Dysregulated CD38 Expression on Peripheral Blood Immune Cell Subsets in SLE

Given its uniformly high expression on plasma cells, CD38 has been considered as a therapeutic target in patients with systemic lupus erythematosus (SLE). Herein, we investigate the distribution of CD38 expression by peripheral blood leukocyte lineages to evaluate the potential therapeutic effect of CD38-targeting antibodies on these immune cell subsets and to delineate the use of CD38 as a biomarker in SLE. We analyzed the expression of CD38 on peripheral blood leukocyte subsets by flow and mass cytometry in two different cohorts, comprising a total of 56 SLE patients. The CD38 expression levels were subsequently correlated across immune cell lineages and subsets, and with clinical and serologic disease parameters of SLE. Compared to healthy controls (HC), CD38 expression levels in SLE were significantly increased on circulating plasmacytoid dendritic cells, CD14⁺⁺CD16⁺ monocytes, CD56⁺ CD16^dim natural killer cells, marginal zone-like IgD⁺CD27⁺ B cells, and on CD4⁺ and CD8⁺ memory T cells. Correlation analyses revealed coordinated CD38 expression between individual innate and memory T cell subsets in SLE but not HC. However, CD38 expression levels were heterogeneous across patients, and no correlation was found between CD38 expression on immune cell subsets and the disease activity index SLEDAI-2K or established serologic and immunological markers of disease activity. In conclusion, we identified widespread changes in CD38 expression on SLE immune cells that highly correlated over different leukocyte subsets within individual patients, but was heterogenous within the population of SLE patients, regardless of disease severity or clinical manifestations. As anti-CD38 treatment is being investigated in SLE, our results may have important implications for the personalized targeting of pathogenic leukocytes by anti-CD38 monoclonal antibodies.     

Sasa quelpaertensis Leaf Extract Inhibits Colon Cancer by Regulating Cancer Cell Stemness in Vitro and in Vivo

A rare subpopulation of cancer cells, termed cancer stem cells (CSCs), may be responsible for tumor relapse and resistance to conventional chemotherapy. The development of a non-toxic, natural treatment for the elimination of CSCs is considered a strategy for cancer treatment with minimal side effects. In the present study, the potential for Sasa quelpaertensis leaf extract (SQE) and its two bioactive compounds, tricin and p-coumaric acid, to exert anti-CSC effects by suppressing cancer stemness characteristics were evaluated in colon cancer cells. CD133⁺CD44⁺ cells were isolated from HT29 and HCT116 cell lines using flow-activated cell sorting (FACs). SQE treatment was found to significantly suppress the self-renewal capacity of both cell lines. SQE treatment was also associated with the down-regulation of β-catenin and phosphorylated GSK3β, while significantly enhancing cell differentiation by up-regulating CK20 expression and blocking the expression of several stem cell markers, including DLK1, Notch1, and Sox-2. In vivo, SQE supplementation suppressed tumor growth in a xenograft model by down-regulating stem cell markers and β-catenin as well as HIF-1α signaling. Compared with two bioactive compounds of SQE, SQE exhibited the most effective anti-CSC properties. Taken together, these results provide evidence that SQE inhibits colon cancer by regulating the characteristics of CSCs.     

Analysis of Circulating Tumor and Cancer Stem Cells Provides New Opportunities in Diagnosis and Treatment of Small Cell Lung Cancer

Current methods for diagnosis and treatment of small cell lung cancer (SCLC) have only a modest efficacy. In this pilot study, we analyzed circulating tumor cells (CTCs) and cancer stem cells (CSCs) in patients with SCLC to search for new diagnostic and prognostic markers and novel approaches to improve the treatment of the disease. In other forms of lung cancer, we showed a heterogeneity of blood CTCs and CSCs populations, as well as changes in other cell populations (ALDH⁺, CD87⁺CD276⁺, and EGF⁺Axl⁺) in smokers. A number of CTCs and CSCs in patients with SCLC have been shown to be resistant to chemotherapy (CT). High cytotoxic activity and resistance to apoptosis of reprogrammed CD3⁺CD8⁺ T-lymphocytes (rTcells) in relation to naive CD3⁺CD8⁺ T-lymphocytes was demonstrated in a smoking patient with SCLC (Patient G) in vitro. The target for rTcells was patient G’s blood CSCs. Reprogramming of CD3⁺CD8⁺ T-lymphocytes was carried out with the MEK1/2 inhibitor and PD-1/PD-L1 pathway blocker nivolumab. The training procedure was performed with a suspension of dead CTCs and CSCs obtained from patient’s G blood. The presented data show a new avenue for personalized SCLC diagnosis and targeted improvement of chemotherapy based on the use of both CTCs and CSCs.     

Cisplatin-Resistant CD44+ Lung Cancer Cells Are Sensitive to Auger Electrons

Cancer stem cells (CSCs) are resistant to conventional therapy and present a major clinical challenge since they are responsible for the relapse of many cancers, including non-small cell lung cancer (NSCLC). Hence, future successful therapy should also eradicate CSCs. Auger electrons have demonstrated promising therapeutic potential and can induce DNA damage while sparing surrounding cells. Here, we sort primary patient-derived NSCLC cells based on their expression of the CSC-marker CD44 and investigate the effects of cisplatin and a thymidine analog (deoxyuridine) labeled with an Auger electron emitter (¹²⁵I). We show that the CD44⁺ populations are more resistant to cisplatin than the CD44⁻ populations. Interestingly, incubation with the thymidine analog 5-[¹²⁵I]iodo-2′-deoxyuridine ([¹²⁵I]I-UdR) induces equal DNA damage, G2/M cell cycle arrest, and apoptosis in the CD44⁻ and CD44⁺ populations. Our results suggest that Auger electron emitters can also eradicate resistant lung cancer CD44⁺ populations.     

Oral Squamous Cell Carcinoma Contributes to Differentiation of Monocyte-Derived Tumor-Associated Macrophages via PAI-1 and IL-8 Production

Tumor-associated macrophages (TAMs) promote cancer cell proliferation and metastasis, as well as anti-tumor immune suppression. Recent studies have shown that tumors enhance the recruitment and differentiation of TAMs, but the detailed mechanisms have not been clarified. We thus examined the influence of cancer cells on the differentiation of monocytes to TAM subsets, including CD163⁺, CD204⁺, and CD206⁺ cells, in oral squamous cell carcinoma (OSCC) using immunohistochemistry, flow cytometry, and a cytokine array. Furthermore, we investigated the effect of OSCC cells (HSC-2, SQUU-A, and SQUU-B cells) on the differentiation of purified CD14⁺ cells to TAM subsets. The localization patterns of CD163⁺, CD204⁺, and CD206⁺ in OSCC sections were quite different. The expression of CD206 on CD14⁺ cells was significantly increased after the co-culture with OSCC cell lines, while the expressions of CD163 and CD204 on CD14⁺ cells showed no change. High concentrations of plasminogen activator inhibitor-1 (PAI-1) and interleukin-8 (IL-8) were detected in the conditioned medium of OSCC cell lines. PAI-1 and IL-8 stimulated CD14⁺ cells to express CD206. Moreover, there were positive correlations among the numbers of CD206⁺, PAI-1⁺, and IL-8⁺ cells in OSCC sections. These results suggest that PAI-1 and IL-8 produced by OSCC contribute to the differentiation of monocytes to CD206⁺ TAMs.     

NK Cells Lose Their Cytotoxicity Function against Cancer Stem Cell-Rich Radiotherapy-Resistant Breast Cancer Cell Populations

Cancer stem cells (CSCs) can be induced from differentiated cancer cells in the tumor microenvironment or in response to treatments and exhibit chemo- and radioresistance, leading to tumor recurrence and metastasis. We previously reported that triple negative breast cancer (TNBC) cells with acquired radioresistance exhibited more aggressive features due to an increased CSC population. Therefore, here, we isolated CSCs from radiotherapy-resistant (RT-R)-TNBC cells and investigated the effects of these CSCs on tumor progression and NK cell-mediated cytotoxicity. Compared to MDA-MB-231 and RT-R-MDA-MB-231 cells, CD24^−/low/CD44⁺ cells isolated from RT-R-MDA-MB-231 cells showed increased proliferation, migration and invasion abilities, and induced expression of tumor progression-related molecules. Moreover, similar to MDA-MB-231 cells, CD24^−/low/CD44⁺ cells recruited NK cells but suppressed NK cell cytotoxicity by regulating ligands for NK cell activation. In an in vivo model, CD24^−/low/CD44⁺ cell-injected mice showed enhanced tumor progression and lung metastasis via upregulation of tumor progression-related molecules and altered host immune responses. Specifically, NK cells were recruited into the peritumoral area tumor but lost their cytotoxicity due to the altered expression of activating and inhibitory ligands on tumors. These results suggest that CSCs may cause tumor evasion of immune cells, resulting in tumor progression.     

Enhancement of Anti-Tumoral Immunity by β-Casomorphin-7 Inhibits Cancer Development and Metastasis of Colorectal Cancer

β-Casomorphin-7 (BCM) is a degradation product of β-casein, a milk component, and has been suggested to affect the immune system. However, its effect on mucosal immunity, especially anti-tumor immunity, in cancer-bearing individuals is not clear. We investigated the effects of BCM on lymphocytes using an in vitro system comprising mouse splenocytes, a mouse colorectal carcinogenesis model, and a mouse orthotopic colorectal cancer model. Treatment of mouse splenocytes with BCM in vitro reduced numbers of cluster of differentiation (CD) 20⁺ B cells, CD4⁺ T cells, and regulatory T cells (Tregs), and increased CD8⁺ T cells. Administration of BCM and the CD10 inhibitor thiorphan (TOP) to mice resulted in similar alterations in the lymphocyte subsets in the spleen and intestinal mucosa. BCM was degraded in a concentration- and time-dependent manner by the neutral endopeptidase CD10, and the formed BCM degradation product did not affect the lymphocyte counts. Furthermore, degradation was completely suppressed by TOP. In the azoxymethane mouse colorectal carcinogenesis model, the incidence of aberrant crypt foci, adenoma, and adenocarcinoma was reduced by co-treatment with BCM and TOP. Furthermore, when CT26 mouse colon cancer cells were inoculated into the cecum of syngeneic BALB/c mice and concurrently treated with BCM and TOP, infiltration of CD8⁺ T cells was promoted, and tumor growth and liver metastasis were suppressed. These results suggest that by suppressing the BCM degradation system, the anti-tumor effect of BCM is enhanced and it can suppress the development and progression of colorectal cancer.     

Tumor Infiltration with CD20+CD73+ B Cells Correlates with Better Outcome in Colorectal Cancer

Immunotherapy has become increasingly important in the treatment of colorectal cancer (CRC). Currently, CD73, also known as ecto-5′-nucleotidase (NT5E), has gained considerable interest as a potential therapeutic target. CD73 is one of the key enzymes catalyzing the conversion of extracellular ATP into adenosine, which in turn exerts potent immune suppressive effects. However, the role of CD73 expression on various cell types within the CRC tumor microenvironment remains unresolved. The expression of CD73 on various cell types has been described recently, but the role of CD73 on B-cells in CRC remains unclear. Therefore, we analyzed CD73 on B-cells, especially on tumor-infiltrating B-cells, in paired tumor and adjacent normal tissue samples from 62 eligible CRC patients. The highest expression of CD73 on tumor-infiltrating B-cells was identified on class-switched memory B-cells, followed by naive B-cells, whereas no CD73 expression was observed on plasmablasts. Clinicopathological correlation analysis revealed that higher CD73⁺ B-cells infiltration in the CRC tumors was associated with better overall survival. Moreover, metastasized patients showed a significantly decreased number of tumor-infiltrating CD73⁺ B-cells. Finally, neoadjuvant therapy correlated with reduced CD73⁺ B-cell numbers and CD73 expression on B-cells in the CRC tumors. As promising new immune therapies are being developed, the role of CD73⁺ B-cells and their subsets in the development of colorectal cancer should be further explored to find new therapeutic options.     

Butylidenephthalide Abrogates the Snail-Induced Cancer Stemness in Oral Carcinomas

Oral cancer is one of the most common cancers worldwide, especially in South Central Asia. It has been suggested that cancer stem cells (CSC) play crucial roles in tumor relapse and metastasis, and approaches to target CSC may lead to promising results. Here, aldehyde dehydrogenase 1 (ALDH1) and CD44 were utilized to isolate CSCs of oral cancer. Butylidenephthalide, a bioactive phthalide compound from Angelica sinensis, was tested for its anti-CSC effects. MTT assay showed that a lower concentration of butylidenephthalide was sufficient to inhibit the proliferation of patient-derived ALDH1⁺/CD44⁺ cells without affecting normal cells. Administration of butylidenephthalide not only reduced ALDH1 activity and CD44 expression, it also suppressed the migration, invasion, and colony formation abilities of ALDH1⁺/CD44⁺ cells using a transwell system and clonogenic assay. A patient-derived xenograft mouse model supported our in vitro findings that butylidenephthalide possessed the capacity to retard tumor development. We found that butylidenephthalide dose-dependently downregulated the gene and protein expression of Sox2 and Snail. Our results demonstrated that overexpression of Snail in ALDH1^-/CD44^- (non-CSCs) cells induced the CSC phenotypes, whereas butylidenephthalide treatment successfully diminished the enhanced self-renewal and propagating properties. In summary, this study showed that butylidenephthalide may serve as an adjunctive for oral cancer therapy.     

The Detection of Immunity against WT1 and SMAD4P130L of EpCAM+ Cancer Cells in Malignant Pleural Effusion

Malignant pleural effusion (MPE) provides a liquid tumor microenvironment model that includes cancer cells and immune cells. However, the characteristics of tumor antigen-specific CD8⁺ T cells have not been investigated in detail. Here, we analyzed MPE samples taken from a patient with pancreatic cancer who received a dendritic cell vaccine targeting Wilms’ Tumor 1 (WT1) antigen over the disease course (two points at MPE^1st and 2^nd, two months after MPE1^st). Epithelial cell adhesion molecule (EpCAM)⁺ cancer cells (PD-L1⁻ or T cell immunoglobulin mucin-3, TIM-3⁻), both PD-1 or TIM-3 positive CD8⁺ T cells, and CD14⁺CD68⁺CD163⁺TIM-3⁺ macrophages increased from the MPE^1st to MPE^2nd. The ratio of WT1-specific cytotoxic lymphocytes (WT1-CTLs) to MPE CD8⁺ T cells and IFN-γ secretion of WT1-CTLs were reduced with disease progression. Coincidentally, the fraction of central memory T (T_CM) of WT1-CTLs was decreased. On the other hand, CD8⁺ T cells in response to SMAD4^P130L, which is homogeneously expressed in EpCAM⁺ cancer cells, were detected using in vitro expansion with the HLA-A*11:01 restrictive SVCVNLYH neoantigen. Furthermore, the CD8⁺ T cell response to SMAD4^P130L was diminished following remarkably decreased numbers of CD8⁺ T_CM in MPE samples. In conclusion, CD8⁺ T cells responding to WT1 or SMAD4^P130L neoantigen expressed in EpCAM⁺ pancreatic cancer cells were detected in MPE. A tumor antigen-specific immune response would provide novel insight into the MPE microenvironment.     

Clinical Implications and Molecular Characterization of Drebrin-Positive,Tumor-Infiltrating Exhausted T Cells in Lung Cancer

T cells express an actin-binding protein, drebrin, which is recruited to the contact site between the T cells and antigen-presenting cells during the formation of immunological synapses. However, little is known about the clinical implications of drebrin-expressing, tumor-infiltrating lymphocytes (TILs). To address this issue, we evaluated 34 surgical specimens of pathological stage I–IIIA squamous cell lung cancer. The immune context of primary tumors was investigated using fluorescent multiplex immunohistochemistry. The high-speed scanning of whole-slide images was performed, and the tissue localization of TILs in the tumor cell nest and surrounding stroma was automatically profiled and quantified. Drebrin-expressing T cells were characterized using drebrin⁺ T cells induced in vitro and publicly available single-cell RNA sequence (scRNA-seq) database. Survival analysis using the propensity scores revealed that a high infiltration of drebrin⁺ TILs within the tumor cell nest was independently associated with short relapse-free survival and overall survival. Drebrin⁺ T cells induced in vitro co-expressed multiple exhaustion-associated molecules. The scRNA-seq analyses confirmed that the exhausted tumor-infiltrating CD8⁺ T cells specifically expressed drebrin. Our study suggests that drebrin-expressing T cells present an exhausted phenotype and that tumor-infiltrating drebrin⁺ T cells affect clinical outcomes in patients with resectable squamous cell lung cancer.     

Bladder Cancer Extracellular Vesicles Elicit a CD8 T Cell-Mediated Antitumor Immunity

Tumor-derived extracellular vesicles (TEVs) play crucial roles in mediating immune responses, as they carry and present functional MHC-peptide complexes that enable them to modulate antigen-specific CD8⁺ T-cell responses. However, the therapeutic potential and immunogenicity of TEV-based therapies against bladder cancer (BC) have not yet been tested. Here, we demonstrated that priming with immunogenic Extracellular Vesicles (EVs) derived from murine MB49 BC cells was sufficient to prevent MB49 tumor growth in mice. Importantly, antibody-mediated CD8⁺ T-cell depletion diminished the protective effect of MB49 EVs, suggesting that MB49 EVs elicit cytotoxic CD8⁺ T-cell-mediated protection against MB49 tumor growth. Such antitumor activity may be augmented by TEV-enhanced immune cell infiltration into the tumors. Interestingly, MB49 EV priming was unable to completely prevent, but significantly delayed, unrelated syngeneic murine colon MC-38 tumor growth. Cytokine array analyses revealed that MB49 EVs were enriched with pro-inflammatory factors that might contribute to increasing tumor-infiltrating immune cells in EV-primed MC-38 tumors. These results support the potential application of TEVs in personalized medicine, and open new avenues for the development of adjuvant therapies based on patient-derived EVs aimed at preventing disease progression.     

Increased Susceptibility of the CD57− NK Cells Expressing KIR2DL2/3 and NKG2C to iCasp9 Gene Retroviral Transduction and the Relationships with Proliferative Potential,Activation Degree,and Death Induction Response

Nowadays, the use of genetically modified NK cells is a promising strategy for cancer immunotherapy. The additional insertion of genes capable of inducing cell suicide allows for the timely elimination of the modified NK cells. Different subsets of the heterogenic NK cell population may differ in proliferative potential, in susceptibility to genetic viral transduction, and to the subsequent induction of cell death. The CD57⁻NKG2C⁺ NK cells are of special interest as potential candidates for therapeutic usage due to their high proliferative potential and certain features of adaptive NK cells. In this study, CD57⁻ NK cell subsets differing in KIR2DL2/3 and NKG2C expression were transduced with the iCasp9 suicide gene. The highest transduction efficacy was observed in the KIR2DL2/3⁺NKG2C⁺ NK cell subset, which demonstrated an increased proliferative potential with prolonged cultivation. The increased transduction efficiency of the cell cultures was associated with the higher expression level of the HLA-DR activation marker. Among the iCasp9-transduced subsets, KIR2DL2/3⁺ cells had the weakest response to the apoptosis induction by the chemical inductor of dimerization (CID). Thus, KIR2DL2/3⁺NKG2C⁺ NK cells showed an increased susceptibility to the iCasp9 retroviral transduction, which was associated with higher proliferative potential and activation status. However, the complete elimination of these cells with CID is impeded.     

Combined Blockade of TIGIT and CD39 or A2AR Enhances NK-92 Cell-Mediated Cytotoxicity in AML

This study aimed to characterize different natural killer (NK) cell phenotypes on bone marrow and peripheral blood cells from acute myeloid leukemia (AML) patients and healthy donors (HDs). Our data show that CD56^dimCD16⁻ and CD56^brightCD16⁻ NK cells represent the predominant NK cell subpopulations in AML, while the CD56^dimCD16⁺ NK cells are significantly reduced compared to HDs. Moreover, TIGIT⁺ and PVRIG⁺ cells cluster on the CD56^dimCD16⁺ subset whereas CD39⁺ and CD38⁺ cells do so on CD56^brightCD16⁻ NK cells in AML. Furthermore, functional effects of (co-)blockade of TIGIT and CD39 or A2AR on NK cell functionality were analyzed. These experiments revealed that the single blockade of the TIGIT receptor results in an increased NK-92 cell-mediated killing of AML cells in vitro. Combined targeting of CD39 or A2AR significantly augments the anti-TIGIT-mediated lysis of AML cells. Our data indicate that distinct NK cell subsets in AML exhibit different immunosuppressive patterns (via the TIGIT/PVRIG receptors and the purinergic pathway). In summary, we conclude that TIGIT, CD39, and A2AR constitute relevant inhibitory checkpoints of NK cells in AML patients. A combinatorial blockade synergistically strengthens NK-92 cell-mediated cytotoxicity. As inhibitors of TIGIT, CD39, and A2AR are clinically available, studies on their combined use could be conducted in the near future.     

B Cell Kinetics upon Therapy Commencement for Active Extrarenal Systemic Lupus Erythematosus in Relation to Development of Renal Flares: Results from Three Phase III Clinical Trials of Belimumab

Renal flares constitute major determinants of poor prognosis in people living with systemic lupus erythematosus (SLE). The aim of the present study was to investigate changes in B cell subsets in relation to renal flares upon initiation of standard therapy (ST) plus belimumab or placebo in patients with SLE. Using data from the BLISS-76, BLISS-SC, and BLISS Northeast Asia trials, we investigated associations of relative to baseline rapid (through week 8) and early (through week 24) percentage changes in circulating CD19⁺ B cell subsets characterised through flow cytometry, anti-dsDNA antibodies, and complement levels with the occurrence of renal flares over one year. Patients who developed renal flares showed more prominent rapid decreases in CD19⁺CD20⁺CD138⁺ short-lived plasma cells (−50.4% vs. −16.7%; p = 0.019) and CD19⁺CD20^-CD27^bright plasmablasts (−50.0% vs. −29.9%; p = 0.020) compared to non-flaring patients, followed by a subsequent return. Less prominent rapid reductions in CD19⁺CD27^-CD24^brightCD38^bright transitional B cells (−42.9% vs. −75.0%; p = 0.038) and CD19⁺CD20^-CD138⁺ peripheral long-lived plasma cells (−11.3% vs. −29.2%; p = 0.019) were seen in belimumab-treated—but not placebo-treated—patients who developed renal flares compared to belimumab-treated patients who did not. Rapid and early changes in anti-dsDNA or complement levels showed no clear association with renal flares. In summary, a rapid drop followed by a subsequent return in circulating short-lived plasma cells and plasmablasts upon treatment for active extra-renal SLE portended renal flares, indicating a need for therapeutic adjustments in patients showing such B cell patterns. Rapid decreases in transitional B cells and peripheral long-lived plasma cells upon belimumab therapy commencement may signify a greater protection against renal flares. B cell kinetics may prove useful in early drug evaluation.     

CD36+ Fibroblasts Secrete Protein Ligands That Growth-Suppress Triple-Negative Breast Cancer Cells While Elevating Adipogenic Markers for a Model of Cancer-Associated Fibroblast

Tumor and stroma coevolve to facilitate tumor growth. Hence, effective tumor therapeutics would not only induce growth suppression of tumor cells but also revert pro-tumor stroma into anti-tumoral type. Previously, we showed that coculturing triple-negative or luminal A breast cancer cells with CD36⁺ fibroblasts (FBs) in a three-dimensional extracellular matrix induced their growth suppression or phenotypic reversion, respectively. Then, we identified SLIT3, FBLN-1, and PENK as active protein ligands secreted from CD36⁺ FBs that induced growth suppression of MDA-MB-231 breast cancer cells and determined their minimum effective concentrations. Here, we have expanded our analyses to include additional triple-negative cancer cell lines, BT549 and Hs578T, as well as HCC1937 carrying a BRCA1 mutation. We show that the ectopic addition of each of the three ligands to cancer-associated fibroblasts (CAFs) elevates the expression of CD36, as well as the adipogenic marker FABP4. Lastly, we show that an agonist antibody for one of the PENK receptors induces growth suppression of all cancer cell lines tested but not for non-transformed MCF10A cells. These results clearly suggest that proteins secreted from CD36⁺ FBs induce not only growth suppression of tumor cells through binding the cognate receptors but also increasing adipogenic markers of CAFs to reprogram tumor stroma.     

Distinct Immune Signatures Indicative of Treatment Response and Immune-Related Adverse Events in Melanoma Patients under Immune Checkpoint Inhibitor Therapy

To identify potential early biomarkers of treatment response and immune-related adverse events (irAE), a pilot immune monitoring study was performed in stage IV melanoma patients by flow cytometric analysis of peripheral blood mononuclear cells (PBMC). Overall, 17 patients were treated with either nivolumab or pembrolizumab alone, or with a combination of nivolumab and ipilimumab every three weeks. Of 15 patients for which complete response assessment was available, treatment responders (n = 10) as compared to non-responders (n = 5) were characterized by enhanced PD-1 expression on CD8⁺ T cells immediately before treatment (median ± median absolute deviation/MAD 26.7 ± 10.4% vs. 17.2 ± 5.3%). Responders showed a higher T cell responsiveness after T cell receptor ex vivo stimulation as determined by measurement of programmed cell death 1 (PD-1) expression on CD3⁺ T cells before the second cycle of treatment. The percentage of CD8⁺ effector memory (CD8⁺CD45RA⁻CD45RO⁺CCR7⁻) T cells was higher in responders compared to non-responders before and immediately after the first cycle of treatment (median ± MAD 39.2 ± 7.3% vs. 30.5 ± 4.1% and 37.7 ± 4.6 vs. 24.0 ± 6.4). Immune-related adverse events (irAE) were accompanied by a higher percentage of activated CD4⁺ (CD4⁺CD38⁺HLADR⁺) T cells before the second treatment cycle (median ± MAD 14.9 ± 3.9% vs. 5.3 ± 0.4%). In summary, PBMC immune monitoring of immune-checkpoint inhibition (ICI) treatment in melanoma appears to be a promising approach to identify early markers of treatment response and irAEs.     

CD38 Correlates with an Immunosuppressive Treg Phenotype in Lupus-Prone Mice

CD38 is a transmembrane glycoprotein expressed by T-cells. It has been reported that patients with systemic lupus erythematosus (SLE) showed increased CD38⁺CD25⁺ T-cells correlating with immune activation and clinical signs. Contrariwise, CD38 deficiency in murine models has shown enhanced autoimmunity development. Recent studies have suggested that CD38⁺ regulatory T-cells are more suppressive than CD38⁻ regulatory T-cells. Thus, we have suggested that CD38 overexpression in SLE patients could play a role in regulating immune activation cells instead of enhancing it. This study found a correlation between CD38 with FoxP3 expression and immunosuppressive molecules (CD69, IL-10, CTLA-4, and PD-1) in T-cells from lupus-prone mice (B6.MRL-Fas^lpr/J). Additionally, B6.MRL-Fas^lpr/J mice showed a decreased proportion of CD38⁺ Treg cells regarding wild-type mice (WT). Furthermore, Regulatory T-Cells (Treg cells) from CD38-/- mice showed impairment in expressing immunosuppressive molecules and proliferation after stimulation through the T-cell receptor (TCR). Finally, we demonstrated an increased ratio of IFN-γ/IL-10 secretion in CD38-/- splenocytes stimulated with anti-CD3 compared with the WT. Altogether, our data suggest that CD38 represents an element in maintaining activated and proliferative Treg cells. Consequently, CD38 could have a crucial role in immune tolerance, preventing SLE development through Treg cells.     

TAMpepK Suppresses Metastasis through the Elimination of M2-Like Tumor-Associated Macrophages in Triple-Negative Breast Cancer

Triple-negative breast cancer (TNBC) accounts for approximately 10–15% of all breast cancer cases and is characterized by high invasiveness, high metastatic potential, relapse proneness, and poor prognosis. M2-like tumor-associated macrophages (TAMs) contribute to tumorigenesis and are promising targets for inhibiting breast cancer metastasis. Therefore, we investigated whether melittin-conjugated pro-apoptotic peptide (TAMpepK) exerts therapeutic effects on breast cancer metastasis by targeting M2-like TAMs. TAMpepK is composed of M2-like TAM binding peptide (TAMpep) and pro-apoptotic peptide d(KLAKLAK)₂ (dKLA). A metastatic mouse model was constructed by injecting 4T1-luc2 cells either orthotopically or via tail vein injection, and tumor burden was quantified using a bioluminescence in vivo imaging system. We found that TAMpepK suppressed lung and lymph node metastases of breast cancer by eliminating M2-like TAMs without affecting the viability of M1-like macrophages and resident macrophages in the orthotopic model. Furthermore, TAMpepK reduced pulmonary seeding and the colonization of tumor cells in the tail vein injection model. The number of CD8⁺ T cells in contact with TAMs was significantly decreased in tumor nodules treated with TAMpepK, resulting in the functional activation of cytotoxic CD8⁺ T cells. Taken together, our findings suggest that TAMpepK could be a novel therapeutic agent for the inhibition of breast cancer metastasis by targeting M2-like TAMs.     

Effects of Aflatoxin B1 on T-Cell Subsets and mRNA Expression of Cytokines in the Intestine of Broilers

This study was conducted to investigate the effects of aflatoxin B₁ (AFB₁) on T-cell subsets and mRNA expression of cytokines in the small intestine of broilers. One hundred and fifty-six one-day-old healthy Cobb broilers were randomly divided into control group (0 mg/kg AFB₁) and AFB₁ group (0.6 mg/kg AFB₁) with three replicates per group and 26 birds per replicate for 21 days, respectively. At 7, 14, and 21 days of age, the duodenum, jejunum and ileum were sampled for analyzing T cell subsets (CD3⁺, CD3⁺CD4⁺ and CD3⁺CD8⁺) by flow cytometry as well as IL-2, IL-4, IL-6, IL-10, IL-17, IFN-γ and TNF-α mRNA expression by qRT-PCR. The percentages of T-cells in the intra-epithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs) of duodenum, jejunum and ileum in the AFB₁ group showed a decreased tendency in comparison to the control group. The mRNA expression of cytokines in the three intestinal segments in the AFB₁ group presented a general decline compared with the control groups. Our data demonstrated that 0.6 mg/kg AFB₁ in the broilers diet could reduce the percentages of T-cell subsets and the expression level of cytokine mRNA in the small intestine, implying that the immune function of the intestinal mucosa might be affected. The reduction of cytokines mRNA expression may be closely associated with the decreased proportions of T cells subsets induced by AFB₁.