Real-world safety of immune checkpoint inhibitor use in patients with non-small cell lung cancer

Baek, Yeonhee Sungkyunkwan university 2021 국내박사

Immune checkpoint inhibitors have emerged as a mainstay in treatment for non-small cell lung cancer. However, the safety profile of immune checkpoint inhibitors remains to be established due to the heterogeneity of real-world populations and dynamic treatment regimens in routine clinical practice compared to selected clinical trials settings. Concerns have been raised regarding adverse treatment outcomes of immune checkpoint inhibitors due to gut microbiome dysbiosis and the risk of immune-related adverse events from off-target toxicity following immune checkpoint inhibitor use. The objectives of this thesis include: 1) to examine the survival outcomes of concomitant use of immune checkpoint inhibitors and proton pump inhibitors (PPIs) which have a potential gut microbiome modulating effect, and 2) to estimate the incidence of serious immune-related adverse events following immune checkpoint inhibitor use and to identify the associated predictors. To provide concrete real-world evidence on the safety of immune checkpoint inhibitors, this thesis used a completely enumerated lung cancer cohort from a nationwide healthcare database in South Korea. The study involved 2,963 patients treated with immune checkpoint inhibitors as second-line or above therapy for stage ≥ IIIB non-small cell lung cancer from August 2017 to September 2018. The first study examines the association between concomitant use of PPIs and immune checkpoint inhibitors and risk of all-cause mortality from national vital statistics. PPI use was assessed up to 30 days before immune checkpoint inhibitor initiation. Upon 1:1 propensity score matching, the concomitant use of PPIs with immune checkpoint inhibitors increased mortality risks compared to non-use, by 28% for prevalent users and 64% for new users, respectively. Subgroup analysis suggests that PPI use was associated with a 2.72-fold higher mortality risk among patients with viral hepatitis. However, the magnitude of association moved toward the null when longer lag-times were applied between cohort entry and outcome. The second study estimates the incidence of serious immune-related adverse events following immune checkpoint inhibitors and identifying predictors. Outcome was defined as simultaneously receiving a pre-defined diagnosis and corticosteroid treatment. When death was treated as a competing risk, the 1-year cumulative incidence of serious immune-related adverse events was 5.6%, which was consistent with findings from clinical trials. Presence of underlying respiratory disease was a predictor for higher incidence of serious immune-related adverse events. In conclusion, the concomitant use of PPIs with immune checkpoint inhibitors increased the risk of early mortality, particularly among patients with viral hepatitis. However, the difference in mortality risk between PPI users and non-users decreased in the later periods of observation, which may imply that PPI use was associated with risk of earlier mortality or alternatively suggest a lack of causal association. This finding necessitates careful interpretation of study results and warrants further investigation. The incidence of serious immune-related adverse events was largely consistent with the findings of clinical trials. However, patients with underlying respiratory disease were susceptible to serious events, and thus require vigilant surveillance and preventive strategies. 면역항암제는 2015년 국내 허가 이후 비소세포폐암의 표준치료로 부상해온 약제이다. 그러나 임상시험이 아닌 실제 임상 환경에서의 면역항암제 안전성에 대한 근거는 부족한 실정이다. 면역항암제 관련 안전성 이슈로는 장내미생물 불균형으로 인한 예후 악화와 치료 표적 외 독성(off-target toxicity)으로 인한 자가면역성 면역관련 이상반응의 위험에 대한 우려가 제기되고 있다. 따라서 본 연구의 목적은 첫째, 장내 미생물 억제 작용이 있다고 알려진 프로톤 펌프 억제제(proton pump inhibitor, PPI)와 면역항암제 병용에 따른 생존율 평가와, 둘째, 면역항암제 사용 후 심각한 면역관련 이상반응의 발생률과 그 위험인자를 추정하였다. 국민건강보험공단의 폐암 전수자료를 활용하여 대표성이 높은 면역항암제 안전성 근거를 창출하고자 하였다. 본 연구의 대상자는 stage IIIB 기 이상 비소세포폐암 환자 중, 2017년 8월부터 2018년 9월 사이에 면역항암제를 2차 치료 이상요법으로 사용한 2,963명의 환자를 대상으로 하였다. 첫째, PPI와 면역항암제 병용에 따른 예후 평가연구에서 PPI 노출은 면역항암제 투여 이전 30일 동안 평가되었으며, 결과변수인 생존율은 통계청 사망자료를 연계하여 활용하였다. 1:1 성향점수 매칭 코호트에서 PPI 병용은 비병용에 비해 사망 위험이 기존 복용자에서는 28%, 신규 복용자에서는 64% 높았다. 하위그룹 분석에서 기저 바이러스성 간염 보유자가 PPI를 면역항암제와 병용할 경우, 2.72배 높은 사망 위험이 나타났다. 그러나, cohort entry와 결과변수 사이에 lag-time을 적용했을 때, lag-time이 길어질수록 관련성의 크기가 감소하는 결과가 나타났다. 둘째, 면역항암제 사용 이후 심각한 면역관련 이상반응 발생률과 위험 요인 평가 연구를 수행하였다. 심각한 면역관련 이상반응은 사전에 정의된 진단코드와 스테로이드 치료를 조합한 알고리즘을 이용하여 정의하였다. 사망을 경쟁위험으로 처리했을 때, 심각한 면역관련 이상반응의 누적 발생률은 5.6% 였으며, 이는 주요 면역항암제 임상시험의 결과와 대체로 일치하였다. 위험 요인분석에서 기저 폐질환 보유는 심각한 면역관련 이상반응의 발생률을 높이는 인자로 나타났다. 결론적으로 PPI와 면역항암제 병용은 조기 사망위험 증가와 관련성을 보였으며, PPI 병용에 따른 위험이 기저 바이러스성 간염 보유자에서 특히 위험의 수준이 높았다. 그러나 lag-time이 길어질수록 PPI 병용에 따른 사망 위험의 관련성이 적어지는 결과를 보였다. 이는 인과적 관련성이 없다는 것을 의미할 수 있으므로, 신중한 해석과 적합한 lag-time을 고려한 후속 연구가 필요하다. 면역항암제 사용 후 면역관련 이상반응 발생률은 임상시험과 비슷한 수준이었으나, 면역관련 이상반응이 기저 폐질환 보유자에서 높게 나타나 취약집단에서의 면밀한 검토와 모니터링이 요구된다.

Immune Enhancing Activity of Maillard Reaction Products of Whey Protein Concentrate and Their Fermented Product using Lactobacillus plantarum LC01

Seol, Hae-Min 고려대학교 대학원 2014 국내석사

Whey protein concentrate (WPC) has a high nutritional value, is a low-cost source of protein. The whey fraction contains a range of known immune-modulatory constituents, including major proteins such as α-lactalbumin and β-lactoglobulin, minor proteins such as lactoperoxidase and lactoferrin and tissue growth factors. WPC also promotes enhanced protection against tumor development in some animal model showing a potential benefit for immune-enhancing activity. The maillard reaction is classified as non-enzymatic browning, which involves sugars, amino acid or proteins that condense and progress into a complex network of reaction products that are known as Maillard reaction products (MRPs). MRPs may influence gut immunity by affecting proliferation, survival rate and adhesion of colonic bacteria. MRPs have an effects related to immune; for example immuno-modulatory and anti-cancer effects. WPC consists of 73.62±0.16% of protein, 7.66±0.14% of lactose and 3.41±0.25% of fat. Conjugated WPC through Maillard reaction or Glycation (G-WPC) for a day at 55℃ and fermentation product of G-WPC hydrolysate using Lactobacillus plantarum LC01 (F-WPC) were prepared. As immune enhancing activity was determined by production of nitric oxide (NO) and cytokines (IL-1β, IL-6, TNF-α and iNOS) in RAW264.7 macrophage. NO and cytokines were increased in order of WPC, G-WPC and F-WPC. F-WPC also promoted the phagocytosis of macrophages than other samples. To measure proliferation of T and B cells, we treated T and B cells with WPC, G-WPC and F-WPC. Proliferations of two cells were not significantly different among WPC, G-WPC and F-WPC. On the other hand, we observed that NK cell contents were increased with the addition of B cell supernatant containing WPC, G-WPC and F-WPC after 6 days and NK cell proliferation in the presence of F-WPC was significantly increased compared to others. Based on anti-tumor properties of WPC and MRPs, we synthesized that WPC, G-WPC and F-WPC has anti-tumor activity and immune-modulating effect in animal models. First of all, 1,000 mg/kg of WPC, G-WPC and F-WPC had no toxicity for SD rats (7 weeks old) and cannot cause death by orally administration for 14-day. After transplanted sarcoma 180 cells subcutaneously into the left groin of BALB/c mice (8 weeks old) and administered with sample for 14 days, we extracted solid tumors and blood samples. The inhibition of tumor cell growth and induction of tumor cell death by activated macrophages were the first function of nitric oxide (NO) in the immune system. NO leads to the prevention of the growth and multiplication of cells or kills tumor cells in vitro. NO production and lysozyme of serum in sample treatment groups, especially F-WPC group, had increased more than or to the level of control group. The significant increase of IFN-γ, TNF-α and IL-1β cytokines in low dose of F-WPC group compared to other groups. Solid tumor cause by injection of sarcoma cells decreased in sample treatment groups compared to NC (negative control) group. The tumor volumes were decreased in the order of WPC, G-WPC and F-WPC when administered low-dose. In conclusion, F-WPC was showed higher immune enhancing effects in vitro and in vivo through an innate immune system. Maillard reaction is a form of non-enzymatic browning that results from a chemical reaction between amino acids and reducing sugars. Whey protein concentrate (WPC) consists of 73.62±0.16% protein, 7.66±0.14% lactose and 3.41±0.25% fat. Conjugated WPC through Maillard reaction or Glycation (G-WPC) for a day at 55℃ and fermentation product of G-WPC hydrolysate using Lactobacillus plantarum LC01 (F-WPC) were prepared, respectively. As immune enhancing activity was determined by production of nitric oxide (NO) and cytokines (IL-1β, IL-6, TNF-α and iNOS) in RAW264.7 macrophage. NO and cytokines were increased in order of WPC, G-WPC and F-WPC. Also F-WPC promoted the phagocytosis of macrophages than other samples. To measure proliferation of T and B cells, we treated T and B cells with WPC, G-WPC and F-WPC and counted the number of two cells every 6 days. Proliferations of two cells were not significantly different among WPC, G-WPC and F-WPC. On the other hand, we observed that NK cell contents were increased with the addition of B cell supernatant containing WPC, G-WPC and F-WPC after 6 days and NK cell proliferation in the presence of F-WPC was significantly increased compared to others. In conclusion, F-WPC was showed higher immune enhancing effects in vitro through an innate immune system. Whey Protein Concentrate (WPC) promotes enhanced protection against tumor development in some animal model system presenting a potential benefit for immune-enhancing activity. The maillard reaction, glycation, is occurred non-enzymatically with sugars and protein. And maillard reaction products (MRPs) have an effects related to immune such as immunomodulatory and anti-cancer effects. Conjugated WPC through Glycation (G-WPC) for a day at 55℃ and fermentation product of G-WPC hydrolysate using Lactobacillus plantarum LC01 (F-WPC) were prepared, respectively. 1,000 mg/kg of WPC, G-WPC and F-WPC had no toxicity for SD rats and cannot cause death by orally administration for 14-day. After transplanted sarcoma 180 cells subcutaneously into the left groin of BALB/c mice and administered with sample for 14 days, we extracted solid tumors and blood samples. NO production and lysozyme from serum in sample treatment groups had increased more than or to the level of control group. When especially administered low-dose of F-WPC, NO production and serum lysozyme significantly increased. Also, the significant increase of IFN-γ, TNF-α and IL-1β cytokines in low-dose of F-WPC group compared to other groups is one of the evidence of an immune-enhancing and anti-tumor activity. Solid tumor cause by injection of sarcoma cells decreased in sample treatment groups compared to NC (negative control) group. The tumor volumes were decreased in the order of WPC, G-WPC and F-WPC when administered low-dose. In conclusion, F-WPC had an anti-tumor and immune-enhancing activity.

Immune enhancing effect of enzymatic hydrolysates of peanut sprout extract

염가희 과학기술연합대학원대학교 2022 국내석사

Immune deficiency is engaged in developing various diseases. Peanuts are high-quality crops containing various nutrients, and they biosynthesize lots of phytochemicals through germination. Peanut sprout extract is known to have an immunomodulatory effect by these physiologically active components. The enzymatic hydrolysis extraction method is known for increasing the extraction yield of these physiologically active components. In part 1, to determine whether the enzyme treatment increases the extraction yield and resveratrol contents of peanut sprouts, cellulase and pectinase were used as the extraction enzymes. Four peanut sprout extracts (PSEs) were prepared by different extraction conditions: peanut sprout non-enzyme extract (PSNE), peanut sprout cellulase extract (PSCE), peanut sprout pectinase extract (PSPE), and peanut sprout cellulase + pectinase extract (PSCPE). In addition, pro or inflammatory mediators (NO, and PGE2), inflammatory cytokines (TNF- α, IL-6, IL-1β, and monocyte chemoattractant protein-1 (MCP-1)) were measured in RAW 264.7 cells to evaluate the immunomodulatory activity of PSEs. The extraction yield of PSEs with different extraction conditions was 17.46% ~ 28.64%. Among the tested PSEs, PSCE showed the highest extraction yield. PSCE showed the highest resveratrol contents (125.17 μg/g, **p < 0.01) among PSEs. All PSEs significantly increased the level of inflammatory mediators (NO, PGE2) especially by PSCE and PSPE in RAW 264.7 cells (***p < 0.001). PSCE and PSPE increased TNF- α and IL-1β production the most among PSEs (***p < 0.001). MCP-1 and IL-6 production were highly increased by PSCPE and PSCE in RAW 264.7 cells (***p < 0.001). To reveal that PSEs could prevent the immune decline in immuno-suppressed BALB/c mice, PSEs were orally administered (200 mg/kg body weight, BW) for 14 days. Cyclophosphamide (CY) was injected intraperitoneally to induce immunosuppression on 14 and 16 days. The spleen, and thymus weight, serum immunoglobulin G (IgG), and mRNA expression of IL-6 and IL-1β were evaluated in immuno-suppressed mice. PSCE showed prevent tendency in weight loss of the spleen (2.52 ± 0.16 to 2.66 ± 0.19 mg/g) and a decrease in serum IgG (40.74 ± 3.59 to 44.08 ± 2.95 μg/mL) due to immune decline (no significance). PSCE prevented a decrease in cytokine expression of IL-6 and IL-1β in mRNA level during immunosuppression. Peanut sprout cellulase extract (PSCE) not only exhibited the highest extraction yield but also showed the highest contents of resveratrol contents. PSCE increased the production of inflammatory mediators and cytokines in RAW 264.7 cells. PSCE also prevented the decline of IL-6 and IL-1β expression in bone marrow due to cyclophosphamide in the BALB/c mice model. These results demonstrate that PSCE could potentially prevent immune decline. Therefore, in part 2, only PSCE was used to determine whether it could enhance immunity. In Part 2, to evaluate whether peanut sprout cellulase extract (PSCE) could stimulate an immune response, iNOS, COX-2, NF-κB, and MAPK signaling pathways were determined by western blot in RAW 264.7 cells. Effect of PSCE on translocation of NF-κB p65 was detected by immunocytochemistry. PSCE significantly activated the iNOS and COX-2 expression at 100 μg/mL (**p < 0.01), phosphorylation of NF-κB p65, and IκBα at 200 μg/mL (***p < 0.001). PSCE also significantly activated phosphorylation of JNK, ERK, and p38 at 100 and 200 μg/mL (***p < 0.001). Furthermore, PSCE increased the translocation of NF-κB p65 into the nucleus in RAW 264.7 cells. These results suggest that PSCE could stimulate an immune response by regulating inflammatory signals. To evaluate the immune-enhancing effect of PSCE in immuno-suppressed BALB/c mice model, 200, 400, and 800 mg/kg body weight (BW) of PSCE were orally administered for 14 days. Cyclophosphamide (CY) was injected intraperitoneally to induce immunosuppression on 8 and 11 days. The body weight, immune organ weight, total leukocyte count, and mRNA expression of IL-6 and IL-1β were evaluated to test immune-stimulatory activities of PSCE in CY-induced immuno-suppressed mice. The weight of mice, which was decreased by cyclophosphamide, was recovered through oral administration with PSCE. In addition, PSCE oral administration significantly increased the number of white blood cells (*p < 0.05), monocytes (***p < 0.001) and lymphocytes (*p < 0.05) at 800 mg/kg BW in immuno-suppressed mice. Oral administration of PSCE at 800 mg/kg BW significantly increased IL-6 and IL-1β expression of bone marrow more than 3-fold (***p < 0.001). In addition, PSCE oral administration increased the number of activated NK cells in splenocytes (**p < 0.01). Our study demonstrated that PSCE had the highest yield and resveratrol contents, also upregulated NF-κB and MAPK signals and activated immune-related TLR4 signals (iNOS, COX-2, and cytokine gene expression) in RAW 264.7 cells. Moreover, the number of white blood cells and natural killer cells was increased by PSCE oral administration in the immuno-suppressed BALB/c mice model. Our results suggest that peanut sprout cellulase extract can be a potential material that could enhance immunity.

Immune Responses to Stimulation of Brucella abortus Antigens

임영빈 서울대학교 대학원 2019 국내박사

Brucellosis caused by Brucella spp. and Brucella abortus has been known as a causative organism of zoonotic disease. B. abortus causes undulant fever, endocarditis, arthritis and osteomyelitis in man and abortion and infertility in cattle. It easily infects not only animals by ingestion or inhalation bacterium in reproductive tissues, fetal fluids, and mammary gland, but also a man by direct or indirect contact with abortion related discharges and ingestion of unpasteurized dairy products from the infected animals. B. abortus which invaded into the hosts through various pathways constructs autologous protection systems to avoid host cell immune system. In addition, the bacterium has been using various factors such as Brucella-containing vacuole (BCV), Type IV secretion system (T4SS), Cyclic β-1,2-glucan (cβG), Brucella-lipopolysaccharide (Br-LPS) to induce survival/replication of the organism in the host cells. Moreover, it interferes with the signaling pathways of phagocytic cells by regulation of the expression of cytokines in the immune cells of the host cells. A number of studies have been carried out for the prevention and eradication of brucellosis that allows such stealthy invasion and survival/replication. In particular, the mechanism of invasion into host cells and the analysis of immunological characteristics are actively investigated. Currently, efforts to detect early infection of B. abortus have been made. Cellular proteins of B. abortus have been received attention as diagnostic antigens through the studies. Based on current knowledge, cellular proteins as recombinant form were used to compare the expression of various genes, especially genes related with cytokines by stimulation of immune cells derived from different host. Also, this study attempted to generate basic information on the development of new diagnostic method(s) and vaccine candidate in B. abortus infection. First, the expressions of IL-1β, IL-4, IL-6, IL-12p40, IFN-γ, TNF-α and iNOS were analyzed in bovine peripheral blood mononuclear cells. Also, the expression of Bax, Bcl-2 and TLR4 was analyzed after stimulation with B. abortus antigens (rMdh, rOMP28, rRocF, rTsf and r0628). Each B. abortus antigens induced different patterns of cytokine expression depending on stimulation time and doses of antigens. Although IL-1β, IL-4, TNF-α and iNOS gene expressions were not observed, the expressions of IL-6, IL-12p40 and IFN-γ were induced in all stimulated B. abortus antigens (P < 0.05). The expression of apoptotic genes was not changed except TLR4 (P < 0.05). Second, RAW 264.7 cells, a mouse macrophage cell-line, and mice were stimulated with B. abortus antigens (rMdh, rOMP10, rOMP19, rOMP28, rRocF and rTsf) to confirm the difference of immune responses in in vitro and in vivo. Changes in cytokine production were examined by stimulating with the cellular proteins in both RAW 264.7 cells and naive splenocytes. The immune response was analyzed by ELISA and ELISpot after immunization of mice with the B. abortus antigens (rMdh, rOMP10, rOMP19, rOMP28, rRocF and rTsf). The production of cytokines such as TNF-α and IL-6 was increased in stimulated RAW 264.7 cells (P < 0.05) while Th1-related cytokines, IFN-γ and IL-2, were induced in naive splenocytes (P < 0.01). In addition, the immune responses induced by the B. abortus antigens were compared by analyzing the in vivo immune response through the difference of IgG secretory cells. As a result, IgG production on the 28th day was significantly increased by immunization with those antigens (P < 0.05). Last, it was studied that the difference in immune responses generated by stimulated with B. abortus antigens (rMdh, rOMP10, rOMP19, rRocF and rTbpA) in THP-1 cells, human-derived leukemic monocytes. In particular, it was investigated to focus on the expression of major immune-regulating factors that play important roles in innate immunity, such as cytokines and Toll-like receptors (TLRs), and acquired immunity in the host. After stimulation of THP-1 cells with B. abortus antigens, intracellular cytokine production and TLR expression were increased at different time points (12 and 24 h), and the differences were analyzed by quantified using ELISA and real-time RT-PCR, respectively (P < 0.05). In the expression of cytokines, it was observed that the production levels of TNF-α and IL-6 were induced at high levels in THP-1 cells stimulated with B. abortus antigens (P < 0.05). In addition, expression of TLR8 was significantly increased at 12 h after stimulation with rOMP19 and rMdh (P < 0.05). These results suggest the need for further studies on how the two B. abortus antigens, rOMP19 and rMdh, are involved in the expression of TLR8 within THP-1 cells. The results of these studies with immune cells derived from different hosts using the B. abortus antigens (rMdh, rTsf, rRocF, r0628, rTbpA, rOMP10, rOMP19 and rOMP28) showed that many of B. abortus cellular antigens showed higher antigenicity in different hosts. It indicated that rMdh and rOMP19 of these B. abortus antigens exhibits higher immunogenicity than other antigens. In particular, rMdh showed the highest immunogenicity to all immune cells used in this study. This study demonstrated the possibility of B. abortus antigen as an intracellular immune response modulator as well as a candidate for high immunogenicity antigens. Therefore, these findings would be useful for understanding basic immune responses, development of diagnostic method and vaccine candidate in B. abortus infection in the future. 브루셀라증은 브루셀라 속균감염에 의해 지속적으로 발생되고 있는 주요한 인수공통전염병 중 하나이다. 특히, Brucella abortus는 소에서 유•사산, 불임등의 번식장애를 사람에서는 파상열, 심내막염, 관절염, 및 골수염을 유발한다. 이러한 B. abortus는 유•사산된 태아, 태반, 양수 및 감염동물의 유선을 통해서 배출된다. B. abortus의 감염은 유•사산물과의 직•간접적인 접촉 또는 멸균되지 않은 유제품의 섭취등에 의해서 이루어질 수 있다. 다양한 경로를 통하여 숙수세포에 침투한 B. abortus는 숙주세포의 면역기전을 회피하기 위한 자가보호시스템을 구축하고 있으며, Brucella-containing vacuole (BCV), Type Ⅳ secretion system (T4SS), Cyclic β-1,2-glucan (cβG), Brucella lipopolysaccharide (Br-LPS)등의 여러가지 요소들을 이용하여 세포내에서의 생존/복제가 가능하다. 또한, 숙주세포에서 면역반응으로 발현되는 사이토카인의 발현을 조절하기 위하여 다양한 대식세포들의 신호전달체계를 방해한다. 이러한 세포내 침입과 생존/복제가 가능한 브루셀라의 예방 및 근절을 위하여 많은 연구가 진행되었다. 특히, 숙주세포 침입기전 분석 및 면역학적 특성 분석에 관한 연구에 많이 집중되었다. 현재 브루셀라의 초기 감염을 진단하기 위한 노력은 지속되고 있으며 B. abortus 감염시 강력한 면역반응을 유발시키는 B. abortus 항원들이 진단항원으로써 주목을 받고 있다. 본 연구에서는 이러한 B. abortus 세포 항원들을 서로 다른 동물유래의 면역세포에서 면역유도능력을 사이토카인과 면역관련 유전자의 발현차이로 비교하여, B. abortus 감염증 예방을 위해 발병기전, 최적의 진단법 및 백신항원후보물질 발굴을 위한 기초 자료를 제공하고자 하였다. 첫번째로, 소의 말초혈액단핵세포(PBMC)의 면역반응 양상을 알아보고자 IL-1β, IL-4, IL-6, IL-12p40, IFN-γ, TNF-α 및 iNOS의 발현을 확인하였다. 또한, 세가지 세포사멸 관련 유전자인 Bax, Bcl-2 및 TLR4의 발현을 B. abortus 항원(rMdh, rOMP28, rRocF, rTsf and r0628) 자극에 의한 면역세포의 반응을 통하여 분석하였다. 각 B. abortus 항원은 자극 시간 및 투여량에 따라 사이토카인 발현양상은 상이하게 나타났다. 즉, IL-1β, IL-4, TNF-α 및 iNOS 유전자 발현은 관찰되지 않았지만, IL-6, IL-12p40 및 IFN-γ의 발현은 모든 B. abortus 항원 자극으로 유도되었다(P < 0.05). 세포사멸 관련 유전자의 발현은 TLR4 외에 변화가 없었다(P < 0.05). 두번째로, 마우스 유래의 면역세포인 RAW 264.7 세포 및 마우스에 B. abortus 항원(rMdh, rOMP10, rOMP19, rOMP28, rRocF and rTsf)을 자극하여 면역반응의 차이를 분석하였다. 사이토카인 발현은 B. abortus 항원으로 RAW 264.7 세포와 비장세포를 자극시킨 후 조사하였다. 면역 반응은 B. abortus 항원(rMdh, rOMP10, rOMP19, rOMP28, rRocF and rTsf)으로 마우스를 면역시킨 후 ELISA 및 ELISpot을 통하여 비교분석하였다. 자극된 RAW 264.7 세포에서는 TNF-α와 IL-6 발현이 증가하였고(P < 0.05), 비장세포에서는 Th1 관련 사이토카인 IFN-γ와 IL-2가 유도되었다(P < 0.01). 또한, 마우스 면역에 따른 생체내 면역반응을 IgG 분비 세포의 발현 차이를 분석함으로 B. abortus 항원에 따라 유도되는 면역반응을 비교한 결과 면역 후 28일째 IgG의 발현이 현저하게 증가하는 것을 관찰할 수 있었다(P < 0.05). 마지막으로, 사람유래 면역세포인 THP-1 세포를 B. abortus 항원(rMdh, rOMP10, rOMP19, rRocF and rTbpA)으로 자극하여 발생하는 면역반응의 차이를 확인하였다. 특히, 사이토카인과 Toll-like receptors (TLRs) 같은 선천성 면역을 조절하고, 감염 후 숙주가 면역을 획득하는데 중요한 역할을 하는 주요 조절인자들의 발현차이에 초점을 맞추어 진행하였다. B. abortus 항원으로 THP-1 세포를 자극 시킨 후, ELISA 및 real-time RT-PCR을 사용하여 사이토카인 생성 및 TLR 발현량을 두시점(12 및 24시간)에서 정량, 비교분석하였다. 사이토카인 생성에 있어서, B. abortus 항원으로 자극된 THP-1 세포에서 TNF-α 및 IL-6의 발현량이 높게 유도되었다(P < 0.05). 또한, TLR8은 rOMP19 및 rMdh로 자극 후 12시간에 대조군에 비해 유의한 발현증가를 나타냈다. 이러한 결과는 두 개의 B. abortus 항원인 rOMP19와 rMdh가 THP-1 세포에서의 TLR8 발현유도에 관한 추가 연구의 필요성을 시사한다. B. abortus 항원(rMdh, rOMP10, rOMP19, rOMP28, rRocF, rTbpA, rTsf and r0628)을 이용한 서로 다른 동물유래 면역세포들의 면역반응 연구결과를 종합하면, 본 연구에 사용된 B. abortus 항원들 중 고면역원성을 나타내는 수종의 B. abortus 세포 항원이 확인되었다. 이들 B. abortus 항원 중 rMdh와 rOMP19는 높은 면역원성을 나타내었다. 특히, rMdh는 본 연구에서 사용한 모든 동물유래 면역세포에 대해 고면역원성을 보였다. 본 연구를 통하여 B. abortus 항원의 세포내 면역반응 조절인자로서의 활성뿐 아니라 고면역원성 항원 후보물질로서의 가능성을 확인할 수 있었으며, 이러한 연구결과는 향후 B. abortus 감염증 예방을 위한 진단법 개발 및 백신후보물질 발굴을 위한 기초자료로 유용할 것으로 사료된다.

IMMUNE-MODULATORY EFFECTS OF HUMAN PLACENTA-DERIVED MESENCHYMAL STEM CELLS ON ACUTE BRAIN INJURY

공태호 차의과학대학교 대학원 2018 국내박사

Human placenta-derived mesenchymal stem cells (hpMSCs) regulate immune responses, and this property can be exploited as cellular sources for cell therapies to treat large spectra of neurologic diseases from acute disorders, including acute stage of stroke and traumatic brain injury (TBI), to chronic aging-related diseases. In neurologic diseases, brain cells are exposed to harsh environment including inflammation and hypoxic conditions. Therefore, investigating how hpMSCs have therapeutic effects in this condition is an important issue. Firstly, we investigated the expression profile of hpMSCs cultured under hypoxic conditions and revealed interesting expression changes in various genes involved in immune regulation. Among them, CD200, a positive regulator of anti-inflammatory TGF-β, was highly expressed under hypoxic conditions. We next investigated whether the CD200-positive hpMSCs influence immunosuppressive effects in vitro. The LPS-stimulated hpMSCs dramatically increased CD200 expression. Immune-modulation factors, including TGF-β, IDO, and IL-10, also exhibited increased expression, thus suggesting that CD200-positive hpMSCs play an important role in regulating inflammatory conditions. To determine whether the immune-modulatory function of hpMSCs is driven by CD200, we used siRNA interference experiments. The siCD200 efficiently silenced CD200 expression in hpMSCs, and this inhibition was maintained after LPS stimulation. In contrast, TGF-β, IDO, and IL-10 expression dramatically decreased after CD200 inhibition and exhibited minimal recovery after LPS stimulation. These results indicated that CD200 may be a key factor for immune-modulation functions in hpMSCs. To confirm the effects of hpMSCs in LPS-stimulated microglial BV2 cells, the cells were primed with LPS and then co-cultured with hpMSCs. CD200-positive hpMSCs exhibited inhibition of pro-inflammatory cytokine expression and NO production in co-cultures with LPS-primed BV2 microglia, and this effect was decreased in CD200-silenced hpMSCs. It suggested that hpMSCs suppress microglial activation and associated NO production through CD200. Therefore, we hypothesized that hpMSCs have strong potentials for ameliorating immune related acute damages such as ischemic stroke by modulating immune responses. We transplanted the CD200-positive hpMSCs into ischemic stroke rat models at 24 h after MCAO and the cells were survived for 1 week after transplantation. To evaluate the immune-modulatory effects of hpMSCs on ischemic stroke brain tissue, we analyzed the mRNA expression of pro-inflammatory and anti-inflammatory factors at 1 day and 1 week after transplantation. Transplantation of hpMSCs showed up-regulation of anti-inflammatory factors including CD200, TGF-b and down-regulation of pro-inflammatory cytokines/ chemokines including IL-1b and TNF-a. These findings suggested that the injected hpMSCs gradually improved the immune-modulatory effects of from 1 day to 1 week in stroke. We determined whether the immune-modulatory effects of hpMSCs might be involved in CD200 expression after transplantation in vivo by using immunohistochemistry analysis. CD200 expression was significantly higher in the region transplanted with hpMSCs than controls at 1 week after transplantation. To determine the effects of cell transplantation on inflammatory responses, we assessed microglia and astrocyte activation in an acute model of stroke by using Iba-1 and GFAP staining. Iba-1 and GFAP-positive cells decreased in the boundary region of hpMSCs-transplanted ischemic brain at 1 week after transplantation. These results demonstrated an hpMSC-dependent decrease of Iba-1 and GFAP in the early phase of stroke, and these effects subsequently contributed to neuroprotection. Altogether, these results demonstrated that hpMSCs are capable of restraining both microglial and astrocytic activation in the early phase of stroke and subsequently protecting neurons against MCAO-related damage. Moreover, at 6 weeks after transplantation, decreases in infarct area and improvements in behavior test were confirmed in hpMSCs-transplanted group when compared with sham group. It demonstrated that the immune-modulatory function of hpMSCs in a stroke rat model underlies the therapeutic recovery potential as a cell therapy. 인간 유래 중간엽 줄기세포는 중추 신경계 질환, 혈관 질환, 퇴행성 질환, 종양을 포함한 많은 질환들을 대상으로 치료 및 저하 된 기능을 향상시키는 능력이 알려지면서 각광받고 있다. 최근 연구들에서 줄기세포는 손상 부위로 이동할 수 있으며, 뛰어난 분화능력, 면역 조절 능력, 분비 인자 등을 바탕으로 손상 부위의 비정상적인 세포를 제거 및 대체 할 수 있을 것이라고 보고되고 있다. 세포치료제 개발에 있어 줄기세포는 중요한 역할을 하고 있으며, 각종 줄기세포에 대한 연구가 활발히 진행되고 있지만 그에 대한 자세한 메커니즘은 잘 알려지지 않은 상태이다. 이러한 한계점 해결을 위하여, 본 실험에서는 중간엽 줄기세포의 하나인 인간 태반 유래 중간엽 세포 (human placenta-derived Mesenchymal Stem Cell; hpMSC) 를 분리 배양하였고, 병리적 환경에서 인간 태반 유래 줄기세포의 치료 메커니즘을 연구하였다. 인간 태반 줄기 세포는 배아 중배엽에서 유래 된 것이며, 대개 출생 후에 폐기되기 때문에 윤리적 문제없이 많은 양의 세포를 얻을 수 있어 세포치료를 위한 훌륭한 후보군이며, 성체 줄기 세포로서 매우 유용하다. 또한, 태반 유래 줄기세포는 HLA 분자를 갖지 않기 때문에 다른 줄기세포들에 비해 면역원성이 낮아 세포 치료제로서 유용한 소스가 될 수 있다. 최근 보고된 연구 결과에서 인간 태반 유래 줄기 세포는 면역 반응을 조절하여 뇌졸중, 외상성 뇌 손상, 알츠하이머 같은 만성 노화 관련 신경 질환 등의 신경계 질환에서 치료 효능이 보여지고 있다. 하지만 염증 및 저산소 상태를 포함한 가혹한 병리적 환경에서 태반 유래 줄기세포가 어떻게 치료 효과를 갖는지에 대한 메커니즘은 명확하지 않다. 본 실험에서는 중간엽 줄기세포의 하나인 인간 태반 유래 중간엽 줄기세포가 병리적 환경에서 어떤 치료 메커니즘을 갖는지 확인하기 위해 저산소 (hypoxic) 조건에서 배양 된 태반 유래 줄기세포를 전사체 분석 (RNA-sequencing) 을 통해 유전자 프로파일을 조사하고 면역 조절과 관련된 다양한 유전자에서 발현 변화를 밝혀냈다. 그 중 CD200은 저산소 상태에서 고도로 발현되었고, 항염증성 인자 TGF-β의 양성 조절 인자로 알려져 있다. 우리는 CD200 양성 태반 유래 줄기세포가 세포 수준에서 면역 조절 효과에 영향을 미치는지 연구했다. 병리적 환경을 위해 LPS로 염증 환경 유도하였으며, 태반 유래 줄기세포는 CD200 발현이 유의적으로 증가 되었다. 항염증 인자로 잘 알려진 TGF-β, IDO, IL-10 또한 증가 된 발현을 나타내어 CD200 양성 태반 유래 줄기세포가 염증 환경 조절에 중요한 역할을 한다는 것을 확인 하였다. 태반 유래 줄기세포의 면역 조절 기능이 CD200에 의해 유도되는지 여부를 확인하기 위해 siRNA 실험을 하였다. siCD200은 태반 유래 줄기세포에서 CD200 발현을 억제 시켰으며, LPS 자극에서도 발현의 억제가 유지되었다. 또한, 항염증 인자 TGF-β, IDO, IL-10 발현도 CD200 억제 후 감소하였고, LPS로 인한 염증 유도 환경에서도 CD200 양성 태반 유래 줄기세포에 비해 항염증 인자들의 발현이 감소되었다. 이러한 결과는 CD200이 태반 유래 줄기세포의 면역 조절 기능의 핵심 요소 일 수 있음을 보여주었다. 다음으로, 마이크로글리아 세포주 (microglia cell line) BV2 세포에서 태반 유래 줄기세포의 효과를 확인하기 위해, LPS로 염증환경을 유도한 뒤 태반 유래 줄기세포와 함께 배양 하였다. CD200 양성 태반 유래 줄기세포는 염증성 사이토카인 발현 및 NO 생산의 억제를 나타내었고, 이 효과는 CD200을 억제 시킨 태반 유래 줄기세포에서 그 효과는 감소되었다. 이 결과는 태반 유래 줄기세포가 CD200을 통해 마이크로글리아 활성화 및 NO 생산을 억제하는 것을 의미한다. 이러한 효과는 태반 유래 줄기세포가 면역 반응을 조절하여 허혈성 뇌졸중과 같은 염증 관련 급성 손상을 치료 할 수 있는 잠재력을 가지고 있다고 할 수 있다. 우리는 허혈성 뇌졸중 모델에서 태반 유래 줄기세포의 염증 조절을 통한 치료 효과를 확인하기 위해 중뇌동맥협착 (Middle Cerebral Artery Occlusion, MCAO) 방법으로 허혈성 뇌졸중 쥐 모델을 만들었으며, 24 시간 후에 CD200 양성 태반 유래 줄기세포를 이식하였다. 병변 주위에 이식된 세포는 뇌 조직 안에서1 주 동안 세포를 생존하는 것을 확인 하였으며, 허혈성 뇌졸중 뇌 조직에서 태반유래 줄기세포의 면역 조절 효과를 평가하기 위해 이식 후 1 일과 1 주일에 염증 인자 및 항염증 인자의 mRNA 발현을 분석 하였다. 태반 유래 줄기세포의 이식은 CD200, TGF-b등의 항염증 인자의 발현을 높였으며, IL-1b 및 TNF-α를 포함하는 염증성 사이토카인/ 케모카인의 발현은 억제시키는 효과를 보였다. 이러한 결과는 태반 유래 줄기세포가 뇌졸중에서 1 일에서 1 주 동안 면역 조절 효과를 점차적으로 향상 시킨다는 것을 의미한다. 다음으로 태반 유래 줄기세포의 면역 조절 효과를 면역조직화학법을 사용하여 뇌 조직에서 CD200 발현을 확인 했다. CD200 발현은 이식 후 1 주 조직에 대조군보다 태반 유래 줄기세포를 이식 한 부위에서 유의하게 높았다. 염증 반응에 대한 세포 이식의 효과를 확인하기 위해 Iba-1 및 GFAP 염색을 하여 급성 뇌졸중 모델에서의 마이크로글리아 및 성상 세포 활성화를 확인했다. Iba-1과 GFAP 는 태반 유래 줄기세포 이식 후 1 주일에서 손상된 병변 부위에서 활성이 감소하였다. 이러한 결과는 뇌졸중의 초기 단계에서 태반유래 줄기세포가 염증 조절 효과를 통해 Iba-1 및 GFAP의 감소를 보여 주었으며, 이러한 효과는 이후 신경 보호 효과에도 영향을 주었다. 결론적으로, 태반 유래 줄기세포는 뇌졸중의 초기 단계에서 마이크로글리아와 성상 세포의 활성화를 억제 할 수 있고, 이후 허혈성 뇌 손상으로부터 신경세포를 보호 할 수 있음을 입증하였다. 또한, 이식 후 6 주째에 태반 유래 줄기세포 이식 군에서는 대조군과 비교하여 경색 면적의 감소 및 행동 검사의 개선이 확인되었다. 이러한 결과는 뇌 손상에서 태반 유래 줄기세포는 면역 조절 기능을 통한 치료제로서의 가능성을 확인하였으며, 면역 조절에 있어서 CD200은 중요한 인자임을 확인 하였다. 또한, 여러 면역 관련 질환에 있어 태반 유래 줄기세포를 이용한 새로운 치료 기전 및 전략을 제공할 수 있어 세포치료제로서의 가능성과 치료법을 한층 더 넓혀주는 기초가 되는 결과이다. 핵심되는 말: 태반 유래 중간엽 줄기세포, 면역 조절, 염증, 뇌졸중, CD200

Immune Polyphony: Dissecting Coordinated Multicellular Networks of Antiviral Immunity

Wilk, Aaron James ProQuest Dissertations & Theses Stanford Universit 2022 해외박사(DDOD)

Viral diseases remain a major cause of morbidity and mortality worldwide and emerging viral pathogens represent a perennial threat to human health. The rapidity, quality, and duration of the immune response to a viral pathogen is a major determinant of disease outcome in viral disease. This immune response to a viral pathogen involves the activity of multiple cell types with distinct and multifaceted functional roles to mount a response that is appropriate in both time and space. An insufficient response may result in unrestrained viral replication while an overly robust response can result in direct tissue damage. Thus, an effectively regulated antiviral immune response requires complex coordination, communication, and regulation between individual immune cells. We coin the term "immune polyphony" to describe these coordinated multicellular networks -- a reference to musical polyphony where multiple individual voices come together to create harmony.Here we present a framework to study and engineer immune polyphony and apply this framework to dissect the features of well-balanced immune responses in multiple viral diseases. First, we examine immune responses in patients with COVID-19 across the full range of the disease severity spectrum to reveal widespread dysregulation of innate immunity in severe COVID-19. Next, we introduce a novel computational framework for the analysis of cell-cell communication pathways at the resolution of the single-cell. We then apply this methodology to analyze inflammatory networks that distinguish immune responses to pathogenic vs. non-pathogenic lentiviruses. Finally, we describe a technique for bio-orthogonal manipulation of primary immune cells, thus providing a pathway for future efforts to engineer polyphonic immune responses to promote effective antiviral immunity.

Siegesbeckiae Herba enhances immune response in forced swimming-induced immunosuppressive mice

송주혜 과학기술연합대학원대학교 한국식품연구원(KFRI) 2024 국내석사

면역 항상성이 무너지면 정상적인 면역 반응을 유지할 수 없어 다양한 질병의 발생 위험이 증가한다. 희첨은 전통 약초로 사용되어 왔으며, 항염증, 항종양, 항혈전, 심근 손상 보호 효과가 있는 것으로 알려져 있지만, 면역 증진 효능은 연구된 바가 없다. 본 연구에서는 희첨 열수 추출물 (SHE)을 면역 증진 소재 후보로 선정하여 생리활성 성분을 분석하고, in vivo 및 ex vivo 실험을 통해 면역 활성 효능을 밝혔다. UPLC-Q-TOF-MS 를 통해 SHE 의 성분을 분석한 결과, 다양한 caffeoylquinic acids 및 quercetin 유도체가 함유된 것으로 나타났다. SHE 의 생체 내 면역 기능 저하 예방 효과를 확인하기 위해, 강제수영을 통해 면역 저하를 유도한 마우스에 SHE (25, 100 mg/kg body weight)를 21일간 경구 투여하였다. 그 결과, SHE 는 강제수영에 의해 저하된 면역 기관 지표, 백혈구 수, 림프구 증식, 면역글로불린 그리고 TNF-α, IL-2, IFN-γ 및 perforin 의 유전자 발현을 회복시켰다. RAW 264.7 대식세포에서 SHE 는 MAPKs (p38, ERK 및 JNK) 신호전달을 활성화시킴으로써 NO 및 전염증성 사이토카인 (IL-6, IL- 1β 및 TNF-α)의 발현과 식작용활성을 증가시켰다. 또한 NK-92 세포에 SHE 를 처리하였을 때, ERK 신호전달 경로의 활성화를 통해 K562 세포에 대한 세포독성과 perforin 및 granzyme B 의 분극화가 증가하였다. 종합적으로, 본 연구를 통해 SHE 가 강제수영에 의해 유도된 억제된 면역 기능을 회복하고 대식세포와 자연살해세포를 활성화시킨다는 것을 확인하였다. 따라서 본 연구는 SHE 가 면역증강 기능성 소재로서의 가치를 가지고 있음을 시사한다. Unbalanced immune responses lead to the disruption of the immune homeostasis, thereby the risk of developing various diseases increases. Siegesbeckiae herba has been used as a traditional medicinal herb and is well known for its anti-inflammatory, anti-tumor, anti-thrombotic, and myocardial damage protection effects. However, its immune-boosting effects have not been reported. In this study, I investigated the immune-enhancing effect of Siegesbeckiae herba water extract (SHE), chosen as a candidate for an immunomodulatory substance, in vivo and in vitro. Through screening of the immune activity evaluation across 23 medicinal plants, SHE demonstrated superior effects on IL-6 production in RAW 264.7 cells and NK cell cytotoxicity in splenocytes. In addition, UPLC-Q-TOF-MS analysis of SHE revealed the presence of multiple caffeoylquinic acids and quercetin derivatives. To confirm the immune-boosting effect of SHE, the SHE (25, 100 mg/kg body weight) was administered orally to forced swimming-induced mice for 21 days. Consequently, SHE recovered forced swimming-induced downregulated organ index, whole blood cell count, lymphocyte proliferation, immunoglobulin, and gene expression such as TNF-α, IL-2, IFN-γ, and perforin. To reveal the immune-boosting mechanism of SHE, I focused on the activities of macrophages and NK cells. In RAW 264.7 macrophages, SHE promoted the production of NO and pro- inflammatory cytokines (IL-6, IL-1β, and TNF-α) expression and phagocytic activity by activating p38, ERK, and JNK signaling pathways. Additionally, SHE led to enhanced cytotoxicity against K562 cells and increased polarization of perforin and granzyme B via activation of the ERK signaling pathway in NK-92 cells. Taken together, this study demonstrated that SHE effectively recovers suppressed immune function induced by forced swimming and promotes the activation of macrophages and NK cells. Therefore, this study proposes that SHE has the potential as an immune-boosting functional material.

Immune dysfunction in Shank2-mutant mice and behavior rescue by immune modulation

정유진 Graduate School, Yonsei University 2023 국내석사

Autistic spectrum disorder (ASD) is a neurodevelopmental disorder that is generally diagnosed in childhood. ASD’s pathogenesis is not clearly established. Recent literature suggests a relationship between immune dysfunction and ASD. Studies show ASD patients have a higher risk of suffering from immune-related dysfunctions (Suzuki et al., 2011; Wasilewska & Klukowski, 2015). Moreover, regulation of immunity and gut microbiota have shown to be effective in alleviating symptoms of ASD in the animal model (Kim et al., 2017; Reed et al., 2020). ASD is accounted for by genetic factors and environmental factors such as maternal immune activation during pregnancy, which has been found to be correlated with immune system abnormalities in ASD animal models (Choi et al., 2016). Until now, studies on ASD caused by single-gene mutation (genetic factors) have focused on abnormalities in brain functions. The effect of a single-gene mutations on the immune system that leads to ASD is unknown. In this study, we highlight to the interaction between genetic factors and the immune system. We investigated the existence of immune system abnormalities in both peripheral organs and the central nervous system in Shank2–/– mice. Moreover, we investigated if it would be possible to lessen the severity of autistic-like behavior through immune modulation. Our findings show abnormalities of the immune system exist in peripheral organs and the central nervous system of Shank2–/– mice. Specifically, interleukin-17a, which plays an important role in sociability (Reed et al., 2020), was significantly decreased in the serum and the brain of Shank2–/– mice. Yet, increasing interleukin-17a levels in the brain through astrocytes-restricted interleukin-17a expression did not lead to recovery of autistic-like behavior in Shank2–/– mice. 이전 연구에서 자폐 스펙트럼 환자의 면역 기능 장애가 보고 되었으며, 면역 및 장내 미생물의 조절은 동물 모델에서 자폐 증상을 완화하는데 효과적인 것으로 나타났음. 자폐는 유전적 요인과 면역 체계 이상을 유발하는 모체 면역 활성화와 같은 환경적 요인에 의해 발생함. 그러나 자폐증이 유전적 요인과 환경적 요인이 복합적으로 작용하여 발생하는지는 알려져 있지 않음. 단일 유전자 돌연변이(유전적 요인)에 의한 자폐 연구는 뇌 기능의 이상에 초점을 맞추어 왔으며, 자폐를 유발하는 단일 유전자 돌연변이가 면역체계에 미치는 영향은 알려져 있지 않음. Shank2 유전자 결핍 마우스에서 말초 기관 및 중추신경계의 면역 체계 이상이 존재하는지 확인했고, 면역 조절을 통해 자폐 유사 행동의 회복이 가능한지 확인 했음. 그 결과 Shank2 유전자 결핍 마우스의 말초 기관 및 중추신경계에서 면역 체계의 이상을 발견 했으며, 구체적으로 자폐 유사 행동에 중요한 역할을 하는 interleukin-17a가 혈청과 뇌에서 유의하게 감소해 있었음. 그러나 별 아교 세포에만 특이적으로 interleukin-17a 발현을 증가 시켜, 뇌 전체적으로 interleukin-17a의 농도를 증가 시켰을 때, Shank2 유전자 결핍 마우스의 자폐 유사 행동이 회복 되지 않았음.

면역관문억제제 사용에 따른 갑상선 관련 이상반응 발생에 관한 연구 : 인구 기반 코호트 및 코호트내 환자-대조군 연구

이종민 중앙대학교 대학원 2024 국내석사

배경 및 목적: 면역관문억제제는 체내의 면역 시스템을 활용하여 암을 사멸시키는 항암제로, 허가 받은 이래 지속적으로 사용량이 늘어왔다. 사용량이 늘어남에 따라 면역관련 이상반응들의 보고가 꾸준히 증가하는 추세에 있어왔다. 대표적으로 보고되는 면역반응 관련 이상반응으로 갑상선 기능 이상과 같은 내분비 관련 면역 이상반응이 보고되고 있다. 본 연구에선 면역관문억제제 사용에 따른 갑상선 기능 이상 발생에 대해 어떤 요인들이 영향을 미치는가에 대해 알아보고자 연구를 수행하였다. 연구방법: 본 연구는 후향적 코호트 연구로, 2016년부터 2022년 사이의 건강보험공단 청구자료를 활용하여 연구를 진행하였다. 2018.01.01 부터 2022.06.30사이에 면역관문억제제 치료를 시작한 환자들을 대상으로 하였으며, 기존에 갑상선 관련 질환 진단 이력이 있는 자들은 제외하였다. 연구기간은 면역관문억제제 치료를 시작한 시점으로부터 최대 6개월로 하였다. 연구기간 내 갑상선 관련 이상반응이 발생한 그룹과 발생하지 않은 그룹으로 나누어 코호트 수준에서 콕스 회귀 분석을 통해 어떤 요인들 (면역관문억제제 기전 및 약제별 영향, 연령, 기저질환 등)이 갑상선 관련 이상반응에 영향을 주는지에 대해 평가하였다. 또한 코호트 내에서 환자군-대조군에 대해 성별, 연령, 폐암 발생에 대해 1:4로 매칭하여 환자-대조군 연구를 진행하였다. 환자-대조군 연구에선 갑상선 관련 이상반응 발생에 영향을 줄 수 있는 요소들을 조건부 로지스틱 회귀분석을 활용하여 그 영향에 대해 평가하였다. 연구결과: 2018년 1월 1일부터 2022년 6월 30일 사이에 면역관문억제제 치료를 시작한 32,186 명 중 제외 사유에 해당하는 사람들을 제외한 18,950명에 대해 연구를 수행하였다. 연구기간 내 갑상선 관련 이상반응이 나타난 환자 수는 672명으로 확인되었다. 코호트 연구에선 확인하지 못하였으나, 환자-대조군 연구를 통해 확인한 바, 면역관문억제제 약제간 및 약제 작용 기전별 갑상선 관련 이상반응 발생에 대한 영향은 nivolumab과 ipilimumab 병용 그룹이 PD-1 그룹 대비 갑상선 관련 이상반응 발생에 영향을 줄 수 있음을 확인하였다 [오즈비: 1.64 (95% confidence interval (CI), 1.01 – 2.66). 면역관문억제제 외에 갑상선 관련 이상반응 발생에 영향을 줄 수 있는 요인들을 확인하고자 하였으나 결과 발생에 영향을 줄 수 있는 요인을 확인하진 못하였다. 결론: 최근 면역관문억제제 사용에 따른 갑상선 관련 이상반응 발생이 지속적으로 보고되고 있고, 면역관문억제제 사용 환자수는 지속 증가 중에 있다. 특히 nivolumab과 ipilimumab 병용 시 갑상선 관련 이상반응 발생 가능성이 타 면역관문억제제 그룹 대비 높음을 확인하였다. 면역관문억제제 사용 환자에 대한 갑상선 관련 이상반응이 나타나는지에 대해 지속적으로 추적관찰 할 필요가 있다. Background: Immune checkpoint inhibitors are anti-cancer drugs that utilize the body's immune system to destroy cancer cells, and their use has been increasing continuously since approval. As their usage increases, reports of immune-related adverse reactions have been steadily rising. Endocrine-related immune adverse reactions, such as thyroid dysfunction, are commonly reported. This study aimed to investigate what factors influence the occurrence of thyroid dysfunction due to the use of immune checkpoint inhibitors. Method: This was a retrospective cohort study using National Health Insurance claims data from 2016 to 2022. The study included patients who started treatment with immune checkpoint inhibitors between January 1, 2018, and June 30, 2022, excluding those with a prior diagnosis of thyroid-related diseases. The study period was set to a maximum of 6 months from the start of treatment with immune checkpoint inhibitors. The cohort was divided into groups based on whether thyroid-related adverse reactions occurred or not, and a Cox regression analysis was conducted to evaluate which factors (mechanism and type of immune checkpoint inhibitors, age, comorbidities, etc.) influence these adverse reactions. In addition, a patient-control study within the cohort was conducted, matching patients and controls based on gender, age, and lung cancer occurrence at a ratio of 1:4. The patient-control study assessed the impact of potential factors influencing thyroid-related adverse reactions using conditional logistic regression analysis. Results: Of the 32,186 people who started treatment with immune checkpoint inhibitors between January 1, 2018, and June 30, 2022, 18,950 individuals were included in the study after excluding those who met the exclusion criteria. A total of 672 patients experienced thyroid-related adverse reactions during the study period. Although not observed in the cohort study, the patient-control study found that the combination of nivolumab and ipilimumab could impact the occurrence of thyroid-related adverse reactions compared to the PD-1 group [Odds Ratios: 1.64 (95% confidence interval (CI), 1.01 – 2.66)]. No factors other than immune checkpoint inhibitors were found to influence the occurrence of thyroid-related adverse reactions. Conclusion: The incidence of thyroid-related adverse events due to the use of immune checkpoint inhibitors has been continuously reported, and the number of patients using these inhibitors is increasing. Particularly, the combination of nivolumab and ipilimumab was found to have a higher likelihood of occurrence than other ICI groups. Continuous monitoring and observation are necessary to track thyroid-related adverse reactions in patients using immune checkpoint inhibitors.

Comprehensive immune profiling and analysis of significance of S100A8-positive immune cells in breast cancer

우지원 서울대학교 대학원 2022 국내박사

Background: Immune microenvironment differs according to hormone receptor (HR) status or tumor progression stage. Its importance is ever-increasing as drugs targeting tumor immunity came up as a game changer in cancer therapeutics. Myeloid-derived suppressor cells (MDSCs) are one of the key players in tumor immunity but not well studied in human tissue because of its phenotypic complexity. They are known to promote tumor progression via various immune-mediated and non-immune-mediated mechanisms. Methods: Differential expression of immune-related genes was evaluated via comprehensive immune profiling according to unsupervised clustering and HR status, and a target gene with significant fold change, possibly MDSC-associated gene, was searched for further analysis. The difference in its expression was validated using tissue microarray of 700 cases of human breast cancer. Its clinicopathological significance and relationship between other tumor infiltrating lymphocytes (TILs) including CD4+, CD8+ and FOXP3+ TILs or PD-L1+ immune cells (ICs) were investigated. Results: In immune profiling analysis, S100A8, a known MDSC marker, showed the most striking fold change in the cluster 2 in unsupervised clustering and HR status dependent grouping. In immunohistochemistry (IHC), S100A8 was stained in both tumor cells and ICs in breast cancer samples. Infiltration of S100A8+ ICs was associated with aggressive clinicopathological features and poor clinical outcome. They were already present in pre-invasive stage of breast cancer and were associated with increased infiltration of CD4+, CD8+, and FOXP3+ TILs and PD-L1+ ICs. Prognostic impact of S100A8+ ICs was stronger in HR-positive, PD-L1+ IC-negative, CD8+ TIL-low and FOXP3+ TIL-low subgroups. When stained with various IHC markers, S100A8+ ICs were mostly revealed as CD33+ CD15- CD163+ cells which are thought to be monocytic-MDSCs derived macrophages. Conclusion: S100A8 is a key molecule that differentiates distinct immune clusters and also highly expressed in HR-negative breast cancers. S100A8 is usually expressed in monocytic-MDSCs derived macrophages and contributes to breast cancer progression from pre-invasive stage. S100A8+ ICs were associated with increase in CD4+, CD8+, and FOXP3+ TILs and PD-L1+ ICs but prognostic impact of S100A8+ ICs was stronger in less immunogenic condition. 서론: 유방암의 면역 미세환경은 호르몬 수용체 발현이나 종양의 진행 단계에 따라 다양한 모습을 보인다. 종양 면역을 대상으로 하는 면역치료제의 효과가 입증됨으로서 유방암 면역에 대한 연구의 중요성은 날로 커지고 있다. 골수유래 면역억제세포는 종양 면역에 중요한 기능을 담당하고 있으나 그 표현형이 복잡하기 때문에 인체 조직에서 연구가 어려운 분야이다. 이 세포들은 면역과 관련된 기전 뿐만 아니라 면역과 관련되지 않은 기전을 통하여서도 다양하게 암의 진행을 촉진시킨다. 방법: 포괄적인 면역 프로파일링을 이용하여 비지도 군집분석과 호르몬 수용체 발현에 따른 면역 관련 유전자들의 발현 차이를 평가하였다. 추가 분석을 위하여 유의미한 발현량 차이를 보이는 유전자로 나타난 골수유래 면역억제세포 관련 유전자를 선택하였다. 700명의 유방암 환자 조직으로 만들어진 조직 마이크로어레이에서 해당 단백질 발현의 차이를 확인하였다. 이 단백질 발현이 가지는 임상병리학적 중요성과 다른 종양 침윤 림프구 (CD4, CD8, FOXP3 양성 림프구), PD-L1 양성 면역세포와의 관계를 분석하였다. 결과: 면역 프로파일링 자료를 비지도 군집분석에서 확인된 ‘군집 2’와 다른 군집들의 비교 및 호르몬 수용체 발현에 따라서 분석한 결과 골수유래 면역억제세포 표지자로 알려진 S100A8이 가장 큰 발현량 차이를 보였다. 면역조직화학염색 결과 S100A8은 유방암 조직의 종양세포와 면역세포 모두에서 염색되었다. 특히 S100A8 양성 면역세포의 침윤은 나쁜 임상병리학적 특성, 나쁜 예후와 관련이 있었다. 이들은 유방암의 침윤 전 단계부터 존재하였고 CD4, CD8, FOXP3 양성 종양 침윤 림프구, PD-L1 양성 면역세포 침윤의 증가와 연관을 보였다. S100A8 양성 면역세포의 예후적 중요성은 호르몬 수용체 양성인 경우, PD-L1 양성 면역세포가 없는 경우, CD8, FOXP3 양성 림프구가 적은 경우에 더 두드러지게 나타났다. 다양한 면역조직화학검사 결과 S100A8 양성 면역세포는 대부분 CD33 양성, CD15 음성, CD163 양성 표현형을 보여 단핵구 계열 골수유래 면역억제세포에서 기원한 대식세포로 생각되었다. 결론: S100A8은 특정 면역 군집을 구분하는 중요한 단백이며 또한 호르몬 음성 유방암에서 증가하였다. 인간 유방암 조직에서 S100A8은 주로 단핵구 계열 골수유래 면역억제세포에서 기원한 대식세포에서 발현되며 침윤 전 단계부터 존재하여 유방암의 진행을 촉진하는 역할을 할 것으로 생각된다. S100A8 양성 면역세포는 CD4, CD8, FOXP3 양성 종양 침윤 림프구와 PD-L1 양성 면역세포의 증가와 연관이 있었으나 그 예후적 중요성은 면역성이 낮은 조건에서 더 두드러지게 나타났다.