High Expression Of CD27 Research Articles

Abstract 2533: In-depth multi-omic profiling of immune cells in cancer patients developing immune-related adverse events

Abstract Background: The clinical efficacy of immune-checkpoint inhibitors is complicated by the risk for immune-related adverse events (irAEs) that can involve any organ systems. To date, (1) we cannot predict irAE risk reliably at patient level and (2) irAEs remain a diagnosis of exclusion as there are no specific clinical or biological markers. The goal of this study was to elucidate intricate transcriptomic and proteomic signatures of immune cells in patients evolving irAEs. Methods: This pilot trial was conducted as part of a prospective multicenter cohort study (NCT04631731). The study cohort consisted of n=71 patients who were treated with ICIs across Blacktown and Westmead Hospitals, NSW, Australia. Peripheral blood mononuclear cells (PBMCs) were collected from both pre-treatment and after 6-8 weeks post-ICI commencement. Samples were further utilized for RNA sequencing, flow cytometry and Chromium 3’ gene expression analysis (10x Genomics). Results: The median age was 67 years old with the 78% (n=56) of Caucasian origin. The predominant number of patients were treated with dual ICIs. N=21 patients experienced irAEs. Firstly, a differential analysis established a set of genes which were significantly (FDR<0.01) dysregulated between patients with (group A) and without irAEs (group B) and were mostly related to cell cycle and regulation. Notably, phospholipase D signaling pathway was exclusively upregulated in group A and is known to be linked to B cell expansion. Secondly, a cell enrichment analysis within the group A revealed a significant increase of CD4+ and CD8+ T central memory (Tcm) cells upon the toxicity onset as compared to baseline stage. Thirdly, the flow cytometry data established that group A had (1) higher expression of CD27 on CD4+ T cells, a member of Traf-linked tumor necrosis factor receptor family essential for T cells’ maturation and (2) lower expression of inhibitory receptor LAG3 (CD223) which is crucial for maintaining homeostasis of immune system through suppression of T cell proliferation. Finally, we sequenced CD45+ cells from n=4 patients from group A and n=4 from group B at both pre- and post-treatment stages. The unsupervised clustering established 17 different clusters in our single cell dataset with cluster 11 was unique only to patients from group A. The differential analysis established that a set of n=62 genes was significantly upregulated (p<0.05) in cluster 11. Functional annotation established that B cell activation and proliferation pathways were enriched by those genes further stressing B cells’ role in irAEs development. Conclusions: Our real-world cohort study established significant molecular and cellular differences between patients with and without irAEs. Further research in larger cohorts is imperative to validate the established findings and to elucidate the precise mechanisms contributing to the development of irAEs. Citation Format: Dmitrii Shek, Bo Gao, Joey Lai, Brian Gloss, Adnan Nagrial, Ines Pires da Silva, Matteo S. Carlino, Scott A. Read, Golo Ahlenstiel. In-depth multi-omic profiling of immune cells in cancer patients developing immune-related adverse events [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 2533.

Compartmentalized spatial profiling of the tumor microenvironment in head and neck squamous cell carcinoma identifies immune checkpoint molecules and tumor necrosis factor receptor superfamily members as biomarkers of response to immunotherapy.

Mucosal head and neck squamous cell carcinoma (HNSCC) are the seventh most common cancer, with approximately 50% of patients living beyond 5 years. Immune checkpoint inhibitors (ICIs) have shown promising results in patients with recurrent or metastatic (R/M) disease, however, only a subset of patients benefit from immunotherapy. Studies have implicated the tumor microenvironment (TME) of HNSCC as a major factor in therapy response, highlighting the need to better understand the TME, particularly by spatially resolved means to determine cellular and molecular components. Here, we employed targeted spatial profiling of proteins on a cohort of pre-treatment tissues from patients with R/M disease to identify novel biomarkers of response within the tumor and stromal margins. By grouping patient outcome categories into response or non-response, based on Response Evaluation Criteria in Solid Tumors (RECIST) we show that immune checkpoint molecules, including PD-L1, B7-H3, and VISTA, were differentially expressed. Patient responders possessed significantly higher tumor expression of PD-L1 and B7-H3, but lower expression of VISTA. Analysis of response subgroups indicated that tumor necrosis factor receptor (TNFR) superfamily members including OX40L, CD27, 4-1BB, CD40, and CD95/Fas, were associated with immunotherapy outcome. CD40 expression was higher in patient-responders than non responders, while CD95/Fas expression was lower in patients with partial response (PR) relative to those with stable disease (SD) and progressive disease (PD). Furthermore, we found that high 4-1BB expression in the tumor compartment, but not in the stroma, was associated with better overall survival (OS) (HR= 0.28, p-adjusted= 0.040). Moreover, high CD40 expression in tumor regions (HR= 0.27, p-adjusted= 0.035), and high CD27 expression in the stroma (HR= 0.2, p-adjusted=0.032) were associated with better survival outcomes. Taken together, this study supports the role of immune checkpoint molecules and implicates the TNFR superfamily as key players in immunotherapy response in our cohort of HNSCC. Validation of these findings in a prospective study is required to determine the robustness of these tissue signatures.

Optimization of therapeutic T cell expansion in G-Rex device and applicability to large-scale production for clinical use

Our center performs experimental clinical studies with advanced therapy medicinal products (ATMPs) based on polyclonal T cells, all of which are currently expanded in standard T-flasks. Given the need to increase the efficiency and safety of large-scale T cell expansion for clinical use, we have optimized the method to expand in G-Rex devices both cytokine-induced killer cells (CIKs) from peripheral or cord blood and blinatumomab-expanded T cells (BETs). We show that the G-Rex reproducibly allowed the expansion of >30 × 106 CD3+ cells/cm2 of gas-permeable membrane in a mean of 10 to 11 days in a single unit, without manipulation, except for addition of cytokines and sampling of supernatant for lactate measurement every 3 to 4 days. In contrast, 21 to 24 days, twice-weekly cell resuspension and dilution into 48 to 72 T-flasks were required to complete expansions using the standard method. We show that the CIKs produced in G-Rex (CIK-G) were phenotypically very similar, for a large panel of markers, to those expanded in T-flasks, although CIK-G products had lower expression of CD56 and higher expression of CD27 and CD28. Functionally, CIK-Gs were strongly cytotoxic in vitro against the NK cell target K562 and the REH pre-B ALL cell line in the presence of blinatumomab. CIK-Gs also showed therapeutic activity in vivo in the Ph+ pre-B ALL-2 model in mice. The expansion of both CIKs and BETs in G-Rex was validated in good manufacturing practices (GMP) conditions, and we plan to use G-Rex for T cell expansion in future clinical studies.

Mass cytometry identifies distinct CD4+ T cell clusters distinguishing HIV-1-infected patients according to antiretroviral therapy initiation.

Recent guidelines recommend antiretroviral therapy (ART) to be administered as early as possible during HIV-1 infection. Few studies addressed the immunological benefit of commencing ART during the acute phase of infection. We used mass cytometry to characterize blood CD4+ T cells from HIV-1-infected patients who initiated ART during acute or chronic phase of infection. Using this method, we analyzed a large number of markers on millions of individual immune cells. The results revealed that CD4+ T cell clusters with high expression of CD27, CD28, CD127, and CD44, whose function involves T cell migration to inflamed tissues and survival, are more abundant in healthy controls and patients initiating ART during the acute phase; on the contrary, CD4+ T cell clusters in patients initiating ART during the chronic phase had reduced expression of these markers. The results are suggestive of a better preserved immune function in HIV-1-infected patients initiating ART during acute infection.

Presence of naive-like HBV-specific CD8+ T cells in chronically infected patients despite ongoing viral replication

In chronically HBV-infected patients, virus-specific CD8+ T cell responses are rarely detectable ex vivo by conventional tetramer staining. This lack of HBV-specific CD8+ T cell responses may be explained by insufficient priming, T cell exhustion and/or deletion. Here, we set out to address this issue by using new techniques for enumerating virus-specific T cells from human peripheral blood based on the combination of tetramer staining, magnetic-bead enrichment, and multiparametric flow cytometry. Indeed, we were able to detect and characterize CD8+ T cell populations specific for 4 well-described HLA-A0201-restricted HBV epitopes (core18 – 27, env183 – 191, env335 – 343 and pol455 – 463) in 24 chronically HBV-infected patients suggesting that these cells are not completely deleted. CD8+ T cells specific for core18 – 27, env335 – 343 and pol455 – 463 were detectable in the majority of analyzed patients (79%, 75% and 100% respectively) while env183 – 191-specific CD8+ T cells were found less frequently (54%). Interestingly, about 20% of analyzed CD8+ T cells had a naive-like phenotype with high expression of CD45RA, CCR7 and CD27 and low expression of CD11a. By contrast 39% of HBV-specific CD8+ T cells showed an effector-memory phenotype characterized by the absence of CD45RA, CCR7 and high expression of CD27 and CD11a indicating viral recognition. Of note, HBV-specific CD8+ T cell populations with naive-like and effector-memory phenotype were detected in parallel for different epitopes within the same patients (9 of 24). Importantly, the observed phenotype did not correlate with any clinical parameter such as HBeAg status, viral load or transaminases and was independent of antiviral treatment. Furthermore, viral sequence analysis revealed the presence of wild-type sequences in patients with naive-like HBV-specific CD8+ T cells indicating that the observed lack of priming is not due to infection with a variant HBV containing mismatched epitopes. Our results indicate that HBV-specific CD8+ T cells are not completely deleted in chronically infected patients, but rather maintained at a very low frequency. Furthermore, a substantial fraction of these cells display a naive-like phenotype despite ongoing viral replication. This suggests, that in addition to T cell exhaustion insufficient priming may also contribute to HBV-specific CD8+ T cell failure in chronically infected patients.

Regulation of tissue-dependent differences in CD8+ T cell apoptosis during viral infection.

Virus-specific CD8+ T cells in the lymphoid organs contract at the resolution of virus infections by apoptosis or by dissemination into peripheral tissues, and those residing in nonlymphoid organs, including the peritoneal cavity and fat pads, are more resistant to apoptosis than those in the spleen and lymph nodes. This stability of memory T cells in the nonlymphoid tissues may enhance protection to secondary challenges. Here, we show that lymphocytic choriomeningitis virus (LCMV)-specific CD8+ T cells in nonlymphoid tissues were enriched for memory precursors (expressing high levels of interleukin-7 receptor and low levels of killer cell lectin-like receptor G1 [IL-7Rhi KLRG1lo]) and had higher expression of CD27, CXCR3, and T cell factor-1 (TCF-1), each a marker that is individually correlated with decreased apoptosis. CD8+ T cells in the peritoneal cavity of TCF-1-deficient mice had decreased survival, suggesting a role for TCF-1 in promoting survival in the nonlymphoid tissues. CXCR3+ CD8+ T cells resisted apoptosis and accumulated in the lymph nodes of mice treated with FTY720, which blocks the export of lymph node cells into peripheral tissue. The peritoneal exudate cells (PEC) expressed increased amounts of CXCR3 ligands, CXCL9 and CXCL10, which may normally recruit these nonapoptotic cells from the lymph nodes. In addition, adoptive transfer of splenic CD8+ T cells into PEC or spleen environments showed that the peritoneal environment promoted survival of CD8+ T cells. Thus, intrinsic stability of T cells which are present in the nonlymphoid tissues along with preferential migration of apoptosis-resistant CD8+ T cells into peripheral sites and the availability of tissue-specific factors that enhance memory cell survival may collectively account for the tissue-dependent apoptotic differences. Most infections are initiated at nonlymphoid tissue sites, and the presence of memory T cells in nonlymphoid tissues is critical for protective immunity in various viral infection models. Virus-specific CD8+ T cells in the nonlymphoid tissues are more resistant to apoptosis than those in lymphoid organs during the resolution and memory phase of the immune response to acute LCMV infection. Here, we investigated the mechanisms promoting stability of T cells in the nonlymphoid tissues. This increased resistance to apoptosis of virus-specific CD8+ T cells in nonlymphoid tissues was due to several factors. Nonlymphoid tissues were enriched in memory phenotype CD8+ T cells, which were intrinsically resistant to apoptosis irrespective of the tissue environment. Furthermore, apoptosis-resistant CD8+ T cells preferentially migrated into the nonlymphoid tissues, where the availability of tissue-specific factors may enhance memory cell survival. Our findings are relevant for the generation of long-lasting vaccines providing protection at peripheral infection sites.

IL-2 regulates tissue-dependent differences in CD8 T cell apoptosis during and after- viral Infection. (110.19)

Abstract Virus-specific CD8 T cell populations in lymphoid organs contract by apoptosis or by dissemination into peripheral tissues, and those residing in non-lymphoid organs, including the peritoneal cavity (PEC: peritoneal exudate cells), are more resistant to apoptosis than those in the lymphoid organs, including the spleen. We questioned whether tissue-dependent apoptotic differences in LCMV-specific T cells were due to environment factors or intrinsic stability of the CD8 T cells. The PEC environment conferred an anti-apoptotic effect, as shown by mixing PEC with excess splenic leukocytes or vice versa. This anti-apoptotic effect correlated with Stat5 phosphorylation induced by IL-2 spontaneously secreted by CD4 T cells. Spontaneous IL-2 production by CD4 T cells occurred in vivo during the contraction and the long term memory stage after LCMV infection in PEC and Fat pads, but much less so in spleen and lymph nodes. Virus-specific CD8 T cells in the PEC were also intrinsically more stable, as most had a memory precursor effector cell (MPEC) phenotype and higher expression of CD27, both correlating with decreased apoptosis. However, regardless of their phenotype, PEC CD8 T cells remained less apoptotic than their spleen cell counterparts. We conclude that long after the resolution of viral infections, IL-2-producing CD4 T cells linger in peripheral sites and locally contribute to the stability of memory CD8 T cells, thereby enhancing resistance to re-infection.

“Immunophenotype Signature of CD8 + T Cells Indicates Impaired Development of a CD26high Memory Subset On Imatinib treatment”.

Abstract 1650Poster Board I-676CD26 is a kind of T cell costimulatory molecule with dipeptidyl peptidase IV activity in its extracellular domain. Based on the expression level of CD26, CD4+ and CD8+ T cells can be divided into 3 (high/int/low) subsets (Figure 1A). The significance of CD26 has been studied mainly on CD4+ T cells, and CD26highCD4+ T cells are considered to represent memory T cells with typical Th1 responses. Furthermore, we reported a significant decrease in number of this subset in CML patients under imatinib (IM) therapy compared with those under IFNa therapy as well as normal volunteers (control). The role of each subset of CD8+ T cells remains to be elucidated. By analogy to a CD4+CD26high subset, we hypothesized that CD8+CD26high cells might represent a memory subset which could be affected by IM treatment. To test this notion, we first tried to define each CD8+ subset based on its expression of 13 well-known surface antigens by multi-parameter flow cytometry (FACS) analysis. FACS data for each CD8+CD26 subset from control group (n=20) were subjected to the unsupervised hierarchical clustering analysis. As a result, the hierarchical clustering revealed three major clusters (cluster I, II and III; Figure1B), which consisted of 20, 26 and 14 data respectively. Cluster I is characterized by high expression of CD127, CD28, CCR5 and CD45RO (a memory profile), to which all 20 data from high subset were categorized. Cluster II is characterized by high expression of CD27, CD62L, CCR7 and CD45RA (a naive profile), to which all 20 data from int subset and 6 of 20 data from low subset were categorized. Cluster III is characterized by high expression of IFNg, perfolin, granzymeB, CD57 and CD11a (a differentiated effector profile), to which 14 of 20 data from low subset was categorized. Thus, we could categorize CD8+ T cells according to the level of CD26 expression.We next investigated the effects of IM on these 3 distinct subsets in CD8+ T cell differentiation program. Then, we compared immunophenotype signature of each CD8+ CD26 subset between IM group and control group (population frequency among CD8+ T cells, absolute cell count and surface antigen profile). We observed a significant decrease of CD8+CD26high subset in IM group (Figure1C), compared with control group. Altered phenotypic profile (decreased CD127/CD28 expression) was also noted in this subset from IM group (Figure1D). Finally, we investigated the impact of IM on primed CD8+ T cells in vitro. Peripheral blood CD8+ T cells were purified from normal subjects, primed with various stimuli and subjected to the grading doses of IM, followed by FACS analysis. CFSE labeling was used for monitoring cell proliferation. Stimuli included anti-CD3/CD28 MAb (aCD3), common g-chain cytokines (IL-7, IL-15), and their combination. Half inhibitory concentrations (IC50) of IM with 95% confidence interval were also calculated (Figure 1E). We found that CD8+ T cells in the presence of therapeutic concentrations of IM showed a reduced activation status (as determined by up-regulation of CD26 expression) and impaired proliferation (as determined by CFSE intensity) in response to TCR engagement as well as cytokine stimulation. Our present results not only propose a potential utility of CD26 as a convenient marker for functional (naïve/memory/effector) compartments of CD8+ T cells, but also offer another evidence for immunomodulatory effects of IM or the significant role of Abl (-related) kinase in memory CD8+ T cell development, possibly in common g-chain cytokine(s)-dependent manner. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Chronic Human Infection with Trypanosoma cruzi Drives CD4+ T Cells to Immune Senescence

Previously we found that the frequency of IFN-gamma-producing CD8(+) T cells specific for Trypanosoma cruzi inversely correlates with disease severity in chronic human Chagas disease along with low levels of IL-2-secreting CD8(+) T cells in all clinical stages. This impairment of the parasite-specific T cell responses was associated with phenotypic features of immune senescence of the CD8(+) T cell compartment. These data prompted us to address the question of whether the CD4(+) T cell compartment also experiences signs of exhaustion. Thus, we performed a functional and phenotypical characterization of T. cruzi-specific and overall CD4(+) T cells in chronically infected subjects with different degrees of cardiac dysfunction. The results show an inverse association between disease severity and the frequency of T. cruzi-specific IFN-gamma-producing CD4(+) T cells. The high expression of CD27 and CD28 with a relative low expression of CD57 found on CD4(+)IFN-gamma(+) T cells suggests that the effector T cell pool in chronic T. cruzi infection includes a high proportion of newly recruited T cells, but a low frequency of long-term memory cells. The total CD4(+) T cell compartment shows signs of senescence and later stages of differentiation associated with more severe stages of the disease. These findings support the hypothesis that long-term T. cruzi infection in humans might exhaust long-lived memory T cells.

Delayed Redistribution of CD27, CD40 and CD80 Positive B Cells and the Impaired In Vitro Immunoglobulin G Production in Patients with Non-Hodgkin Lymphoma Treated with Rituximab.

Rituximab (RT) has been proven to be very effective in depleting normal and malignant B lymphocytes in vivo and it is widely used for the treatment of B cell malignancies, particularly B cell non-Hodgkin's lymphoma (NHL). RT alone does not appear to cause severe hypogammaglobulinemia according to initial clinical trials. However, recent studies revealed that patients who received RT as an adjuvant to stem cell transplantation (SCT) demonstrated an increased risk of developing severe hypogammaglobulinemia. We have found such hypogammaglobulinemia to be due to the delayed recovery of CD27 positive memory B cells and an impaired isotype expression. (Nishio et al. Eur J Haematol, 2006). This finding suggests that RT can influence not only the quantity, but also the quality of B-cell redistribution. Nevertheless, to our knowledge, precisely how the B-cell repertoire regenerates after anti-CD20-mediated transient B-cell depletion in patients with NHL remains to be elucidated. To clarify this, we performed a phenotypical analysis of B cells.A total of 22 patients with NHL who received RT combined with autologous SCT (n=17) or CHOP (n=5) were evaluated to identify their immunophenotype. The median period after the last administration of RT was 33.5 months (range from 12 to 56 months). We investigated the expression of various markers, including CD27, CD38, CD40, CD80, CD86 and CD95 on B cells by immunofluorescence staining with a flowcytometry analysis. A statistically significant difference was noted in three of the six surface antigens when the expressions of those antigens were compared with those in the healthy control populations (N=14). The most striking differences we found was the expression levels of CD27. The healthy control group had a much higher expression of CD27 in comparison to those of the patients treated with RT (28.1±14.1% vs 8.2±6.1%, p<0.001). In addition, significant differences in the expression of CD40 and CD80 were also noted. While the positive rates of CD80 and CD40 on B cells from healthy controls were 21.5±10.8% and 80.5±16.7%, those of patients treated with RT were 9.9±6.9% and 49.7±33.5%, respectively (p<0.01 and p<0.05).Since CD40-CD40L and CD80-CD28 pathways between B and T cells are necessary for the development of CD27 positive polyclonal B-cell activation and immunoglobulin production, we hypothesized that the B cells from patients treated with RT thus had a reduced ability to differentiate into plasma cells and immunoglobulin production in vitro. To test this hypothesis, we purified the B cells from ten patients with NHL treated with RT and then cultured them upon the engagement of immunoglobulin receptor and CD40 in the presence of IL-2 and IL-10. After eight days of stimulation, the supernatants of the culture were harvested and the concentrations of immunoglobulin were measured by ELISA. As a result, the IgG production was found to be significantly impaired in patients with NHL in comparison to those from the healthy controls.The observation of a delayed recovery of the memory B cells with an abnormal cell marker expression and function demonstrates that naive B cells may therefore be responsible for their failure to differentiate into plasma cells after RT therapy.

Expression of CD27–CD70 on Early B Cell Progenitors in the Bone Marrow: Implication for Diagnosis and Therapy of Childhood ALL

CD27, a member of the TNF receptor family, plays an important role in lymphoid proliferation, differentiation and apoptosis. This study addresses the expression of CD27 and its ligand, CD70, in children with acute lymphoblastic leukemia (ALL) and the possible role of this receptor-ligand pair in the pathogenesis of ALL. Expression of CD27 and CD70 was evaluated with three-color flow cytometry in blood and bone marrow (BM) samples in children with ALL and controls. The biological role of these molecules on leukemic cell proliferation was studied in an in vitro culture system. The expression of the membrane bound CD27, as well as membrane bound CD70, on CD19(+) cells in the BM was significantly increased in ALL children compared to the expression found in the controls. Importantly, a substantial reduction in the in vitro proliferation of leukemic cells could be observed when the leukemic cells were cultured in presence of a blocking anti-human CD70 monoclonal antibody. The level of soluble CD27 (sCD27) in serum was also investigated and found to be significantly elevated in leukemic children as compared to healthy children. The high expression of CD27 and CD70 on ALL cells may represent an amplification of the normal CD27-CD70 expression present on early B cell progenitors. Our finding suggests that interference with CD27-CD70 interaction may represent novel treatment opportunities in ALL. Further studies are required to pin-point the role of this receptor-ligand pair in normal and malignant hematopoiesis.