Low PD-1 Expression Research Articles

The prognostic and therapeutic value of the tumor microenvironment and immune checkpoints in pancreatic neuroendocrine neoplasms

Pancreatic neuroendocrine neoplasms (Pan-NEN) represent a group of highly heterogeneous cancers, characterized by complex and diverse biological behavior. The objective of this study was to systematically investigate the immunological features of the tumor microenvironment (TME) in Pan-NEN, aiming to identify prognostic biomarkers and explore the therapeutic potential of immunotherapy for Pan-NEN. Tumor and adjacent normal tissues were collected from 56 patients with Pan-NEN. Immunohistochemical analysis was conducted on tumor tissues using a panel of monoclonal antibodies targeting key immune markers. The expression levels of these markers were quantitatively assessed and correlated with clinicopathological features and overall survival. Low expression of CD3, CD8, CD4, CD68, CD163, Foxp3, CD56, CD69, GZMB, HLA-1, HLA-II, PD-1, and PD-L1 were observed in the majority of Pan-NEN patient samples. PD-1 expression was positively correlated with CD4 and Foxp3 expression, while PD-L1 expression was positively correlated with CD68, CD163, and Foxp3 expression; HLA-II expression was positively correlated with GZMB expression. Infiltration of lymphocytes (CD3 + or CD8+) and macrophages (CD68 + or CD163+) and expression of PD-1/PD-L1 were more pronounced in poorly differentiated neuroendocrine carcinoma (Pan-NEC) compared to well-differentiated neuroendocrine tumors (Pan-NET), while CD68 and PD-L1 correlated with advanced disease stage. Conversely, HLA-I antigen expression was commonly downregulated in Pan-NEC. Univariate Cox proportional hazard analysis demonstrated that tumor grade, stage; CD4+, CD68+, and CD163 + cell count; and expression of PD-1 and PD-L1 were significantly associated with poor survival outcomes, while the positive expression of HLA-I was correlated with a more favorable survival prognosis. Furthermore, multivariate Cox proportional hazard analyses revealed that tumor grade, stage, and PD-1 expression are independent prognostic factors. The immunological landscape of Pan-NEN offers potential prognostic value and therapeutic targets. The findings suggest that immunotherapy, particularly targeting the PD-1/PD-L1 pathway, may serve as a promising strategy for the treatment of Pan-NEN, especially for Pan-NEC patients.

Higher frequency of peripheral blood CD103+CD8+ T cells with lower levels of PD-1 and TIGIT expression related to favorable outcomes in leukemia patients.

Leukemia is a prevalent pediatric life-threatening hematologic malignancy with a poor prognosis. Targeting immune checkpoints (ICs) to reverse T cell exhaustion is a potentially effective treatment for leukemia. Tissue resident memory T (TRM) cells have been found to predict the efficacy of programmed death receptor-1 inhibitor (anti-PD-1) therapy in solid tumors. However, the IC characteristics of TRM cells in leukemia and their relationship with prognosis remain unclear. We employed multi-color flow cytometry to evaluate the frequencies of CD103+CD4+ and CD103+CD8+ T cells in the peripheral blood (PB) of patients with acute myeloid leukemia and B-cell acute lymphoblastic leukemia compared to healthy individuals. We examined the expression patterns of PD-1 and T cell immunoreceptor with immunoglobulin and ITIM domain (TIGIT) within the circulating CD103+ T cell subsets affected by leukemia. To further elucidate the immunological landscape, we assessed the differentiation status of CD103+ T cells across various disease states in patients with leukemia. Our findings showed a significant increase in the frequency of CD103+CD8+ T cells in the PB of patients with leukemia who had achieved complete remission (CR) compared to those in the de novo (DN) and relapsed/refractory (RR) stages. This increase was accompanied by a notable decrease in the expression levels of PD-1 and TIGIT in CD103+CD8+ T cells in the CR stage. Additionally, our analysis revealed a higher proportion of CD103+CD8+ T cells in the central memory (TCM) and effector memory (TEM) subsets of the immune profile. Notably, the proportions of CD103+ naïve T cells, CD103+ TEM, and CD103+ terminally differentiated T cells within the CD8+ T cell population were significantly elevated in patients with CR compared to those in the DN/RR stages. The data indicate that circulating higher frequency of CD103+CD8+ T cells with lower expression of PD-1 and TIGIT are associated with favorable outcomes in patients with leukemia. This suggests a potential role of TRM cells in leukemia prognosis and provides a foundation for developing targeted immunotherapies.

OBX-115, an interleukin 2 (IL2)-sparing engineered tumor-infiltrating lymphocyte (TIL) cell therapy, in patients (pts) with immune checkpoint inhibitor (ICI)-resistant unresectable or metastatic melanoma.

9515 Background: Investigational unengineered TIL cell therapy has shown promising activity in ICI-resistant metastatic melanoma but requires systemic high-dose IL2, which has well-described high-grade toxicity frequently requiring specialized management and limiting pt eligibility. OBX-115 autologous engineered TIL cell therapy does not require co-administration of IL2 due to its regulatable expression of membrane-bound IL15 (mbIL15) using the FDA-approved small-molecule drug acetazolamide (ACZ) to provide cytokine support for TIL expansion and persistence. Methods: This Phase 1 study (NCT05470283) assesses the OBX-115 regimen in pts with ICI-resistant unresectable/metastatic melanoma. OBX-115 is manufactured from the pt’s tumor tissue (surgical excision or core needle biopsy [CNB]). After lymphodepletion (cyclophosphamide, fludarabine), pts receive OBX-115 followed by ACZ once daily for up to 7 days; ACZ redosing (up to 7 days) is permitted at Wk 6 for non-responders. No systemic IL2 is administered. Peripheral blood and tumor tissue is collected for longitudinal immune profiling. Results: As of 02 Jan 2024, 9 pts (median age, 50 y) with ICI-resistant metastatic melanoma were infused with OBX-115 (median study follow up, 17 wks; range, 2–58 wks). Median lines of prior therapy was 3 (range, 1–6). OBX-115 was successfully manufactured for all pts, including from CNB tumor tissue (n = 5). The infusion product had a high proportion of CD8+ cells (≥96%) and stem-like cells (CD8+CD39-CD69-; median 76%) with very low PD-1 expression in the CD8+ population ( < 1%). Post-infusion safety included no DLTs, 3 Gr 3 nonhematologic TEAEs in 2 pts (abdominal pain, ALT elevation, syncope), and no Gr 4 nonhematologic TEAEs. ACZ was redosed and well-tolerated in 4 of 5 eligible pts. Among patients with minimum 12-wk follow-up (n = 6), ORR per RECIST v1.1 was 50% (2 CR, 1 PR, 3 SD); all responses occurred between Wk 6–18 and were ongoing, with longest response sustained > 12 mo. One pt developed new metastatic disease (liver) and progressed at Wk 24, despite continued target lesion reduction; no pt developed brain metastasis. Post-infusion ctDNA was not detectable in any of the responders at Day 14 or 42. Though OBX-115 had minimal NK cells ( < 1%), post-infusion peripheral blood and tumor biopsies showed NK cell expansion, consistent with trans-presentation of mbIL15 to circulating NK cells. Updated efficacy data on all infused pts will be presented. Conclusions: OBX-115regulatableengineeredTIL cell therapy was well-tolerated and produced consistently deepening and durable responses, indicating that OBX-115 may mediate CRs and durable clinical benefit in ICI-resistant metastatic melanoma without high-dose IL2. OBX-115 investigation continues in this and an ongoing Phase 1/2 multicenter study (NCT06060613). Clinical trial information: NCT05470283 .

Abstract B008: High expression of the exhaustion markers PD1 and PD-L1 in non-muscle invasive bladder cancer is associated with poor outcome following Bacillus Calmette-Guérin immunotherapy

Abstract Introduction: The recommended treatment for high-risk non-muscle invasive bladder cancer (NMIBC) is intravesical instillations of Bacillus Calmette-Guérin (BCG). However, 40 % experience recurrence within 5 years despite completing BCG treatment. T cell exhaustion has been associated with poor outcome following treatment with BCG. Here we investigated if T cell exhaustion, characterized by protein expression of PD1 and PD-L1 measured in paired samples obtained before and after BCG treatment could further explain BCG response and help predict outcome in patients with NMIBC. Methods: We included 111 patients with NMIBC, from which we had 183 TURB-T samples obtained before and after BCG treatment. Using immunohistochemistry (IHC) staining of sections from tissue microarrays (TMAs) with triplicate core biopsies, we investigated the protein expression of the exhaustion markers PD1 (clone EP239) and PD-L1 (clone 22c3). Data was analyzed using digital pathology software (Visiopharm®), where tissue areas were defined as either carcinoma or stroma based on PanCK staining. Cells were scored as positive when detection of a nucleus was combined with a positive PD1 or PD-L1 stain, which was based on visually defined thresholds to differentiate between positive and unspecific staining. In total, 167 samples from 105 patients were considered suitable for further analysis (cell count > 200 in carcinoma and stromal areas) with 2-3 cores per sample, resulting in a total of 441 tissue cores. The average cell count was 5732 per core (defined by positive nuclei stain). Stratification of samples into high or low expression was based on a median split of the positive cell fraction. Results: When investigating the number of PD1 and PD-L1 positive cells in the tumors (carcinoma and stroma combined), we observed that PD-1 expression significantly increased with tumor stage in pre- (p=0.002) and post-BCG (p=0.006) tumor samples. PD1 expression was also increased in high-grade tumors compared to low-grade tumors in pre-BCG samples (p = 0.001). Furthermore, we observed a significant association between tumors of higher stage and high PD-L1 expression in the pre-BCG samples (p = 0.007). Patients with low expression of PD1 and PD-L1 in the pre-BCG tumor samples had a superior high-grade recurrence-free survival (HG-RFS) compared to patients with high PD1 (p=0.002) and PD-L1 (p=0.021) expression. In addition, low expression of PD-L1 in pre-BCG tumors was correlated with a better progression-free survival compared to the patients with high PD-L1 expression (p = 0.013). Conclusion: Protein expression of PD1 and PD-L1 in pre-BCG tumor samples were correlated to higher stage and grade as well as worse HGFRS, indicating that T cell exhaustion plays an important role in resistance to BCG treatment. Citation Format: Tine G. Andreasen, Trine Strandgaard, Jørgen B. Jensen, Lars Dyrskjøt. High expression of the exhaustion markers PD1 and PD-L1 in non-muscle invasive bladder cancer is associated with poor outcome following Bacillus Calmette-Guérin immunotherapy [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr B008.

The effects of photodynamic therapy on the immune response of women with cervical high-grade squamous intraepithelial lesions

ObjectiveTo evaluate and elucidate the effects and mechanism of photodynamic therapy (PDT) on the immune response of women with cervical high-grade squamous intraepithelial lesions (HSILs). Materials and methodsImmunofluorescence staining was used to compare immune infiltration before and after PDT in 23 patients with cervical HSILs. The infiltration of immune cells into the cervical tissues of patients with different outcomes was also compared at the 6-month follow-up period. Immune cell counts in samples collected before and after treatment were compared. ResultsWe found an increased number of CD8+ T cell infiltration, an increased proportion of CD8+ T cells expressing Granzyme B (GrB), Chemokine receptor 3 (CXCR3), and CD8+ tissue-resident memory T (TRM) cells, and a decreased proportion of CD8+ T cells expressing PD-1 in patients with cervical HSIL compared to that before PDT. Moreover, at the 6-month follow-up, there was higher infiltration of CD8+ T and CD8+ TRM cells, higher expression of GrB and CXCR3, and lower expression of PD-1 on CD8+ T cells in the HPV and cervical HSIL clearance groups than in the persistent HPV infection and cervical HSIL regression groups. However, no significant difference was observed in the number of CD8+ TSCM following PDT. ConclusionPDT activates immune responses mediated by CD8+ T cells. The degree of the immune response is correlated with the prognosis of cervical HSIL.

Increased PD-1 Expression on Circulating T Cells Correlates with Inferior Outcome after Autologous Stem Cell Transplantation

High-dose chemotherapy followed by autologous stem cell transplantation (auto-SCT) is a well-established treatment option for multiple myeloma and malignant lymphoma patients. It is able to induce long-term progression-free survival (PFS) in both patient groups and even provide a cure in patients with aggressive lymphoma. However, relapse is common and has been associated with the pace and quality of immunologic reconstitution after transplantation, as well as with immune cell exhaustion and immunometabolic defects. We aimed to analyze the dynamics of the prototypical exhaustion marker PD-1 on immune cells during reconstitution on high-dose chemotherapy followed by auto-SCT and its impact on PFS. We performed a comprehensive analysis of exhaustion and metabolic markers on immune cells from myeloma and lymphoma patients undergoing auto-SCT using flow cytometry and NanoString technologies. The expression levels of PD-1 were increased during early reconstitution after transplantation on T cells and natural killer (NK) cells, as well as on monocytes. However, while PD-1 expression in NK cells and monocytes normalized over time, PD-1 expression on T cells demonstrated a variable course. Of note, lymphoma patients with continuously increasing PD-1 expression on T cells after auto-SCT had an inferior median PFS of only 146 days, whereas the median PFS was not reached in the lymphoma patients without such a PD-1 expression pattern. T cells from patients with increased PD-1 expression after auto-SCT exhibited an immunometabolic (over)activation and exhausted phenotype compared to T cells from patients with a low PD-1 expression after transplantation, including higher levels of the glycolytic pacemaker enzyme hexokinase 2 and the inhibitory receptor CTLA-4. In addition, proliferating Ki-67+ T cells were more abundant in patients with high PD-1 expression on T cells compared to those with low expression after auto-SCT (11.9% versus 4.2%). PD-1 expression on T cells might serve as an adverse biomarker for lymphoma patients undergoing auto-SCT; however, further validation by larger prospective studies is required.

Bariatric Surgery Induces Alterations in the Immune Profile of Peripheral Blood T Cells.

Low-grade inflammation is closely linked to obesity and obesity-related comorbidities; therefore, immune cells have become an important topic in obesity research. Here, we performed a deep phenotypic characterization of circulating T cells in people with obesity, using flow cytometry. Forty-one individuals with obesity (OB) and clinical criteria for bariatric surgery were enrolled in this study. We identified and quantified 44 different circulating T cell subsets and assessed their activation status and the expression of immune-checkpoint molecules, immediately before (T1) and 7-18 months after (T2) the bariatric surgery. Twelve age- and sex-matched healthy individuals (nOB) were also recruited. The OB participants showed higher leukocyte counts and a higher percentage of neutrophils. The percentage of circulating Th1 cells were negatively correlated to HbA1c and insulin levels. OB Th1 cells displayed a higher activation status and lower PD-1 expression. The percentage of Th17 and Th1/17 cells were increased in OB, whereas the CD4+ Tregs' percentage was decreased. Interestingly, a higher proportion of OB CD4+ Tregs were polarized toward Th1- and Th1/17-like cells and expressed higher levels of CCR5. Bariatric surgery induced the recovery of CD4+ Treg cell levels and the expansion and activation of Tfh and B cells. Our results show alterations in the distribution and phenotype of circulating T cells from OB people, including activation markers and immune-checkpoint proteins, demonstrating that different metabolic profiles are associated to distinct immune profiles, and both are modulated by bariatric surgery.

CD4 T cell-activating neoantigens enhance personalized cancer vaccine efficacy.

Personalized cancer vaccines aim to activate and expand cytotoxic antitumor CD8+ T cells to recognize and kill tumor cells. However, the role of CD4+ T cell activation in the clinical benefit of these vaccines is not well defined. We previously established a personalized neoantigen vaccine (PancVAX) for the pancreatic cancer cell line Panc02, which activates tumor-specific CD8+ T cells but required combinatorial checkpoint modulators to achieve therapeutic efficacy. To determine the effects of neoantigen-specific CD4+ T cell activation, we generated a vaccine (PancVAX2) targeting both major histocompatibility complex class I- (MHCI-) and MHCII-specific neoantigens. Tumor-bearing mice vaccinated with PancVAX2 had significantly improved control of tumor growth and long-term survival benefit without concurrent administration of checkpoint inhibitors. PancVAX2 significantly enhanced priming and recruitment of neoantigen-specific CD8+ T cells into the tumor with lower PD-1 expression after reactivation compared with the CD8+ vaccine alone. Vaccine-induced neoantigen-specific Th1 CD4+ T cells in the tumor were associated with decreased Tregs. Consistent with this, PancVAX2 was associated with more proimmune myeloid-derived suppressor cells and M1-like macrophages in the tumor, demonstrating a less immunosuppressive tumor microenvironment. This study demonstrates the biological importance of prioritizing and including CD4+ T cell-specific neoantigens for personalized cancer vaccine modalities.

Biomarkers of Pathologic Complete Response to Neoadjuvant Immunotherapy in Mismatch Repair-Deficient Colorectal Cancer.

Immune checkpoint inhibitors (ICI) have become the standard of care for patients with mismatch repair-deficient/microsatellite instability-high (dMMR/MSI-H) colorectal cancer. However, biomarkers of response to ICI are still lacking. Forty-two patients with dMMR colorectal cancer treated with neoadjuvant PD-1 blockade were prospectively enrolled. To identify biomarkers of pathologic complete response (pCR) to neoadjuvant therapy, we analyzed genomic and transcriptomic profiles based on next-generation sequencing, and immune cell density based on multiplex immunofluorescence (mIF) staining. An integrated analysis of single-cell RNA sequencing from our previous study and GSE178341, as well as mIF was performed to further explore the significance of the tumor microenvironment (TME) on pCR response. The tumor mutation burden of both tumor tissue and plasma blood samples was comparable between the pCR and non-pCR groups, while HLA-DQA1 and HLA-DQB1 were significantly overexpressed in the pCR group. Gene signature enrichment analysis showed that pathways including T-cell receptor pathway, antigen presentation pathway were significantly enriched in the pCR group. In addition, higher pre-existing CD8+ T-cell density was associated with pCR response (767.47 per.mm2 vs. 326.64 per.mm2, P = 0.013 Wilcoxon test). Further integrated analysis showed that CD8+ T cells with low PD-1 expression (PD-1lo CD8+ T cells) expressing high levels of TRGC2, CD160, and KLRB1 and low levels of proliferated and exhausted genes were significantly associated with pCR response. Immune-associated transcriptomic features, particularly CD8+ T cells were associated with pCR response to ICI in dMMR colorectal cancer. Heterogeneity of TME within dMMR colorectal cancer may help to discriminate patients with complete response to neoadjuvant ICI.

The effects of 5-aminolevulinic acid photodynamic therapy on the local immune response of women with cervical intraepithelial neoplasia grade 2.

To evaluate and elucidate the effects and mechanism of 5-aminolevulinic acid photodynamic therapy (ALA-PDT) on the local immune response of women with cervical intraepithelial neoplasia grade 2 (CIN2). Immunofluorescence staining was used to compare immune cells infiltration before and after ALA-PDT in 23 patients with CIN2. The infiltration of immune cells into the cervical tissues of patients with different outcomes was also compared at the 6-month follow-up period. Immune cell counts in samples collected before and after treatment were compared. We found an increased number of CD8+ T cell infiltration, an increased proportion of CD8+ T cells expressing Granzyme B (GrB), Chemokine receptor 3 (CXCR3), and CD8+ tissue-resident memory T (TRM) cells, and a decreased proportion of CD8+ T cells expressing PD-1 in patients with CIN2 compared to that before ALA-PDT. Moreover, at the 6-month follow-up, there was higher infiltration of CD8+ T and CD8+ TRM cells, higher expression of GrB and CXCR3, and lower expression of PD-1 on CD8+ T cells in the HPV clearance and CIN2 disappearance groups than in the HPV-positive and CIN2 regression groups. However, no significant difference was observed in the number of CD8+ TSCM following ALA-PDT. ALA-PDT could activate CD8+ T cell responses by modulating the expression of CXCR3 and PD-1 in CD8+ T cells and increasing the infiltration of CD8+ TRM cells. And the infiltration of CD8+ T cells is correlated with the prognosis of CIN2.

Neoantigen-specific stem cell memory-like CD4+ T cells mediate CD8+ T cell-dependent immunotherapy of MHC class II-negative solid tumors.

CD4+ T cells play key roles in a range of immune responses, either as direct effectors or through accessory cells, including CD8+ T lymphocytes. In cancer, neoantigen (NeoAg)-specific CD8+ T cells capable of direct tumor recognition have been extensively studied, whereas the role of NeoAg-specific CD4+ T cells is less well understood. We have characterized the murine CD4+ T cell response against a validated NeoAg (CLTCH129>Q) expressed by the MHC-II-deficient squamous cell carcinoma tumor model (SCC VII) at the level of single T cell receptor (TCR) clonotypes and in the setting of adoptive immunotherapy. We find that the natural CLTCH129>Q-specific repertoire is diverse and contains TCRs with distinct avidities as measured by tetramer-binding assays and CD4 dependence. Despite these differences, CD4+ T cells expressing high or moderate avidity TCRs undergo comparable in vivo proliferation to cross-presented antigen from growing tumors and drive similar levels of therapeutic immunity that is dependent on CD8+ T cells and CD40L signaling. Adoptive cellular therapy (ACT) with NeoAg-specific CD4+ T cells is most effective when TCR-engineered cells are differentiated ex vivo with IL-7 and IL-15 rather than IL-2 and this was associated with both increased expansion as well as the acquisition and stable maintenance of a T stem cell memory (TSCM)-like phenotype in tumor-draining lymph nodes (tdLNs). ACT with TSCM-like CD4+ T cells results in lower PD-1 expression by CD8+ T cells in the tumor microenvironment and an increased frequency of PD-1+CD8+ T cells in tdLNs. These findings illuminate the role of NeoAg-specific CD4+ T cells in mediating antitumor immunity via providing help to CD8+ T cells and highlight their therapeutic potential in ACT.

Approach to relapsed CLL including Richter Transformation

Approach to relapsed CLL including Richter Transformation

Double Positive CD4+CD8+ (DP) T-Cells Display Distinct Exhaustion Phenotype in Chronic Hepatitis C.

In chronic hepatitis C (CHC), characterized by exhaustion of T-cell function, increased frequencies of double-positive (DP) (CD4+CD8+) cells are present in peripheral blood. We compared the exhaustion phenotype between DP and single positive (SP) T-cells, including HCV-specific cells, and assessed the effect of successful HCV treatment on inhibitory receptors expression. Blood samples from 97 CHC patients were collected before and six months post-treatment. PD-1 (programmed cell death protein 1) and Tim-3 (T-cell immunoglobulin and mucin domain-containing molecule-3) expression was assessed by flow cytometry. DP T-cells displayed significantly higher PD-1 expression, lower Tim-3 expression than CD8+ SP T-cells and lower percentages of PD-1-Tim-3- cells than CD4+ SP T-cells, both before and after treatment. PD-1+Tim-3+ DP T-cells decreased following treatment. HCV-specific cells were more frequent among DP than SP T-cells, both before and after treatment. HCV-specific DP T-cells were characterized by lower PD-1 expression, higher PD-1 and Tim-3 co-expression, and lower percentages of PD-1-Tim-3- cells (both before and after treatment) and higher post-treatment Tim-3 than HCV-specific SP T-cells. Their percentages decreased following treatment, but the exhaustion phenotype remained unchanged. DP T-cells in CHC exhibit a distinct exhaustion phenotype from SP T-cells, and these changes mostly persist following successful treatment.

456 CD39 T Cells With Low PD-1 Expression are Markers for Durable Immunotherapy Response to Anti-PD-1 in Murine Glioblastoma

INTRODUCTION: Clinical trials investigating anti-PD-1 therapy for glioblastoma (GBM) have failed to demonstrate increased overall survival, unlike in other cancers such as non-small cell lung cancer (NSCLC). Despite the immunosuppressive characteristics of GBM, a small subset of patients in these trials have shown durable anti-PD-1 response. Recently, CD39+ T cells have been implicated as infiltrating lymphocytes that, once activated, demonstrate robust anti-tumor activity. Understanding the unique immunosuppressive milieu of GBM including this cell population may help elucidate why some patients show treatment response. METHODS: Mice orthotopically implanted with GL261 had blood collection prior to anti-PD-1 exposure with t-SNE mapping of T cells. The molecular profiles of mice with long-term survival were compared with those that did not. Due to a lack of GBM patient samples that have shown immunotherapy response, we examined whether these molecular patterns recapitulated in the blood of NSCLC patients responded to anti-PD-1. RESULTS: The blood of long-term survivor mice implanted with GBM demonstrated a significantly greater proportion of CD39+ CD8 T cells with low PD-1 expression (PD-1lo) with subsequent increased representation of this population in the tumor as well (P < 0.05). This CD39+ CD8+ PD-1lo population was also highly represented in the blood of treatment-naÏve NSCLC patients who responded to anti-PD-1 (P < 0.05). Furthermore, once exposed to anti-PD-1 in vivo, this CD39+ CD8+ PD-1lo population showed high immune activity with robust IFN-y expression (P < 0.05). CONCLUSIONS: Mice with CD39+ CD8+ PD-1lo T cells are strongly associated with anti-PD-1 response. This has implications for exploring this cell population as a blood biomarker and an active anti-tumor population that may be mobilized with adoptive cell transfer and CAR-T cell strategies.

Paeonol inhibits melanoma growth by targeting PD1 through upregulation of miR-139-5p

The abnormal immune response mediated by malignant melanoma is related to PD1. Paeonol has pharmacological antitumor activity. Previous studies have indicated that paeonol induces tumor cell apoptosis, but its underlying mechanism in tumor immunity remains unknown. In this study, malignant melanoma was established in normal and thymectomized mice to determine the important role of the thymus in the antitumor effects of paeonol. Paeonol-treated thymocytes were cocultured with melanoma cell spheres to further evaluate the regulatory role of thymocytes in tumor immune dysfunction. Studies have shown that PD1 may be targeted by miR-139–5p. Our results revealed that tumor-induced thymic atrophy was significantly accompanied by high PD1 expression and low miR-139–5p expression. Interestingly, paeonol significantly reversed thymic atrophy and largely protected thymocytes against low PD1 expression and high miR-139–5p expression. Dual-luciferase assays indicated that miR-139–5p interacted with the 3′ untranslated region (3′-UTR) of PD1. These results showed that paeonol alleviates PD1-mediated antitumor immunity by reducing miR-139–5p expression and demonstrated a novel mechanism for melanoma immunotherapy.

Immune checkpoint analysis of T-cell responses to pp65 and IE-1 antigens in end-stage lung diseases.

Lung transplant (LTX) patients are at high risk of cytomegalovirus (CMV) infection, which is often associated with high mortality and morbidity. Reactivation of CMV causes cell injury due to the cytopathic effect of viral replication and triggering of T cell immunity. The aim of this study was to compare expression of immune checkpoints (ICs) (PD-1, CTLA-4, LAG-3 and TIGIT) in CD4, CD8 and CD56 and activation markers CD137, CD154 and CD69 of end-stage patients awaiting lung transplant. Eighteen pre-LTX positive for anti-CMV IgG titres and 18 healthy subjects were enrolled. IC and activation markers have been evaluated through flow cytometric analysis in HC and pre-LTX patients. Reactive (QF+) and unreactive (QF-) patients were stratified according to QuantiFERON-CMV assays. ICs' and activation markers' expression were determined before and after in vitro stimulation with pp-65 and IE-1 antigens. Lower expression of PD-1 was observed in CD4 and CD8 cells of pre-LTX patients than controls, whereas CTLA4 appeared upregulated in CD56 and CD8 cells. TIGIT is increased on the surface of CD4, CD8 and NK cells after peptide stimulation in QF-negative patients and PD-1 is only downregulated after stimulation in the QF-positive patients. This study provides new evidence of immune dysregulation in patients with end-stage lung disorders, particularly in relation to immune checkpoint cell biology. The change in QF+ mostly happens on cytotoxic cells NK and CD8, while the changes in QF- were observed in adaptive immune cells, including CD4 and CD8.

Large Scale Ex Vivo Expansion of γδ T cells Using Artificial Antigen-presenting Cells.

Higher γδ T cell counts in patients with malignancies are associated with better survival. However, γδ T cells are rare in the blood and functionally impaired in patients with malignancies. Promising results are reported on the treatment of various malignancies with in vivo expansion of autologous γδ T cells using zoledronic acid (zol) and interleukin-2 (IL-2). Here we demonstrated that zol and IL-2, in combination with a novel genetically engineered K-562 CD3scFv/CD137L/CD28scFv/IL15RA quadruplet artificial antigen-presenting cell (aAPC), efficiently expand allogeneic donor-derived γδ T cells using a Good Manufacturing Practice (GMP) compliant protocol sufficient to achieve cell doses for future clinical use. We achieved a 633-fold expansion of γδ T cells after day 10 of coculture with aAPC, which exhibited central (47%) and effector (43%) memory phenotypes. In addition, >90% of the expanded γδ T cells expressed NKG2D, although they have low cell surface expression of PD1 and LAG3 inhibitory checkpoint receptors. In vitro real-time cytotoxicity analysis showed that expanded γδ T cells were effective in killing target cells. Our results demonstrate that large-scale ex vivo expansion of donor-derived γδ T cells in a GMP-like setting can be achieved with the use of quadruplet aAPC and zol/IL-2 for clinical application.

First-in-Class Histone Acetyltransferase (HAT) Activator, YF2, Modulates Immune Evasion in DLBCL, Enhancing the Effects of Immune Checkpoint Blockade

We reported the discovery of YF2, a EP300 (p300) and CREBBP (CBP) HAT activator which demonstrates selective cytotoxicity in HAT-mutated DLBCL cell lines and induces histone acetylation in vitro and in vivo. Recently, we have focused on the role of HAT induction in regulating immune response in DLBCLs. CBP has been shown to function as an enhancer that regulates GC B-cell fate by controlling antigen presentation and terminal differentiation. HAT mutations are linked to reduced MHC-II HLA transcripts due to decreased acetylation at the promoter regions which leads to decreased GC B:T cell entanglement and derangements in the GC reaction. Numerous studies have reported the effect of epigenetic drugs on antigen presentation. This response is caused by disruption of the BLC6/SMRT/HDAC3 repressor complex which co-occupies enhancers in the MHC Class II loci. Previously, we demonstrated YF2 induced p300 mediated acetylation of BCL6; abrogating its activity. More recently, we demonstrated through RNA-seq that prolonged treatment of YF2 upregulated the expression of several MHC Class I-II genes resulting in the upregulation of the immune regulation signaling pathway. Further, treatment with YF2 up-regulated MHC-I and -II expression in a dose dependent manner. In addition to regulating immune response, BCL6 is a master regulator of GC formation. We hypothesize that induction of HATs will modulate immune response in splenic and circulating B and T-cells. Immunized BALB/c mice were treated with saline or 50mg/kg YF2 for 10 days. Spleen and blood samples were analyzed by flow cytometry. We observed a significant reduction in splenic weight, B-cell frequency, and GCs formation in YF2 treated mice. YF2 treatment increased the frequency of mature and transitional B-cells in the spleen. Further, YF2 treatment reduced light zone and increased dark zone cells in the spleen. YF2 treatment also increased the frequency of plasma cells and plasmablast. MHC-I and -II expression was significantly upregulated in YF2 treated mice. YF2 exposure resulted in an increase of PD-L1hi B-cells. Lastly, circulating B-cells showed significant increase in MHC-II expression following YF2 treatment. In splenic T-cells, we observed a reduction in CD4+ helper T-cells and an increase in CD8+ cytotoxic T-cells in YF2 treated mice. YF2 caused a reduction in naïve CD4+ cells and an increase in the frequency of central and effector memory cells Lastly, we examined the frequency of inhibitory molecules PD1, TIGIT, CD226, Lag3, and VISTA. PD-1, TIGIT, and CD226 were upregulated with YF2 treatment in CD4+ cells and CD226 was upregulated in CD8+ cells. Together, these results demonstrate YF2 is able to modulate immune response in B and T-cells. DLBCLs are known to have limited sensitivity to checkpoint blockade with PD-1 and PD-L1 inhibitors due to low PD-L1 and PD-1 expression which is in part driven by HAT mutations and overexpression of BCL6; which regulates PD-L1. We hypothesize that combination treatment of YF2 and PD-1 inhibitor in the immunocompetent syngeneic A20 mouse tumor xenograft model will result in decreased tumorigenicity and improved survival in mice. Xenografted and immunized BALBc mice were treated with saline, YF2, PD-1 inhibitor, or a combination of YF2 and PD-1 inhibitor for 28 days. We observed, a significant reduction in tumor volume in both the PD-1 and combination treated groups. Kaplan-Meier analysis showed an increase in median survival for all treatment with the combination treatment resulting in the greatest increase in median survival vs. control (Fig A). Spatial analysis was performed using the 10x Visium platform and sc-RNA-seq to examine the tumor microenvironment in control and YF2 treated mouse tumor samples (Fig B). We observed a more aggressive phenotype in the untreated tumors as well as an increase in the number of distinct clusters indicating a more heterogenous tumor population. Lastly, PBMCs from patient samples were isolated and treated with YF2. Compared to healthy donor patients, YF2 induced cytotoxicity across various lymphoma subtype. Normal PBMCs were not affected by treatment. Based on the success of YF2 in the preclinical setting, the use of this first-in-class drug for treatment of lymphoma was approved to proceed in clinical studies in patients with relapsed/refractory lymphoma. Patient samples will be collected and analyzed for immune response. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Content of PD-1+ and PD-L1+ peripheral blood lymphocytes in individuals with pollen sensitization before and after allergen-specific immunotherapy

The available data on the contribution of the PD-1 and its ligands to immunoregulatory processes suggest their involvement into development of tolerance during immunotherapy. Currently, allergen-specific immunotherapy (ASIT) is the single treatment option that can influence the outcome of allergic diseases. Our purpose was to evaluate the PD-1/PD-L1 expression on the immune cells in patients with confirmed sensitization to plant pollen allergens in comparison with healthy controls before and after ASIT. The patients with bronchial asthma (BA) (n = 5, age 33.82.7), allergic rhinitis (AR) (n = 7, age 31.62.8), and healthy donors (n = 12, age 32.81.8) were included. Venous blood samples were obtained from the patients three times: before starting ASIT, upon completion of the ASIT course, and during the period of seasonal exacerbation. In patients with AR, the number of B lymphocytes was decreased, and the expression of PD-L1 by B lymphocytes increased after ASIT in comparison with donor parameters. At the same time, B lymphocyte counts were increased in BA patients before ASIT and returned to normal after ASIT. In AR, the CD8+PD-1+T lymphocyte count was reduced before ASIT, however, returning to normal values after ASIT was completed. Meanwhile, the reduced number of CD4+PD-1+T lymphocytes returned to normal only during the pollination season following ASIT. In BA patients, both before or after ASIT, PD-1 and PD-L1 expression on CD4+ and CD8+T lymphocytes did not differ from the donor parameters. The PD-1 expression in the T regulatory cells (Tregs) was decreased comparing with donors before ASIT in BA, and in the patients with AR, both before and after treatment. It was shown earlier that low PD-1 expression in the circulating CD4+ T cells is associated with high specific IgE concentrations. Thus, low PD-1 levels on CD4+ and CD8+T lymphocytes and regulatory T cells may indicate their functional disorders in allergic pathology. In summary, our results show a regulatory role of PD-1/PD-L1 axis in the immune response during ASIT and reflect differences in pathogenesis of allergic disorders, which are associated with imbalance of the cell activation and suppression. Further studies are required to establish the role of PD-1/PD-L1 interactions in the process of ASIT-induced modification of allergic responses.

Postacute sequelae and adaptive immune responses in people with HIV recovering from SARS-COV-2 infection.

Limited data are available on the long-term clinical and immunologic consequences of SARS-CoV-2 infection in people with HIV (PWH). We measured SARS-CoV-2-specific humoral and cellular responses in people with and without HIV recovering from COVID-19 ( n = 39 and n = 43, respectively) using binding antibody, surrogate virus neutralization, intracellular cytokine staining, and inflammatory marker assays. We identified individuals experiencing postacute sequelae of SARS-CoV-2 infection (PASC) and evaluated immunologic parameters. We used linear regression and generalized linear models to examine differences by HIV status in the magnitude of inflammatory and virus-specific antibody and T-cell responses, as well as differences in the prevalence of PASC. Among PWH, we found broadly similar SARS-CoV-2-specific antibody and T-cell responses as compared with a well matched group of HIV-negative individuals. PWH had 70% lower relative levels of SARS-CoV-2-specific memory CD8 + T cells ( P = 0.007) and 53% higher relative levels of PD-1+ SARS-CoV-2-specific CD4 + T cells ( P = 0.007). Higher CD4 + /CD8 + ratio was associated with lower PD-1 expression on SARS-CoV-2-specific CD8 + T cells (0.34-fold effect, P = 0.02). HIV status was strongly associated with PASC (odds ratio 4.01, P = 0.008), and levels of certain inflammatory markers (IL-6, TNF-alpha, and IP-10) were associated with persistent symptoms. We identified potentially important differences in SARS-CoV-2-specific CD4 + and CD8 + T cells in PWH and HIV-negative participants that might have implications for long-term immunity conferred by natural infection. HIV status strongly predicted the presence of PASC. Larger and more detailed studies of PASC in PWH are urgently needed.