Blood T-cell Research Articles

Cholinergic regulation of vascular endothelial function by human ChAT+ T cells

Endothelial dysfunction and impaired vasodilation are linked with adverse cardiovascular events. T lymphocytes expressing choline acetyltransferase (ChAT), the enzyme catalyzing biosynthesis of the vasorelaxant acetylcholine (ACh), regulate vasodilation and are integral to the cholinergic antiinflammatory pathway in an inflammatory reflex in mice. Here, we found that human T cell ChAT mRNA expression was induced by T cell activation involving the PI3K signaling cascade. Mechanistically, we identified that ChAT mRNA expression was induced following the attenuation of RE-1 Silencing Transcription factor REST-mediated methylation of the ChAT promoter, and that ChAT mRNA expression levels were up-regulated by GATA3 in human Tcells. In functional experiments, T cell-derived ACh increased endothelial nitric oxide-synthase activity, promoted vasorelaxation, and reduced vascular endothelial activation and promoted barrier integrity by a cholinergic mechanism. Further, we observed that survival in a cohort of patients with severe circulatory failure correlated with their relative frequency of ChAT +CD4+ Tcells in blood. These findings on ChAT+ human Tcells provide a mechanism for cholinergic immune regulation of vascular endothelial function in human inflammation.

HIV-1 DNA and Immune Activation Levels Differ for Long-Lived T-Cells in Lymph Nodes, Compared with Peripheral Blood, during Antiretroviral Therapy.

Elucidating the mechanisms underlying the persistence and location of the HIV reservoir is critical for developing cure interventions. While it has been shown that levels of T-cell activation and the size of the HIV reservoir are greater in rectal tissue and lymph nodes (LN) than in blood, the relative contributions of T-cell subsets to this anatomic difference are unknown. We measured and compared HIV-1 DNA content, expression of the T-cell activation markers CD38 and HLA-DR, and expression of the exhaustion markers programmed cell death protein 1 (PD-1) and T-cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domains (TIGIT) in naive, central memory (CM), transitional memory (TM), and effector memory (EM) CD4+ and CD8+ T-cells in paired blood and LN samples among 14 people with HIV who were receiving antiretroviral therapy. HIV-1 DNA levels, T-cell immune activation, and TIGIT expression were higher in LN than in blood, especially in CM and TM CD4+ T-cell subsets. Immune activation was significantly higher in all CD8+ T-cell subsets, and memory CD8+ T-cell subsets from LN had higher levels of PD-1 expression, compared with blood, while TIGIT expression levels were significantly lower in TM CD8+ T-cells. The differences seen in CM and TM CD4+ T-cell subsets were more pronounced among participants with CD4+ T-cell counts of <500 cells/μL within 2 years after antiretroviral therapy initiation, thus highlighting increased residual dysregulation in LN as a distinguishing feature of and a potential mechanism for individuals with suboptimal CD4+ T-cell recovery during antiretroviral therapy. IMPORTANCE This study provides new insights into the contributions of different CD4+ and CD8+ T-cell subsets to the anatomic differences between LN and blood in individuals with HIV who have optimal versus suboptimal CD4+ T-cell recovery. To our knowledge, this is the first study comparing paired LN and blood CD4+ and CD8+ T-cell differentiation subsets, as well as those subsets in immunological responders versus immunological suboptimal responders.

New Generation Dielectrophoretic-Based Microfluidic Device for Multi-Type Cell Separation

This study introduces a new generation of dielectrophoretic-based microfluidic device for the precise separation of multiple particle/cell types. The device features two sets of 3D electrodes, namely cylindrical and sidewall electrodes. The main channel of the device terminates with three outlets: one in the middle for particles that sense negative dielectrophoresis force and two others at the right and left sides for particles that sense positive dielectrophoresis force. To evaluate the device performance, we used red blood cells (RBCs), T-cells, U937-MC cells, and Clostridium difficile bacteria as our test subjects. Our results demonstrate that the proposed microfluidic device could accurately separate bioparticles in two steps, with sidewall electrodes of 200 µm proving optimal for efficient separation. Applying different voltages for each separation step, we found that the device performed most effectively at 6 Vp-p applied to the 3D electrodes, and at 20 Vp-p and 11 Vp-p applied to the sidewall electrodes for separating RBCs from bacteria and T-cells from U937-MC cells, respectively. Notably, the device's maximum electric fields remained below the cell electroporation threshold, and we achieved a separation efficiency of 95.5% for multi-type particle separation. Our findings proved the device's capacity for separating multiple particle types with high accuracy, without limitation for particle variety.

Radiotherapy combined with anti-CEACAM1 immunotherapy to induce survival advantage in glioma

BackgroundWe aimed to observe the effect of radiotherapy on the expression of immune checkpoint molecule CEACAM1 in patients with glioma and the therapeutical effect of radiotherapy combined with blockade of CEACAM1 in mice with intracranial gliomas.MethodsThe expression of CEACAM1 on T-lymphocytes in the peripheral blood of patients with glioma was detected before and after radiotherapy; GL261 murine glioma cells (stably transfected with the luciferase gene) were implanted in the right caudate nucleus of C57BL/6 mice, and tumour growth was observed using the small animal in vivo imaging system. Mice were divided into 4 groups: (1) the isotype control; (2) the radiotherapy; (3) the anti-CEACAM1 treatment; and (4) the combination therapy. The survival of mice after treatment was recorded; the expression of CEACAM1 on murine glioma cells was detected by immunohistochemistry before and after radiotherapy; flow cytometry was adopted to detect CD8+ T-cells (Treg) (CD4+FoxP3+CD25+) among mouse brain-infiltrating T-cells; serum levels of IFN-γ and IL-10 were detected by ELISA; proliferation and apoptosis were observed by immunohistochemistry; Retrospective RNA-seq data analysis was conducted in a cohort of 325 patients with glioma in the Chinese Glioma Genome Atlas (CGGA) database and 702 patients in The Cancer Genome Atlas (TCGA) database.ResultsThe expression of CEACAM1 on CD4+ and CD8+ T-cells in the peripheral blood of patients with glioma was significantly higher 1 week after radiotherapy than before radiotherapy and was further increased 1 month after radiotherapy. Combined therapy notably inhibited the growth of intracranial tumours in mice and prolonged their survival time, with some mice being capable of surviving long-term (> 90 d). Immunohistochemistry revealed that the expression of CEACAM1 in murine glioma tissues after radiotherapy was elevated in a time-dependent manner. Flow cytometry analysis showed an increase in mouse brain-infiltrating CD8+ T-lymphocytes, a decrease in Treg cells, and an increase in CD8+ T/Treg cells after treatment. ELISA demonstrated the elevated levels of IFN and decreased levels of IL-10 in the serum of mice in the combination therapy group.ConclusionsRadiotherapy combined with CEACAM1 inhibitors resulted in strong and durable anti-tumour immune responses against murine glioma and long-term survival of some mice. Hence, this study is expected to offer new effective immunotherapy strategies against glioma.

Correlation analysis of the peripheral blood lymphocyte count and occurrence of pneumonia after lung transplantation

BackgroundInfections are the most common complication in patients after lung transplantation and the main cause of death at all stages after transplantation; therefore, awareness regarding the occurrence of infectious pneumonia after lung transplantation is vital. This study aimed to explore the correlation between the absolute lymphocyte and T-lymphocyte subpopulation counts in the peripheral blood and the occurrence of pneumonia after lung transplantation and to predict the risk of pneumonia development after lung transplantation. MaterialsPatients who underwent lung transplantation with long-term follow-up between June 2018 and December 2021 were prospectively included. The patients were divided into pneumonia and non-pneumonia groups. Demographic and clinical characteristics, and the levels of leukocytes, neutrophils, platelets, C-reactive protein (CRP), procalcitonin (PCT), serum albumin, peripheral blood T lymphocytes, and CD4+ and CD8+ T cells in the peripheral blood were measured in both groups. ResultsWe included 22 patients with post-lung transplants in the analysis. Of the 104 collected samples, 26 (56.5%) were pathogenically positive, 16 (61.5%) had bacterial infections, 7 samples (26.9%) had fungal infections, and 8 (30.8%) had viral infections. Patients with pneumonia had higher levels of peripheral blood neutrophils (P = 0.01), platelets (P = 0.03), and CRP (P < 0.001) than did those without pneumonia. Logistic regression analysis showed that the levels of peripheral blood neutrophils, total T lymphocytes, CRP, and PCT were associated with the development of pneumonia after transplantation (P < 0.05), as documented by their area under the curve (AUC) values of 0.702, 0.792, 0.899, and 0.789, respectively. The AUC for the combined receiver operating characteristic curve for predicting the development of pneumonia was 0.943, with a sensitivity of 91.3% and specificity of 93.1%. There was no significant difference in T-lymphocyte counts in patients with lung transplants between the pneumonia and non-pneumonia groups who were treated with two anti-rejection agents. In contrast, the absolute lymphocyte, total T-lymphocyte, and CD4+ and CD8+ T-cell counts in patients who developed pneumonia after treatment with three anti-rejection agents were lower than those in patients who did not develop pneumonia (P < 0.05). ConclusionBacterial pneumonia is more common after lung transplantation than after fungal or viral infections. Peripheral blood T-lymphocyte counts combined with neutrophil, CRP, and PCT levels had good predictive value for the development of pneumonia after lung transplantation. Monitoring of patients should be strengthened by implementing peripheral blood T-lymphocyte counts to improve the early identification and prevention of pneumonia after lung transplantation.

A219 BIOLOGIC USE DURING PREGNANCY IN WOMEN WITH INFLAMMATORY BOWEL DISEASE AND ASSOCIATED NEONATAL OUTCOMES: A RETROSPECTIVE CHART REVIEW

Abstract Background Biologics are the mainstay of therapy for patients with advanced inflammatory bowel disease (IBD). Most biologics readily undergo placental transfer during the second trimester and remain detectable in infant serum for up to 12 months. Therefore, most clinical guidelines currently recommend avoiding live vaccines in the first 6 to 12 months for exposed infants. In Canada, the rotavirus vaccine is the only live vaccine administered before 6 months. Emerging evidence is suggesting that the vaccine may safely be given to infants with normal immune function, even if serum biologic level is detectable. Specialist assessment of infant immune function may help guide decision-making about rotavirus vaccine administration, but there remains a large gap in our knowledge of the impact of biologic exposure on the immune function of infants, necessitating further study. Purpose In this single-centre retrospective chart review, we aim to study the clinical outcomes, immune function, and rotavirus vaccine recommendations for infants exposed to biologics in utero during their first year of life. Method The study included mothers seen at the Pregnancy IBD Clinic at Mount Sinai Hospital in Toronto, ON, who were offered a referral to the Special Immunization Clinic (SIC) at The Hospital for Sick Children due to biologic exposure in utero. Data was collected on the recommendations made by paediatric specialists at SIC, based on complete blood count, lymphocyte phenotyping, and T-cell receptor excision analysis. Data was obtained on adverse neonatal outcomes in the first year of life, including prematurity, congenital malformations, and infections based on post-partum surveys completed by mothers and follow-up letters from SIC. Result(s) 43 patients were referred to and seen by paediatric specialists at SIC between 2 to 12 months of age. 18 infants (42%) were exposed to Adalimumab in utero, 16 (37%) to Infliximab, 5 (12%) to Vedolizumab, and 4 (9%) to Ustekinumab. The rotavirus vaccine was recommended to 34 infants (81%) and not recommended to 3 (7%) for reasons including gastrointestinal illness, neutropenia, and low lymphocyte counts. Recommendation is pending for 6 infants (14%). Two infants (5%) had premature births. Four infants (9%) were admitted to the NICU for reasons including respiratory distress, and prematurity (9%). One infant (2%) had a congenital malformation, specifically bilateral sensorineural hearing loss. Six infants (14%) had upper respiratory tract infections, none of which required hospital admission or antibiotics. Conclusion(s) This study is currently in progress. Further data is required to assess whether biologic exposure in utero has a significant impact on neonatal immune function, especially beyond the first year of life, and whether there is a significant difference in vaccine recommendations and response based on the type of biologic received. Disclosure of Interest None Declared

Cell-mediated and humoral immunity during COVID-19 in the Republic of Crimea

The COVID-19 (coronavirus disease 2019) pandemic has spurred the development of highly effective quantitative methods for assessing the adaptive immune response to the SARS-CoV-2 (severe acute respiratory syndrome-related coronavirus 2) virus. In order to assess the humoral component of the immune response, various methods for detecting immunoglobulins A, M, G are widely used. ELISPOT seems to be the most accessible and effective method to assess the level of T cells that specifically respond to the SARS-CoV-2 virus antigens.The aim. To assess cell-mediated and humoral immunity in COVID-19 in residents of the Republic of Crimea.Methods. The study was performed on 24 volunteers: the presence of coronavirus antibodies was determined by ELISA method, and the presence of contact with coronavirus proteins – by the ELISPOT “TigraTest® SARS-CoV-2” method (Generium, Russia). For retrospective study of humoral immunity in the population, we assessed 10 000 ELISA tests (ECOlab IgM and IgG, Russia) performed in our laboratory for the period from July 2020 to January 2022.Results. The results show the effectiveness of using the ELISPOT method to detect latent forms of coronavirus infection. It is important to note that there is statistically significant relationship between the timing of the disease and the number of spots in both antigen panels. After vaccination against SARS-CoV-2, cell-mediated immunity lasts up to 6 months or more.Conclusions. As a result of the study, it was found that during 2021, the level of immunization of the population of the Republic of Crimea against COVID-19 has significantly increased; the proportion of residents who have positive IgG test has increased from 27 % to 87 %. The results of ELISPOT studies using a set of reagents for in vitro detection of blood T-lymphocytes that specifically respond to SARS-COV-2 virus antigens (“TigraTest® SARS-CoV-2”) showed that this method is more sensitive than ELISA in detecting latent diseases.

Dependence of the Translocation Frequency in Blood Lymphocytes on the Dose and Age at the Onset of Exposure in Residents of the Techa Riverside Settlements

Evaluation of age effect on the frequency of radiation-induced translocations, registered using FISH in circulating T-lymphocytes in the long-term period after exposure, is both of theoretical and practical interest for the purposes of biodosimetry. The objective of our study was to analyze the dose dependence of the translocation frequency in the peripheral blood T-lymphocytes in donors of different age who were exposed in the Techa Riverside settlements (n = 197). In cytogenetic studies, whole chromosome painting probes were used to stain three pairs of chromosomes. A total of 104,721 genome equivalents (GE) were calculated and 2,540 translocations were found. For each donor, the individual absorbed doses in organs and tissues at the time of blood sampling were calculated using the Techa River Dosimetry System. In addition, doses to T-lymphocytes and their progenitors were calculated using the innovative modelling approach with due account of age related-dynamics of T-lymphocytes. The age dependence of the translocation frequency was associated particularly with these doses. The main sources of donor exposure were 89,90Sr, accumulating in bones and irradiating the bone marrow almost locally. To assess the parameters of the dose-effect relationship, linear regression model was used. After taking into account background values, the lowest frequency of translocations per 1000 GE per Gy was found in donors aged 0–5 years at the time of exposure (9.3 ± 1.3), which is statistically significantly lower than in children aged 6–18 years (15.3 ± 1.5), but not in adults (11.9 ± 2.9). The value for adults (18 years) was characterized by the maximum scatter, but was close to the values obtained in an international study of nuclear enterprise personnel after external exposure (11.6 ± 1.6). The values of the background translocation frequencies registered in various age groups correspond to the published data obtained in a joint international study on unexposed donors. We have also confirmed the absence of sex-effect on the frequency of translocations.

LOW DOSE OF ESMOLOL ATTENUATES SEPSIS-INDUCED IMMUNOSUPPRESSION VIA MODULATING T-LYMPHOCYTE APOPTOSIS AND DIFFERENTIATION.

Background: Immunosuppression caused by immune cell apoptosis and an imbalance of T helper 2 cells (T H 2) and T helper 1 cells (T H 1), is associated with poor outcomes in septic patients. Esmolol was reported to improve survival by modulating immune responses in septic shock. Whether esmolol could alleviate sepsis-induced immunosuppression and the optimal dose are unclear. Methods: Four hours after cecal ligation and puncture (CLP), Wistar rats were randomized into CLP, CLP + E-5 (esmolol: 5 mg·kg -1 ·h -1 ) and CLP + E-18 (esmolol: 18 mg·kg -1 ·h -1 ) groups. Eight rats were underwent sham operation. Eighteen hours after CLP, hemodynamics and organ histological injuries were evaluated, peripheral blood mononuclear cells apoptosis and T-lymphocyte subsets counts were determined by flow cytometry, and the expression of p-Akt, Bcl-2, cleaved Caspase-3, and p-Erk1/2 in splenic CD4 + T-lymphocytes was determined by western blot and immunohistochemistry. β 1 -Adrenoreceptor expressions were evaluated using real-time polymerase chain reaction and immunohistochemistry. Results: Cecal ligation and puncture induced tachycardia, hypotension, hyperlactatemia, and multiple organ injury. Heart rate was unchanged in the CLP + E-5 group but decreased in the CLP + E-18 group. Hypotension, lactatemia, and multiple organ injuries were improved only in the CLP + E-5 group. T-lymphocyte apoptosis and T H 2/T H 1 ratio was decreased in CLP + E-5 but not in CLP + E-18. p-Akt and Bcl-2 expressions were increased, while cleaved Caspase-3 and p-Erk1/2 expressions were decreased in CLP + E-5. β 1 -Adrenoreceptor expressions were unchanged in both CLP + E-5 and CLP + E-18 groups. Conclusions: Low dose of esmolol reduced T-lymphocyte apoptosis and restored T H 2/T H 1 ratio in septic shock. Esmolol might modulate Akt/Bcl-2/Caspase-3 pathway to relieve T-lymphocyte apoptosis and inhibit Erk1/2 activity to decrease T H 0 differentiation to T H 2. Esmolol may be a potential immunoregulator of septic shock.

Study of the action of peripheral blood T-lymphocytes on renal cell carcinoma cells in model systems

Background. The introduction of immunotherapy based on immune checkpoint inhibitors has significantly improve the effectiveness of kidney cancer treatment. Nevertheless, not all patients respond to such treatment and there are no reliable predictive markers. Therefore, the development of a model system for assessing the cellular immune response to a tumor seems to be an urgent task.Aim. Development of a model to assess the T-cell immune response was the focus of this study.Materials and methods. Primary tumor cell culture and peripheral blood T-cell fraction were obtained under standard sterile conditions. T-cell activation were perform via anti-CD3 and anti-CD28 antibodies. The cell index was assessed using the RTCA xCELLigence biosensor technology (ACEA Biosciences, USA).Results. Tumor and T-cells from the same patient were cultured together to assess the growth rate of the tumor cell population. Measurements were taken at 30-minute intervals. The duration of observation was 24 hours. It has been shown that non-activated T-cells do not affect the proliferative properties of cultured cancer cells. On the contrary, activated T-cells suppressed the proliferative properties of cancer cells, which was associated with an increase in the proportion of Т-cells carrying the HLA-DR receptor (CD3+HLA–DR+) because of activation. Tumor-specific T-cell activity can lead to three consequences: lack of effect, partial suppression of proliferative properties, and complete death of tumor cells. In the latter case, the absence of such cells was determined by flow cytometry.Conclusion. The developed approach makes it possible to evaluate the cytotoxic properties of T cells in relation to tumor cells in a particular patient. The advantage of this method is that the measurement can be carried out in the presence of immune checkpoint inhibitors. The proposed method may be useful for evaluating the treatment regimen within the framework of personalized therapy.

Avelumab in Men With Metastatic Castration-Resistant Prostate Cancer, Enriched for Patients Treated Previously With a Therapeutic Cancer Vaccine.

Therapeutic cancer vaccines including sipuleucel- T , a prostatic acid phosphatase (PAP) targeted vaccine that improves survival in metastatic castration-resistant prostate cancer (mCRPC), can produce immune responses that translate to clinical benefit. The effects of sequential checkpoint inhibitors after therapeutic vaccine on immune responses are unknown. Avelumab is an anti-programmed death ligand-1 monoclonal antibody evaluated in patients with mCRPC in the JAVELIN solid tumor phase 1 trial expansion cohort, enriched for patients with a previous therapeutic prostate cancer-targeted vaccine. mCRPC patients received intravenous avelumab 10 mg/kg every 2 weeks with imaging every 6 weeks. Peripheral blood T-cell responses to PAP and to PA2024, the peptide containing PAP utilized by the vaccine, were evaluated pre and posttreatment. Eighteen patients enrolled, and previous treatments included abiraterone or enzalutamide in 14 (78%), therapeutic cancer vaccine in 14 (78%), and chemotherapy in 4 (22%). Avelumab had a manageable safety profile. There were no sustained prostate specific antigen decreases. Of 17 patients evaluable for best overall response by RECISTv1.1, 12 had stable disease (SD) and 5 had progressive disease. Seven patients had SD for >24 weeks posttreatment. Fourteen patients had previously received therapeutic cancer vaccines. Eleven (79%) had SD as the best overall response. Of these 14 patients, 9 had previously received sipuleucel T . Analysis of antigen-specific T-cell responses pre and postavelumab treatment did not demonstrate changes in interferon-γ production or proliferation in response to PAP or PA2024. This unplanned analysis does not support the use of sequential therapeutic cancer vaccine therapy followed by programmed death ligand-1 inhibition in mCRPC.

Evaluation of T-lymphocytes in Peripheral Blood of Diabetic Patients

Our study includes the estimation of absolute number of T-lymphocytes by using (E-rosette) test in diabetic patients with hyperglycemia. The study included the collection of (20 )blood samples from individuals Type I ,II diabetes mellitus, ten of them were of type one I others of type II, with age range between (8-55)years and of both sexes. Blood samples from healthy individuals as control samples were used as well. The study showed a significant decrease in absolute numbers of T-lymphocytes in diabetic patients when compared with control and also a significant reduction in the absolute number in type I and type II diabetic patients compared with control, our result suggest that there is a defect in T-lymphocytes numbers in diabetic patients with hyperglycemia.

Microenvironment-responsive anti-PD-L1 × CD3 bispecific T-cell engager for solid tumor immunotherapy

Bispecific T-cell Engager (BiTE) antibodies can redirect T-cells to tumor cells, and turn on the targeted lysis of tumor cells. However, BiTE has been challenging in solid tumors due to short plasma half-life, "off-target" effect, and immunosuppression via PD-1/PD-L1 axis. This study designed a safe, long-acting, and highly effective Protease-Activated PSTAGylated BiTE, named PAPB, which includes a shielding polypeptide domain (PSTAG), a protease-activated linker, and a BiTE core. The BiTE core consists of two scFvs targeting PD-L1 and CD3. BiTE core bound PD-L1 and CD3 in a dose-dependent manner, and PAPB can release BiTE core in response to MMP2 in the tumor microenvironment to exert antitumor activity. The plasma half-life of PAPB in mice was significantly prolonged from 2.46h to 6.34h of the BiTE core. In mice bearing melanoma (A375) xenografts, PAPB significantly increased infiltration of T lymphocytes in tumor tissue, and inhibited tumor proliferation without activating T-cells in the peripheral blood. Overall, the engineering protein PAPB could be a promising drug candidate for solid tumor immunotherapy.

Bovine blood and milk T-cell subsets in distinct states of activation and differentiation during subclinical Staphylococcus aureus mastitis

T-lymphocytes are key mediators of adaptive cellular immunity and knowledge about distinct subsets of these cells in healthy and infected mammary gland secretions remains limited. In this study, we used a multiplex cytometry panel to show that staphylococcal mastitis causes the activation of CD4+, CD8+ and γδ T-cells found in bovine milk. We also highlight remarkable differences in the proportions of naïve and memory T-cells subsets found in blood and milk. These observations will contribute to a better understanding of cell-mediated immune mechanisms in the udder and to the development of new therapeutic and preventive strategies targeting mastitis.

T-Cell Monoclonality in the Blood and the Skin Correlates With Poor Response to Treatment in Mycosis Fungoides.

The prognostic value of skin and blood T-cell receptor clonality in mycosis fungoides is a matter of debate. Our aim was to ascertain the relation between the presence of a monoclonal T-cell population in the blood and in the skin with response to treatment in patients with mycosis fungoides. Clinical features and follow-up data were retrospectively collected and analyzed in 94 patients with mycosis fungoides seen at a cutaneous lymphoma clinic in a single tertiary center. All patients had results of polymerase chain reaction analysis of T-cell receptor gamma gene rearrangement in lesional skin and in peripheral blood at time of diagnosis. Association of response to treatment with clonality in the tissue and in the blood was assessed. T-cell monoclonality was detected in the skin in 30 of 94 patients, in the blood in 12 of 94 cases and the same clone was found in both tissues in 6 of 94 patients. The presence of a polyclonal T-cell population in the circulation was associated with complete response (P=.006). Lack of response to treatment (stable disease or progression of disease) was associated with T-cell clonality in skin (P=.009), in blood (P=.002) and in both tissues (P < .001). A multivariate analysis showed that T-cell monoclonality in the skin is independently associated with lack of response of mycosis fungoides to therapy. Blood and skin should be studied for T-cell clonality as part of the routine initial workup, even in patients with early-stage disease.

Abstract PR004: Older adult-specific microbes correlate with treatment response and markers of T-cell senescence in NSCLC

Abstract The gut microbiome changes with age and affects many aspects of human health, including response to cancer treatments. Cancer survival rates have improved with new treatment options, including immune checkpoint blockade (ICB); however, the objective response rate remains low. Manipulation of the microbiome is a promising approach to improving cancer outcomes, but the effect of age is understudied. Here, we sought to understand whether (1) specific microbes are associated with treatment response in older adults with non-small cell lung cancer (NSCLC) and (2) whether these microbes are the same as for younger adults. Next, we explored the causal effects of the microbiome on ICB response in mouse models and the relationship with blood-based markers of T-cell senescence. We conducted a prospective cohort study of adults ≥60 years with a new diagnosis of NSCLC who received any treatment modality. Stool was collected, and metagenomic whole-genome shotgun sequencing was performed. Blood T-cells were isolated, the RNA purified and then assessed for markers of senescence by nanostring. Response to treatment was determined by RECIST v1.1 criteria. Generalized linear regression was used to relate baseline microbiome abundances to treatment response and non-parametric correlations associated with CDKN2A (p16) expression to microbe abundances. To assess the causal role of the gut microbiome in ICB response, we gavaged gut microbiome samples from responders and non-responders into C57BL/6 mice to create human-microbiome avatar models. The mice were then injected with MC38 cancer cells and treated with anti-PD1 or isotype control antibodies, and tumor volume was measured over time. Biospecimens and best response data at three months were captured from 23 patients, of which five had a complete response, eight had a partial response, eight had stable disease, and two had progressive disease. Over 50 microbes were associated with a response after p-value adjustment. Responder stool was enriched for microbes associated with youth and ICB response (Bifidobacterium adolescentis, p = 2.64e-20). However, microbial taxa associated with response differed from those reported in younger populations (Firmicutes sp. CAG 145, p = 1.58e-20, Oscillibacter sp. 57-20, p = 7.96e-24). Stool from non-responders (NRs) was enriched in taxa previously linked to treatment-related toxicities and shorter progression-free survival (Streptococcus lutetiensis, p = 4.55E-24) but also contained microbes previously linked to response in younger adults (e.g., Roseburia sp. CAG 309, p = 5.16e-15). The T cell senescence marker, p16, correlated with the most enriched taxon in non-responders NRs (Streptococcus thermophilus, r = 0.45, p = 0.02), suggesting a connection between immune aging and the microbiome. Preliminary fecal transplant studies in mice showed improved ICB response in mice engrafted with stool from responders versus non-responders. Together, these data identify potential differences in the gut microbiomes of young and older adult NSCLC patients who respond to ICB. Citation Format: Daniel Spakowicz, Rebecca Hoyd, Caroline E. Wheeler, Nyelia Williams, Amna Bibi, Marium Husain, Srichandhana Rajamouli, Shankar Suman, Joseph Amann, Madison Grogan, Pooja Vibhakar, Dwight H. Owen, David P. Carbone, Ashley Rosko, Christin E. Burd, Carolyn J. Presley. Older adult-specific microbes correlate with treatment response and markers of T-cell senescence in NSCLC [abstract]. In: Proceedings of the AACR Special Conference: Aging and Cancer; 2022 Nov 17-20; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_1):Abstract nr PR004.

Neo-adjuvant immunotherapy emerges as best medical practice, and will be the new standard of care for macroscopic stage III melanoma

Survival of macroscopic stage III melanoma is poor. Five-year overall survival and relapse-free survival rates for surgery alone range from 40 to 59% and 30 to–39%, respectively. The current standard of care is therapeutic lymph node dissection (TLND) followed by a year of adjuvant systemic therapy. Multiple phase 2 trials have shown that neo-adjuvant immunotherapy induces major pathologic response (MPR) rates (pathologic complete response (pCR): pCR, 0% viable tumour cells; near-pCR, <10% viable tumour cells), which translate into durable relapse free survival rates. Single agent anti-PD-1 achieves 20–30% MPR, the combination of ipilimumab and nivolumab doubles the MPR rates to 50–60%. The OpACIN trial demonstrated that neoadjuvant immunotherapy induced both higher numbers and a broader repertoire of tumour-resident T-cells in peripheral blood compared with adjuvant immunotherapy. Very recently, the randomised phase 2 trial S1801 reported its first interim results. S1801 compared TLND, followed by 18 courses of adjuvant pembrolizumab, to three courses of neoadjuvant pembrolizumab, followed by surgery and 15 adjuvant doses. With a median follow-up of 14 months, a 23% EFS rate benefit was observed. The ongoing phase 3 NADINA trial randomises patients between TLND + one year of adjuvant nivolumab (control arm) or 2 courses of neoadjuvant therapy with ipilimumab + nivolumab, followed by adjuvant therapy only for non-MPR patients. There is rapid, consistent, and accumulating evidence generated from all phase 1 and phase 2 trials, indicating clinical superiority of neoadjuvant immunotherapy over adjuvant systemic therapy for macroscopic stage III melanoma. Therefore, payers should consider neoadjuvant immunotherapy for reimbursement as this approach is the best option for our patients, and classify it as the best medical practice.

TGF-β2 antisense oligonucleotide enhances T-cell mediated anti-tumor activities by IL-2 via attenuation of fibrotic reaction in a humanized mouse model of pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers, with high mortality and recurrence rate. In this study, we generated a human immune system mouse model by transplanting human peripheral blood mononuclear cells into NSG-B2m mice followed by xenografting AsPC-1 cells, after which we assessed the role of transforming growth factor-β2 (TGF-β2) in T-cell-mediated anti-tumor immunity. We observed that inhibiting the TGF-β2 production by TGF-β2 antisense oligonucleotide (TASO) combined with IL-2 delays pancreatic cancer growth. Co-treatment of TASO and IL-2 had little effect on the SMAD-dependent pathway, but significantly inhibited the Akt phosphorylation and sequentially activated GSK-3β. Activation of GSK-3β by TASO subsequently suppressed β-catenin and α-SMA expression and resulted in attenuated fibrotic reactions, facilitating the infiltration of CD8 + cytotoxic T lymphocytes (CTLs) into the tumor. TGF-β2 inhibition suppressed the Foxp3 + regulatory T-cells in peripheral blood and tumors, thereby enhancing the tumoricidal effects of CTLs associated with increased granzyme B and cleaved caspase-3. Moreover, changes in the T-cell composition in peripheral blood and at the tumor site by TASO and IL-2 induced the increase of pro-inflammatory cytokines such as IFN-γ and TNF-α and the decrease of anti-inflammatory cytokines such as TGF-βs. These results indicate that the TGF-β2 inhibition by TASO combined with IL-2 enhances the T-cell mediated anti-tumor immunity against SMAD4-mutated PDAC by modulating the tumor-associated fibrosis, suggesting that TASO in combination with IL-2 may be a promising immunotherapeutic intervention for PDAC.

Conditioned medium from human cord blood mesenchymal stem cells attenuates age-related immune dysfunctions.

Aging is accompanied with progressive deterioration of immune responses and tissue's function. Using 12-month-old mice as model, we showed that conditioned medium of human cord blood mesenchymal stem cells (CBMSC-CM) significantly reduced the population percentage of CD3-CD335+ NK and CD4+CD25+ regulatory T-cells in peripheral blood. The CBMSC-CM administration also increased naïve T-cells number and restored the ratio of naïve to memory T-cells in CD4+ T-cells population. These results indicated that CBMSC-CM improved the immune response efficiency of aged mice. Moreover, we also found CBMSC-CM treatment significantly reduced the number of senescenT-cells in kidney tissues. Finally, we demonstrated that CBMSC-CM remarkably attenuated hydrogen peroxide triggered T-cell response and ameliorated oxidative stress induced cellular senescence. All of these data suggest a prominent anti-aging effect of secretome of CBMSCs.

Effect of budesonide and azithromycin on the chemotaxis of blood monocytes and lymphocytes in patients with chronic obstructive pulmonary disease

Objective. To evaluate the ability of a combination of budesonide and azithromycin to influence the migration of blood monocytes and lymphocytes in patients with chronic obstructive pulmonary disease (COPD).Materials and methods. Peripheral blood mononuclear cells from patients with COPD (n=8) were incubated with glucocorticoid budesonide (10 nM), macrolide antibiotic azithromycin (10 µg/mL), or their combination, and then transferred to chemotaxis chambers containing chemokines RANTES (CCL5, 10 nM) or IP-10 (CXCL10, 10 nM). Cells migrated to the lower compartment of the chamber were collected, stained with monoclonal antibodies to CD3, CD14, CD19, CD45 and counted on a flow cytometer.Results. Azithromycin alone and in combination with budesonide inhibited the migration of blood T-lymphocytes and B-cells and enhanced the migration of blood monocytes to RANTES and IP-10. The combination of azithromycin and budesonide had a more suppressive effect on the chemotaxis of blood T- and B-lymphocytes to RANTES and IP-10 than budesonide alone. The combination of azithromycin and budesonide had an effect similar to azithromycin alone on the migration of blood T- and B-lymphocytes, as well as monocytes in patients with COPD.Conclusion. The results of the study demonstrate the ability of azithromycin alone to modulate the chemotaxis of peripheral blood monocytes and lymphocytes in patients with COPD and the lack of advantages of its combination with budesonide.