CD8-positive T-cell Research Articles

Efficacy of anti-CD19 CAR-T therapy after bispecific monoclonal antibody (CD19/CD3) therapy in a patient with relapsed Ph-positive acute lymphoblastic leukemia with a massive extramedullary component

Introduction. Patients with relapsed/refractory (r/r) Ph-positive acute lymphoblastic leukemia (ALL) have a poor prognosis and limited treatment options. New promising immunotherapies using CAR T cells or monoclonal antibodies (blinatumomab, inotuzumab/ozogamicin) in combination with tyrosine kinase inhibitors are being used for this group of patients. However, long-term results have not been studied when relapse or refractoriness develops after the use of one of these methods.Aim: to present a clinical observation of the use of anti-CD19 CAR T therapy after blinatumomab therapy in a patient with a relapsed course of Ph-positive acute lymphoblastic leukemia.Main findings. A 28-year-old patient was first diagnosed with Ph-positive B-ALL (transcript p210) in 2014. In the 1st line of treatment he was treated with therapy according to the protocol ‘ALL-2009’ with imatinib and allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, 2 months later the first molecular relapse was detected, and imatinib was replaced by dasatinib. In November 2016, a second relapse was diagnosed. In 2017, a third relapse with testicular involvement and T315I mutation was diagnosed, and therapy with ponatinib was initiated. In 2022, a 4th medullary relapse developed, and therapy with blinatumomab (5 courses) in combination with ponatinib was performed. After the 5th course of therapy, the appearance of leukaemides was noted, and asciminib was added to the therapy. However, no clinical effect was noted. The patient was treated with anti-CD19 CAR T therapy. A complete response to CAR T-cell therapy was confirmed on day 28 after CAR T administration. Further follow-up, over the course of one year, demonstrated stable clinical response and persistence of CD3-positive CAR T-cells. This case report of anti-CD19 CAR T cell therapy in a patient with CD19-positive combined relapse with massive extramedullary component after blinatumomab therapy demonstrated long-term efficacy with a 1-year follow-up.

CD248 Cleaved Form in Human Colorectal Cancer Stroma: Implications for Tumor Behavior and Prognosis

CD248 (endosialin/tumor endothelial marker 1) is upregulated in cancer, including colorectal cancer (CRC), but its exact role in tumor progression remains to be elucidated. Previous studies have shown that the extracellular domain of CD248 mediates the interaction between tumor cells and extracellular matrix proteins and that interfering with this interaction may reduce tumor invasion and migration activities. We have examined the expression of CD248 in 117 human CRC samples by immunohistochemistry and investigated the association with various clinicopathologic features, including the occurrence of metastasis, intratumoral immune cell density and overall survival. Of the 117 specimens analyzed, 76.1% (89/117) exhibited CD248-high stromal expression, whereas 23.9% (28/117) demonstrated CD248-low stromal expression. Interestingly, we detected the presence of a cleaved form of CD248, which appears to accumulate in the stromal extracellular matrix. A higher metastasis rate (lymph node and distant) was observed in the CD248-low group (21/28, 75.0% vs 44/89, 49.4%; P = .02). In addition, CD248-low tumors had fewer CD163-positive macrophages and FoxP3-positive regulatory T cells (P < .05) with no significant difference in CD8-positive T-cell infiltration and PD-L1 expression between the groups (P > .05). Finally, overall survival was lower in CD248-low tumors than in CD248-high tumors, with 5-year survival rates of 35.7% and 57.3%, respectively (P = .01). In a multivariate analysis, the hazard ratio of CD248-low tumors vs CD248-high tumors was 1.93 (95% CI, 1.09-3.41; P = .02). Our findings suggest that CD248 stromal expression may influence the tumor microenvironment, impacting tumor behavior and prognosis, and can serve as a promising prognostic biomarker in CRC.

The Short-Term Efficacy and Safety of Brentuximab Vedotin Chemotherapy Combined with Chidamide in the Treatment of CD30-Positive Peripheral T-Cell Lymphoma

The Short-Term Efficacy and Safety of Brentuximab Vedotin Chemotherapy Combined with Chidamide in the Treatment of CD30-Positive Peripheral T-Cell Lymphoma

Primary Cutaneous Acral-type CD8-positive T-cell Lymphoproliferations: Widely Divergent Clinical Outcomes Despite Nearly Identical Histomorphology

Abstract Introduction/Objective Non-epidermotropic and acral CD8(+) T-cell proliferations have recently been reclassified as lymphoproliferative disorders (LPDs) due to their indolent course clinical. Here, we describe two cases with similar histomorphology but disparate clinical outcomes, highlighting the diagnostic challenges. Methods/Case Report Case 1: An 89-year-old male developed a 2 cm erythematous papule on the forehead. Biopsy showed a non-epidermotropic pan-dermal lymphocytic infiltrate, a distinct grenz zone, and areas of necrosis. Case 2: A 53-year-old male presented with a rapidly growing, ulcerating 8.9 cm scalp lesion. Biopsy showed a deep dermal lymphoid infiltrate with complete epidermal sparing. In both cases neoplastic cells were positive by immunohistochemistry for CD2, CD3, CD5, CD7 (partial), CD8, granzyme B, TIA-1, and CD68 (perinuclear dot-like pattern). The Ki-67 proliferation index varied modestly between cases (50% in case 1 and 70% in case 2, respectively). Despite treatment, within the span of a few months, case 2 developed adjacent, widely distributed scalp nodules with extensive local tissue destruction. Pathology was consistent with recurrent disease (flow cytometry demonstrating T-cells, co-expressing CD8, CD2, CD3, CD5, CD7 and monotypic TRBC1). Results (if a Case Study enter NA) NA Conclusion CD8+ T-cell lymphoproliferative disorders, specifically primary cutaneous acral CD8+ T-cell lymphoproliferative disorder, is a rare cutaneous T-cell disorder that was re-classified, owing to its indolent clinical course, low risk of systemic dissemination, and favorable prognosis. We report two cases of nearly identical isolated cutaneous lesions that demonstrated a CD8(+)-cytotoxic phenotype with CD68-dot-like positivity. Although all morphologic findings were supportive of the diagnosis, the aggressive clinical progression in case 2 highlights the potential for the disease to defy the classically described indolent behavior. Given that few cases with aggressive clinical courses have been published, we present these cases to underscore their complexity and propose that the classification incorporate clinicopathological correlation to guide management.

CD8-Positive T-Cells Are Key Immune Cells for Predicting the Therapeutic Effect of Neoadjuvant Chemotherapy in Triple-negative Breast Cancer.

Patients with triple-negative breast cancer (TNBC) who obtain a pathological complete response (pCR) after neoadjuvant chemotherapy have an improved prognosis. Lymphocyte-predominant breast cancer is more likely to respond to neoadjuvant chemotherapy. Here, we investigated the correlation between tumor-infiltrating lymphocytes (TILs) in pre-treatment biopsy specimens from patients with TNBC in relation to response to NAC. The level of infiltration by immune cells expressing immune cell lineage surface markers (CD8, CD4, CD19, CD14, CD11c, and CD11b) in biopsy specimens from 52 patients with TNBC was examined using multispectral immunofluorescent labelling. The level of CD8-positive TILs was significantly higher in patients with a pCR (p=0.045). The Cox proportional hazard model confirmed that lymph node involvement was associated with poorer disease-free survival (p=0.008). A high level of CD8-positive TILs was related to significantly prolonged disease-free survival in patients with node-positive TNBC (p=0.018). Assessing infiltration by CD8-positive TILs in the primary tumor is a useful biomarker to predict pCR and improved outcome in patients with node-positive TNBC.

Common variable immunodeficiency disorder (CVID)-related liver disease: assessment of the main histological aspects using novel semiquantitative scoring systems, image analysis and correlation with clinical parameters of liver stiffness and portal hypertension

AimsWe aimed to investigate the relationship between T-cell-mediated sinusoidal injury, nodular regenerative hyperplasia like changes (NRH-LC) and fibrosis, clinical measures of fibrosis and portal hypertension, and progression rate in common...

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.

Paneth cell differentiation associated with neoadjuvant therapy in esophageal adenocarcinoma.

Paneth cells and Paneth cell metaplasia are well-known in pathology as foundational components in the gastrointestinal system. When within malignant cells (Paneth cell differentiation [PCD]), however, the function and significance of these cells is less well understood. Here, we present findings from the first study focused on PCD in postneoadjuvant esophageal adenocarcinoma (EAC) resection specimens. Patients with EAC treated with neoadjuvant chemoradioation and followed by esophagectomy between 2012 and 2018 in our institution were retrospectively evaluated. A tissue microarray was constructed, and special and immunohistochemical stains were performed. A total of 64 cases were collected, of which 8 had PCD, as highlighted by periodic acid-Schiff with diastase staining. Adenocarcinomas with PCD were more commonly seen in patients 60 to 70 years of age and typically had a poorly differentiated morphology, observationally fewer stromal mucinous changes, and less lymph node metastasis. β-catenin activation induced by neoadjuvant therapy was more frequent in the PCD-positive cases. Patients with PCD-positive disease had low programmed cell death 1 ligand 1 levels, no positive or equivocal ERBB2 (HER2) expression, and low CD8-positive T-cell infiltration; they were also mismatch repair proficient. Patients with PCD-positive disease showed a survival pattern inferior to that of patients with PCD-negative disease. When induced by neoadjuvant therapy in EAC, PCD is associated with high β-catenin activation, less expression of targetable biomarkers, and a potentially worse clinical prognosis.

Phase I/II clinical trial of brentuximab vedotin for pretreated Japanese patients with CD30-positive cutaneous T-cell lymphoma.

Brentuximab vedotin (BV), a conjugate of anti-CD30 antibody and monomethyl auristatin E, has emerged as a promising treatment option for refractory CD30+ mycosis fungoides (MF) and primary cutaneous anaplastic large-cell lymphoma (pcALCL). BV has been shown to be safe and effective in treating Hodgkin's lymphoma and peripheral T-cell lymphoma. This multicenter, prospective, single-arm phase I/II study evaluated the efficacy of BV in Japanese patients with CD30+ cutaneous lymphomas, namely CD30+ cutaneous T-cell lymphoma. Participants were divided into two groups: those with CD30+ MF or pcALCL (cohort 1, n = 13) and those with CD30+ lymphoproliferative disorders other than those in cohort 1 (cohort 2, n = 3). The studied population included the full analysis set (FAS), modified FAS (mFAS), and safety analysis set (SAF). These sets were identified in cohorts 1 and 1 + 2 and labeled FAS1 and FAS2, mFAS1 and mFAS2, and SAF1 and SAF2, respectively. Each treatment cycle lasted 3 weeks, and BV was continued for up to 16 cycles after the third cycle based on treatment response. The primary endpoint was the 4-month objective response rate (ORR4) determined by the Independent Review Forum (IRF). ORR4 was 69.2% for FAS1 and 62.5% for FAS2 (P < 0.0001). Secondary endpoints of ORR, assessed using the global response score (53.8% in FAS1) and modified severity-weighted assessment tool (62.5% in FAS1), using the IRF, provided results comparable to the primary findings. The incidence of ≥grade 3 adverse events (≥15%) in SAF1 was peripheral neuropathy in three patients (23%) and fever and eosinophilia in two patients (15%). In conclusion, BV showed favorable efficacy, tolerability, and safety profile in Japanese patients with relapsed or refractory CD30+ primary cutaneous T-cell lymphoma. The trial was registered with University Hospital Medical Information Network Clinical Trials Registry, Japan (protocol ID: UMIN000034205).

Brentuximab vedotin plus cyclophosphamide, doxorubicin, etoposide, and prednisone followed by brentuximab vedotin consolidation in CD30-positive peripheral T-cell lymphomas: a multicentre, single-arm, phase 2 study

CD30 expression is universal in anaplastic large-cell lymphoma and is expressed in some other peripheral T-cell lymphoma subtypes. Incorporation of brentuximab vedotin into initial therapy for people with CD30-positive peripheral T-cell lymphomas prolonged progression-free survival, but there is room for improvement, especially for people with non-anaplastic large-cell lymphoma subtypes. We conducted a multicentre, international, single-arm, phase 2 trial to evaluate the safety and activity of CHEP-BV (cyclophosphamide, doxorubicin, prednisone, brentuximab vedotin, and etoposide) followed by brentuximab vedotin consolidation in patients with CD30-expressing peripheral T-cell lymphomas across five academic centres in the USA and Canada. Adults aged 18 years or older with newly diagnosed, untreated CD30-positive peripheral T-cell lymphomas, Eastern Cooperative Oncology Group score of 0-2, and adequate organ function were eligible to receive six planned cycles of CHEP-BV (ie, 1·8 mg/kg brentuximab vedotin intravenously on day 1, cyclophosphamide 750 mg/m2 intravenously on day 1, doxorubicin 50 mg/m2 intravenously on day 1, etoposide 100 mg/m2 daily intravenously on days 1-3, and prednisone 100 mg daily orally on days 1-5) with prophylactic G-CSF. Patients who responded to the treatment could receive brentuximab vedotin consolidation for up to ten additional cycles either after autologous haematopoietic stem-cell transplantation (HSCT) or directly after CHEP-BV. The primary endpoints were unacceptable toxicity during a 3-plus-3 safety lead-in in participants who received study treatment and completed the safety evaluation period (to confirm the recommended phase 2 dose of brentuximab vedotin in CHEP-BV) and the complete response rate after CHEP-BV induction therapy in participants who received study treatment and had response evaluation. The study was registered at ClinicalTrials.gov (NCT03113500), and this cohort completed the trial. The trial is ongoing with the enrolment of a new cohort. 54 patients were screened for eligibility and 48 were eligible for the study. The participants (18 [38%] women and 30 [63%] men; 34 [71%] White, four [8%] Black, five [10%] Asian, ten [21%] Hispanic, and 37 [77%] non-Hispanic people) were recruited and enrolled between Dec 4, 2017, and June 14, 2021, and followed up until Aug 25, 2023, when the database was locked for analysis. 48 participants were evaluable for toxicity, and 47 were evaluable for response (one participant died from COVID-19 before response assessment). During the safety lead-in, one of six participants had an unacceptable toxicity (ie, platelet count <10 000 per mm3 in a participant with extensive bone marrow involvement), and the proposed phase 2 dose of 1·8 mg/kg brentuximab vedotin in CHEP-BV was confirmed. At completion of CHEP-BV, 37 of 47 participants had complete response, yielding a complete response rate of 79% (95% CI 64-89). The most common CHEP-BV-related toxicities of grade 3 or higher were neutropenia (14 [29%] of 48), leukopenia (11 [23%]), anaemia (ten [21%]), febrile neutropenia (ten [21%]), lymphopenia (nine [19%]), and thrombocytopenia (nine [19%]). There were no treatment-related deaths. In patients with mostly CD30-expressing peripheral T-cell lymphomas other than non-anaplastic large-cell lymphoma, CHEP-BV (with or without autologous HSCT) followed by brentuximab vedotin consolidation was safe and active. SeaGen, Leukemia and Lymphoma Society, Lymphoma Research Foundation, and the National Cancer Institute of the National Institutes of Health.

Clinicopathological features of primary mucosal CD30-positive T-cell lymphoproliferative disorders

Objective: To investigate the clinicopathological features and differential diagnosis of primary mucosal CD30-positive T-cell lymphoproliferative disorders (pmCD30+TLPD). Methods: Eight cases of pmCD30+TLPD diagnosed from 2013 to 2023 at the Department of Pathology, Beijing Friendship Hospital Affiliated to Capital Medical University and Beijing Ludaopei Hospital were retrospectively collected. The immunophenotype, EBV infection status and T-cell receptor (TCR) clonability of tumor cells were examined. The clinicopathological features were analyzed and related literatures were reviewed. Results: There were 5 females and 3 males, aged 28 to 73 years, without B symptoms, lack of trauma and autoimmune diseases. Seven cases occurred in oral mucosa and one in anal canal mucosa. Submucosal nodules with ulcerations were presented in all cases except one, which only submucosal nodule. Morphologically, there was different distribution of allotypic lymphocytes in inflammatory background. Four cases showed "kidney-shaped", "embryonic" and "horseshoe-shaped" cells, and one case resembled Hodgkin and Reed/Sternberg (HRS) cells. Allotypic lymphocytes expressed CD3 (7/8), CD4+/CD8-(7/8) and CD4-/CD8-(1/8). CD30 was uniformly strongly positive while ALK and CD56 were negative. In situ hybridization of EBER was negative in five cases (5/5). Clonal TCR gene rearrangement was positive in two cases. Four patients did not receive radiotherapy or chemotherapy. All the seven patients survived without disease except one died due to concurrent leukopenia. Conclusions: pmCD30+TLPD had a broad morphological spectrum and could be easily confused with primary cutaneous CD30+TLPD and systemic ALK-negative anaplastic large cell lymphoma involving mucosa, which may lead to misdiagnosis. Although the majority of the cases had a favorable prognosis, a few cases relapsed or progressed to lymphoma.

DUSP22-rearranged primary cutaneous CD30-positive T-cell lymphoproliferative disorders and adult T-cell leukemia/lymphoma frequently share the LEF1+/TIA1− immunophenotype

DUSP22 rearrangements are genetic alterations observed in a subset of systemic anaplastic large cell lymphoma (S-ALCL), primary cutaneous anaplastic large cell lymphoma (C-ALCL), and lymphomatoid papulosis (LyP). Previous investigations have shown that the LEF1+/TIA1− immunoprofile and MSC E116K mutations are highly associated with DUSP22 rearrangement in ALCL. However, the existing literature primarily focuses on S-ALCL. Our understanding of the LEF1/TIA1 immunoprofile and MSC mutation status in C-ALCL/LyP is still limited. In this study, we aimed to assess LEF1/TIA1 expression and MSC mutations in a cohort of 23 C-ALCL/LyP cases, along with a control group of histological mimickers. DUSP22 rearrangements were detected by fluorescence in situ hybridization in eight cases (6/10 C-ALCL, 2/13 LyP). We found LEF1 expression in five out of eight (63%) DUSP22-rearranged cases (3/6 C-ALCL, 2/2 LyP), and none of the 15 cases lacking DUSP22 rearrangements. Furthermore, we also found frequent LEF1 expression in adult T-cell leukemia/lymphoma (ATLL; 10 of 11, 91%) within the control group. TIA1 expression was consistently negative in all DUSP22-rearranged C-ALCL/LyP and ATLL cases tested. MCS E116K mutation was identified in one of five DUSP22-rearranged C-ALCL cases. RNA sequencing of a DUSP22-rearranged C-ALCL revealed a novel DUSP22::SNHG fusion coexisting with a CD58::WNT2B fusion. In conclusion, our findings demonstrated a lower rate of LEF1 expression in DUSP22-rearranged C-ALCL/LyP compared to previous reports that predominantly focused on S-ALCL. Moreover, we observed that the majority of ATLL cases also expressed LEF1, suggesting that the LEF1+/TIA1− immunoprofile does not differentiate DUSP22-rearranged C-ALCL/LyP from ATLL.

Etoposide addition and brentuximab vedotin consolidation in first-line treatment of CD30-positive peripheral T-cell lymphoma: can we improve BV–CHP?

Etoposide addition and brentuximab vedotin consolidation in first-line treatment of CD30-positive peripheral T-cell lymphoma: can we improve BV–CHP?

Morphology, immunophenotype, and suggested diagnostic criteria of TCL1 family–negative T-prolymphocytic leukemia

Abstract Objectives We sought to investigate the morphologic and immunophenotypic characteristics of TCL1 family–negative T-cell prolymphocytic leukemia (T-PLL) Methods Twenty cases of TCL1 family–negative T-PLL were studied. Results The doubling time of leukemic cells ranged from less than 2 days to more than 5 years, with a median of 5.5 months. Leukemic cells were small to medium-sized, with round to irregular nuclei, variably condensed chromatin, and small amounts of agranular cytoplasm. A visible nucleolus was identified in 11 (55%) cases. Cytoplasmic blebs/protrusions were identified in all cases, but their occurrence was highly variable from case to case. Bone marrow biopsy showed an interstitial pattern in 90% of cases and a diffuse pattern in the remaining 10% of cases. Flow cytometric immunophenotypic analysis showed that the leukemic cells in all cases were CD4 positive; 3 (15%) also showed concurrent CD8 expression. All cases were positive for CD2 and CD5. Surface CD3 and CD7 were positive in 19 of 20 (95%) cases, and all CD3-positive cases expressed the T-cell receptor αβ. Compared with prototypic T-PLL cases, these 2 groups shared many immunophenotypic findings, except CD8 and CD26, both of which were more commonly expressed in prototypic T-PLL cases. Conclusions TCL1 family–negative T-PLL cases have morphologic and immunophenotypic features that are similar to prototypic T-PLL. They are characterized by neoplastic proliferation of small to medium-sized mature T cells with CD4-positive T-cell receptor αβ phenotype. Tumor cells frequently maintain pan-T antigen expression. Recognizing these morphologic and immunophenotypic features will aid in accurately diagnosing this rare subset of T-PLL.

CD24 is expressed in HNSCC and is correlated with a dampened immune response

Purpose:Recently, CD24 on tumor cells was identified as a phagocytic inhibitor contributing to an immunosuppressive tumor microenvironment via binding to Siglec-10 on macrophages. Head and neck squamous cell carcinoma (HNSCC) are highly immunogenic tumors, but little is known about the expression of CD24 in HNSCC. We aimed to evaluate the expression of CD24 and its binding partner Siglec-10 as well as the infiltration density of macrophages and CD8 positive T-cells. Material and methodsA previously described and characterized cohort of HNSCC (n = 156) was semiquantitatively analyzed by immunohistochemistry for CD24, Siglec-10, CD8, CD68, CD163 and CD80 expression. The expression data was correlated to clinico-pathological parameters. ResultsCD24 was expressed in 81.6 % of cases, showing an inverse correlation with the CD68 and CD8 infiltration density, indicating an immunosuppressive feature of CD24. Also, CD24 expression was higher in HNSCC with a negative HPV status. Nodal positive HNSCC showed significantly increased infiltration density of CD68+ cells. Furthermore, the cell density of CD68 and CD163 positive cells was associated with HPV/p16 positive HNSCC. ConclusionCD24 is commonly expressed in HNSCC and may contribute to an immunosuppressive tumor microenvironment in HNSCC. Its role as a putative therapy target should be clarified in further studies.

Abstract PS07-07: The RXR agonist, IRX4204, delays the formation of Brca1 mutant mammary tumors via modulation of the anti-tumor immune response

Abstract Background: Women with germline BRCA1 mutations are at an increased risk for developing breast cancer in their lifetime, often at a young age with more aggressive tumors. At present, prophylactic bilateral mastectomy is the most effective strategy for reducing breast cancer risk. However, this invasive procedure is irreversible and associated with potential complications. We and others have found that PARP inhibitors can delay Brca1-mutant tumor formation in mice and could be beneficial for the prevention of breast cancer. However, currently available PARP inhibitors are associated with modest toxicities that may not be acceptable to women without cancer. Thus, there remains an urgent need for the development of safe and effective therapies for the prevention of breast cancer. Here, we present data demonstrating the activity of IRX4204, a minimally toxic and highly specific agonist of the nuclear retinoid X receptor (RXR), to delay the formation of mammary tumors in a Brca1-deficient mouse model. This inhibitory effect on tumor growth is due, in part, to the role of IRX4204 in stimulating the anti-tumor immune response. Methods: We used the established MMTV-Cre, conditional Brca1 gene knockout, p53 heterozygous loss mouse model (BRCA1co/co; MMTV-Cre+/+; p53+/-) and selected for mutant female pups using PCR genotyping. At 16 weeks of age, all mice were separated into 4 treatment groups (n=10 per group): (1) sesame oil control; (2) the novel RXR agonist, IRX-4204 (10mg/kg); (3) high-dose IRX4204 (20 mg/kg); and (4) the RXR agonist, 9-cis-UAB-30 (5 mg/kg). All treatments were given by oral gavage five days per week, and mice were observed daily for tumor formation and toxicity. At the study endpoint, tumors and normal mammary glands were collected for additional analyses. Immunohistochemical staining was used to quantify CD8a, Ki-67 and cleaved caspase 3 expression in Brca1-deficient tumors. Oil Red O staining was used to measure changes in lipid accumulation in Brca1-deficient cell lines treated with IRX4204. qPCR was used to quantify the changes in gene expression of lipid metabolism-associated genes upon treatment with IRX4204 in vitro. Results: Vehicle-treated Brca1-deficient mice had a median time-to-tumor formation (TTF) of 211 days, with 100% developing tumors by 330 days. Mice treated with UAB 5 mg/kg had an improved median TTF of 261 days, whereas mice treated with IRX4204 10mg/kg or 20mg/kg had a median TTF of 347 and 304 days, respectively (p &amp;lt; 0.01). In addition, 60% of mice treated with IRX4204 10 mg/kg remained tumor-free at 330 days. IRX4204-treated tumors showed an increased infiltration of CD8-positive T-cells over vehicle-treated tumors (p &amp;lt; 0.05). Treatment of Brca1-deficient cell lines with IRX4204 in vitro resulted in a significant increase in lipid accumulation accompanied by a 2-fold increase of Srebf1 expression (a key transcription factor that regulates lipid homeostasis) within 24 hours of treatment (p &amp;lt; 0.05). Conclusion: These data demonstrate a novel use of the RXR agonist, IRX4204, to delay the formation of Brca1-deficient mammary tumors. We have found that IRX4204 treatment modulates the tumor immune response through increased infiltration of cytotoxic CD8-positive T-cells in Brca1-deficient mammary tumors in vivo. We have also determined that IRX4204 modulates lipid metabolism in breast cancer cell lines in vitro. It is known that lipid-derived antigens can stimulate T-cell activity. Our findings suggest that RXR agonists may alter lipid antigen production to activate an anti-tumor response. Additional immune and lipidomic studies are on-going. This work was supported by NCI-PREVENT grant (to PB and AM HHSN26100008) and CFP Foundation (Odyssey Fellowship to CM). Citation Format: Cassandra Moyer, Darian Coleman, Jamal Hill, Lana A. Vornik, Michelle Savage, Martin Sanders, Sei Shizuko, Altaf Mohammed, Powel Brown, Abhijit Mazumdar. The RXR agonist, IRX4204, delays the formation of Brca1 mutant mammary tumors via modulation of the anti-tumor immune response [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS07-07.

Development of a T-cell activation panel using image cytometry in rheumatoid arthritis patient samples

Abstract Rheumatoid arthritis (RA) is an autoimmune disease that results in cartilage damage and bone loss due to chronic inflammation of joints. Treatments for RA include disease-modifying antirheumatic drugs and nonsteroidal anti-inflammatory drugs, as well as the inhibition of activated T-cells. Consequently, there is a need to screen RA patient samples rapidly and effectively to monitor treatment efficacy, disease progression, immune response, and predictive markers. In this work, we developed and demonstrated a multiplex detection method to monitor T-cell activation in a panel consisting of CD3, CD69, CD25, and CD279 (PD-1) surface markers using the Cellaca™ PLX Image Cytometer (Revvity). First, a dosage titration of PHA-P was performed on PBMCs to stimulate T-cell activation. Once stimulated, CD3 positive T-cells were evaluated for CD69, CD25, and the checkpoint molecule CD279. After 24 hours, the percent surface marker positive T-cells and the level of marker expression (median fluorescent intensity values) were used to determine optimal PHA-P dosage. Results showed concentration dependent increases in the expression levels with a calculated EC50 value for PHA-P ranging from 20.16-30.78 µg/mL with a 95% CV based on the CD279 titration curve. The proposed image cytometric surface marker detection method may prove to be an efficient tool to rapidly screen RA patient samples for T-cell activation in the setting of clinical disease treatment or progression.

Prognostic Significance of T-Cells and Macrophages in the Tumour Microenvironment of Nodal DLBCL.

Revised International Prognostic (R-IPI) score is used widely for risk stratification of DLBCL cases, yet some patients belonging to same risk category tend to exhibit different outcomes. The role of T-cells and macrophages in prognostication of lymphomas has been a point of interest of late. We aimed to study the association of FOXP3 positive T-regulatory cells, cytotoxic T-cells and macrophages with the immunophenotypic subtypes, clinicopathological characteristics, treatment response and survival in nodal diffuse large B-cell lymphoma (DLBCL) patients. The clinicopathological and treatment data of 83 DLBCL patients diagnosed and treated at our institute from January 2015 to December 2018 were collected and followed up till June 2020. CD8, FOXP3 and CD68 immunostains were performed to highlight the cytotoxic T-cells, T-regulatory cells and macrophages respectively on the lymph node biopsies and the distribution of these cells and their association with clinico-pathological factors, treatment response and survival was analyzed. DLBCL cases with higher percentage of CD3 positive T-cells and CD8 positive cytotoxic T-cells had significant association with attainment of complete response to treatment. In addition, CD8 positive T-cells of more than 6.5% proved to be an independent predictor of treatment outcome (p = 0.022). Multivariate Cox regression survival analysis revealed that cases with 'good' R-IPI prognostic score and 'high CD68 positive macrophages in tumor microenvironment' had a significantly longer overall survival. Increased number of cytotoxic T-cells was significantly associated with complete response to treatment and higher number of macrophages correlated significantly with better overall survival signifying their antitumor effects.

Carrier-free mRNA vaccine induces robust immunity against SARS-CoV-2 in mice and non-human primates without systemic reactogenicity

Carrier-free naked mRNA vaccines may reduce the reactogenicity associated with delivery carriers; however, their effectiveness against infectious diseases has been suboptimal. To boost efficacy, we targeted the skin layer rich in antigen-presenting cells (APCs) and utilized a jet injector. The jet injection efficiently introduced naked mRNA into skin cells, including APCs in mice. Further analyses indicated that APCs, after taking up antigen mRNA in the skin, migrated to the lymph nodes (LNs) for antigen presentation. Additionally, the jet injection provoked localized lymphocyte infiltration in the skin, serving as a physical adjuvant for vaccination. Without a delivery carrier, our approach confined mRNA distribution to the injection site, preventing systemic mRNA leakage and associated systemic proinflammatory reactions. In mouse vaccination, the naked mRNA jet injection elicited robust antigen-specific antibody production over 6months, along with germinal center formation in LNs and the induction of both CD4- and CD8-positive Tcells. By targeting the SARS-CoV-2 spike protein, this approach provided protection against viral challenge. Furthermore, our approach generated neutralizing antibodies against SARS-CoV-2 in non-human primates at levels comparable to those observed in mice. In conclusion, our approach offers a safe and effective option for mRNA vaccines targeting infectious diseases.

Abstract 11: Development of innovative off-the-shelf cancer immunotherapies with iPSC-derived CD8-positive T-cells

Abstract Chimeric antigen receptor (CAR) based T-cell immunotherapies have transformed the oncology landscape and evolved as part of the standard of care for several hematological malignancies. To generate CAR-T-cell products, autologous patient-derived αβT-cells are engineered with a CAR for tumor cell targeting. However, generating autologous T-cell products fails in a significant number of cases due to the poor quality and quantity of blood-derived T-cells and is associated with manufacturing and logistical complexity as well as high product costs. Therefore, it is crucial to develop strategies for off-the-shelf T-cell products as otherwise many patients are left without this treatment option. Manufacturing allogeneic T-cells from induced pluripotent stem cells (iPSCs) has a great potential to generate cell products with consistently high quality and scalable quantities. In addition, high fidelity gene-editing can be performed in iPSCs to address tumor resistance mechanisms and to prevent exhaustion of the T-cells. We have established a feeder-free differentiation protocol that enables robust production of iPSC-derived αβT-cells (iαβT). Flow cytometry and single cell transcriptome analysis ensure stringent monitoring of all process stages. To demonstrate functional activity of iαβT, cytotoxicity and cytokine release assays were performed. We compared the differentiation potential of iPSC lines derived from T-cells (t-iPSCs) to a hematopoietic stem cell-derived iPSC line with an αβTCR knock-in. Hematopoietic progenitor cells were induced from the different iPSC lines and differentiated into iαβT. During the differentiation process, surface markers CD45, CD5 and CD7 were displayed, and cells started to express αβTCRs. Importantly, iαβT derived from the different iPSC lines expressed CD8α and CD8β which is crucial for the function of cytotoxic T-cells. The CD4/CD8 double positive stage is an important step in T-cell development and was observed only when T-cells were generated from t-iPSC, but not from the TCR knock-in cells. Functional analysis of iαβT-cells confirmed their cytotoxic activity and potential to release cytokines after activation. Our scalable process for iαβT differentiation generates CD8-positive T-cells that secrete cytokines and show cytotoxic activity against tumor cells, demonstrating their potential as a promising cell source for TCR-T or CAR-T cancer immunotherapies. Citation Format: Riga Tawo, Michela Mirenda, Olivia Cypris, Philip Hublitz, Nadja Wagner, Michael Epstein, Michael Esquerre, Audrey Holtzinger, Monika Braun, Markus Dangl, Rodrigo Grandy, Daniel Sommermeyer. Development of innovative off-the-shelf cancer immunotherapies with iPSC-derived CD8-positive T-cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 11.