. In vivo depletion of Tregs in foxp3-DTR mice prior to tumor excision reversed the suppression. In vivo modulation of Tregs by GITR ligation had a similar effect, reducing the number of intra-tumor Tregs leading to ex-vivo tumor killing. Using neutralizing antibodies, we found that blocking TGF-β reversed the suppression. In addition, soluble factors from collagen-fibrin gel tumors do not inhibit killing suggesting that suppression is contact or proximity dependent. The CD8 T cells recovered from these gels exhibit a decrease in Granzyme B expression and an increase in expression of T cell exhaustion marker PD-1. These findings support the conclusion that intra-tumor contact with Tregs during the effector phase of the immune response is responsible for inhibiting anti-melanoma immunity in a TGF-β dependent manner shedding light into novel ways to inhibit intratumoral Tregs.

Blockade of Treg derived TGF-β abrogates suppression of effector T cell function within the tumor microenvironment

Regulatory T cells (Treg) pl ay ar ole in suppression of anti-melanoma immunity; however, the exact mechanism is poorly understood. Through intravital two photon microscopy, we found that Pmel-1 effectors engage in cell-cell interactions with tumor resident Tregs. To determine if contact between Tregs and T effectors (Teff) hinders killing of tumor cells in vivo, we utilized ex-vivo three-dimensional collagen-fibrin gel cultures of B16 melanoma cells. Collagen-fibrin gel cultures recapitulated the in vivo suppression, rendering the dissociated tumor resistant to killing by in vitro activated antigen specific Teff. In vivo depletion of Tregs in foxp3-DTR mice prior to tumor excision reversed the suppression. Additionally, In vivo modulation of intra-tumor Tregs suppressive function by GITR ligation had a similar effect, leading to ex-vivo tumor killing. Using neutralizing antibodies, we found that blocking TGF-b reversed the suppression. In addition, soluble factors from collagen-fibrin gel tumors do not inhibit killing suggesting that suppression is contact or proximity dependent. The CD8 Teff recovered from these gels exhibit a decrease in Granzyme B expression and an increase in expression of T cell exhaustion marker PD-1. These findings support the conclusion that intra-tumor contact with Tregs during the effector phase of the immune response is responsible for inhibiting anti-melanoma immunity in a TGF-b dependent manner, elucidating a novel way to target intratumoral Tregs.

A novel anti-EMMPRIN function-blocking antibody reduces T cell proliferation and neurotoxicity: relevance to multiple sclerosis

BackgroundExtracellular matrix metalloproteinase inducer (EMMPRIN; CD147, basigin) is an inducer of the expression of several matrix metalloproteinases (MMPs). We reported previously that blocking EMMPRIN activity reduced neuroinflammation and severity of disease in an animal model of multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE).MethodsTo improve upon EMMPRIN blockade, and to help unravel the biological functions of EMMPRIN in inflammatory disorders, we have developed several anti-EMMPRIN monoclonal antibodies.ResultsOf these monoclonal antibodies, a particular one, clone 10, was efficient in binding mouse and human cells using several methods of detection. The specificity of clone 10 was demonstrated by its lack of staining of EMMPRIN-null embryos compared to heterozygous and wild-type mouse samples. Functionally, human T cells activated with anti-CD3 and anti-CD28 elevated their expression of EMMPRIN and the treatment of these T cells with clone 10 resulted in decreased proliferation and matrix metalloproteinase- 9 (MMP-9) production. Activated human T cells were toxic to human neurons in culture and clone 10 pretreatment reduced T cell cytotoxicity correspondent with decrease of granzyme B levels within T cells. In vivo, EAE mice treated with clone 10 had a markedly reduced disease score compared to mice treated with IgM isotype control.ConclusionsWe have produced a novel anti-EMMPRIN monoclonal antibody that blocks several aspects of T cell activity, thus highlighting the multiple roles of EMMPRIN in T cell biology. Moreover, clone 10 reduces EAE scores in mice compared to controls, and has activity on human cells, potentially allowing for the testing of anti-EMMPRIN treatment not only in EAE, but conceivably also in MS.

Ziram activates mitogen-activated protein kinases and decreases cytolytic protein levels in human natural killer cells

Human natural killer (NK) cells are central in immune defense with their ability to lyse tumor cells and virally infected cells. Tumor formation and viral infection may increase if NK cytotoxic function is disrupted. Ziram (zinc dithiocarbamate) is used as an accelerating agent in the production of latex and to protect various fruits and vegetables from fungal infection. Previously, we have shown that exposure to ziram inhibits NK lytic function. Butyltin environmental contaminants, which also inhibit NK lytic function, cause rapid activations of mitogen-activated protein kinases (MAPKs) and decreases in expression of the cytolytic proteins granzyme B and perforin (after 24 h) in exposed NK cells. MAPKs are important regulators of the lytic response of NK cells, and spurious activation of these enzymes by contaminants would leave the NK cells unable to respond to appropriate targets. This study examined the effects of ziram exposures on MAPKs (p44/42, p38, and c-jun-N-terminal kinase) and on levels of cytolytic proteins. Ten-minute to 6-h exposures of NK cells to ziram caused activation of MAPKs, p44/42, and p38. Exposure to ziram for 24 h caused a decrease in granzyme B and perforin levels. MAPK inhibitors were able to prevent these ziram-induced decreases in granzyme B and perforin. These results suggest that ziram-induced MAPK activation is at least in part responsible for decreased cytolytic function in ziram-exposed NK cells. Furthermore, the results indicate that these changes are in common with other environmental contaminants that have been shown to decrease NK lytic function.

Role of p44/42 activation in TBT‐ induced losses of granzyme B and perforin in human natural killer cells

Natural killer (NK) cells are key players in innate immunity and are responsible for early defense against tumors and viruses. It is well known that both perforin and granzyme B are released from NK cells upon target cell contact. The environmental pollutant, tributyltin chloride (TBT) has been reported to decrease human NK cell lytic function with accompanying activation of p44/42 and decreased levels of granzyme B and perforin. The current studies are designed to examine the role of the mitogen activated protein kinase (MAPK), p44/42, activation in TBT‐induced losses of granzyme B and perforin. p44/42 activation was blocked by the MEK inhibitor, PD98059. NK cells were exposed to PD98059 for 1 h followed by 1 h exposure to 200 or 100 nM TBT. Following the incubation period, NK cells were washed twice with media and incubated for 24 h period in compound‐free media. Western blot analysis using an antibody to granzyme B revealed that PD98059 was able to block 37% of the 200 nM TBT‐induced loss of granzyme B. Moreover, western blot analysis using an antibody to perforin revealed that PD98059 was able to block 42 % of the 200 nM TBT‐induced loss of perforin. Thus our data show a role for TBT‐induced activation of p44/42 in the decreases in granzyme B and perforin in NK cells and that the TBT‐induced p44/42 activation is not solely responsible for the losses of granzyme B and perforin protein levels.

Induced Liver Allograft Immunological Tolerance in Rats by Intramuscular Injection of Recombinant Adeno-Associated Virus–Human Cytotoxic T-lymphocyte-associated Antigen-4 Immunoglobulin (rAAV-hCTLA4Ig)

Induction of liver allograft immunological tolerance was performed in rats by intramuscular injection of recombinant adeno-associated virus-human cytotoxic T-lymphocyte-associated antigen-4 immunoglobulin (rAAV-hCTLA4Ig). Dark Agouti and Lewis rats were liver allograft donors and recipients, respectively, in four groups: (A) syngeneic control, (B) blank control, (C) rAAV-enhanced green fluorescent protein negative control, (D) rAAV-hCTLA4Ig. Gene transfers occurred 6 weeks before transplantation. Group D had a significantly longer liver graft survival time (> 100 days) than groups B (11.9 +/- 1.3 days) and C (11.6 +/- 1.1 days). Groups B and C showed severe rejection responses and large amounts of CD4(+) and CD8(+) T-lymphocyte infiltration, while only a mild response and few T-lymphocytes were observed in group D. There were no significant differences in interleukin-2 and interferon-gamma levels in liver grafts between groups D and C, but there were significant decreases in granzyme B and lymphotoxin beta levels in group D compared with group C. It is concluded that immunological tolerance to liver allograft could be achieved by gene transfer of rAAV-hCTLA4Ig through intramuscular injection.

Enhanced Levels of Costimulation Lead to Reduced Effector/Memory CD8+ T Cell Functionality

The role of different levels of costimulation in conjunction with signal 1 in the activation of memory CD8+ T cells remains elusive. In this study, we demonstrate, in a mouse model with the influenza nucleoprotein epitope NP68, that mouse early memory (effector/memory) CD8+ T cells that were generated with high levels of costimulation have reduced CTL functionality compared with those that were generated with low levels of costimulation. This reduction is associated with increased phosphorylation of the negative regulatory site 292 on Zap70 and a decrease in granzyme B levels. Furthermore, we show that enhanced costimulation reduces proliferation and cytokine production of effector/memory CD8+ T cells in response to intermediate and weak TCR stimulation, in contrast to previously described positive effects of costimulation on naive CD8+ T cells. This effect is associated with the expression of ICAM-1 on APCs. Together, our results indicate that enhanced costimulation can lead to reduced functionality in effector/memory CD8+ T cells. This compromised effector function of effector/memory CD8+ T cells in response to high levels of costimulation can have important implications for designing immunotherapeutic strategies to enhance immune responses.

Effects of interleukins 2 and 12 on the levels of granzyme B and perforin and their mRNAs in tributyltin-exposed human natural killer cells

Natural killer (NK) cells are a subset of lymphocytes that are capable of killing tumor cells, virally infected cells and antibody coated cells. Tributyltin (TBT) is a toxic chemical used for various industrial purposes such as: slime control in paper mills, disinfection of circulating industrial cooling waters, anti-fouling agents, and the preservation of wood. TBT can be found in edible items such as fish. A previous study showed that a 1 h exposure of NK cells to TBT caused persistent inhibition of NK-cell ability to destroy tumor cells in the 24 and 48 h periods following exposure and that this loss of function could be significantly prevented and/or reversed if the NK-stimulatory interleukins (IL) 2 or 12 were present during the 24 and 48 h periods. We had also shown that TBT exposure was able to significantly decrease the protein and mRNA levels of the cytotoxic proteins, granzyme B and perforin, and the phosphorylation of cAMP-response-element-binding protein (CREB) under these conditions. In this study we address the effects of IL-2 and IL-12 on the TBT-induced decreases in NK-cell levels of the cytotoxic proteins, their mRNAs, and CREB phosphorylation. IL-2 appeared to prevent/reverse TBT-induced declines in perforin protein levels and the mRNA for perforin seen in the 24 h period following a 1 h exposure to 300 nM TBT. However, the TBT-induced decreases in the levels of perforin and perforin mRNA seen in the 48 h period following a 1 h exposure to TBT were not statistically significantly prevented/reversed by IL-2. Additionally, the TBT-induced decreases in granzyme B, granzyme B mRNA, and CREB phosphorylation were not statistically significantly reversed by either IL-2 or IL-12 after 24 or 48 h.

Dibutyltin exposure decreases granzyme B and perforin in human natural killer cells

Natural killer (NK) cells are a subset of lymphocytes that are capable of killing tumor and virally-infected cells. Dibutyltin (DBT) is a catalyst in the production of PVC plastics and a breakdown product of tributyltin (TBT). DBT is a significant environmental contaminant. This study investigates the mechanism by which DBT exposure decreases the immune function of human NK cells. NK cells destroy their target cells by releasing cytotoxic proteins, perforin, and granzyme B. We examined the effect of DBT exposures on the levels of cytotoxic proteins and their mRNAs. Exposure of NK cells to DBT for 1 h caused significant decreases in the mRNAs for granzyme B and perforin but not in protein levels. A 24 h exposure to DBT decreased mRNAs as well as protein levels for both granzyme B and perforin. Exposure to DBT for 1 h followed by either a 24 or 48 h period in DBT-free media, decreased levels of granzyme B and perforin. The results indicate that decreases in granzyme B and perforin levels in NK cells are consequences of DBT exposure. Additionally, DBT causes rapid decreases in mRNAs for perforin and granzyme B, suggesting decreases in transcription and/or increases in mRNA degradation.