Positive CD4 Research Articles

Simple Analysis Using Immunohistochemical Staining for Tumor-Infiltrating Lymphocytes in Brain Metastasis of Small Cell Lung Cancer

Background: We investigated the distribution of tumor-infiltrating lymphocytes (TILs) and the expression of programmed cell death-ligand 1 (PD-L1) in patients with brain metastasis (BM) from small cell lung cancer (SCLC). Methods: A retrospective analysis was performed on 12 surgical specimens of BMs from SCLC at our institute for 5 years. The Immunofluorescence-based Tissue Microenvironment Analysis Panel (MAP) was utilized for the detection of TILs, including CD3, CD8, PD-1, and PD-L1, in pathological archival specimens of BMs. The correlation between the overall survival (OS) and the above-mentioned markers was analyzed in the patients.Results: Positive rates of CD3+ TILs in the tumor parenchyma versus tumor stroma were 0.60±0.94% versus 1.76±2.72% (p=0.010), respectively; positive rates of CD8+ TILs in the tumor parenchyma versus tumor stroma were 0.80±0.78% versus 2.46±3.72% (p=0.016), respectively. There was no co-expression of CD8+ and PD-1+ TILs in the tumor parenchyma of 11 cases, and the infiltration density of co-expressed CD3+ and PD-1+ TILs was more than 10/mm2 in only 1 case. There was no co-expression of CD3+ and PD-1+ TIL in the stroma of 10 cases, and the infiltration density of CD8+ and PD-1+ TILs was more than 10/mm2 in 2 cases. Immunohistochemistry was used to detect the expression of PD-L1 in 12 cases of BMs, and 3 cases (25%) were positive. Survival analysis showed that patients with positive CD3+ TILs had significantly longer OS (p=0.040). Conclusions: The distribution of TILs in BM of SCLC is low and mainly distributed in the stroma, with the low expression of PD-L1 in the tumor tissues.

T cell and soluble co-inhibitory receptor expression in patients with visceral leishmaniasis are markers of treatment response and clinical outcome

Abstract Background Co-inhibitory receptors (immune checkpoints) regulate activated immune cells. Their expression on T cells can limit host defense. We hypothesized that chronic Leishmania donovani infection in patients with visceral leishmaniasis (VL) leads to expression of co-inhibitory receptors that could be markers of treatment response and clinical outcome. Method A prospective cohort of 21 subjects with VL (7 with HIV coinfection) and 10 controls was established to measure T cell expression of co-inhibitory receptors (PD-1, Tim-3, LAG-3, CTLA-4, and TIGIT) by flow cytometry in discarded remnants of diagnostic splenic or bone marrow aspirates and peripheral blood collected before and after treatment. Plasma levels of soluble co-inhibitory proteins (sPD-1, sTim-3, sLAG-3, and sCTLA-4), and selected cytokines were determined by immunoassay. Results Expression of co-inhibitory receptors in peripheral blood T cells generally reflected findings in spleen and bone marrow aspirates. PD-1 and Tim-3 were upregulated in CD4+ T cells in HIV-negative and HIV-positive subjects with VL compared to controls. CD8+ T cells from HIV-negative subjects with VL displayed a similar pattern. Plasma levels of sPD-1 and sTim-3 were also greater in VL patients than controls. CD8+ and CD4+ T cells co-expressing PD-1 and Tim-3 showed considerable decline with treatment. Mortality in HIV-negative VL patients was associated with increased CD8+ T cells co-expressing Tim-3 and PD-1, triple positive CD4+ and CD8+ T cells (PD-1+Tim-3+LAG-3+), and elevated sLAG3. Conclusion Tim-3 and PD-1 expression on CD4+ and CD8+ T cells, and increased plasma sLAG-3, were markers of treatment response and clinical outcome in patients with VL.

Immunoproteomics Reveal Different Characteristics for the Prognostic Markers of Intratumoral-Infiltrating CD3+ T Lymphocytes and Immunoscore in Colorectal Cancer

Tumor-infiltrating lymphocytes (TILs) and immunoscoring based on densities of CD3+ and CD8+ TILs are both favorable prognostic markers in colorectal cancer (CRC). However, determination of the molecular features of TILs, particularly their immunoproteomic signatures would require the development of large scale in situ spatiotemporal technologies. Recently, a multiplex in situ digital spatial proteomic profiling (DSP) tool GeoMx DSP has been applied to identify biomarkers predictive of therapeutic responses and to understand disease mechanisms and progression. Taking advantage of this tool, we simultaneously characterized the spatial distribution and interactions of 42 immune proteins in tumor cells (TCs), CD3+ T stromal TILs (sTILs), and CD20+ B sTILs using tissue microarrays, and further studied their associations with CD3+ T TILs and immunoscores in CRC. First, our data showed that well-known immune checkpoints, such as PD-L1, PD-L2, and LAG3, were expressed at low levels, whereas some other immune proteins, such as CD11c, CD68, STING, and CD44, were highly expressed. Second, 8 spatial interactions were identified, including 5 interactions between TC and CD20+ B sTILs, 2 interactions between CD3+ T sTILs and CD20+ B sTILs, and 1 interaction among TC, CD3+ T sTILs, and CD20+ B sTILs. Third, the differential immune microlandscape in the spatial compartments was identified in tissues with positive CD3+ T intratumoral TILs and high immunoscores. Collectively, to our knowledge, our study is the first to provide in situ spatial immune characteristics at the proteomic level. Moreover, our findings provide direct evidence supporting the infiltration of CD3+ T sTILs from stoma to TC and shed important insights into better understanding and treating CRC patients related to different immune prognostic markers.

P53 ABNORMAL ENDOMETRIAL CANCERS – ANALYSIS OF PD-1 CO-EXPRESSION BY CD4 HELPER AND CD8 CYTOTOXIC T-CELL SUBTYPES IN THE TUMOR MICROENVIRONMENT

Abstract Introduction/Objective Endometrial cancer is classified into four molecular subtypes of which the p53 abnormal variant is biologically aggressive. Programmed cell death protein 1 (PD-1) is a cell surface receptor and a vital inhibitor of the immune response. Programmed death ligand 1 (PD-L1) is a trans-membrane co-inhibitory factor of the immune response. PD-1 / PDL-1 interaction inhibits the activation and survival of T cells and decreases cytokine secretion. Some cancer cells express PD-L1, which then bind to PD-1 expressed on tumor specific T cells, and escape destruction by the T cells. The CD4/CD8 T cell immune response in the p53 abnormal variant has not been adequately evaluated with respect to the PD-1/PD-L1 pathway. Understanding this immune microenvironment is important to assess the potential use of anti-PD-1 targeted tumor therapy. Methods/Case Report Twenty cases of p53 abnormal endometrial cancers were obtained. All cases were PDL-1 +ve by immunohistochemistry. Two dual stains were performed - PD-1/CD4 and PD-1/CD8 antibodies. Six high-power fields (40x objective) with the greatest concentration of lymphocytes were counted. The total number of CD4 or CD8-only cells was recorded for each of the two stained slides for each case. The total number of PD- 1/CD4 and PD-1/CD8 positive cells were counted and recorded. The results were tabulated, and a paired t-test statistical analysis was performed to compare the total number of CD4 and CD8 cells and the percentage of PD-1/CD4 cells with the percentage of PD-1/CD8 positive cells. Results (if a Case Study enter NA) A statistically significant increase in the number of CD8 (Mean = 383) compared to CD4 cells (Mean = 317) was seen in the tumor microenvironment (p-value=0.0023). Evaluating PD-1 expression, the converse was true, and the percentage of PD-1/CD4 cells (Mean = 13.035%) was increased compared to the percentage of PD-1/CD8 cells (Mean = 9.205%), achieving statistical significance (p-value=0.0116). Conclusion The statistically significant increase in CD8 cells in the tumor and its stroma suggests that a robust cytotoxic response is being mounted to this high-risk molecular variant. The concurrent increase in CD4/PD-1 positive lymphocytes provides an appropriate microenvironment for the use of anti-PD-1 immunotherapy, since blockade of PD-1 on CD4 helper T cells may augment the tumor ablative cytotoxic properties of the CD8 positive lymphocytes. This includes reinvigoration of exhausted CD8+ T cells. Anti-PD-1 may also prevent the inhibition of PD-1 positive CD8 cells.

Long-Term Human Immune Reconstitution, T-Cell Development, and Immune Reactivity in Mice Lacking the Murine Major Histocompatibility Complex: Validation with Cellular and Gene Expression Profiles.

Humanized mice transplanted with CD34+ hematopoietic cells (HPCs) are broadly used to study human immune responses and infections in vivo and for testing therapies pre-clinically. However, until now, it was not clear whether interactions between the mouse major histocompatibility complexes (MHCs) and/or the human leukocyte antigens (HLAs) were necessary for human T-cell development and immune reactivity. We evaluated the long-term (20-week) human hematopoiesis and human T-cell development in NOD Scid Gamma (NSG) mice lacking the expression of MHC class I and II (NSG-DKO). Triplicate experiments were performed with HPCs obtained from three donors, and humanization was confirmed in the reference strain NOD Rag Gamma (NRG). Further, we tested whether humanized NSG-DKO mice would respond to a lentiviral vector (LV) systemic delivery of HLA-A*02:01, HLA-DRB1*04:01, human GM-CSF/IFN-α, and the human cytomegalovirus gB antigen. Human immune reconstitution was detectable in peripheral blood from 8 to 20 weeks after the transplantation of NSG-DKO. Human single positive CD4+ and CD8+ T-cells were detectable in lymphatic tissues (thymus, bone marrow, and spleen). LV delivery harnessed the detection of lymphocyte subsets in bone marrow (αβ and γδ T-cells and NK cells) and the expression of HLA-DR. Furthermore, RNA sequencing showed that LV delivery increased the expression of different human reactome pathways, such as defense responses to other organisms and viruses. Human T-cell development and reactivity are independent of the expression of murine MHCs in humanized mice. Therefore, humanized NSG-DKO is a promising new model for studying human immune responses, as it abrogates the xenograft mouse MHC interference.

Evaluation of CD56 and CD117 Double-positive as a Predictor of Poor Prognosis in Multiple Myeloma Patients: A Retrospective Analysis.

Despite advancements in treatment, multiple myeloma (MM) remains a challenging hematologic malignancy. It is crucial to stratify risk and perform prognostic assessment through various markers, including the cluster of differentiation 56 (CD56) and thecluster of differentiation 117 (CD117) expression. However, the relationship of these markers with MM-related survival remains unclear. In this context, the objective of this study isto investigate the prognostic implications of CD56 and CD117 expression and associated clinical features in MM patients. The population of this retrospective single-center study consisted of adult MM patients whose CD56 and CD117 expressionswere analyzed. Patients were divided into four groups according to their immunophenotypes: CD56+ CD117-, CD56-CD117+, CD56+CD117+, and CD56-CD117-.These groups were compared in terms of demographic and clinical characteristics, response to treatment, and survival outcomes. Of the 168 MM patients included in the study, CD56 positive, CD117 positive,CD56, and CD117 double-positive and double negative were observed in 57.1%, 38.1%, 21.4%, and 26.2%, respectively. Patients with double positivehad significantly higher cytogenetic risk and significantly lower overall response rate (ORR) compared toother patients(p<0.001 for both cases). ORR and overall survival (OS) were significantly lower in CD56 positivepatients than those CD56 negative (p=0.017 and p=0.004, respectively). Mortality rates were significantly higher in CD56 positive patients and CD117 positive than in those with double negative(p<0.001 and p=0.002). Double negative patients had significantly lower ORR and OS and higher mortality than others (p=0.001, p=0.002, and p<0.001, respectively). The high cytogenetic risk was found to bean independent predictor of shorter OS (p>0.001). The study's findings revealed that CD56 and CD117 double- positive MM patients had poorer prognosis, lower ORR, shorter OS, and higher mortality.

CD8 + T-cells restrict the development of peritoneal metastasis and support the efficacy of hyperthermic intraperitoneal chemotherapy (HIPEC)

Multimodal therapy for peritoneal metastasis (PM) including cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC) provides long-term survival in highly selected colorectal cancer patients. Mechanisms behind HIPEC are unknown and may include induction of adaptive immunity. We therefore analyzed human PM samples and explored the impact of HIPEC in experimental models. Human samples from colorectal primary tumors (n = 19) and PM lesions (n = 37) were examined for the presence of CD8 + T-cells and their association with disease free (DFS) and overall survival (OS). CD8 + T cell response after HIPEC was assessed using an in-vivo PM mouse model, tumor cell lines and patient-derived tumor organoids. Patients with high intraepithelial CD8 + T cell counts showed longer DFS and OS. In the mouse model, HIPEC controlled growth of PM and increased numbers of functional granzyme positive CD8 + T cells within tumors. Cell lines and human organoids that were treated with heated chemotherapies showed immunogenic changes, reflected by significantly higher levels of MHC-class I molecules and expression of Cancer Testis Antigens Cyclin A1 and SSX-4. Using in-vitro co-culture assays, we noticed that cancer cells treated with heated chemotherapy primed dendritic cells, which subsequently enhanced effector functions of CD8 + T cells. The presence of CD8 + T-cells within PM lesions is associated with prolonged survival of patients with PM. Data from PM mouse model and in-vitro assay show that heated chemotherapies induce immunogenic changes on cancer cells leading to induction of CD8 + T-cells mediated immunity, which seems to control growth of PM lesions in mice after HIPEC.

Mycosis fungoides type d’emblee after henna dye - case report and an update

Mycosis fungoides is a cutaneous T-cell lymphoma, arising from peripheral epidermotropic T-cells that express a CD4+ immunophenotype, with a high CD4/CD8 ratio. Initially manifesting as patches, the condition progresses into infiltrated plaques and can eventually lead to tumor formations. Double positive immunophenotypes, although rare, could be considered as potential predictors for better survival rates. Environmental exposure to radiation and chemicals, along with other risk factors, may be potentially linked to the development of Mycosis fungoides. We present a 74-year-old female with potentially henna-induced Multicentric Mycosis Fungoides type tumor d’emblee, staged T3N0M0B1 lA. Immunohistochemistry and flow cytometry revealed a double positive immunophenotype CD4+/CD8+. Throughout her routine follow-ups, two biopsies were conducted to confirm the diagnosis, with several changes of the therapeutic regimen, all resulting in disease progression. On her latest follow-up, we recommended initiating therapy with methotrexate. We will briefly review the clinical presentation and treatment approaches for a rare and treatment-resistant case of MF, with a primary focus on two newly emerged hypotheses: 1) the significance of double immunophenotypes as prognostic indicators, and 2) the potential chemical contamination of henna with different carcinogens / mutagens and its subsequent role in MF development and progression. Key words: multicentric mycosis fungoides; tumor d’emblee; double positive immunophenotype; henna dye; tattoos; methotrexate

Lymphomatoid Papulosis Type D in a Mestizo-Ancestry Man.

Lymphomatoid papulosis (LyP) belongs to the CD30+ skin lymphoproliferative disorders; it is defined as a chronic, recurrent, self-healing eruption of papules and small nodules with the histopathologic features of a cutaneous T-cell lymphoma. It is classified according to histopathology into subtypes A to F and with chromosomal rearrangement 6p25.3. Type D is characterized by epidermotropism of atypical CD8+ and CD30+ lymphocytes, small to medium size, forming papules and nodules with erosion and necrosis, which represents a formidable challenge in the differential diagnosis with aggressive cutaneous cytotoxic lymphomas. We present the clinical case of a 22-year-old man with subacute dermatosis, who underwent a skin biopsy with a report of LyP. Immunohistochemistry showed negative CD4, CD5, granzyme-B markers and positive CD3, CD30, CD8, CD56, and (T-cell intracellular antigen 1) TIA-1 markers, concluding the diagnosis of type D LyP. The course of the disease is recurrent; however, the prognosis is good with a 10-year survival of 100%. We present the case of a mestizo-ancestry patient who developed a type-D LyP, and, to the best of our knowledge, there are no publications of type D LyP from Latin-American authors or about mestizo-ancestry (or hispanic) patients; therefore, we consider of relevance to inform about these findings.

CD8 positive T-cells decrease neurogenesis and induce anxiety-like behaviour following hepatitis B vaccination.

Mounting evidence indicates the involvement of peripheral immunity in the regulation of brain function, influencing aspects such as neuronal development, emotion, and cognitive abilities. Previous studies from our laboratory have revealed that neonatal hepatitis B vaccination can downregulate hippocampal neurogenesis, synaptic plasticity and spatial learning memory. In the current post-epidemic era characterized by universal vaccination, understanding the impact of acquired immunity on neuronal function and neuropsychiatric disorders, along with exploring potential underlying mechanisms, becomes imperative. We employed hepatitis B vaccine-induced CD3 positive T cells in immunodeficient mice to investigate the key mechanisms through which T cell subsets modulate hippocampal neurogenesis and anxiety-like behaviours. Our data revealed that mice receiving hepatitis B vaccine-induced T cells exhibited heightened anxiety and decreased hippocampal cell proliferation compared to those receiving phosphate-buffered saline-T cells or wild-type mice. Importantly, these changes were predominantly mediated by infiltrated CD8+ T cells into the brain, rather than CD4+ T cells. Transcriptome profiling of CD8+ T cells unveiled that C-X-C motif chemokine receptor 6 positive (CXCR6+) CD8+ T cells were recruited into the brain through microglial and astrocyte-derived C-X-C motif chemokine ligand 16 (CXCL16). This recruitment process impaired neurogenesis and induced anxiety-like behaviour via tumour necrosis factor-α-dependent mechanisms. Our findings highlight the role of glial cell derived CXCL16 in mediating the recruitment of CXCR6+CD8+ T cell subsets into the brain. This mechanism represents a potential avenue for modulating hippocampal neurogenesis and emotion-related behaviours after hepatitis B vaccination.

EP110 - Chyloperitoneum in Follicular lymphoma – presenting as an inguinal hernia

Abstract Introduction Follicular lymphoma is one of the subtypes of slow growing Non-Hodgkin Lymphoma. Rarely, follicular lymphoma can present with chyloperitoneum or chylous ascites which is characterized by a milky-white coloured fluid with raised triglyceride levels. Case Report We present a case of a 60-year-old diabetic male, complaining of a right sided inguinal swelling for 1 year. Examination revealed a reducible right inguinal swelling with a positive cough impulse. The laboratory workup was unremarkable. Ultrasound scan of anterior abdominal wall showed a defect of 2.1 cm in right inguinal region with protrusion of abdominal contents and a positive cough impulse. A diagnosis of right sided reducible direct inguinal hernia was made. Patient was scheduled for Lichtenstein repair. Per-operatively, the hernial sac contained healthy omentum with milky-white ascitic fluid. Omentum was reduced and suture repair was performed. A drain was placed intra-peritoneally. The postoperative course of the patient was uneventful. Ascitic fluid analysis showed high triglyceride levels and no evidence of malignant cells, suggesting chylous ascites. Contrast enhanced CT scan of abdomen and pelvis revealed a large homogenous minimally enhancing soft tissue density retroperitoneal mass in peri aortic and paravertebral location with abdominal lymphadenopathy suggesting retroperitoneal lymphoma. Excisional biopsy of an enlarged inguinal lymph node confirmed follicular lymphoma with positive BCL2 and CD10 markers. Conclusion Follicular lymphoma presents in a variety of ways including chylous ascites in an inguinal hernia and a high index of suspicion is required to diagnose it.

High proportion of PD-1 and CD39 positive CD8+ tissue resident T lymphocytes correlates with better clinical outcome in resected human oesophageal adenocarcinoma

ObjectiveTo understand the CD8+ tumour infiltrating lymphocyte (TIL) compartment of oesophageal adenocarcinoma (OAC) with regards to markers of lymphocyte exhaustion, tissue residency and to identify possible reasons behind differential responses to therapy.DesignTumour samples from 44 patients undergoing curative resection for OAC were assessed by flow cytometry for presence of antigen-experienced TILs and markers of activation and exhaustion. Populations of PD-1 and CD39 positive OAC TILs were sorted, and bulk RNA sequencing undertaken using a modified SmartSeq2 protocol. Flow cytometric assessment of functionality was completed.ResultsA higher proportion of antigen experienced CD8+ OAC TILs was associated with improved survival following surgery; while, high double positivity (DP) for PD-1 and CD39 among these TILs also correlated significantly with outcome. These DP TILs possess a minority population which is positive for the markers of exhaustion TIM3 and LAG3. Transcriptomic assessment of the PD-1 and CD39 DP TILs demonstrated enrichment for a tissue resident memory T lymphocyte (TRM) phenotype associated with improved survival in other cancers, reinforced by positivity for the canonical TRM marker CD103 by flow cytometry. This population demonstrated maintained functional capacity both in their transcriptomic profile, and on flow cytometric assessment, as well as preserved proliferative capacity.ConclusionResected OAC are variably infiltrated by PD-1 and CD39 DP TILs, an abundance of which among lymphocytes is associated with improved survival. This DP population has an increased, but still modest, frequency of TIM3 and LAG3 positivity compared to DN, and is in keeping with a functionally competent TRM phenotype.

540. INFILTRATION OF RESECTED OESOPHAGEAL ADENOCARCINOMA WITH ANTIGEN-EXPERIENCED PD-1 AND CD39 POSITIVE CD8+ TISSUE RESIDENT MEMORY CELLS CORRELATE WITH IMPROVED SURVIVAL

Abstract Background The development of novel treatments for oesophageal adenocarcinoma (OAC) represents an area of significant unmet need. Despite the application of immune checkpoint blockade alongside chemotherapy as standard of care for some patients with metastatic OAC, responses to immunotherapies are modest and 5-year survival is 17%. The reasons for this remains unclear and while tumour infiltration with CD8+ T lymphocytes (TILs) at the time of resection is associated with improved outcomes, this heterogeneous population remains incompletely characterised in OAC. Tissue resident memory cells (TRM) are increasingly understood as key mediators of immunotherapy response but their role in OAC is poorly understood. Methods Tumour samples were accessed by punch biopsy from the central portion of cancers resected from 44 patients undergoing curative surgery for OAC. Samples were assessed by multi-parametric flow cytometry for the presence of antigen-experienced TILs and markers of activation and exhaustion. Populations of antigen-experienced PD-1 and CD39 positive CD8+ OAC TILs were sorted, and bulk RNA sequencing (RNAseq) undertaken using a modified SmartSeq2 protocol allowing differential gene expression analysis and gene set enrichment analysis. Flow cytometric assessment of functionality was completed. Results Resected OAC are often highly infiltrated with antigen experienced CD8+ TILs expressing high levels of PD-1 and CD39 and an abundance of this population correlates with improved survival (%PD-1/CD39+ &amp;gt;Median, OS HR 0.12). RNAseq of sorted TILs identified this PD-1+ and CD39+ lymphocyte population as enriched for precursor exhausted-like CD8+ TRM (NES 2.01 p=0.00). This is correlated by their CD103+ and TIM3- predominance on flow cytometry in keeping with TRMsassociated with immunotherapy response in other cancers. This population demonstrated a maintained proliferative potential, an ability to degranulate and produce the key effector molecules IFN-γ, TNF-α and Granzyme B. Conclusions An abundance of a PD-1 and CD39 positive CD8+ TIL population, consistent with a precursor exhausted-like TRM population with maintained proliferative and functional capacity, is observed to correlate with improved outcomes following curative surgery for OAC. This TRM population is consistent with analogous populations observed to be responsible for immunotherapy response in other cancers and their identification may predict benefit from such approaches in OAC and allow better patient selection for treatment with immune checkpoint inhibitors.

Impact of positive CD4 cells on event-free survival in follicular lymphoma patients.

Previous results about prognostic value of CD4+ T cells in follicular lymphoma (FL) remain controversial. Immunohistochemistry was used to examine expression of positive CD4 cells in 103 patients with FL 1-3A. Early failure was described as failing to achieve event-free survival (EFS) at 12 or 24 months. There were 49 (47.6%) male and 54 (52.4%) females, with a median age of 54 years. Compared to patients with <20% of positive CD4 cells, patients with ≥20% of positive CD4 cells exhibited a significant lower risk of early failure (2-year EFS rate: 56.7% vs 73.5%, p = 0.047). When patients were stratified based on positive CD4 cell combined with FLIPI, the median EFS (p = 0.002) and median OS (p = 0.007) were significantly different. This study demonstrated that higher expression of positive CD4 cells predicts lower risk of early failure in follicular lymphoma, and combination analysis of CD4 and FLIPI could better predict disease relapse and survival outcome.

Lymphocyte subsets in epicardial, thymic and subcutaneous adipose tissue during advanced coronary atherosclerosis in patients with coronary artery disease

The important role of epicardial (EAT) and thymic (TAT) adipose tissue in the development of atherosclerosis in patients with coronary artery disease (CAD) is widely discussed. The purpose of the study was to investigate the lymphocyte subsets and FoxP3+Treg lymphocytes in epicardial, thymic and subcutaneous adipose tissue depending on the severity of coronary atherosclerosis in patients with chronic CAD. We examined 24 patients with CAD (21 men; mean age 65.0 (58.0-68.0) years) scheduled for open-heart surgery. In samples of EAT, TAT and subcutaneous adipose tissue (SAT), the content of CD4+, CD8+, B lymphocytes, NK and NKT cells, CD4+CD25hiFoxP3+ and CD4+CD25lowFoxP3+T regulatory lymphocytes (Treg) and a proportion of Tregs with FoxP3 nuclear translocation was determined by imaging flow cytometry. Depending on the severity of atherosclerosis, assessed according to Gensini Score, patients were divided into groups: group 1 – patients with Gensini Score 65; group 2 – patients with Gensini Score ≥ 65. Patients in group 2 had higher frequency of EAT CD4+CD25lowTreg with FoxP3nuclear translocation, TAT CD8+T lymphocytes and NK cells, a lower content of TAT double positive CD4+CD8+T lymphocytes, and a tendency towards a decrease of frequency of TAT CD4+CD25hiTreg with FoxP3 nuclear translocation compared to patients in group 1. The level of nuclear translocation of FoxP3 in CD4+CD25hiTreg cells in TAT was inversely related to the proportion of CD8+T lymphocytes (rs = -0.653; p = 0.012) and NK cells (rs = -0.723; p = 0.003) in TAT, and directly – to the proportion of double positive CD4+CD8+T lymphocytes in TAT (rs = 0.567; p = 0.034) and the value of the waist-to-hip ratio (rs = -0.474; p = 0.041). Further research is required to study the molecular mechanisms of these relationships in patients with coronary atherosclerosis and chronic coronary artery disease.

Abstract We059: Age-associated Alterations in T cell Output and Functions

The elderly have a higher risk for a wide range of diseases, including infection, tumor, and cardiovascular disease. This could be, at least partially, attributed to T lymphocyte changes during aging. The goal of this project was to characterize age-related changes in T cell output and functions. In this study, young (3-6 months old) and aged (18-24 months old) male and female C57BL/6J mice were utilized. Our data showed that compared to young mice, aged mice demonstrated significantly smaller thymus and fewer developing thymocytes, including double negative CD4 - CD8 - , double positive CD4 + CD8 + , as well as single positive CD4 + and CD8 + thymocytes, which implies that aged mice generate less T cells. Accordingly, circulating T cell count was significantly lower in aged mice than young controls. To investigate how aging affects T cell functions, we isolated splenic T cells and stimulated them with PMA (100 nM) + ionomycin (1 µM) (P+I) or vehicle for 4 hours. At the steady state, aged T cells expressed higher levels of Ifng , Tnfα , granzyme b , and perforin than young T cells, suggesting that aged T cells show a senescence-associated secretory phenotype (SASP). After stimulation with P+I, both young and aged T cells expressed dramatically higher levels of Ifng , Tnfα , Il2 , Il4 , Il17 , and granzyme b , indicative of T cell activation. Interestingly, aged T cells had higher expression of Ifng, Il17, and granzyme b, but lower Tnfα and Il2 levels. Moreover, aged T cells expressed higher levels of exhaustion markers, such as Pd1 , Ctla4 , Lag3 , and Tigit , indicating that aged T cells exhibit an exhaustion phenotype. In conclusion, our study demonstrates that aged mice show smaller thymus and fewer thymocytes and blood T cells than young mice. In addition, aged T cells exhibit a SASP and exhaustion phenotype. Upon activation, aged T cells might be more cytotoxic by secreting higher levels of IFN-γ, IL-17, and granzyme B.

Correlation Between the Hepatitis C Virus NS3 Protein and CD30 Expression in Diffuse Large B-cell Lymphoma.

One of the etiologies of non-Hodgkinlymphoma (NHL) is chronic infection related to lymphoma pathogenesis, with a high prevalence ofhepatitis C virus (HCV) infection seen. In determining the treatment and prognosis of NHL, cluster of differentiation 30 (CD30) immunohistochemical staining plays an important role. High levels of CD30are found in patients with HCV infection. This study aimed to determine the prevalence of CD30 and HCV expression and its correlation with clinicopathological characteristics of Indonesian diffuse large B-cell lymphoma (DLBCL) patients. A total of 86 formalin-fixed paraffin-embedded (FFPE) samples of DLBCL cases were collected over the course of two years from the Anatomical Pathology department at Dr. Sardjito General Hospital in the special region of Yogyakarta, Indonesia. Immunohistochemistry was performed to detect the two markers (CD30 and HCV). Chi-square tests were used to investigate the correlations between CD30 expression and clinicopathological features in DLBCL patients. The positivity rate of CD30 expression in 86 DLBCL samples was 25.6% (22/86) when using a 0% cut-off, and 7.0% (6/86) while using a 20% cutoff. The positivity rate of HCV expression in DLBCL samples was 34.9% (30/86). Positive CD30 expression, HCV expression and clinicopathological features (age, sex, Ann Arbor stage, extranodal involvement, and morphological variations) did not have statistically significant relationships (p>0.05). There was no statistically significant correlation between CD30 immunoreactivity (cut-off >0% or >20%) and HCV NS3 expression and clinicopathological features (age, sex, Ann Arbor stage, extranodal involvement, lactate dehydrogenase, Eastern Cooperative Oncology Group status and morphological variants) in DLBCL.

Comparison of the infrapatellar and subcutaneous adipose tissue biopsy material as a source of mesenchymal stromal cells for regenerative medicine in traumatology

Objective. The use of infrapatellar fat pad (IPFP) as the source of mesenchymal stem cells (MSCs), show an age-independent proliferation and differentiation potential. In addition, the pronounced chondrogenic potential of IPFP-ASCs makes them promising candidates for research for use in other methodsof regenerative therapy. Methods. A direct immunohistochemical study was carried out in serial paraffin sections of the biopsy material of Hoffa`s fat pad and subcutaneus fat tissue, using monoclonal antibodies. The minimum criteria established by the International Society for Cell Therapy to ensure the identity of MSCs use CD73 and CD105 as positive markers and CD34, CD31, CD45 as a negative. Results. According to the results of histological, immunohistochemical, morphometric and statistical studies, it was found that in the biopsy of both tissues the relative number of cells with an immunoprofile CD105+, CD73+, CD34–, CD31–, CD45– in the standard field of view (×200) was: 7.25 (5.42; 8.89 ) %, and 11.11 (8.46; 13.45) %, no statistically significant difference was found between comparison groups (р &gt; 0,05). Conclusion. The therapeutic effect of mesenchymal stem cells of subcutaneous adipose tissue has been proven, thus the identification of similar cells in the biopsy material and relatively similar density in the Hoffa body makesit an important source of adipose-derived stem cells that can be used for regenerative engineering tissue.

Significance of Combined Immuno-histochemical Expression of P63 &amp; CD56 in Papillary and Follicular Thyroid Carcinoma

Background: Thyroid neoplasm is the commonest endocrine neoplasm. More than 80% of thyroid malignancies are papillary thyroid carcinoma (PTC) followed by follicular carcinoma (FC). The diagnosis of papillary thyroid carcinoma is based on characteristic nuclear morphology of a thyroid neoplasm. In contrast, follicular variant of PTC may cause, if the nuclear features of PTC are insufficiently appreciated, severe problems in differentiating from follicular thyroid carcinoma. Several immunohistochemical (IHC) markers such as P63 and CD56 have been recommended to differentiate between this two thyroid malignancies with overlapping histomorphology. Objectives: Our aim was to identify the possible diagnostic role of P63 and CD56 immunoexpression that distinguish PTC, including the follicular variant from follicular thyroid carcinoma. Methods: This cross-sectional descriptive study was conducted in the Department of Pathology, Rajshahi Medical College from March 2020 to February 2022. A total of 44 cases, histologically confirmed as papillary and follicular thyroid carcinoma were included in the study. Immunohistochemistry was done for P63 and CD56 from selected paraffin blocks. Results: Histologically, 39 cases were diagnosed as papillary thyroid carcinoma (27 were classical PTC &amp; 12 were FVPTC) and the rest 5 were follicular thyroid carcinoma. In this study, mean age of the patients was 40.86 ± 6.57 years (SD) and male to female ratio was 1:2.4. P63 showed 74.4% positivity with papillary thyroid carcinoma. Positive immunoreactivity of P63 was highly significant in distinguishing papillary from follicular thyroid carcinoma (P=0.003). Combined P63 positive CD56 negative (P=0.048) and P63 negative CD56 negative immunoexpression (P=0.039) were statistically significant in differentiating papillary from follicular thyroid carcinoma. Conclusion: This study suggested that the use of P63 and CD56 may be helpful in the diagnosis of papillary and follicular thyroid carcinoma along with histopathological examination and their combination may also help in this purpose.

(130) PREVALENCE OF CONDITIONS INVOLVING ABERRANT MAST CELL ACTIVITY IN PATIENTS WITH NEUROPROLIFERATIVE VESTIBULODYNIA

Abstract Introduction Neuroproliferative vestibulodynia (NPV) is a provoked vestibular pain shown to be associated with excess immunohistochemical staining for CD117 and PGP9.5, consistent with mast cells and nerves, respectively. NPV is distinguished from control tissue by having &amp;gt;8 mast cells/high power field. Objective In patients with a histopathologically confirmed diagnosis of NPV, we wished to assess the prevalence of concomitant conditions involving other aberrant mast cell activity. Methods Medical records from people with vulvas who underwent vestibulectomy between June 1, 2019 and October 31, 2023 at a single facility were retrospectively reviewed. All had histopathologically confirmed NPV, having observed &amp;gt; 8 positive CD117 stained cells/high power field in the sub-epithelial stroma. As part of their history, each patient provided a list of medical conditions which included disorders where aberrant mast cell activity has been reported. The following eight conditions were included in medical history forms: asthma, chronic sinusitis, endometriosis, food allergy/intolerance, interstitial cystitis, irritable bowel syndrome, migraine, and postural orthostatic tachycardia syndrome. Results A total of 88 NPV patients were included in this chart review. A total of 34 (39%) reported no other aberrant mast cell activity, however 54 (61%) had at least one concomitant condition. The most common conditions were migraine (n=21; 24%), interstitial cystitis (n=17; 19%) and endometriosis (n=15; 17%), followed by food allergy/intolerance (n=12; 14%), irritable bowel syndrome (n=9; 10%), asthma (n=9; 10%), chronic sinusitis (n=5; 6%) and postural orthostatic tachycardia syndrome (n=2; 2%). Among these 88 patients, a total of 33 (38%), 13 (15%), 3 (6%), 1 (1%) and 2 (2%) had one, two, three, four and five other conditions involving aberrant mast cell activity, respectively. There was no correlation between the number of concomitant conditions with aberrant mast cell activity reported and the density of CD117-immunopositive staining in the overall NPV cohort, whether diagnosed with lifelong or acquired NPV. Conclusions Patients with histopathologically confirmed NPV have a high prevalence of concomitant conditions associated with aberrant mast cell activity, possibly due to a genetic predisposition. While more studies are needed, NPV is likely part of a broader systemic collection of conditions associated with aberrant mast cell activity. This finding is relevant in terms of future diagnostic and treatment options for patients with NPV. Disclosure No.