Evaluation of the Homogenous Mobility Shift Assay for Infliximab and Adalimumab Anti-drug Antibody Detection in the Clinical Laboratory.

BACKGROUND: Detecting antidrug antibodies (ADAs) against infliximab or adalimumab is useful for therapeutic drug monitoring. Various ADA detection methods exist, and antibody titer is an output in some algorithms. Homogenous mobility shift assay (HMSA) measures relative ADA concentration and determines drug-ADA complex size in vitro. However, the relevance of complex size determination in drug monitoring remains unclear. Hence, the association between complex size, ADA concentration, and sample detectable neutralizing activity was evaluated. METHODS: Sera from infliximab-treated and adalimumab-treated patients who tested positive for ADA in the National Screening Service were analyzed using 3 ADA assays. HMSA determined the relative ADA concentrations and complex sizes, competitive ligand-binding assay evaluated the sample neutralizing capacity, and enzyme-linked immunosorbent assay detected immunoglobulin (Ig)G4 ADA. RESULTS: Most ADA-positive samples (>80%) formed drug-ADA dimer complexes, whereas 17% had dimer and multimer complexes, and 3% had multimeric complexes. Multimer presence had 100% positive predictive value for detectable neutralizing activity. ADA concentration and detectable neutralizing activity were moderately correlated (r = 0.65) in adalimumab-treated patients and strongly correlated (r = 0.81) in infliximab-treated patients. In adalimumab-treated patients, multimer presence was a stronger predictor of neutralizing activity than ADA concentration was, but not in infliximab-treated patients. However, in infliximab-treated patient samples, multimer presence revealed a distinct subset with high ADA concentrations, neutralizing activity, and IgG4 ADA. CONCLUSIONS: Multimers detected using HMSA had a strong positive predictive value for competitive ligand-binding assay detectable neutralizing activity. Multimeric IgG4-containing ADA-drug complexes revealed a distinct subset of infliximab-treated patient samples, whose clinical relevance merits further investigation.

Four-year review of New Zealand laboratory infliximab and adalimumab concentration results indicating potential for improved dosing.

A review of laboratory results across New Zealand for therapeutic drug monitoring (TDM) of infliximab and adalimumab concentrations and antidrug antibodies (ADAs) over 4 years was completed. Of 6591 results, the median serum concentration for infliximab was 5.7 mg/L and for adalimumab was 5.5 mg/L. Subtherapeutic drug concentrations (<7 mg/L) were measured in 54% of samples. Drug concentrations <2 mg/L were measured in 23% of samples, with ADAs detected in 51% of these. The high number of samples with subtherapeutic drug concentrations and common ADA detection is consistent with failing therapy but could also suggest that standard dosing is frequently too low for patients. These results reinforce the value of antitumour necrosis factor drug TDM in making decisions to adjust dosing or switch agents in patients taking infliximab and adalimumab.

Levels and in vitro functional effects of circulating anti-hinge antibodies in melanoma patients receiving the immune checkpoint inhibitor pembrolizumab.

The efficacy of PD-1 monoclonals such as pembrolizumab can be modulated by the signals delivered via their Fc region. Tumour/inflammation associated proteases can generate F(ab')2 fragments of therapeutic monoclonals, and subsequent recognition of F(ab')2 epitopes by circulating anti-hinge antibodies (AHA) can then, potentially, link F(ab')2 binding to the target antigen with novel Fc signalling. Although elevated in inflammatory diseases, AHA levels in cancer patients have not been investigated and functional studies utilising the full repertoire of AHA present in sera have been limited. AHA levels in pembrolizumab treated melanoma patients (n = 23) were therefore compared to those of normal donors and adalimumab treated patients. A subset of melanoma patients and the majority of adalimumab patients had elevated levels of AHA reactive with F(ab')2 fragments of IgG4 anti-PD-1 monoclonals (nivolumab, pembrolizumab) and IgG1 therapeutic monoclonals (rituximab, adalimumab). Survival analysis was restricted by the small patient numbers but those melanoma patients with the highest levels (>75% percentile, n = 5) of pembrolizumab-F(ab')2 reactive AHA had significantly better overall survival post pembrolizumab treatment (p = 0.039). In vitro functional studies demonstrated that the presence of AHA+ sera restored the neutrophil activating capacity of pembrolizumab to its F(ab')2 fragment. Neither pembrolizumab nor its F(ab')2 fragments can induce NK cell or complement dependent cytotoxicity (CDC). However, AHA+ sera in combination with pembrolizumab-F(ab')2 provided Fc regions that could activate NK cells. The ability of AHA+ sera to restore CDC activity was more restricted and observed using only one pembrolizumab and one adalimumab patient serum in combination with rituximab- F(ab')2. This study reports the presence of elevated AHA levels in pembrolizumab treated melanoma patients and highlight the potential for AHA to provide additional Fc signaling. The issue of whether tumour associated proteolysis of PD-1 mAbs and subsequent AHA recognition impacts on treatment efficacy requires further study.

Detection of Chitinase 3-Like 1 in Symptomatic Primary Care Patient Faecal Samples is Not a Reliable Biomarker of Colonic Lesions.

BACKGROUND: The current gold standard non-invasive test for detecting pre-cancerous changes is the faecal immunochemical test (FIT). However, this test can lack sensitivity and specificity and testing for another biomarker may address these limitations. Chitinase 3-like 1 (CHI3L1) is emerging as a potential biomarker of inflammation-associated carcinogenic changes in epithelial cells.  In this study CHI3L1 levels were analysed in patients and controls to determine their ability to improve detection of early CRC either alone or in combination with a FIT. METHODS: CHI3L1 levels were measured by ELISA in serum and stool samples from cohorts of CRC and healthy donors as well as stool samples from a cohort of symptomatic primary care patients. Faecal haemoglobin was also analysed in the same primary care samples using FIT. RESULTS: CHI3L1 levels were a good discriminatory marker of CRC, with no significant difference between levels detected in the stool and serum samples.  ROC curves that determined the optimal cut-point however identified that stool samples gave higher sensitivity (83% versus 69%) and specificity (89% versus 74%) than matched serum samples. Faecal CHI3L1 levels in the primary care patients were not significantly different (p=0.193) from those detected in the healthy controls. ROC curve analysis confirmed that faecal CHI3L1 levels had limited ability to discriminate between patients who did or didn't have evidence of lesions (AUC=0.52, p=0.74). Similarly, CHI3L1 levels did not reliably identify those symptomatic primary care patients who subsequently presented with early-stage disease (polyps and adenomas) or CRC. The discriminatory power of FIT was not increased by incorporating the CHI3L1 results in this setting. CONCLUSION: There was no evidence that measurement of faecal CHI3L1 has the potential to increase diagnostic accuracy, either alone or in combination with a FIT, in symptomatic primary care patients.

CD36 down regulation by the macrophage antioxidant 7,8-dihydroneopterin through modulation of PPAR-γ activity.

CD36 is the key scavenger receptor driving the formation of cholesterol-loaded foam cells, the principal cellular component of atherosclerotic plaques. CD36 is down regulated by 7,8-dihydroneopterin, a potent superoxide and hypochlorite scavenging antioxidant generated by interferon-γ stimulated macrophages. 7,8-dihydroneopterin downregulates CD36 mRNA and protein levels so inhibiting macrophage foam cell formation in vitro. We examined the mechanism of 7,8-dihydroneopterin downregulation of CD36 by measuring CD36 and PPAR-γ levels by Western blot analysis, in the monocyte-like U937 cells with a range of PPAR-γ stimulants and inhibitors. Lipoxygenase activity was measured by monitoring linoleic acid oxidation at 234 nm for diene formation. Between 100 and 200 µM, 7,8-dihydroneopterin decreased CD36 levels by 50% within 12 h with levels dropping below 25% by 24 h. CD36 levels returned to basal levels after 24 h. Inhibition of protein synthesis by cycloheximide shows 7,8-dihydroneopterin had no effect on CD36 degradation rates. PPAR-γ levels were not altered by the addition of 7,8-dihydroneopterin. MAP Kinase, P38 and NF-κB pathways inhibitors SP600125, PD98059, SB202190 and BAY 11-7082, respectively, did not restore the CD36 levels in the presence of 7,8-dihydroneopterin. The addition of the lipophilic PPAR-γ activators rosiglitazone and azelaoyl-PAF prevented the CD36 downregulation by 7,8-dihydroneopterin. 7,8-dihydroneopterin inhibited soybean lipoxygenase and reduced U937 cell basal levels of cellular lipid oxides as measured by HPLC-TBARS analysis. The data show 7,8-dihydroneopterin down regulates CD36 expression by decreasing the level of lipid oxide stimulation of PPAR-γ promotor activity, potentially through lipoxygenase inhibition.

Functional effects of immune complexes formed between pembrolizumab and patient-generated anti-drug antibodies.

The PD-1-targeting IgG4 antibody pembrolizumab has significant anti-tumor activity in a proportion of stage IV melanoma patients. A subset of patients develop anti-drug antibodies (ADA) which can form immune complexes (IC) with pembrolizumab. Although IC can induce powerful, Fc-mediated, immune-regulatory effects, their functional impact during pembrolizumab treatment is unclear. The functional effects of IC generated in vitro using pembrolizumab and patient-derived ADA was, therefore, investigated. Screening identified a patient whose trough serum samples from three treatment cycles contained both ADA with neutralizing activity and low levels of pembrolizumab. This patient responded well to therapy over 2 years and had ongoing, infusion-related, hypersensitivity reactions despite the later absence of detectable ADA. The components of IC were mimicked by forming a complex of pembrolizumab by absorption onto a solid phase with or without subsequent exposure to the ADA+ patient sera. Complexes comprised of pembrolizumab alone significantly inhibited TLR4 (LPS)-driven IL-10 production by PBMC and stimulated the generation of reactive oxygen species by granulocytes. In contrast, soluble and solid-phase F(ab´)2 fragments of pembrolizumab had no effect demonstrating the requirement for cross-linked Fc regions. IC containing pembrolizumab and ADA could additionally induce complement and NK activation. The results of this study demonstrate that, when oligomerized, the Fc region of pembrolizumab alone can provide immuno-regulatory signals. Furthermore, IC containing both pembrolizumab and patient-generated ADA can induce additional signals. These Fc-mediated signals may modulate both hypersensitivity reactions and anti-tumor responses associated with pembrolizumab therapy.

Neopterin formation through radical scavenging of superoxide by the macrophage synthesised antioxidant 7,8-dihydroneopterin.

Clinical measurement of neopterin has been extensively used as a marker of inflammation but the in vivo mechanism generating neopterin is poorly understood. Neopterin is described as the oxidation product of 7,8-dihydroneopterin, a potent antioxidant generated by monocyte/macrophages in response to interferon-γ. While peroxyl and hydroxyl scavenging generates dihydroxanthopterin, hypochlorite efficiently oxidises 7,8-dihydroneopterin into neopterin, but this reaction alone does not explain the high levels of neopterin seen in clinical data. Here, we examine whether superoxide scavenging by 7,8-dihydroneopterin generates neopterin. U937 cells incubated with oxLDL showed a time dependent increase superoxide and 7,8-dihydroneopterin oxidation to neopterin. Neopterin generation in oxLDL or phorbol ester treated U937 cells or human monocytes was inhibited by apocynin and PEG-SOD. Addition of the myeloperoxidase inhibitor 4-aminobenzoic acid hydrazide (ABAH) had no effect of the superoxide generation or neopterin formation. 7,8-Dihydroneopterin reacted with superoxide/hydroxy radical mixtures generated by X-ray radiolysis to give neopterin. Formation of neopterin by superoxide derived from the xanthine/xanthine oxidase system was inhibited by superoxide dismutase. Neopterin formation was inhibited by apocynin in phorbol ester treated human carotid plaque rings in tissue culture. These results indicate that 7,8-dihydroneopterin scavenges superoxide and is subsequently oxidised into neopterin in cellular and cell-free experimental systems.

Development of a competitive binding homogeneous mobility shift assay for the quantification of adalimumab levels in patient serum.

Adalimumab is a TNF specific monoclonal widely used therapeutically. Monitoring adalimumab levels is important for guiding treatment strategies and is predominantly performed using an ELISA. The homogeneous mobility shift assay (HMSA) has many advantages over an ELISA for adalimumab monitoring but current HMSA methodologies do not discriminate between adalimumab and other TNF specific monoclonals such as infliximab. The development and validation of a competitive binding HMSA (cHMSA) specific for adalimumab is reported here. The cHMSA had a lower limit of quantitation of 1.25 µg/ml and the intra-assay and inter-assay coefficents of variation (CV) were <20%. No signal was detected in adalimumab naïve control serum including those containing rheumatoid factor or infliximab. The majority (14/20) of adalimumab patient samples containing anti-adalimumab antibodies gave a cHMSA signal >3 standard deviations lower than the controls. The performance of the cHMSA and an ELISA was compared using adalimumab patient samples (n = 82). There was a strong correlation between the assays (r = 0.91) and the intra-class correlation coefficient (0.88) was indicative of good-excellent inter-assay reliability. Bland-Altman plots showed little overall bias and comparison of the sub-groups defined using cut-points (1.25 or 7.3 µg/ml) gave percent agreement (>90%) and Cohens kappa (95% CI: 0.61-0.93) values indicative of substantial-almost perfect agreement. These results demonstrate that cHMSA provides an accurate and specific method for monitoring adalimumab levels and can additionally provide an initial screen for the presence of anti-adalimumab antibodies.

Impact of increased access to novel agents on the survival of multiple myeloma patients treated at a single New Zealand centre.

BACKGROUND: The impact of changes in novel agent (NA) usage on the survival of multiple myeloma (MM) patients in real-world hospital settings is unclear. In New Zealand (NZ) in 2011, frontline bortezomib became available and thalidomide availability was expanded. AIM: This retrospective study analyses the impact these change had on the survival of MM patients treated at a NZ hospital. METHODS: Clinical and overall survival (OS) data were collected on MM patients who were treated at Christchurch Hospital during 2000-2009 (pre-cohort, n = 337) and 2011-2017 (post-cohort, n = 343). Outcomes were compared using pre-cohort data truncated at 2011. RESULTS: Patients in the post-cohort had significant increases (P < 0.001) in not only NA usage (85 vs 55%) and OS (median = 56 vs 44 months) but also the proportion (74 vs 49%) of young patients (age < 70) who received an autologous stem cell transplant (ASCT). Separate analysis of older patients demonstrated that those in the post-cohort had significantly longer OS (median OS 28 vs 17, P < 0.001) although 5-year relative survival remained less than 50%. Separate analysis of young patients demonstrated that those in the post-cohort had significantly increased initial OS with the survival curves converging at 5 years. Although ASCT-treated patients had similar OS in each cohort, their progression-free survival (PFS) was significantly increased in the post-cohort (median 40 vs 20 months, P < 0.0001). CONCLUSION: In the setting of a NZ hospital the increased availability of NA was associated with a significant improvement in both the OS of older patients and the PFS of ASCT patients.

Discrimination of Anti-drug Antibodies With Neutralizing Capacity in Infliximab- and Adalimumab-Treated Patients: Comparison of the Homogeneous Mobility Shift Assay and the Affinity Capture and Elution Assay.

BACKGROUND: The measurement of anti-drug antibody (ADA) levels in adalimumab (ADAL)-treated and infliximab (IFX)-treated patients is critical for guiding therapeutic strategies. The homogeneous mobility shift assay (HMSA) and affinity capture elution (ACE) assay provide effective, drug-tolerant formats for measuring total ADA levels. However, their ability to discriminate between ADA from samples with or without neutralizing capacity is unclear and therefore was analyzed in this study. METHODS: Sera from ADAL and IFX patients with low drug levels (<1 mcg/mL) were analyzed by ACE, HMSA, and bridging assay. Neutralizing capacity was determined by competitive ligand-binding assay. RESULTS: HMSA and ACE detected high ADA levels in all ADAL (19/42) and IFX (27/64) samples with neutralizing capacity. ADA was also detected in most of the samples without neutralizing capacity, but levels were significantly lower (P < 0.0001). Receiver operator characteristic curve analysis demonstrated that for both assays, ADA levels were a strong discriminatory marker of neutralizing ADA (area under the curve > 0.9, P < 0.0001). Using a signal >8× background as a cut-point, neutralizing ADA could be identified with high specificity (HMSA > 95%, ACE > 85%) and sensitivity (HMSA > 70%, ACE > 80%). The detection of multimeric drug-ADA complexes after HMSA was also a highly specific marker (specificity > 95%) of neutralizing ADA in both ADAL and IFX patients. Results using ACE and HMSA were highly correlated. CONCLUSIONS: Results obtained after HMSA and ACE analysis are strongly correlated, and in both assays, high ADA levels are a specific marker of neutralizing capacity. The detection of multimeric complexes by HMSA also selectively identifies sera with neutralizing capacity. These data support the use of these assays as quantitative rather than simple qualitative measures of ADA.

Helicobacter pylori outer membrane vesicles inhibit human T cell responses via induction of monocyte COX-2 expression.

The modulation of T cell responses by Helicobacter pylori is thought to potentiate both H. pylori persistence and development of gastric pathologies including cancer. Release of outer membrane vesicles (OMV) by H. pylori provides a potential vehicle for modulation of the immune system. Although OMV are thought to have T cell suppressive activity, this has not yet been demonstrated. Their suppressive activity was investigated in this study using the responses of peripheral blood mononuclear cells (PBMC) to T cell stimuli as a readout. We demonstrate that addition of OMV to PBMC significantly inhibits subsequent T cell proliferation in a cyclo-oxygenase-2 (COX-2)-dependent manner. Addition of OMV did not significantly modulate PBMC apoptosis, but induced strong expression of COX-2 by the monocytes present and significantly increased levels of PGE2 and IL-10. These effects were independent of vacuolating cytotoxin expression. Together, these findings demonstrate that OMV can suppress human T cell responses and that the predominant mechanism is not through a direct effect on the T cells but results from the induction of COX-2 expression in monocytes. This increased COX-2 activity may modulate not only H. pylori-directed immune responses but also wider immune responses.

Idelalisib and caffeine reduce suppression of T cell responses mediated by activated chronic lymphocytic leukemia cells.

Chronic lymphocytic leukemia (CLL) is associated with T cell dysfunction. Activated CLL cells are found within the lymphoid tumor micro-environment and overcoming immuno-suppression induced by these cells may improve anti-CLL immune responses. However, the mechanisms by which activated CLL cells inhibit T cell responses, and reagents targeting such mechanisms have not been identified. Here we demonstrate that the ability of in vitro activated CLL cells to suppress T cell proliferation is not reversed by the presence of ecto-nuclease inhibitors or blockade of IL-10, PD-1 and CTLA-4 pathways. Caffeine is both an adenosine receptor antagonist and a phosphatidylinositol-3-kinase, p110δ (PI3Kδ) inhibitor and, at physiologically relevant levels, significantly reversed suppression. Significant reversal of suppression was also observed with the PI3Kδ specific inhibitor Idelalisib but not with adenosine receptor specific antagonists. Furthermore, addition of caffeine or Idelalisib to activated CLL cells significantly inhibited phosphorylation of AKT, a downstream kinase of PI3K, but did not affect CLL viability. These results suggest that caffeine, in common with Idelalisib, reduces the immuno-suppressive activity of activated CLL cells by inhibiting PI3Kδ. These findings raise the possibility that these compounds may provide a useful therapeutic adjunct by reducing immuno-suppression within the tumor micro-environment of CLL.

Effect of 7,8-dihydroneopterin mediated CD36 down regulation and oxidant scavenging on oxidised low-density lipoprotein induced cell death in human macrophages.

The role of CD36 in oxidised low-density lipoprotein (oxLDL) mediated cell death was examined by down regulating the receptor level with the macrophage generated antioxidant 7,8-dihydroneopterin. Down regulation of CD36 protein levels in human monocyte derived macrophages by 7,8-dihydroneopterin corresponded to a decrease in CD36-mRNA. The oxidation products of 7,8-dihydroneopterin, dihydroxanthopterin and neopterin did not significantly down regulate CD36. The CD36 down regulation resulted in a decrease in oxLDL uptake measured as 7-ketocholesterol accumulation. Though less oxLDL was taken up by the macrophages as a result of the 7,8-dihydroneopterin induced down regulation in CD36 levels, the cytotoxicity of the oxLDL was not decreased. Addition of 7,8-dihydroneopterin to oxLDL treated macrophages decreased the concentration of intracellular oxidants. In the presence of oxLDL, 7,8-dihydroneopterin was oxidised to neopterin showing that the 7,8-dihydroneopterin was scavenging intracellular oxidants generated in response to the oxLDL. The results show CD36 down regulation does not protect human macrophages form oxLDL cytotoxicity but 7,8-dihydroneopterin intracellular oxidant scavenging is protective.

Development of an ELISA-Based Competitive Binding Assay for the Analysis of Drug Concentration and Antidrug Antibody Levels in Patients Receiving Adalimumab or Infliximab.

BACKGROUND: There is increasing interest in measuring both drug and antidrug antibody (ADA) levels in patients receiving anti-tumor necrosis factor treatment as part of algorithms for guiding therapeutic strategies. Many of the current assays for ADA detection have limitations with respect to specificity, sensitivity, and/or laboratory requirements. Specific identification of ADA based on their inhibitory activity in a simple competitive binding assay remains problematic. The development of an enzyme-linked immunosorbent assay (ELISA)-based method for detection of both drug and ADA in patients receiving either adalimumab or infliximab would widen availability of monitoring for these patients. METHODS: An ELISA for the specific detection of adalimumab and infliximab using widely available reagents was developed. A simple modification for the detection of ADA capable of competitively inhibiting the in vitro binding of drug to solid phase tumor necrosis factor was also developed. Drug and ADA levels were analyzed in patients with rheumatoid arthritis and inflammatory bowel disease. RESULTS: The ELISA specifically detected drug concentrations in patient sera with no evidence of positive or negative interference by rheumatoid factor positive control sera. A subset of those patients with low drug concentrations had detectable levels of ADA with inhibitory activity in a competitive binding assay. Spiking with both drugs confirmed the specificity of the ADA detected. CONCLUSIONS: A modified ELISA protocol can be used to for the detection of both drug concentrations and ADA in patients receiving either adalimumab or infliximab. The ELISA incorporates those features identified in the literature as important for the accurate analysis of these antibodies and uses laboratory facilities and reagents that are widely available. It therefore provides a relatively simple and low cost assay for therapeutic drug monitoring of inpatients receiving adalimumab or infliximab.

Dynamic changes in myeloid derived suppressor cell subsets following renal transplant: A prospective study.

BACKGROUND: Myeloid-derived suppressor cells (MDSC) are powerful suppressors of immune responses. MDSC numbers are increased in some renal transplant recipients (RTR) and there is increasing interest in their potential role in promoting both transplant tolerance and transplant associated malignancy. However the factors influencing MDSC mobilisation are unknown, and the relative temporal changes in the granulocytic (G-MDSC) and monocytic (Mo-MDSC) subsets of MDSC have not been defined. METHODS: The circulating frequencies of MDSC and dendritic cells (DC) were analysed by multicolour flow cytometry in RTR (n = 8) prior to transplant and at regular intervals out to 1 year post-transplant. RESULTS: In RTRs, numbers of both G-MDSC and Mo-MDSC increased rapidly following transplant and peaked within 8 days, whilst DC numbers decreased. A second peak in G-MDSC numbers was observed in 7/8 patients within 3 months and 2 patients had a further 3rd peak in G-MDSC numbers which, in one patient, corresponded to a rejection event. Mo-MDSC numbers underwent less fluctuation and a subsequent peak in numbers was observed in only 2/8 patients. Respective kidney donors (n = 5) underwent only small transient increases in MDSC following surgery. Overall, there was little correlation between increases in MDSC and the occurrence of detectable clinical events or treatment changes. CONCLUSIONS: In RTRs, MDSC numbers increase rapidly and peak following commencement of immunosuppression, but then fluctuate in response to as yet undefined stimuli. Further studies are required to identify the factors modulating MDSC mobilisation.

Suppression of CD3/CD28 antibody stimulated responses by human granulocytic myeloid-derived suppressor cells: fact or artefact?

T cell responses to CD3/CD28 antibodies are widely used to demonstrate the immunosuppressive activity of added human granulocytic myeloid-derived suppressor cells (G-MDSC). Granulocytic populations have the well established capability to chemically modify antibody structure and/or, phagocytose stimulatory CD3/CD28 antibody coated beads. However the possibility that the suppression observed in CD3/CD28 antibody based assays may result from the effects of the G-MDSC on the stimulatory antibodies rather than the T cells is not routinely controlled for experimentally. In the absence of controls to evaluate potential antibody associated artefacts considerable caution should be applied to the use and interpretation of this assay system as a means of defining suppressive G-MDSC populations.

Effect of activated human polymorphonuclear leucocytes on T lymphocyte proliferation and viability.

Human polymorphonuclear leucocytes (PMN) are thought to be immunosuppressive. The suppressive mechanism(s) used by PMN are, however, not well defined and in this study they were analysed using T-cell responses to CD3(+) CD28 monoclonal antibodies (mAb) as a readout. We demonstrate that in vitro activated PMN (PMN(act)) can, without any T-cell interaction, induce apparent T-cell suppression by inhibiting the stimulatory capacity of the CD3 mAb. However, a cell-directed suppression of T-cell proliferation was observed when PMN(act) were added to pre-activated T cells that are already committed to polyclonal proliferation. This suppression was partially reversed by catalase addition (P < 0·01) and largely reversed by addition of exogenous interleukin-2 (P < 0·001) but was not significantly reduced by nitric oxide synthase inhibition, myeloperoxidase inhibition or addition of excess arginine. Following removal of PMN(act) , suppressed T cells could respond normally to further stimulation. In addition to suppressing proliferation, co-culture with PMN(act) also induced a significant decrease in T-cell viability that was reversed by catalase addition (P < 0·05). The addition of the arginase inhibitor N-hydroxy-nor-l-arginine induced both a further significant, catalase-sensitive, loss in T-cell viability and increased nitrite release (P < 0·001). These data demonstrate that PMN, when activated, can both induce T-cell death and reversibly inhibit proliferation of activated T cells. The mechanisms underlying these distinct processes and the effects of arginase inhibitors on PMN induced cytotoxicity merit further investigation.

Renal transplant recipients have elevated frequencies of circulating myeloid-derived suppressor cells.

BACKGROUND: Cancer, particularly cutaneous squamous cell carcinoma (SCC), is a major cause of mortality in renal transplant recipients (RTRs). Myeloid-derived suppressor cells (MDSC) play a central role in suppressing cancer immunosurveillance but their potential mobilisation in RTRs and levels relative to those of other immunoregulatory dendritic cell (DC) populations have not been analysed. METHODS: The circulating frequencies of MDSC and DC were analysed by multicolour flow cytometry in immunocompetent patients without (n = 13) or with (ICI-SCC(Pos), n = 14) current SCC, normal donors (NDs, n = 34), chronic kidney disease patients (CKD patients, n = 22) and RTRs (n = 31). RESULTS: Compared to NDs, RTRs had significantly elevated levels of both CD14(Neg) and CD14(Pos) MDSC subsets (P < 0.001), while CKD patients and ICI-SCC(Pos) had significantly elevated levels of only the CD14(Neg)-MDSC subset. DC frequencies were significantly decreased in RTRs and CKD patients but were at normal levels in ICI-SCC(Pos). The MDSC/DC ratio was significantly elevated (P < 0.05) in RTRs (median = 5.7), CKD patients (median = 3.2) and ICI-SCC(Pos) (median = 3.5) relative to NDs (median = 0.7). The use of immunosuppressive drugs in CKD patients and past/current occurrence of SCC in RTRs was associated with significantly increased CD14(Neg)-MDSC frequencies. MDSC enriched from RTRs, when co-cultured with activated NDs T cells significantly suppressed extracellular IL-10 levels and can, when activated with formyl-methionyl-leucyl-phenylalanine, inhibit T-cell proliferation. CONCLUSIONS: RTRs, CKD patients and ICI-SCC(Pos) have increased MDSC frequencies and MDSC/DC ratios. These changes may impact on cancer immunosurveillance. Therefore, MDSC represent both a potential therapeutic target and prognostic marker in these patients, with respect to the development of SCC and other malignancies.