Acute malaria suppresses the B lymphocytic niche in the bone marrow through the alteration of CXCL12-abundant reticular cells.

Bone marrow is a dynamic organ composed of stem cells that constantly receive signals from stromal cells and other hematopoietic cells in the niches of the bone marrow to maintain hematopoiesis and generate immune cells. Perturbation of the bone marrow microenvironment by infection and inflammation affects hematopoiesis and may affect immune cell development. Little is known about the effect of malaria on the bone marrow stromal cells that govern the hematopoietic stem cell (HSC) niche. In this study, we demonstrate that the mesenchymal stromal CXCL12-abundant reticular (CAR) cell population is reduced during acute malaria infection. The reduction of CXCL12 and interleukin-7 signals in the bone marrow impairs the lymphopoietic niche, leading to the depletion of common lymphoid progenitors, B cell progenitors, and mature B cells, including plasma cells in the bone marrow. We found that interferon-γ (IFNγ) is responsible for the upregulation of Sca1 on CAR cells, yet the decline in CAR cell and B cell populations in the bone marrow is IFNγ-independent. In contrast to the decline in B cell populations, HSCs and multipotent progenitors increased with the expansion of myelopoiesis and erythropoiesis, indicating a bias in the differentiation of multipotent progenitors during malaria infection. These findings suggest that malaria may affect host immunity by modulating the bone marrow niche.

MyD88 in osteoclast- and osteoblast-lineages differentially controls bone remodeling in homeostasis and malaria.

Chronic bone loss is an under-recognized complication of malaria, the underlying mechanism of which remains incompletely understood. We have previously shown that persistent accumulation of Plasmodium products in the bone marrow leads to chronic inflammation in osteoblast (OB) and osteoclast (OC) precursors causing bone loss through MyD88, an adaptor molecule for diverse inflammatory signals. However, the specific contribution of MyD88 signaling in OB or OC precursors in malaria-induced bone loss remains elusive. To assess the direct cell-intrinsic role of MyD88 signaling in adult bone metabolism under physiological and infection conditions, we used the Lox-Cre system to specifically deplete MyD88 in the OB or OC lineages. Mice lacking MyD88 primarily in the maturing OBs showed a comparable decrease in trabecular bone density by microcomputed tomography (µCT) to that of controls after PyNL infection. In contrast, mice lacking MyD88 in OC precursors showed significantly less trabecular bone loss than controls, suggesting that malaria-mediated inflammatory mediators are primarily controlled by MyD88 in the OC lineage. Surprisingly, however, depletion of MyD88 in OB, but not in OC precursors, resulted in reduced bone mass with decreased bone formation rates in the trabecular areas of femurs under physiological conditions. Notably, IGF-1, a key molecule for OB differentiation, was significantly lower locally and systemically when MyD88 was depleted in OBs. Thus, our data demonstrate an indispensable intrinsic role for MyD88 signaling in OB differentiation and bone formation, while MyD88 signaling in OC lineages plays a partial role in controlling malaria-induced inflammatory mediators and following bone pathology. These findings may lead to the identification of novel targets for specific intervention of bone pathologies, particularly in malaria-endemic regions.

Inhaled Fine Particles Induce Alveolar Macrophage Death and Interleukin-1α Release to Promote Inducible Bronchus-Associated Lymphoid Tissue Formation.

Particulate pollution is thought to function as an adjuvant that can induce allergic responses. However, the exact cell types and immunological factors that initiate the lung-specific immune responses are unclear. We found that upon intratracheal instillation, particulates such as aluminum salts and silica killed alveolar macrophages (AMs), which then released interleukin-1α (IL-1α) and caused inducible bronchus-associated lymphoid tissue (iBALT) formation in the lung. IL-1α release continued for up to 2 weeks after particulate exposure, and type-2 allergic immune responses were induced by the inhalation of antigen during IL-1α release and iBALT formation, even long after particulate instillation. Recombinant IL-1α was sufficient to induce iBALTs, which coincided with subsequent immunoglobulin E responses, and IL-1-receptor-deficient mice failed to induce iBALT formation. Therefore, the AM-IL-1α-iBALT axis might be a therapeutic target for particulate-induced allergic inflammation.

TBK1 and IKKε act like an OFF switch to limit NLRP3 inflammasome pathway activation.

NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) inflammasome activation is beneficial during infection and vaccination but, when uncontrolled, is detrimental and contributes to inflammation-driven pathologies. Hence, discovering endogenous mechanisms that regulate NLRP3 activation is important for disease interventions. Activation of NLRP3 is regulated at the transcriptional level and by posttranslational modifications. Here, we describe a posttranslational phospho-switch that licenses NLRP3 activation in macrophages. The ON switch is controlled by the protein phosphatase 2A (PP2A) downstream of a variety of NLRP3 activators in vitro and in lipopolysaccharide-induced peritonitis in vivo. The OFF switch is regulated by two closely related kinases, TANK-binding kinase 1 (TBK1) and I-kappa-B kinase epsilon (IKKε). Pharmacological inhibition of TBK1 and IKKε, as well as simultaneous deletion of TBK1 and IKKε, but not of either kinase alone, increases NLRP3 activation. In addition, TBK1/IKKε inhibitors counteract the effects of PP2A inhibition on inflammasome activity. We find that, mechanistically, TBK1 interacts with NLRP3 and controls the pathway activity at a site distinct from NLRP3-serine 3, previously reported to be under PP2A control. Mutagenesis of NLRP3 confirms serine 3 as an important phospho-switch site but, surprisingly, reveals that this is not the sole site regulated by either TBK1/IKKε or PP2A, because all retain the control over the NLRP3 pathway even when serine 3 is mutated. Altogether, a model emerges whereby TLR-activated TBK1 and IKKε act like a "parking brake" for NLRP3 activation at the time of priming, while PP2A helps remove this parking brake in the presence of NLRP3 activating signals, such as bacterial pore-forming toxins or endogenous danger signals.

Anti-tumor immunity by transcriptional synergy between TLR9 and STING activation.

Agonists for TLR9 and stimulator of IFN genes (STING) offer therapeutic applications as both anti-tumor agents and vaccine adjuvants, though their clinical applications are limited; the clinically available TLR9 agonist is a weak IFN inducer and STING agonists induce undesired type 2 immunity. Yet, combining TLR9 and STING agonists overcame these limitations by synergistically inducing innate and adaptive IFNγ to become an advantageous type 1 adjuvant, suppressing type 2 immunity, in addition to exerting robust anti-tumor activities when used as a monotherapeutic agent for cancer immunotherapy. Here, we sought to decipher the immunological mechanisms behind the synergism mediated by TLR9 and STING agonists and found that their potent anti-tumor immunity in a Pan02 peritoneal dissemination model of pancreatic cancer was achieved only when agonists for TLR9 and STING were administered locally, and was via mechanisms involving CD4 and CD8 T cells as well as the co-operative action of IL-12 and type I IFNs. Rechallenge studies of long-term cancer survivors suggested that the elicitation of Pan02-specific memory responses provides protection against the secondary tumor challenge. Mechanistically, we found that TLR9 and STING agonists synergistically induce IL-12 and type I IFN production in murine APCs. The synergistic effect of the TLR9 and STING agonists on IL-12p40 was at protein, mRNA and promoter activation levels, and transcriptional regulation was mediated by a 200 bp region situated 983 bp upstream of the IL-12p40 transcription initiation site. Such intracellular transcriptional synergy may hold a key in successful cancer immunotherapy and provide further insights into dual agonism of innate immune sensors during host homeostasis and diseases.

Using a new three-dimensional CUBIC tissue-clearing method to examine the brain during experimental cerebral malaria.

Cerebral malaria (CM) is a life-threatening complication of the malaria disease caused by Plasmodium falciparum infection and is responsible for the death of half a million people annually. The molecular pathogenesis underlying CM in humans is not completely understood, although sequestration of infected erythrocytes in cerebral microvessels is thought to play a major role. In contrast, experimental cerebral malaria (ECM) models in mice have been thought to be distinct from human CM, and are mainly caused by inflammatory mediators. Here, to understand the spatial distribution and the potential sequestration of parasites in the whole-brain microvessels during a mouse model of ECM, we utilized the new tissue-clearing method CUBIC (Clear, Unobstructed, Brain/Body Imaging Cocktails and Computational analysis) with light-sheet fluorescent microscopy (LSFM), and reconstructed images in three dimensions (3D). We demonstrated significantly greater accumulation of Plasmodium berghei ANKA (PbANKA) parasites in the olfactory bulb (OB) of mice, compared with the other parts of the brain, including the cerebral cortex, cerebellum and brainstem. Furthermore, we show that PbANKA parasites preferentially accumulate in the brainstem when the OB is surgically removed. This study therefore not only highlights a successful application of CUBIC tissue-clearing technology to visualize the whole brain and its microvessels during ECM, but it also shows CUBIC's future potential for visualizing pathological events in the whole ECM brain at the cellular level, an achievement that would greatly advance our understanding of human cerebral malaria.

ZBP1 governs the inflammasome-independent IL-1α and neutrophil inflammation that play a dual role in anti-influenza virus immunity.

Influenza A virus (IAV) triggers the infected lung to produce IL-1 and recruit neutrophils. Unlike IL-1ß, however, little is known about IL-1α in terms of its mechanism of induction, action and physiological relevance to the host immunity against IAV infection. In particular, whether Z-DNA-binding protein 1 (ZBP1), a key molecule for IAV-induced cell death, is involved in the IL-1α induction, neutrophil infiltration and the physiological outcome has not been elucidated. Here, we show in a murine model that the IAV-induced IL-1α is mediated solely by ZBP1, in an NLRP3-inflammasome-independent manner, and is required for the optimal IL-1ß production followed by the formation of neutrophil extracellular traps (NETs). During IAV infection, ZBP1 displays a dual role in anti-IAV immune responses mediated by neutrophils, resulting in either protective or pathological outcomes in vivo. Thus, ZBP1-mediated IL-1α production is the key initial step of IAV-infected NETs, regulating the duality of the consequent lung inflammation.

Heparin induces neutrophil elastase-dependent vital and lytic NET formation.

Heparin is used extensively as an anticoagulant in a broad range of diseases and procedures; however, its biological effects are not limited to coagulation and remain incompletely understood. Heparin usage can lead to the life-threatening complication known as heparin-induced thrombocytopenia (HIT), caused by the development of antibodies against heparin/PF4 complexes. Here, we demonstrate the ability of heparin to induce neutrophil extracellular traps (NETs). NETs occurred with cell lysis and death, but live neutrophils releasing extracellular DNA strands, known as vital NETs, also occurred abundantly. Formation of NETs was time and dose dependent, and required reactive oxygen species and neutrophil elastase. Other compounds related to heparin such as low molecular weight heparin, fondaparinux and heparan sulfate either failed to induce NETs, or did so to a much lesser extent. Our findings suggest the ability of heparin to directly induce NET formation should be considered in the context of heparin treatment and HIT pathogenesis.

B cell-intrinsic MyD88 signaling controls IFN-γ-mediated early IgG2c class switching in mice in response to a particulate adjuvant.

Adjuvants improve the potency of vaccines, but the modes of action (MOAs) of most adjuvants are largely unknown. TLR-dependent and -independent innate immune signaling through the adaptor molecule MyD88 has been shown to be pivotal to the effects of most adjuvants; however, MyD88's involvement in the TLR-independent MOAs of adjuvants is poorly understood. Here, using the T-dependent antigen NIPOVA and a unique particulate adjuvant called synthetic hemozoin (sHZ), we show that MyD88 is required for early GC formation and enhanced antibody class-switch recombination (CSR) in mice. Using cell-type-specific MyD88 KO mice, we found that IgG2c class switching, but not IgG1 class switching, was controlled by B cell-intrinsic MyD88 signaling. Notably, IFN-γ produced by various cells including T cells, NK cells, and dendritic cells was the primary cytokine for IgG2c CSR and B-cell intrinsic MyD88 is required for IFN-γ production. Moreover, IFN-γ receptor (IFNγR) deficiency abolished sHZ-induced IgG2c production, while recombinant IFN-γ administration successfully rescued IgG2c CSR impairment in mice lacking B-cell intrinsic MyD88. Together, our results show that B cell-intrinsic MyD88 signaling is involved in the MOA of certain particulate adjuvants and this may enhance our specific understanding of how adjuvants and vaccines work.

Silica crystals and aluminum salts regulate the production of prostaglandin in macrophages via NALP3 inflammasome-independent mechanisms.

Particulates such as silica crystal (silica) and aluminum salts (alum) activate the inflammasome and induce the secretion of proinflammatory cytokines in macrophages. These particulates also induce the production of immunoglobulin E via a T helper 2 (Th2) cell-associated mechanism. However, the mechanism involved in the induction of type 2 immunity has not been elucidated. Here, we showed that silica and alum induced lipopolysaccharide-primed macrophages to produce the lipid mediator prostaglandin E2 (PGE2) and interleukin-1ß (IL-1ß). Macrophages deficient in the inflammasome components caspase 1, NALP3, and ASC revealed that PGE2 production was independent of the NALP3 inflammasome. PGE2 expression was markedly reduced in PGE synthase-deficient (Ptges⁻/⁻) macrophages, and Ptges⁻/⁻ mice displayed reduced antigen-specific serum IgE concentrations after immunization with alum or silica. Our results indicate that silica and alum regulate the production of PGE2 and that the induction of PGE2 by particulates controls the immune response in vivo.

A distinct subpopulation of CD25- T-follicular regulatory cells localizes in the germinal centers.

T-follicular helper (Tfh) cells differentiate through a multistep process, culminating in germinal center (GC) localized GC-Tfh cells that provide support to GC-B cells. T-follicular regulatory (Tfr) cells have critical roles in the control of Tfh cells and GC formation. Although Tfh-cell differentiation is inhibited by IL-2, regulatory T (Treg) cell differentiation and survival depend on it. Here, we describe a CD25- subpopulation within both murine and human PD1+CXCR5+Foxp3+ Tfr cells. It is preferentially located in the GC and can be clearly differentiated from CD25+ non-GC-Tfr, Tfh, and effector Treg (eTreg) cells by the expression of a wide range of molecules. In comparison to CD25+ Tfr and eTreg cells, CD25- Tfr cells partially down-regulate IL-2-dependent canonical Treg features, but retain suppressive function, while simultaneously up-regulating genes associated with Tfh and GC-Tfh cells. We suggest that, similar to Tfh cells, Tfr cells follow a differentiation pathway generating a mature GC-localized subpopulation, CD25- Tfr cells.

Rapid Quantification of NETs In Vitro and in Whole Blood Samples by Imaging Flow Cytometry.

Neutrophil extracellular trap (NET) formation involves the release of DNA outside the cell to neutralize pathogens. Techniques such as live microscopy, flow cytometry, and intravital imaging allow the characterization of NETs, but these either cannot be applied in vivo, lack specificity or require invasive procedures. We developed an automated analysis method to rapidly acquire and characterize cells as NETs or NET precursors, as opposed to cells undergoing other forms of cell death, using imaging flow cytometry. NETs were maintained in solution using a novel three-dimensional cell culture system in which cells are suspended at the interface of two liquids of different density. Critically, we identify NETs using an image analysis algorithm based on morphological data showing the extrusion of DNA beyond the cell boundaries. In vitro, we used this technique to demonstrate different requirements for NET formation in human and mouse neutrophils. We also measured NETs in whole blood during infection of mice with the malaria parasite Plasmodium yoelii. We expect this technique will provide a valuable approach to better understand the process of NET formation and its importance in disease. © 2019 International Society for Advancement of Cytometry.

Unforeseen pathologies caused by malaria.

Individuals from malaria-endemic regions often acquire partial immunity after multiple repeated infections throughout their lives. This partial immunity prevents them from developing severe complications and they often remain asymptomatic with a persistent, low parasite density in the blood, and therefore the necessity for treatment is neglected. These patients with chronic, asymptomatic malaria serve as a reservoir for Plasmodium parasite transmission, becoming a major obstacle for eradication efforts. The constant exposure to malaria infection may have benefits in the short term by conferring protection from acute, severe malaria; however, it may cause substantially more harm in the long term. Rather than the parasite burden itself, the complications induced by the dysregulated immune responses and the tissue damage done by the parasites and their products can cause chronic and irreversible suffering. Furthermore, the complete clearance of parasites in the body may not lead to complete recovery from the disease as complications can still persist. The fact that there are chronic pathologies caused by malaria that mostly remain obscure and have the potential to cause a serious burden has recently been gaining attention. Here, we present and discuss the evidence of unforeseen pathologies and the risks associated with malaria.

Does it take three to tango? An unsuspected multimorbidity of CD8+ T cell lymphoproliferative disorder, malaria, and EBV infection.

BACKGROUND: Malaria is known to cause acute and deadly complications. However, malaria can cause unforeseen pathologies due to its chronicity. It increases the risk of endemic Burkitt Lymphoma development by inducing DNA damage in germinal centre (GC) B cells, and leading higher frequency of Epstein-Barr virus (EBV)-infected cells in GCs. EBV is well known for its tropism for B cells. However, less is known about EBV's interaction with T cells and its association with T cell lymphoma. CASE PRESENTATION: A 43-year-old Sudanese male admitted to hospital in Istanbul, Turkey, a non-endemic country, with hyperpigmented painful skin rashes on his whole body. A complete blood count and a peripheral blood smear during admission revealed large granular lymphocytes (LGLs) with abnormally higher CD8 T cell numbers. Additional skin biopsy and pathology results were compatible with CD8+ T cell lymphoproliferative disorder with skin involvement. Patient was treated and discharged. However, a pathologist noticed unusual structures in skin tissue samples. Careful evaluation of skin biopsy samples by polarized microscopy revealed birefringent crystalloid structures resembling malarial haemozoin mainly loaded in macrophages and giant histiocytes. After purification of DNA from the skin biopsy samples, nested PCR was performed for the detection of Plasmodium parasites and Plasmodium falciparum DNA was amplified. Because, the co-presence of EBV infection with malaria is a well-known aetiology of lymphoma, EBV-early RNA (EBER) transcripts were investigated in paraffin-embedded tissue samples and found to be positive in macrophage-like histiocytes. CONCLUSIONS: This is a unique case of malaria and EBV infection in a T-LGL lymphoma patient who presented in a non-endemic country. This case emphasizes the clinical importance of EBV monitoring in T-LGL patients with skin involvement. Notably, Plasmodium infection should be examined in patients from malaria endemic regions by pathological and molecular investigations.

Hydroxypropyl-ß-cyclodextrin spikes local inflammation that induces Th2 cell and T follicular helper cell responses to the coadministered antigen.

Cyclodextrins are commonly used as a safe excipient to enhance the solubility and bioavailability of hydrophobic pharmaceutical agents. Their efficacies and mechanisms as drug-delivery systems have been investigated for decades, but their immunological properties have not been examined. In this study, we reprofiled hydroxypropyl-ß-cyclodextrin (HP-ß-CD) as a vaccine adjuvant and found that it acts as a potent and unique adjuvant. HP-ß-CD triggered the innate immune response at the injection site, was trapped by MARCO(+) macrophages, increased Ag uptake by dendritic cells, and facilitated the generation of T follicular helper cells in the draining lymph nodes. It significantly enhanced Ag-specific Th2 and IgG Ab responses as potently as did the conventional adjuvant, aluminum salt (alum), whereas its ability to induce Ag-specific IgE was less than that of alum. At the injection site, HP-ß-CD induced the temporary release of host dsDNA, a damage-associated molecular pattern. DNase-treated mice, MyD88-deficient mice, and TBK1-deficient mice showed significantly reduced Ab responses after immunization with this adjuvant. Finally, we demonstrated that HP-ß-CD-adjuvanted influenza hemagglutinin split vaccine protected against a lethal challenge with a clinically isolated pandemic H1N1 influenza virus, and the adjuvant effect of HP-ß-CD was demonstrated in cynomolgus macaques. Our results suggest that HP-ß-CD acts as a potent MyD88- and TBK1-dependent T follicular helper cell adjuvant and is readily applicable to various vaccines.

Nonagonistic Dectin-1 ligand transforms CpG into a multitask nanoparticulate TLR9 agonist.

CpG DNA, a ligand for Toll-like receptor 9 (TLR9), has been one of the most promising immunotherapeutic agents. Although there are several types of potent humanized CpG oligodeoxynucleotide (ODN), developing "all-in-one" CpG ODNs activating both B cells and plasmacytoid dendritic cells forming a stable nanoparticle without aggregation has not been successful. In this study, we generated a novel nanoparticulate K CpG ODN (K3) wrapped by the nonagonistic Dectin-1 ligand schizophyllan (SPG), K3-SPG. In sharp contrast to K3 alone, K3-SPG stimulates human peripheral blood mononuclear cells to produce a large amount of both type I and type II IFN, targeting the same endosome where IFN-inducing D CpG ODN resides without losing its K-type activity. K3-SPG thus became a potent adjuvant for induction of both humoral and cellular immune responses, particularly CTL induction, to coadministered protein antigens without conjugation. Such potent adjuvant activity of K3-SPG is attributed to its nature of being a nanoparticle rather than targeting Dectin-1 by SPG, accumulating and activating antigen-bearing macrophages and dendritic cells in the draining lymph node. K3-SPG acting as an influenza vaccine adjuvant was demonstrated in vivo in both murine and nonhuman primate models. Taken together, K3-SPG may be useful for immunotherapeutic applications that require type I and type II IFN as well as CTL induction.

Label-free Raman imaging of the macrophage response to the malaria pigment hemozoin.

Hemozoin, the 'malaria pigment', is engulfed by phagocytic cells, such as macrophages, during malaria infection. This biocrystalline substance is difficult to degrade and often accumulates in phagocytes. The macrophage response to hemozoin relates to the severity of the disease and the potential for malaria-related disease complications. In this study we have used Raman spectroscopy as a label-free method to investigate the biochemical changes occurring in macrophages during the first few hours of hemozoin uptake. We found a number of distinct spectral groups, spectrally or spatially related to the presence of the hemozoin inside the cell. Intracellular hemozoin was spectrally identical to extracellular hemozoin, regardless of the location in the cell. A small proportion of hemozoin was found to be associated with lipid-based components, consistent with the uptake of hemozoin into vesicles such as phagosomes and lysosomes. The spatial distribution of the hemozoin was observed to be inhomogeneous, and its presence largely excluded that of proteins and lipids, demonstrating that cells were not able to break down the biocrystals on the time scales studied here. These results show that Raman imaging can be used to answer some of the open questions regarding the role of hemozoin in the immune response. How different combinations of hemozoin and other molecules are treated by macrophages, whether hemozoin can be broken down by the cell, and more importantly, which co-factors or products are involved in the subsequent cell reaction are the expected issues to be elucidated by this technique.

TANK-binding kinase-1 delineates innate and adaptive immune responses to DNA vaccines.

Successful vaccines contain not only protective antigen(s) but also an adjuvant component that triggers innate immune activation and is necessary for their optimal immunogenicity. In the case of DNA vaccines, this consists of plasmid DNA; however, the adjuvant element(s) as well as its intra- and inter-cellular innate immune signalling pathway(s) leading to the encoded antigen-specific T- and B-cell responses remain unclear. Here we demonstrate in vivo that TANK-binding kinase 1 (TBK1), a non-canonical IkappaB kinase, mediates the adjuvant effect of DNA vaccines and is essential for its immunogenicity in mice. Plasmid-DNA-activated, TBK1-dependent signalling and the resultant type-I interferon receptor-mediated signalling was required for induction of antigen-specific B and T cells, which occurred even in the absence of innate immune signalling through a well known CpG DNA sensor-Toll-like receptor 9 (TLR9) or Z-DNA binding protein 1 (ZBP1, also known as DAI, which was recently reported as a potential B-form DNA sensor). Moreover, bone-marrow-transfer experiments revealed that TBK1-mediated signalling in haematopoietic cells was critical for the induction of antigen-specific B and CD4(+) T cells, whereas in non-haematopoietic cells TBK1 was required for CD8(+) T-cell induction. These data suggest that TBK1 is a key signalling molecule for DNA-vaccine-induced immunogenicity, by differentially controlling DNA-activated innate immune signalling through haematopoietic and non-haematopoietic cells.

The Programmed Cell Death Ligand 1 and Lipocalin 2 Expressions in Primary Breast Cancer and Their Associations with Molecular Subtypes and Prognostic Factors.

Purpose: Breast cancers exhibit molecular heterogeneity, leading to diverse clinical outcomes and therapeutic responses. Immune checkpoint inhibitors targeting PD-L1 have shown promise in various malignancies, including breast cancer. Lipocalin 2 (LCN2) has also been associated with tumor aggressiveness and prognostic potential in breast cancers. However, the expression of PD-L1 and LCN2 in breast cancer subtypes and their prognostic implications remains poorly investigated. Methods: A retrospective analysis of 89 primary breast cancer cases was conducted to assess PD-L1 and LCN2 expressions using immunohistochemistry. Cases were classified into four different molecular subtypes based on ER, PR, HER2, and Ki-67 status. Associations between PD-L1 and LCN2 expressions and various prognostic factors were examined. Results: Although low expression of LCN2 (Allred score of <3) was observed even in normal breast tissue, LCN2 expression with increasing Allred score (≥3) positively correlated with the histological grade, high Ki-67 proliferation index, and ER/PR negativity. Significant elevations of LCN2 and PD-L1 expressions were observed in triple-negative and HER2-positive breast cancers. Conclusion: The results of the study highlight the association of LCN2 with known prognostic factors and molecular subtypes. To identify potential immunotherapy recipients, it would be useful to evaluate LCN2 as well as PD-L1 immune targets in different subgroups of breast cancer patients. Further studies with larger patient numbers are warranted to validate these observations and establish standardized scoring criteria for LCN2 expression assessment.