It has been noted by the World Health Organisation that cases of tuberculosis in 2022 globally numbered 10.6 million, resulting in 1.3 million deaths, such that TB is one of the infectious diseases causing the greatest morbidity and mortality worldwide. Since as early as 1918, there has been an ongoing debate as to the relationship between cigarette smoking and TB. However, numerous epidemiological studies, as well as meta-analyses, have indicated that both active and passive smoking are independent risk factors for TB infection, development of reactivation TB, progression of primary TB, increased severity of cavitary disease, and death from TB, among several other considerations. With this considerable body of evidence confirming the association between smoking and TB, it is not surprising that TB control programmes represent a key potential preventative intervention. In addition to coverage of the epidemiology of TB and its compelling causative link with smoking, the current review is also focused on evidence derived from clinical- and laboratory-based studies of disease pathogenesis, most prominently the protective anti-mycobacterial mechanisms of the alveolar macrophage, the primary intracellular refuge of M. tuberculosis. This section of the review is followed by an overview of the major strategies utilised by the pathogen to subvert these antimicrobial mechanisms in the airway, which are intensified by the suppressive effects of smoke inhalation on alveolar macrophage function. Finally, consideration is given to a somewhat under-explored, pro-infective activity of cigarette smoking, namely augmentation of antibiotic resistance due to direct effects of smoke per se on the pathogen. These include biofilm formation, induction of cellular efflux pumps, which eliminate both smoke-derived toxicants and antibiotics, as well as gene modifications that underpin antibiotic resistance.

Introduction: Increasing drug resistance and the absence of a cure necessitates exploration of novel treatment strategies for people living with HIV (PLWH). Targeting of soluble co-inhibitory immune checkpoint molecules (sICMs) represents a novel, potentially effective strategy in the management of HIV. Methods: In this retrospective, longitudinal, observational study, the plasma levels of five prominent co-inhibitory sICMs—CTLA-4, LAG-3, PD-1 and its ligand PD-L1, as well as TIM-3—were quantified in 68 PLWH—before and one year after antiretroviral therapy (ART)—and compared with those of 15 healthy control participants. Results: Relative to control participants, PLWH had substantially elevated pre-treatment levels of all five co-inhibitory sICMs (p < 0.0001–p < 0.0657), which, over the 12-month period of ART, remained significantly higher than those of controls (p < 0.0367–p < 0.0001). PLWH with advanced disease, reflected by a CD4+ T cell count <200 cells/mm3 before ART, had the lowest levels of CTLA-4 and LAG-3, while participants with pre-treatment HIV viral loads ≥100,000 copies/mL had higher pre-treatment levels of TIM-3, which also persisted at 12 months. Conclusions: Plasma levels of CTLA-4, LAG-3, PD-1, PD-L1 and TIM-3 were significantly elevated in treatment-naïve PLWH and remained so following one year of virally-suppressive ART, possibly identifying LAG-3 and TIM-3 in particular as potential targets for adjuvant immunotherapy.

The recent global resurgence of severe infections caused by the Group A streptococcus (GAS) pathogen, Streptococcus pyogenes, has focused attention on this microbial pathogen, which produces an array of virulence factors, such as the pore-forming toxin, streptolysin O (SOT). Importantly, the interactions of SOT with human neutrophils (PMN), are not well understood. The current study was designed to investigate the effects of pretreatment of isolated human PMN with purified SOT on several pro-inflammatory activities, including generation of reactive oxygen species (ROS), degranulation (elastase release), influx of extracellular calcium (Ca2+) and release of extracellular DNA (NETosis), using chemiluminescence, spectrophotometric and fluorimetric procedures, respectively. Exposure of PMN to SOT alone caused modest production of ROS and elastase release, while pretreatment with the toxin caused significant augmentation of chemoattractant (fMLP)-activated ROS generation and release of elastase by activated PMN. These effects of treatment of PMN with SOT were associated with both a marked and sustained elevation of cytosolic Ca2+(2+)concentrations and significant increases in the concentrations of extracellular DNA, indicative of NETosis. The current study has identified a potential role for SOT in augmenting the Ca2+-dependent pro-inflammatory interactions of PMN, which, if operative in a clinical setting, may contribute to hyper-activation of PMN and GAS-mediated tissue injury.

Aside from their key protective roles in hemostasis and innate immunity, platelets are now recognized as having multifaceted, adverse roles in the pathogenesis, progression and outcome of many types of human malignancy. The most consistent and compelling evidence in this context has been derived from the notable association of elevated circulating platelet counts with the onset and prognosis of various human malignancies, particularly lung cancer, which represents the primary focus of the current review. Key topics include an overview of the association of lung cancer with the circulating platelet count, as well as the mechanisms of platelet-mediated, pro-tumorigenic immunosuppression, particularly the role of transforming growth factor beta 1. These issues are followed by a discussion regarding the pro-tumorigenic role of platelet-derived microparticles (PMPs), the most abundant type of microparticles (MPs) in human blood. In this context, the presence of increased levels of PMPs in the blood of lung cancer patients has been associated with tumor growth, invasion, angiogenesis and metastasis, which correlate with disease progression and decreased survival times. The final section of the review addresses, firstly, the role of cancer-related platelet activation and thrombosis in the pathogenesis of secondary cardiovascular disorders and the associated mortality, particularly in lung cancer, which is second only to disease progression; secondly, the review addresses the potential role of antiplatelet agents in the adjunctive therapy of cancer.

e14546 Background: Checkpoint proteins regulate the immune system. Breast cancer is associated with up-regulation or down-regulation of these proteins to evade anti-tumor immune responses. Soluble forms of immune checkpoint molecules (sICMs) can be measured in human plasma. The study aimed to measure the systemic levels of a series of co-stimulatory and co-inhibitory ICMs at diagnosis, post-neo-adjuvant chemotherapy (NAC) and post-surgery in newly- diagnosed BC patients (pts) and compare the biological behaviour of these sICMs between TNBC and non-TNBC patients. Methods: Soluble ICMs were measured using multiplex bead array technology in plasma from 72 BC pts and 45 healthy controls. Data were prospectively obtained and levels were compared between pre-treatment, post-NAC, and post-surgery using non-parametric tests (Mann-Whitney & Kruskal-Wallis). Results: Following NAC, the plasma levels of six soluble co-stimulatory checkpoints (CD28, CD40, ICOS, CD27, CD80, GITR), all involved in the activation of CD8+ cytotoxic T cells, were significantly increased (p < 0.04-p < 0.00001). Four of the soluble co-inhibitory checkpoints (LAG-3, PDL1, TIM-3 and HVEM) increased significantly post-NAC, reaching levels significantly greater than those of the control group. PD-1 remained unchanged, while BTLA and CTLA-4 decreased significantly (p < 0.03 and p < 0.00001, respectively). Normalization of soluble co-stimulatory immune checkpoints seemingly indicates a reversal of systemic immune dysregulation following the administration of NAC in early BC. In contrast, recovery of immune homeostasis may explain the increased levels of several negative checkpoint proteins, albeit with the exceptions of CTLA-4 and PD-1. No significant differences were detected between the changes of 16 sICMs levels at pre-NAC, post-NAC and post-surgery when comparing the TNBC and non-TNBC patients. Conclusions: This study demonstrates low levels of co-stimulatory and co-inhibitory sICMs in newly-diagnosed, non-metastatic BC pts. Following treatment with NAC, the sICMs levels increase substantially, except CTLA-4. In the case of co-stimulatory sICMs, these novel findings are indicative of an immune-restorative mechanism. The pattern of co-inhibitory sICMs (elevated levels of PD-L1, LAG-3, TIM-3 and HVEM), might be indicative of immune-therapeutic resistance, underscoring the augmentative immune-therapeutic promise of targeting these molecules, either individually or in combination, to improve the outcome of pts with early BC. These differences were not significant between TNBC and non-TNBC patients.

Neoadjuvant chemotherapy (NAC) may alter the immune landscape of patients with early breast cancer (BC), potentially setting the scene for more effective implementation of checkpoint-targeted immunotherapy. This issue has been investigated in the current study in which alterations in the plasma concentrations of 16 soluble co-stimulatory and co-inhibitory, immune checkpoints were measured sequentially in a cohort of newly diagnosed, early BC patients (n=72), pre-treatment, post-NAC and post-surgery using a Multiplex® bead array platform. Relative to a group of healthy control subjects (n=45), the median pre-treatment levels of five co-stimulatory (CD27, CD40, GITRL, ICOS, GITR) and three co-inhibitory (TIM-3, CTLA-4, PD-L1) soluble checkpoints were significantly lower in the BC patients vs. controls (p<0.021-p<0.0001; and p<0.008-p<0.00001, respectively). Following NAC, the plasma levels of six soluble co-stimulatory checkpoints (CD28, CD40, ICOS, CD27, CD80, GITR), all involved in activation of CD8+ cytotoxic T cells, were significantly increased (p<0.04-p<0.00001), comparable with control values and remained at these levels post-surgery. Of the soluble co-inhibitory checkpoints, three (LAG-3, PD-L1, TIM-3) increased significantly post-NAC, reaching levels significantly greater than those of the control group. PD-1 remained unchanged, while BTLA and CTLA-4 decreased significantly (p<0.03 and p<0.00001, respectively). Normalization of soluble co-stimulatory immune checkpoints is seemingly indicative of reversal of systemic immune dysregulation following administration of NAC in early BC, while recovery of immune homeostasis may explain the increased levels of several negative checkpoint proteins, albeit with the exceptions of CTLA-4 and PD-1. Although a pathological complete response (pCR) was documented in 61% of patients (mostly triple-negative BC), surprisingly, none of the soluble immune checkpoints correlated with the pCR, either pre-treatment or post-NAC. Nevertheless, in the case of the co-stimulatory ICMs, these novel findings are indicative of the immune-restorative potential of NAC in early BC, while in the case of the co-inhibitory ICMs, elevated levels of soluble PD-L1, LAG-3 and TIM-3 post-NAC underscore the augmentative immunotherapeutic promise of targeting these molecules, either individually or in combination, as a strategy, which may contribute to the improved management of early BC.

Background Electronic cigarettes (ECs) are electronic aerosol delivery systems composed of nicotine and various chemicals, which are widely used to facilitate smoking cessation. Although ECs are considered safer than cigarettes, they do, however, contain chemical toxicants, some of which may interact with cells of the host's innate immune system of which neutrophils constitute a key component. Methods The current study was designed to compare the effects of aqueous EC aerosol extracts (ECEs; with or without nicotine) with those of cigarette smoke extract (CSE) on neutrophil and platelet reactivity in vitro. Neutrophil reactivity is characterised by the generation of reactive oxygen species (ROS), degranulation (elastase release) and the release of extracellular DNA (neutrophil extracellular trap (NET) formation: NETosis), which were measured using chemiluminescence, spectrophotometric and microscopic procedures, respectively. Platelet reactivity was measured according to the magnitude of upregulated expression of the adhesion molecule CD62P on activated cells using a flow cytometric procedure. Results Exposure of neutrophils to either ECEs or CSE caused a significant inhibition of ROS generation and elastase release by N-formyl-l-methionyl-l-leucyl-l-phenylalanine (1 µM)-activated neutrophils. Pre-treatment of neutrophils with CSE also resulted in a marked attenuation of phorbol 12-myristate 13-acetate (6.25 nM)-mediated release of extracellular DNA, which was unaffected by the ECEs. Similarly, CSE, but not the ECEs, inhibited the expression of CD62P by platelets activated with ADP (100 µM). Conclusions These observations suggest that ECE aerosols may inhibit some of the immuno-protective activities of neutrophils such as ROS production and elastase release by activated cells, the effect of which was not enhanced by inclusion of nicotine. The inhibitory effects of CSE were significantly more pronounced than those of ECEs, especially so for suppression of NET formation and platelet activation. If operative in vivo, these harmful immunosuppressive effects of ECEs may compromise intrinsic pulmonary antimicrobial defence mechanisms, albeit less so than cigarette smoke.

Background: Checkpoint proteins regulate the immune system. Breast cancer (BC) cells can up-regulate or down-regulate these proteins to evade anti-tumor immune responses. Soluble forms of immune checkpoint molecules (ICMs) can be measured in human plasma. The study aimed to measure the systemic levels of a series of positive and negative ICMs at diagnosis, post-neo-adjuvant chemotherapy (NAC) and post-surgery in newly- diagnosed BC patients (pts) relative to those of a healthy control group. Method: Soluble ICMs were measured using Multiplex® bead array technology in plasma from 72 BC patients and 45 healthy controls. Data was prospectively obtained and levels compared between pre-treatment, post-NAC, and post-surgery using non-parametric tests (Mann-Whitney & Kruskal-Wallis). Results: Pre-treatment, soluble stimulatory molecules viz. GITR (p<0.0000), GITRL (p< 0.0199), CD27 (p< 0.0243), CD40 (p< 0.0209), ICOS (p< 0.0087), as well as the inhibitory molecules PD-L1 (p< 0.0000), CTLA-4 (p< 0.005), TIM-3 (p< 0.0004), HVEM (p< 0.0004) levels were significantly lower in early BC pts compared to controls. Post treatment, there were significant increases in most ICM levels (Table 1), with the exception of CTLA-4 which decreased significantly following treatment. A pCR was documented in 65% of patients (mostly TNBC). There was no correlation between pre-treatment ICM levels and pCR. Conclusion: We identified low levels of stimulatory and inhibitory ICMs in newly-diagnosed, non-metastatic BC patients compared to healthy controls. Following treatment, with the exception of CTLA-4, most of these pre-treatment abnormalities of systemic ICM levels corrected. NAC is associated with upregulation of sPD-L1 and most other ICMs. These results indicate that early BC is associated with down-regulation of soluble stimulatory and inhibitory ICMs. Newly-diagnosed early BC patients appear to have generalized immune-suppression independent of subtype and stage. To our knowledge, this is the first study to describe the effect of treatment on systemic ICMs in early BC patients. Table 1.The effect of treatment on soluble, systemic ICMsICMControlDiagnosis (Group A)Post-NAC (Group B)Post-surgery (Group C) Group A vs Group BComparing pre-treatment levels vs post-treatment levelsMedian (pg/ml)p-valueBTLA18147130229987127770,0366CD8023291678304836110,0000CD8614297115859922124390,2789CTLA-4261815665986870,0000LAG31504161312754648805001330,0000PD-L133421647479452150,0000PD-1149171230513350150760,8888TIM350473897997596150,0000CD2745773342535154270,0000CD28461353291444277500580,0415CD4019771523203020540,0002GITR37971497403544340,0000GITRL71515886533959270,4938ICOS265061512326586297460,0001HVEM22901865404739500,0000TLR2304772683133837370420,0257 Citation Format: Bernardo Leon Rapoport, Helen C Steel, Carol Ann Benn, Simon Nayler, Teresa Smit, Liezl Heyman, Annette Theron, Nomsa Hlatswayo, Luyanda Kwofie, Pieter Meyer, Ronald Anderson. Dysregulation of immune checkpoint proteins in newly- diagnosed early breast cancer patients [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-04-11.

Dolutegravir is a highly potent HIV integrase strand transfer inhibitor that is recommended for first-line anti-retroviral treatment in all major treatment guidelines. A recent study has shown that people taking this class of anti-retroviral treatment have a substantially higher risk of early-onset cardiovascular disease, a condition shown previously to be associated with increased platelet reactivity. To date, few studies have explored the effects of dolutegravir on platelet activation. Accordingly, the current study was undertaken with the primary objective of investigating the effects of dolutegravir on the reactivity of human platelets in vitro. Platelet-rich plasma, isolated platelets, or buffy coat cell suspensions prepared from the blood of healthy adults were treated with dolutegravir (2.5–10 µg/ml), followed by activation with adenosine 5’-diphosphate (ADP), thrombin, or a thromboxane A2 receptor agonist U46619. Expression of platelet CD62P (P-selectin), formation of heterotypic neutrophil:platelet aggregates, and calcium (Ca2+) fluxes were measured using flow cytometry and fluorescence spectrometry, respectively. Dolutegravir caused dose-related potentiation of ADP-, thrombin- and U46619-activated expression of CD62P by platelets, as well as a significant increases in formation of neutrophil:platelet aggregates. These effects were paralleled by a spontaneous, receptor-independent elevation in cytosolic Ca2+ that appears to underpin the mechanism by which the antiretroviral agent augments the responsiveness of these cells to ADP, thrombin and U46619. The most likely mechanism of dolutegravir-mediated increases in platelet cytosolic Ca2+ relates to a combination of lipophilicity and divalent/trivalent metal-binding and/or chelating properties of the anti-retroviral agent. These properties are likely to confer ionophore-type activities on dolutegravir that would promote movement of Ca2+ across the plasma membrane, delivering the cation to the cytosol where it would augment Ca2+-dependent intracellular signaling mechanisms. These effects of dolutegravir may lead to hyper-activation of platelets which, if operative in vivo, may contribute to an increased risk for cardiometabolic co-morbidities.

Breast cancer cells exploit the up-regulation or down-regulation of immune checkpoint proteins to evade anti-tumor immune responses. To explore the possible involvement of this mechanism in promoting systemic immunosuppression, the pre-treatment levels of soluble co-inhibitory and co-stimulatory immune checkpoint molecules, as well as those of cytokines, chemokines, and growth factors were measured in 98 newly diagnosed breast cancer patients and compared with those of 45 healthy controls using multiplex bead array and ELISA technologies. Plasma concentrations of the co-stimulatory immune checkpoints, GITR, GITRL, CD27, CD28, CD40, CD80, CD86 and ICOS, as well as the co-inhibitory molecules, PD-L1, CTLA-4 and TIM-3, were all significantly lower in early breast cancer patients compared to healthy controls, as were those of HVEM and sTLR-2, whereas the plasma concentrations of CX3CL1 (fractalkine), CCL5 (RANTES) and those of the growth factors, M-CSF, FGF-21 and GDF-15 were significantly increased. However, when analyzed according to the patients' breast cancer characteristics, these being triple negative breast cancer (TNBC) vs. non-TNBC, tumor size, stage, nodal status and age, no significant differences were detected between the plasma levels of the various immune checkpoint molecules, cytokines, chemokines and growth factors. Additionally, none of these biomarkers correlated with pathological complete response. This study has identified low plasma levels of soluble co-stimulatory and co-inhibitory immune checkpoint molecules in newly diagnosed, non-metastatic breast cancer patients compared to healthy controls, which is a novel finding seemingly consistent with a state of systemic immune dysregulation. Plausible mechanisms include an association with elevated levels of M-CSF and CCL5, implicating the involvement of immune suppressor cells of the M2-macrophage/monocyte phenotype as possible drivers of this state of systemic immune quiescence/dysregulation.

Background Neoadjuvant chemotherapy (NAC) may alter the immune landscape of patients with early breast cancer (BC), potentially setting the scene for more effective implementation of checkpoint-targeted immunotherapy, albeit by largely unexplored mechanisms. Methods This study investigated which alterations in the plasma concentrations of 16 soluble co-stimulatory and co-inhibitory, immune checkpoints were measured in a cohort of newly diagnosed, early BC patients (n=72) pre-treatment, post-NAC, and post-surgery using a Multiplex® bead array platform. Results Relative to a group of healthy control subjects (n=45), the median pre-treatment levels (table 1) of 5 co-stimulatory (CD27, CD40, GITRL, ICOS, GITR) and 3 co-inhibitory (TIM-3, CTLA-4, PD-L1) soluble checkpoints were significantly lower in the BC patients vs. controls (p<0.021—p<0.0001; and p<0.008—p<0.00001, respectively). Following NAC (table 2), the plasma levels of 6 soluble co-stimulatory checkpoints (CD28, CD40, ICOS, CD27, CD80, GITR), all involved in activation of CD8+ cytotoxic T cells, were significantly increased relative to pre-treatment levels (p<0.04—p<0.00001), being comparable with control values and remaining at these levels post-surgery (table 3). Of the soluble co-inhibitory checkpoints, 3 (LAG-3, PD-L1, TIM-3) increased significantly post-NAC, while PD-1 was unchanged and BTLA and CTLA-4 were significantly decreased (p<0.03 and p<0.00001, respectively). A pathological complete response was documented in 65% of patients (mostly TNBC). Conclusions Normalization of soluble co-stimulatory immune checkpoints is seemingly indicative of reversal of systemic immune dysregulation following administration of NAC in early BC, independent of response to treatment, while recovery of immune homeostasis may explain the increased levels of several negative checkpoint proteins, albeit with the exceptions of CTLA-4 and PD-1. Ethics Approval Ethics approval was granted by The Research Ethics Committee, Faculty of Health Sciences, University of Pretoria (Ethics Committee Approval Numbers 517/2017.

There is increasing awareness of an association between the uptake of the HIV integrase inhibitor, dolutegravir, in first-line antiretroviral regimens with unusual weight gain and development of the metabolic syndrome, particularly in African women. Although seemingly unexplored, the development of systemic inflammation linked to the putative pro-inflammatory activity of dolutegravir represents a plausible pathophysiological mechanism of this unusual weight gain. This possibility was explored in the current study undertaken to investigate the effects of dolutegravir (2.5–20 μg/mL) on several pro-inflammatory activities of neutrophils isolated from the blood of healthy, adult humans. These activities included the generation of reactive oxygen species (ROS), degranulation (elastase release) and alterations in the concentrations of cytosolic Ca2+ using chemiluminescence, spectrophotometric and fluorimetric procedures, respectively. Exposure of neutrophils to dolutegravir alone resulted in the abrupt, dose-related, and significant (p < 0.0039–p < 0.0022) generation of ROS that was attenuated by the inclusion of the Ca2+-chelating agent, EGTA, or inhibitors of NADPH oxidase (diphenyleneiodonium chloride, DPI), phospholipase C (U733122), myeloperoxidase (sodium azide) and phosphoinositol-3-kinase (wortmannin). In addition, exposure to dolutegravir augmented the release of elastase by stimulus-activated neutrophils. These pro-inflammatory effects of dolutegravir on neutrophils were associated with significant, rapid, and sustained increases in the concentrations of cytosolic Ca2+ that appeared to originate from the extracellular compartment, seemingly consistent with an ionophore-like property of dolutegravir. These findings are preliminary and necessitate verification in the clinical setting of HIV infection. Nevertheless, given the complex link between inflammation and obesity, these pro-inflammatory interactions of dolutegravir with neutrophils may contribute to unexplained weight gain, possibly via the development of insulin resistance.

Systemic biomarkers of inflammation, including cytokines and chemokines, are potentially useful in the management of both HIV infection and non-AIDS-defining disorders. However, relatively little is known about the utility of measurement of circulating biomarkers of platelet activation as a strategy to monitor the efficacy of combination antiretroviral therapy (cART), as well as the persistence of systemic inflammation following virally-suppressive therapy in HIV-infected persons. These issues have been addressed in the current study to which a cohort consisting of 199 HIV-infected participants was recruited, 100 of whom were cART-naïve and the remainder cART-treated and virally-suppressed. Fifteen healthy control participants were included for comparison. The study focused on the effects of cART on the responsiveness of three biomarkers of platelet activation, specifically soluble CD40 ligand (sCD40L), sCD62P (P-selectin), and platelet-derived growth factor-BB (PDGF-BB), measured using multiplex suspension bead array technology. Most prominently sCD40L in particular, as well as sCD62P, were significantly elevated in the cART-naïve group relative to both the cART-treated and healthy control groups. However, levels of PDGF-BB were of comparable magnitude in both the cART-naïve and –treated groups, and significantly higher than those of the control group. Although remaining somewhat higher in the virally-suppressed group relative to healthy control participants, these findings identify sCD40L, in particular, as a potential biomarker of successful cART, while PDGF-BB may be indicative of persistent low-level antigenemia.

556 Background: Checkpoint proteins regulate the immune system. Breast cancer (BC) cells exploit the up-regulation or down-regulation of these proteins to evade anti-tumor immune responses. Soluble forms of immune checkpoint molecules (ICM) can be measured in human plasma, however their biological and clinical significance remains essentially unknown. The aim of the present analysis was to measure the pre-treatment ICM in newly- diagnosed BC patients (pts) and compare them to healthy controls. Methods: Soluble forms of ICM, as well as cytokines and chemokines, were measured using Multiplex bead array and ELISA technologies. Plasma samples from 98 BC pts and 45 healthy controls were analyzed for each protein. Data was prospectively obtained. Measured levels were compared between BC pts and healthy controls using a non-parametric test (Mann-Whitney). P-values below 0.05 were considered statistically significant. Results: Soluble stimulatory molecules GITR (p < 0,000002), GITRL (p < 0,007), CD27 (p < 0,002), CD28 (p < 0,003), CD40 (p < 0,003), CD80 (p < 0,009), ICOS (p < 0,0006), as well as inhibitory molecules PD-L1 (p < 0.0000001), CTLA-4 (p < 0,005), TIM-3 (p < 0,00006), HVEM (p < 0,00002) TLR-2 (p < 0,05), levels were significantly lower in early BC pts compared to healthy controls. When analyzed according to BC characteristics (TNBC vs. non-TNBC, tumor size, stage, nodal status and age) no significant difference was detected between the soluble levels of these ICM between the different subsets. Additionally, serum CXCL5 (p < 0,000001), CCL23 (p < 0,04), IL-16 (p < 0,00005), interferon-α (p < 0,03) and IL1-RA (p < 0,03) were significantly lower compared to healthy controls. Serum CX3CL1 or fractalkine (p < 0,024465) was significantly higher compared with healthy controls. Serum interferon-γ (p < 0,2), IL-6 (p < 0.6) and IL-2 (p < 0.6) levels were not significantly different between the BC pts and the healthy controls. Conclusions: We identified low levels of both the stimulatory and inhibitory immune checkpoint molecules, in newly-diagnosed, non-metastatic BC pts compared to healthy controls. These results indicate that early BC is associated with a down-regulation of both soluble stimulatory and inhibitory immune-checkpoint pathways. Newly-diagnosed early BC pts have a generalized immune-suppression independent of subtype (TNBC vs non-TNBC) and stage, which, to our knowledge, is the first study to describe soluble immune checkpoints in early BC pts.

Neutrophils are important components of the innate immune system that mediate pathogen defense by multiple processes including phagocytosis, release of proteolytic enzymes, production of reactive oxygen species, and neutrophil extracellular trap formation. Abnormalities of neutrophil count and function have been described in the setting of HIV infection, with the majority of antiretroviral agents (ARVs), excluding zidovudine, having been reported to correct neutropenia. Questions still remain, however, about their impact on neutrophil function, particularly the possibility of persistent neutrophil activation, which could predispose people living with HIV to chronic inflammatory disorders, even in the presence of virally-suppressive treatment. In this context, the effects of protease inhibitors and integrase strand transfer inhibitors, in particular, on neutrophil function remain poorly understood and deserve further study. Besides mediating hemostatic functions, platelets are increasingly recognized as critical role players in the immune response against infection. In the setting of HIV, these cells have been found to harbor the virus, even in the presence of antiretroviral therapy (ART) potentially promoting viral dissemination. While HIV-infected individuals often present with thrombocytopenia, they have also been reported to have increased platelet activation, as measured by an upregulation of expression of CD62P (P-selectin), CD40 ligand, glycoprotein IV, and RANTES. Despite ART-mediated viral suppression, HIV-infected individuals reportedly have sustained platelet activation and dysfunction. This, in turn, contributes to persistent immune activation and an inflammatory vascular environment, seemingly involving neutrophil-platelet-endothelium interactions that increase the risk for development of comorbidities such as cardiovascular disease (CVD) that has become the leading cause of morbidity and mortality in HIV-infected individuals on treatment, clearly underscoring the importance of unraveling the possible etiologic roles of ARVs. In this context, abacavir and ritonavir-boosted lopinavir and darunavir have all been linked to an increased risk of CVD. This narrative review is therefore focused primarily on the role of neutrophils and platelets in HIV transmission and disease, as well as on the effect of HIV and the most common ARVs on the numbers and functions of these cells, including neutrophil-platelet-endothelial interactions.

Although bedaquiline has advanced the treatment of multidrug-resistant tuberculosis (TB), concerns remain about the cardiotoxic potential of this agent, albeit by unexplored mechanisms. Accordingly, we have investigated augmentation of the reactivity of human platelets in vitro as a potential mechanism of bedaquiline-mediated cardiotoxicity. Platelet-rich plasma (PRP) or isolated cells prepared from the blood of healthy, adult humans were treated with bedaquiline (0.625–10 µg/ml), followed by activation with adenosine 5’-diphosphate (ADP), thrombin or the thromboxane A2 receptor agonist (U46619). Expression of platelet CD62P (P-selectin), platelet aggregation, Ca2+ fluxes and phosphorylation of Akt1 were measured using flow cytometry, spectrophotometry, fluorescence spectrometry, and by ELISA procedures, respectively. Exposure to bedaquiline caused dose-related inhibition of ADP-activated, but not thrombin- or U46619-activated, expression of CD62P by platelets, achieving statistical significance at a threshold concentration of 5 µg/ml and was paralleled by inhibition of aggregation and Ca2+ mobilization. These ADP-selective inhibitory effects of bedaquiline on platelet activation were mimicked by wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3-K), implicating PI3-K as being a common target of both agents, a contention that was confirmed by the observed inhibitory effects of bedaquiline on the phosphorylation of Akt1 following activation of platelets with ADP. These apparent inhibitory effects of bedaquiline on the activity of PI3-K may result from the secondary cationic amphiphilic properties of this agent. If operative in vivo, these anti-platelet effects of bedaquiline may contribute to ameliorating the risk of TB-associated cardiovascular disease, but this remains to be explored in the clinical setting.

Abstract Background: Checkpoint proteins regulate the immune system. Breast cancer (BC) cells exploit the up-regulation or down-regulation of these proteins to evade anti-tumor immune responses. Soluble forms of immune checkpoint molecules (ICM) can be measured in human plasma. However, their biological and clinical significance remains mostly unknown. The aim of the present analysis was to measure the levels of pre-treatment ICM in newly diagnosed BC patients (pts) and compare them to healthy controls. Method: Soluble forms of ICM, as well as cytokines and chemokines, were measured using Multiplex® bead array and ELISA technologies. Plasma samples from 98 BC pts and 45 healthy controls were analyzed for each protein. Data was prospectively obtained. Measured levels were compared between BC pts and healthy controls using a non-parametric test (Mann-Whitney). Results: Soluble stimulatory molecules GITR (p < 0.000002), GITRL (p < 0.007), CD27 (p < 0.002), CD28 (p < 0.003), CD40 (p < 0.003), CD80 (p < 0.009), ICOS (p < 0.0006) as well as inhibitory molecules PD-L1 (p < 0.0000001), CTLA-4 (p < 0.005), TIM-3 (p < 0.00006), HVEM (p < 0.00002) and TLR-2 (p < 0.05) levels were significantly lower in early BC pts compared to healthy controls. When analyzed according to BC characteristics (TNBC vs. non-TNBC, tumor size, stage, nodal status and age) no significant difference was detected between the soluble levels of these ICM and between the different subsets. Additionally, serum levels of CXCL5 (p < 0.000001), CCL23 (p < 0.04), IL-16 (p < 0.00005), interferon-α (p < 0.03) and IL1-RA (p < 0.03) were significantly lower compared to healthy controls. Serum CX3CL1 or fractalkine (p < 0.024465) was significantly higher compared to healthy controls. Conclusion: In the current study, we identified low levels of both stimulatory and inhibitory soluble immune checkpoint molecules in newly diagnosed, non-metastatic BC pts compared to healthy controls. These results indicate that early BC is associated with a down-regulation of both soluble stimulatory and inhibitory immune-checkpoint pathways. Newly diagnosed early BC pts have a generalized immune-suppression independent of subtype and stage, which, to our knowledge, is the first study to describe soluble immune checkpoints in early BC pts. Citation Format: Bernardo Rapoport, Helen Steel, Teresa Smit, Liezl Heyman, Annette Theron, Nomsa Hlatswayo, Luyanda Kwofie, Lidia Jooste, Farhana Moosa, Carol Ann Benn, Simon Nayler, Ronald Anderson. Dysregulation of soluble immune checkpoint proteins in newly - diagnosed early breast cancer patients [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-36.

High mobility group box 1 (HMGB1) is an extremely versatile protein that is located predominantly in the nucleus of quiescent eukaryotic cells, where it is critically involved in maintaining genomic structure and function. During cellular stress, however, this multifaceted, cytokine-like protein undergoes posttranslational modifications that promote its translocation to the cytosol, from where it is released extracellularly, either actively or passively, according to cell type and stressor. In the extracellular milieu, HMGB1 triggers innate inflammatory responses that may be beneficial or harmful, depending on the magnitude and duration of release of this pro-inflammatory protein at sites of tissue injury. Heightened awareness of the potentially harmful activities of HMGB1, together with a considerable body of innovative, recent research, have revealed that excessive production of HMGB1, resulting from misdirected, chronic inflammatory responses, appears to contribute to all the stages of tumorigenesis. In the setting of established cancers, the production of HMGB1 by tumor cells per se may also exacerbate inflammation-related immunosuppression. These pro-inflammatory mechanisms of HMGB1-orchestrated tumorigenesis, as well as the prognostic potential of detection of elevated expression of this protein in the tumor microenvironment, represent the major thrusts of this review.

Aim: We investigated the prognostic potential of pretherapy measurement of the neutrophil/lymphocyte ratio (NLR) in patients (n = 56) with non-small-cell lung cancer deemed suitable for treatment with nivolumab. Materials & methods: This was a multicenter, noninterventional, retrospective data analysis, involving five oncology centers. Results: Patients with prenivolumab NLR values of <5 and ≥5 had respective median overall survival (OS) values of 14.5 and 7.02 months (p = 0.0026). Patients with ≤2 and >2 metastatic sites had median OS values of 11.4 and 6.1 months, respectively (p = 0.0174). A Cox multiple regression model revealed baseline NLR ≥5 as the only variable significantly associated with decreased OS (p < 0.0447). Conclusion: Pretreatment elevated NLR values are associated with poor outcomes in patients with recurrent metastatic non-small-cell lung cancer treated with nivolumab.

3044 Background: Checkpoint proteins regulate the immune system. Breast cancer (BC) cells can up-regulate or down-regulate these proteins to evade anti-tumor immune responses. Soluble forms of immune checkpoint molecules (ICMs) can be measured in human plasma. The study aimed to measure the systemic levels of a series of co-stimulatory and co-inhibitory ICMs at diagnosis, post-neo-adjuvant chemotherapy (NAC) and post-surgery in newly- diagnosed BC patients (pts) relative to those of a healthy control group. Methods: Soluble ICMs were measured using multiplex bead array technology in plasma from 72 BC pts and 45 healthy controls. Data was prospectively obtained and levels compared between pre-treatment, post-NAC, and post-surgery using non-parametric tests (Mann-Whitney & Kruskal-Wallis). Results: Pre-treatment levels of the soluble stimulatory molecules viz. GITR (p<0.0001), GITRL (p< 0.020), CD27 (p< 0.024), CD40 (p< 0.021), ICOS (p< 0.009), as well as the inhibitory molecules PD-L1 (p< 0.0001), CTLA-4 (p< 0.005), TIM-3 (p< 0.0004), HVEM (p< 0.0004) were significantly lower in early BC pts compared to controls. Post-treatment, there were significant increases in most ICM levels (Table), with the exception of CTLA-4, which decreased significantly following treatment. On the other hand, pre-treatment plasma concentrations of CCL5 (RANTES) (84.22 vs. 48.72 pg/mL, p<0.0001), M-CSF (84.41 vs 13.34 pg/mL, p<0.0001), FGF-21 (24.36 vs. 8.64 pg/mL, p<0.001) and GDF-15 (806.82 vs. 430.03 pg/mL, p<0.0001) were significantly increased in the breast cancer pts compared to healthy controls. A pathological complete response (pCR) was documented in 65% of pts (mostly TNBC). There were no correlations between pre-treatment ICM levels, CCL5, M-CSF, FGF-21 and GDF-15 and pCR. Conclusions: We identified low levels of stimulatory and inhibitory ICMs in newly-diagnosed, non-metastatic BC pts compared to healthy controls. Following treatment, with the exception of CTLA-4, most of these pre-treatment abnormalities of systemic ICM levels corrected. NAC is associated with upregulation of sPD-L1 and most other ICMs. These results indicate that early BC is associated with down-regulation of soluble stimulatory and inhibitory ICMs. Newly-diagnosed early BC pts appear to have generalized immune-suppression independent of subtype and stage. To our knowledge, this is the first study to describe the effect of treatment on systemic ICMs in early BC pts. [Table: see text]