T-cell Depletion Research Articles

Low Risk of Prolonged SARS-CoV-2 Shedding and Molecular Evolution in Kidney Transplant Recipients during the Omicron Era: A Prospective Observational Study

The aim of this prospective study was to assess the duration of culture-viable SARS-CoV-2 and to monitor the emergence of mutations in a cohort of 23 kidney transplant recipients (KTRs) from June 2022 to June 2023. Combined nares/oropharyngeal swabs were performed weekly starting as soon as possible after symptom onset. The time from symptom onset to a negative culture was (11 days (IQR 8-14), while time to negative RT-qPCR was 18 days (IQR 15-30). 21.7% had a positive culture beyond the first swab and 8.7% replicated viable virus for longer than 30 days. T-cell depletion (rate ratio 2.5, 95% CI 1.9, 3.3; p<0.001) and time from transplantation (rate ratio 0.93, 95% CI 0.90, 0.97; p=0.006) were associated with time of viable virus shedding. A cycle threshold (Ct) value of 24.2 demonstrated a 91.3% negative predictive value of viability (95% CI 76, 100). The odds of viability decreased by 69% per week of infection (OR 0.31, 95% CI 0.12, 0.76). Overall, RNA sequencing did not show accelerated molecular evolution though mutation rate could be increased in molnupiravir-treated KTRs. In conclusion, viable SARS-CoV-2 is eliminated rapidly, risk of virus evolution is low, and prolonged self-isolation is generally unnecessary for most KTRs.

COVID-19 Pneumonia Diagnosed by Bronchoalveolar Lavage Fluid, With CD4 T-cell Depletion Contributing to Prolonged Infection: Two Case Reports

COVID-19 Pneumonia Diagnosed by Bronchoalveolar Lavage Fluid, With CD4 T-cell Depletion Contributing to Prolonged Infection: Two Case Reports

Dual inhibition of HERs and PD-1 counteract resistance in KRASG12C-mutant head and neck cancer

BackgroundBasket clinical trials targeting the KRASG12C-mutation in solid tumors have shown initial promise, including in orphan KRASG12C head and neck cancer (HNC). However, development of resistance to KRASG12C-mutant-specific inhibitors (KRASG12Ci) remains a major obstacle. Here, we investigated the intrinsic (tumor-cell autonomus) and tumor-microenvironment (TME) mechanisms of resistance to the KRASG12Ci—MRTX849 and AMG510 in a unique syngenic murine KRASG12C-mutated HNC cell line.MethodsWestern-blotting was used for protein abundance and activation, overexpression, and ligand activation studies to verify the intrinsic mechanism of resistance to KRASG12Ci in KRASG12C-mutated HNC cell line, 4NQO-L. In vitro KRASG12C-acquired-resistant cells were developed from 4NQO-L (4NQO-L-AcR). MRTX849/lapatinib combination efficacy, and CD8+ T-cells depletion, were assessed in C57BL/6 J mice and supplementation of anti-PD-1 (αPD-1) to MRTX849/lapatinib was also performed in 4NQO-L– KRASG12Ci-senisitve and 4NQO-L-AcR tumors. Immunohistochemistry (IHC) and Immunoflourescence (IF) analyses were performed to profile the TME and programmed death-ligand 1 (PD-L1) expression in tumors.ResultsActivation and upregulation of EGFR and HER2/3 (pan-HERs) are the intrinsic mechanism of resistance to KRASG12Ci in 4NQO-L cells, and blocking pan-HERs signaling with lapatinib enhanced MRTX849 efficacy in vitro by inhibiting the MAPK and AKT/mTOR pathways. 4NQO-L-AcR upregulated the expression of pan-HERs, and lapatinib treatment re-sensitized 4NQO-L-AcR to MRTX849. In mice, MRTX849 showed a slight anti-tumor effect, but in combination with lapatinib a significant tumor growth delay was observed, but all tumors progressed over time. Histopathology analysis of the TME revealed infiltration of CD8+ T-cells after treatment combination, and these CD8+ T-cells play a key role in MRTX849/lapatinib efficacy. MRTX849/lapatinib treatment upregulated PD-L1 overexpression in both stromal and tumor cells, which presumably suppressed CD8+ T-cells and enabled immune escape and tumor progression. Supplementation of αPD-1 prolonged the progression-free survival of 4NQO-L-bearing mice treated with MRTX849/lapatinib. MRTX849/lapatinib treatment delayed tumor growth of 4NQO-L-AcR in mice; however, the percentages of CD8+ T-cells in 4NQO-L-AcR were low, and supplementation of MRTX849/lapatinib with αPD-1 did not improve the outcome.ConclusionsOur study highlights the critical need for blocking both intrinsic and extrinsic mechanisms of resistance for the prolonged response and shows that such treatment is ineffective in KRASG12Ci-AcR tumors.

Abstract A003: The role of aging in promoting immune mediated reactivation from metastatic melanoma dormancy

Abstract Metastasis is the primary cause of melanoma death. A major problem with the vast majority of previous studies is that young mice (8-weeks old, equivalent to 20 human years) are used to investigate the mechanisms of metastatic outgrowth, whereas the median age of melanoma diagnosis in humans is ∼65 years. Advanced age also correlates with metastasis, mortality, and immune dysfunction. Moreover, most studies often only focus on lung metastases, despite melanoma commonly metastasizing to sites such as the liver in patients, which are significantly more resistant to checkpoint inhibitors (ICIs) compared to patients with lung metastases. We have begun addressing these core limitations by developing novel syngeneic mouse models of melanoma metastasis and colonization in young (8-week), middle-aged (12-month), and geriatric (18-22 month) mice, in which metastatic colonization/dissemination can be targeted to either the lung or liver alone, or to both simultaneously. Our data now show that aging promotes reactivation of dormant tumor cells and subsequent outgrowth of lung and liver metastases. We have found that frontline innate lymphocytes such as NK-cells and γδ T-cells are decreased in the pre-metastatic lung and livers of aged mice relative to young mice and remain decreased during early and late-stage metastasis. Using inducible KO mouse models and antibody depletion methods in young growth restrictive mice, we find that depletion of either NK-cells or γδ T-cells is sufficient to promote reactivation from dormancy and resistance to anti-PD1 therapy. Secondly, we further delineated changes in the post-metastatic lung and liver niche. We find that PROS1 secretion by reactivated melanoma cells expressing high levels of the MER tyrosine kinase receptor (MER) in the aged metastatic lung and liver increases CD11b myeloid cells expressing the immunosuppressive factor ARG1. Within this CD11b subset, we see an increase in polymorphonuclear myeloid derived suppressor cells (PMN MDSCs). Treatment with recombinant ARG1 promotes reactivation from dormancy in young mice whilst depletion of PMN MDSCs inhibits age induced outgrowth in the lung and liver. Overall, our study highlights common immune-mediated age-related changes in the pre and post metastatic lung and liver which contribute to aggressive metastatic initiation, reactivation from dormancy, progression, and resistance to anti-PD1 Citation Format: Kelly Coutant, Jhon Passamonte, Christopher Price, Pulkit Datt, Mitchell Fane. The role of aging in promoting immune mediated reactivation from metastatic melanoma dormancy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr A003.

Herbacetin ameliorates lipopolysaccharide-elicited inflammatory response by suppressing NLRP-3/AIM-2 inflammasome activation, PI3K/Akt/MAPKs/NF-κB redox inflammatory signalling, modulating autophagy and macrophage polarization imbalance.

Herbacetin, a flavonol abundant in traditional medicines, is documented as an anti-inflammatory agent. However, information regarding its attributes on lipopolysaccharide (LPS)-induced inflammatory immunopathies has not been delineated yet. The present study aimed to comprehend herbacetin effects on LPS-induced aspects of unwarranted, non-resolving inflammation, particularly via targeting the vicious circle of oxi-inflammatory stress, autophagy-apoptosis, macrophages polarization, impaired inflammasome activation, and inflammatory cascades. In-vitro model of LPS-stimulated RAW 264.7 macrophage was recapitulated to investigate different inflammatory anomalies using enzyme-linked immunosorbent assay, qRT-PCR (Real-Time Quantitative Reverse Transcription PCR), immunoblotting. Concanavalin A challenged splenocytes and in silico studies were performed to measure Tregs population and binding affinity, respectively. Herbacetin administration caused remarkable reduction in nitric oxide, reactive oxygen species, mitochondrial membrane potential hyperpolarization, tumor necrosis factor-α, interferon-γ, interleukin-6, inducible nitric oxide synthase and ratio of M1/M2 markers (inducible nitric oxide synthase/arginase-1/macrophage scavenger receptor-1/mannose receptor C type-1) in in vitro model of persistent inflammation. Suppression of interleukins-5,17 and matrix metalloproteinases-2,3,9,13 and proliferating cell nuclear antigen, signifies its anti-inflammatory attributes. Noticeable decline in monodansylcadaverine-Lysotracker staining, caspase-6, and enhanced p62, B-cell lymphoma-2 expression indicates apoptosis-autophagosome accumulation inhibition and lysosomal destabilization. These were accompanied by reduced NLRP3 activation, caspase-1, AIM-2 expression, and interleukin-1β release. Subsequently, up-regulated activation of TLR-4, NF-κB, PI3K, Akt, ERK1/2, and JNK was decisively thwarted by herbacetin. In silico investigation signified the interaction of herbacetin with these targets. Decreased cytokines and enhanced Tregs conferred its role in extenuating inflammation facilitated by T-cells depletion. Collectively, these findings comprehend attributes of herbacetin as an alternative therapeutic strategy in relieving LPS-associated chronic inflammatory disorders.

α9-Containing Nicotinic Acetylcholine Receptors Are Required for RgIA-5474 Attenuation of Chemotherapy-Induced Neuropathic Pain.

Nicotinic acetylcholine receptors containing the α9 subunit have been mechanistically implicated in alleviating chemotherapy-induced neuropathic pain. However, the cell types that underlie these effects are currently unknown. RgIA-5474 is a recently developed, synthetic α-conotoxin analog that is a potent antagonist of human α9α10 nAChRs. We used germline α9 subunit knockout mice, CD3+ T-cell depletion, and conditional knockdown of the α9 subunit in immune cells to examine the role of α9-containing nAChRs that mediate RgIA-5474 alleviation of oxaliplatin-induced neuropathic pain. RgIA-5474 potently and selectively blocked mouse α9α10 nAChRs. A one-time oxaliplatin injection resulted in cold allodynia that was reversed by RgIA-5474 administration in the wild type but not in α9 germline knockout mice. RgIA-5474 also failed to produce analgesia in CD3+ T-cell-depleted male and female animals. Conditional knockdown of the α9 subunit in immune cells of mice by the CreloxP system also eliminated the therapeutic effects of RgIA-5474 in both male and female mice. These results indicate that the α9 nAChR subunit is necessary for the analgesic effects of RgIA-5474 and implicate α9-containing nAChRs in immune cells as a nonopioid target for treating neuropathic pain.

Impact of Systemic Antibiotics Use on Outcomes after Allogeneic Hematopoietic Stem Cell Transplantation

Introduction: Post-transplant complications lead to significant morbidity and mortality in the management of patients undergoing allogeneic hematopoietic stem cell transplant (allo-HCT). Broad-spectrum systemic antimicrobials and better prophylaxis strategies have reduced infection-related morbidity and mortality in allo-HCT patients. However, broad-spectrum antibiotic use disrupts the microbiome and immunome, which may result in poor post-transplant outcomes. We aimed to evaluate the impact of systemic antibiotics on allo-HCT outcomes. Methods: A retrospective multicenter analysis was conducted, including allo-HCT patients in the publicly available Center for International Blood and Marrow Transplant (CIBMTR) registry from 2013 to 2018 using P5646 data by Ramathan et al. Chi-square and t-tests were used to compare categorical and continuous baseline demographics. Multivariate Cox regression analyses were used to calculate hazard ratios (HR) with 95% confidence intervals (CI) for overall survival (OS), disease-free survival (DFS), relapse, non-relapse mortality (NRM), acute graft-versus-host disease (aGVHD) and chronic GVHD (cGVHD), engraftment, and infection-related mortality between the two groups. Statistical analysis was conducted using R version 4.16, with statistical significance defined as p &amp;lt;0.05. Results: Among 7524 allo-HCT patients, 5779 were administered broad-spectrum systemic antibiotics (SA) while 1745 were not given systemic antibiotics (NSA). The median age was 58.58 years (range: 2.03-82.67) in the SA group and 56.18 years (range: 2.00 - 77.86) in the NSA group, and 58% were men in both groups. Ethnicities were Caucasian (76%), Hispanic (8%), African American (6%), and others (10%). The primary disorders included acute myeloid leukemia (47%), myelodysplastic syndrome (35%), and acute lymphoblastic leukemia (18%). Myeloablative conditioning was used in 53% of patients. Donor types were matched unrelated (49.5%), matched related (34%), and cord blood (16%). Graft sources were bone marrow (15%), peripheral blood stem cells (68%), and cord blood (16%). GVHD prophylaxis regimens included Tacrolimus and Cyclosporine-based (77%), post-transplant Cyclophosphamide-based (9%), Ex vivo T-cell depletion or CD34 selection (3%), and others (11%). In the multivariate regression analyses, systemic antibiotics independently predicted poor OS (55% SA vs 57% NSA; HR 1.13, 95% CI 1.01-1.22, p=0.026). Higher incidence of NRM (19% SA vs 15% NSA; HR 1.32, 95% CI 1.12-1.55, p=0.001), grade 2-4 aGVHD (41% SA vs 34% NSA; HR 1.24, 95% CI 1.12-1.38, p&amp;lt;0.001), and cGVHD (40% SA vs 39% NSA; HR 1.12, 95% CI 1.01-1.24, p=0.040) were observed with SA use. The two groups had no significant difference in DFS, relapse, infection-related mortality, and engraftment rates. The outcomes analyses were adjusted for the significant predictors identified in the univariate analyses. Conclusion: Systemic antibiotics use in allo-HCT patients is associated with higher morbidity and mortality. Judicious use and de-escalation of systemic antibiotics when safe may help to improve post-transplant outcomes. Future research exploring microbiome restoration could result in better post-transplant outcomes.

A phase 1 study of interleukin-15 in combination with mogamulizumab in relapsed and refractory T-cell malignancies

IntroductionRecombinant human interleukin-15 (rhIL-15) is an immunotherapeutic which enhances NK-cells to augment the antibody directed cellular cytotoxicity (ADCC) of monoclonal antibodies. Mogamulizumab is a CCR4 directed monoclonal antibodies that exerts cytotoxicity through ADCC and depletes regulatory T-cells within the tumor microenvironment. MethodsWe conducted a phase 1 clinical trial of rhIL-15 combined with mogamulizumab. Patients with relapsed or refractory adult T-cell leukemia/lymphoma (ATLL), mycosis fungoides (MF) and Sezary syndrome (SS) received fixed dose mogamulizumab combined with escalating doses of rhIL-15 to identify the maximum tolerated dose (MTD). ResultsSix patients were enrolled, 4 with ATLL and 2 with MF/SS. The most common adverse events were rash, infection and fever (67%, in all). Two patients (33%) had grade 4 acute kidney injury and 25% of cycles had grade ≥3 anemia. The MTD was dose level 1. One patient with ATLL had a partial response despite receiving only 4 cycles due to grade 4 myositis. Circulating NK cells were increased in all patients during the first cycle and a rapid reduction in tumor cells within the peripheral circulation was noted. Ex vivo assessment demonstrated increased NK cell activation and increased cell lysis in the presence of monoclonal antibodies after only 5 days. DiscussionOur small study suggests that rhIL-15 combined with mogamulizumab leads to effector NK cell activation and regulatory T-cell depletion but has an unfavorable safety profile. Future development of combinations of immunotherapy that target the microenvironment in relapsed or refractory T-cell lymphomas remain rational. NCT04185220

Post-transplant cyclophosphamide separates graft-versus host disease and graft versus leukemia effects after HLA-matched stem-cell transplantation for acute myeloid leukemia.

The association of graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL) effects after allogeneic stem-cell transplantation (SCT) is well-established but was not confirmed in the modern era and following post-transplant cyclophosphamide (PTCy). We assessed GVHD/ GVL association in AML patients following HLA-matched SCT with standard calcineurin-based (n = 12,653, 57% with additional in-vivo T-cell depletion) or PTCy-based (n = 508) GVHD prophylaxis. Following standard prophylaxis, acute GVHD grade II-IV and III-IV, chronic GVHD, and extensive chronic GVHD rates were 23.8%, 7.5%, 37.0%, and 16.3%, respectively. Acute GVHD grade II and III-IV were associated with lower relapse [hazard-ratio (HR) 0.85, P = 0.002; HR 0.76, P = 0.003, respectively)], higher non-relapse mortality (NRM) (HR 1.5, P < 0.001; HR 6.21, P < 0.001) and lower overall survival (OS) (HR 1.49, P < 0.001; HR 6.1, P < 0.001). Extensive chronic GVHD predicted lower relapse (HR 0.69, P < 0.001), higher NRM (HR 2.83, P < 0.001), and lower OS (HR 2.74, P < 0.001). Following PTCy, GVHD rates were 22.8%, 6.2%, 35.5%, and 17.7%, respectively. Acute GVHD was not associated with relapse (HR 1.37, P = 0.15) but predicted higher NRM (HR 3.34, P < 0.001) and lower OS (HR 1.92, P = 0.001). Chronic GVHD was not prognostic for these outcomes. In conclusion, GVHD and GVL are strongly associated with contemporary SCT. However, following PTCy, GVHD is not associated with reduced relapse.

Antiviral immune response against HTLV-1 invalidates T-SPOT.TB® results in patients with HTLV-1-positive rheumatic diseases.

T-SPOT.TB®, one of the screening tests for latent tuberculosis infection (LTBI), yields invalid results in human T-cell leukemia virus type 1 (HTLV-1)-positive patients with rheumatoid arthritis. However, the detailed mechanisms behind this invalidation are unclear. Additionally, it remains unclear whether T-SPOT.TB® or QuantiFERON-TB (QFT) is more useful in HTLV-1-positive patients with rheumatic disease (RD). Among all of the HTLV-1-positive RD patients who visited our department between August 2012 and December 2022, 44 patients who were screened using T-SPOT.TB® were included in the analysis. QFT testing was performed in 33 of the 44 patients, and the results were compared with that of T-SPOT.TB®. Furthermore, we performed a culture experiment mimicking T-SPOT.TB® using peripheral blood mononuclear cells (PBMCs) obtained from HTLV-1-positive patients with RD. Additionally, T-cell subsets with autonomous product IFN-γ were analyzed using a flow cytometer. Of the included patients, 13 (29.5%) were invalid for T-SPOT.TB® because of the increased number of negative control spots. The median HTLV-1 proviral load in the invalid group was higher than that in the valid group (2.45 vs. 0.49 copies/100 PBMCs, respectively, p = 0.002). QFT was performed in all 33 patients, including 13 patients who were invalid in T-SPOT.TB®. The main source of IFN-γ production was CD8+ T-cells in the T-SPOT.TB® mimic experiment. Furthermore, Tax-expressing CD4+ T-cells and Tax-specific cytotoxic CD8+ T-cells were more frequently observed in patients with invalid results than in patients with valid results. CD4+ T-cell depletion in the T-SPOT.TB® mimic experiment reduced the population of IFN-γ producing CD8+ T cells. T-SPOT.TB® may be invalidated by the interaction between Tax-expressing CD4+ T-cells and cytotoxic CD8+ T-cells. Moreover, HTLV-1-associated immune reactions due to contact between these cells may be unlikely to occur in QFT using whole blood. Therefore, our results reveal the superiority of QFT over T-SPOT.TB® as a screening test for LTBI in HTLV-1-positive patients with RD.

Hematological Cancer and Viral Infection

Hematological cancer patients are particularly vulnerable to the morbidity and death caused by viral infections. But, it is not well known how common viral infections are or what effects they have on patients undergoing traditional nontransplant treatment. How severe and how long T-cell-mediated immune suppression is determines the variation in viral infection incidence and prognosis between patient groups. Topics covered in this mini-review article include late CMV infection, new viral pathogens (human herpesvirus-6, BK virus, adenovirus, and human metapneumovirus), advancements in molecular diagnostics, and the possibility of novel agents for viral prophylaxis (maribavir) or preemptive therapy (valganciclovir). The infections caused by these viruses have been extensively studied. If we want to understand the range of these viral diseases and come up with effective ways to prevent and cure them, we need well-designed prospective trials. Patients undergoing nontransplant treatment for hematological malignancies are at greater risk for viral infections; this is especially important given the rising use of drugs like alemtuzumab, which cause significant T-cell depletion.

Targeted degradation of the HPV oncoprotein E6 reduces tumor burden in cervical cancer.

Human Papilloma Virus (HPV)-related cancers are a global health burden, yet there are no targeted therapies available for chronically infected patients. The HPV protein E6 is essential for HPV-mediated tumorigenesis and immune evasion, making it an attractive target for antiviral drug development. In this study, we developed an E6-targeting Proteolysis Targeting Chimera (PROTAC) that inhibits the growth of HPV(+) tumors. To develop E6 antagonists, we generated a panel of nanobodies targeting E6 proteins derived from the oncogenic HPV16 subtype. The highest affinity E6 nanobody, A5, was fused to Von Hippel Lindau protein (VHL) to generate a PROTAC that degrades E6 (PROTACE6). Mutational rescue experiments validated specific degradation via the CRL2VHL E3 ligase. Intralesional administration of the PROTACE6 using a clinically viable DNA vaccine reduced tumor burden in an immunocompetent mouse model of HPV(+) cancer. The inhibitory effect of the PROTACE6 was abrogated by CD4+ and CD8+ T-cell depletion, indicating that the antitumor function of the PROTACE6 relies in part on a host immune response. Overall, these results suggest that the targeted degradation of E6 inhibits its oncogenic function and stimulates a robust immune response against HPV(+) tumors, opening new opportunities for virus-specific therapies in the treatment of HPV-related cancers.

From Gut to Blood: Redistribution of Zonulin in People Living with HIV.

Gastrointestinal mucosal damage due to human immunodeficiency virus (HIV) infection leads to microbial translocation and immune activation, contributing to the development of non-infectious comorbidities (NICM) in people living with HIV (PLWH). Additionally, persistent proviral HIV-1 in the gut-associated lymphatic tissue (GALT) can trigger immunological changes in the epithelial environment, impacting the mucosal barrier. However, the role of zonulin, a modulator of epithelial tight junctions in GALT during HIV infection, remains poorly understood. We measured zonulin in serum and intestinal tissue sections from five treatment-naive (HIV+NAIVE) and 10 cART-treated (HIV+cART) HIV+ individuals, along with 11 controls (CTRL). We compared zonulin levels with clinical characteristics, inflammatory markers (IFN-α, CXCR3, and PD-1), and the viral reservoir in peripheral blood (PB) and terminal ileum (TI). Upon HIV infection, TI was found to harbor more HIV DNA than PB. Circulating zonulin levels were highest in HIV+NAIVE compared to HIV+cART or CTRL. Surprisingly, in the gut tissue sections, zonulin levels were higher in CTRL than in HIV+ individuals. Elevated circulating zonulin levels were found to be correlated with CD4+T-cell depletion in PB and TI, and with intestinal IFN-α. The findings of this study indicate a shift in zonulin levels from the gut to the bloodstream in response to HIV infection. Furthermore, elevated systemic zonulin levels are associated with the depletion of intestinal CD4+ T cells and increased gut inflammation, suggesting a potential link between systemic zonulin and intestinal damage. Gaining insight into the regulation of gut tight junctions during HIV infection could offer valuable understanding for preventing NICM in PLWH.

The unseen spread: a case of disseminated tuberculosis with renal manifestation in a healthy adult

Disseminated tuberculosis (TB), resulting from the hematogenous spread of tubercle bacilli, typically affects immunocompromised individuals, such as those infected with the human immunodeficiency virus. However, risk factors in immunocompetent populations are not well understood. Here, we report a rare case of disseminated TB with CD4+ T-cell depletion in a previously healthy 35-year-old man. The patient presented with a 2-month history of intermittent gross hematuria, dysuria, loose stools, and weight loss. His medical history was unremarkable except for a herpes zoster infection 4 years prior to presentation. Laboratory tests revealed microscopic hematuria and pyuria; however, the urine culture was negative. Urine specimens tested positive for TB-polymerase chain reaction. Abdominal computed tomography revealed a focal filling defect in the left kidney, segmental wall thickening of the terminal ileum, and multiple enlarged lymph nodes with central necrosis. Chest computed tomography revealed active pulmonary TB. Colonoscopy confirmed intestinal TB in the terminal ileum and ileocecal valve, with positive TB-polymerase chain reaction results from sputum and ileal ulcer tissue. The patient was diagnosed with disseminated TB and was treated with standard anti-TB drugs. Although the human immunodeficiency virus test results were negative, the patient’s CD4+ T-cell count was significantly low (278/μL). Follow-up tests after 1 month showed negative TB cultures; however, the patient’s CD4+ T-cell depletion persisted, with counts remaining low after 1 year. This case highlights the rare occurrence of disseminated TB in immunocompetent individuals with CD4+ T-cell depletion and emphasizes the importance of CD4+ T-cell assessment in healthy patients presenting with disseminated TB.

The CCR6-CCL20 Axis Promotes Regulatory T-cell Glycolysis and Immunosuppression in Tumors.

Regulatory T cells (Treg) are important players in the tumor microenvironment. However, the mechanisms behind their immunosuppressive effects are poorly understood. We found that CCR6-CCL20 activity in tumor-infiltrating Tregs is associated with greater glycolytic activity and ablation of Ccr6 reduced glycolysis and lactic acid production while increasing compensatory glutamine metabolism. Immunosuppressive activity toward CD8+ T cells was abrogated in Ccr6-/- Tregs due to reduction in activation-induced glycolysis. Furthermore, Ccr6-/- mice exhibited improved survival across multiple tumor models compared to wild-type mice and Treg and CD8+ T-cell depletion abrogated the improvement. In addition, Ccr6 ablation further promoted the efficacy of anti-PD-1 therapy in a preclinical glioma model. Follow-up knockdown of Ccl20 with siRNA also demonstrated improvement in antitumor efficacy. Our results unveil CCR6 as a marker and regulator of Treg-induced immunosuppression and identify approaches to target the metabolic determinants of Treg immunosuppressive activity.

Exploring the Impact of Meditation Therapy on CD4 Levels in HIV/AIDS Patients at Karawang Hospital

Background: The HIV/AIDS epidemic remains a significant global health concern, with increasing prevalence in Indonesia's Karawang Regency. People living with HIV/AIDS face numerous challenges, including a decline in immune function and CD4 T-cell depletion. While antiretroviral therapy (ART) is the primary treatment, there is a growing interest in complementary therapies to support immune function and overall well-being. Objective: This study aimed to investigate the effect of meditation therapy on CD4 levels in HIV/AIDS patients at Karawang Regional General Hospital. Methods: A quasiexperimental study with a prepost design was conducted on 15 HIV/AIDS patients with CD4 levels ranging from 200–500 cells/mm3. The participants engaged in meditation sessions twice a week, twice daily, for one month. CD4 levels were measured before and after the intervention. Demographic data were collected, and statistical analyses were performed via paired t tests (p&lt;0.05). Results: Most of the participants were male (80%), aged 31-37 years (46.7%), and had a vocational high school education (40%). Preintervention CD4 levels ranged from 226 to 459 cells/mm3 (mean: 334.87±57.583), whereas postintervention levels ranged from 220 to 467 cells/mm3 (mean: 340.93±62.721). A paired t test revealed no statistically significant effect of meditation therapy on CD4 levels (p=0.150). Conclusion: This study revealed no significant increase in CD4 levels following a one-month meditation intervention in HIV/AIDS patients. While this research does not support the use of short-term meditation therapy to increase CD4 levels in HIV/AIDS patients, it underscores the need for further investigation of complementary therapies for this population.

Evaluation of risk for bronchiolitis obliterans syndrome after allogeneic hematopoietic cell transplantation with myeloablative conditioning regimens

Bronchiolitis obliterans syndrome (BOS), as chronic manifestation of graft-versus-host disease (GVHD), is a debilitating complication leading to lung function deterioration in patients after allogeneic hematopoietic cell transplantation (allo-HCT). In the present study, we evaluated BOS development risk in patients after receiving myeloablative conditioning (MAC) regimens. We performed a retrospective analysis of patients undergoing allo-HCT, who received MAC with busulfan/cyclophosphamid (BuCy, n = 175) busulfan/fludarabin (FluBu4, n = 29) or thiotepa/busulfan/fludarabine (TBF MAC, n = 37). The prevalence of lung disease prior allo-HCT, smoking status, GvHD prophylaxis, HCT-CI score, EBMT risk score and GvHD incidence varied across the groups. The cumulative incidence of BOS using the NIH diagnosis consensus criteria at 2 years after allo-HCT was 8% in FluBu4, 23% in BuCy and 19% in TBF MAC (p = 0.07). In the multivariate analysis, we identified associated factors for time to BOS such as FEV1<median (99% of predicted) (HR = 2.39, p = 0.004), CMV patient serology positivity (HR = 2.11, p = 0.014), TLC < 80% of predicted (HR = 0.12, p = 0.02) and GvHD prophylaxis with in vivo T-cell depletion (HR = 0.29, p = 0.001) as predictors of BOS. In summary, we identified risk factors for BOS development in patients receiving MAC conditioning. These findings might serve to identify patients at risk, who might benefit from closely monitoring or early therapeutic interventions.

Abstract B043: Synergy of Subasumstat and anti-CD40 improves survival by augmenting tumor macrophage infiltration

Abstract Background: Identification of effective immunomodulatory strategies remains a major unmet need in pancreatic ductal adenocarcinoma (PDAC). Agonistic anti-CD40 antibody (aCD40) exhibits direct cytotoxicity on tumor cells and augments antigen presentation. Subasumstat(SUB), a sumoylation inhibitor, augments innate and adaptive immune responses by increasing interferon signaling. We hypothesized that SUB+aCD40 combination would promote an effective antitumor immune response in an aggressive orthotopic model of PDAC. Methods: 2000 KPC 46 cells were implanted in the pancreas of four groups of F1 hybrid (initial survival, CD8+ T-cell depletion, and clodronate liposome depletion) and nude mice, enrolled when tumors had reached 3-5mm. The vehicle (veh) group received 80ul SUB vehicle (q48 hrs) and 100 ug blank IgG (qwk); SUB only group received 15mg/kg q48h; aCD40 only group received 100ug qwk; SUB+aCD40 group received 15mg/kg SUB q48h and 100 ug aCD40 qwk. Single cell RNA sequencing (scRNASeq) was conducted on n=2 tumors from each group. Immunohistochemistry (IHC) was performed on veh and SUB+aCD40 tumors for macrophage infiltration using F4/80. Flow cytometry (FC) was employed for the presence of lymphoid and myeloid markers in veh, SUB+aCD40, and clodronate depleted SUB+aCD40 (SUB+aCD40+clod) groups. Results: Mice receiving SUB+aCD40 had a significantly improved median survival over those receiving monotherapy or vehicle (24.5d vs. SUB:15.5d, aCD40:20d, veh: 19d). scRNASeq revealed increased tumor infiltrating CD8+ T-cell and myeloid populations when comparing veh and monotherapy to SUB+aCD40. F4/80 IHC confirmed increased macrophage infiltration in SUB+aCD40 vs veh (p = 0.01). The survival advantage conferred by SUB+aCD40 was preserved in both CD8+ T-cell depleted mice (p=0.025) and nude mice (p=0.037), while clodronate eliminated the treatment survival advantage (p=0.39). FC showed increased tumor macrophage/monocyte infiltration in SUB+aCD40 vs veh (veh:11.9%, SUB+aCD40:22.5%, p=0.03) and reduction in SUB+aCD40+clod (12.6%, p=0.01). CD8+ T-cell CD69 expression decreased in the SUB+aCD40+clod group (SUB+aCD40:19%, SUB+aCD40+clod:6%, p&amp;lt; 0.01). CD8 T-cell infiltration (SUB+aCD40:4.95%, SUB+aCD40+clod:3.85%, p=0.27) and CD4/CD8 T-cell ratio (veh: 1.26, SUB+aCD40:0.52, SUB+aCD40+clod:0.69, p=0.09) were not affected by clodronate. Conclusion: SUB+aCD40 therapy confers a survival advantage in the KPC46 model of PDAC in a T-cell independent manner by increasing macrophage/monocyte infiltration. SUB+aCD40 promotes CD8+ T-cell infiltration and activation, but they do not contribute to survival advantage, possibly due to exhaustion. Future work will include addition of therapeutics to mitigate/reverse T-cell exhaustion to harness both innate and adaptive immunity in treating PDAC. Citation Format: Asimina Courelli, Kevin Li, James Lee, Herve Tiriac, Yuan Chen, Andrew Lowy. Synergy of Subasumstat and anti-CD40 improves survival by augmenting tumor macrophage infiltration [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B043.

Abstract A052: Silencing MICAL2 Expression in Pancreatic Cancer Cells rewires the tumor microenvironment through the IL1-a/p38 MAP kinase/STAT-3 axis and Sensitizes Tumors to Immune Checkpoint Blockade therapy

Abstract Introduction: PDAC is marked by a fibroinflammatory stroma and thrives on interactions between cancer cells and their microenvironment, including CAFs and immune cells, driving disease progression and treatment resistance. Using ChIP-seq on resected human PDAC samples, we identified MICAL2 as a super-enhancer-associated gene and its role in tumor progression. MICAL2 is a flavin monooxygenase that promotes actin depolymerization and SRF transcription. Here, we evaluated how tumor cell-expressed MICAL2 rewires the PDAC microenvironment through the IL1-a/p38 MAP kinase/STAT3 axis. Methods: KPC-Shcontrol (ShCNT) and MICAL2 (M2KD) were orthotopically injected to assess tumor growth. Sc-RNA seq, immunophenotyping, and immunohistochemistry were performed on ShCNT and M2KD tumors. Atomic force microscopy was done to assess tissue stiffness. CD8+ specific antibodies were used for T-cell depletion. Adoptive transfer of CD8+ T cells enriched from M2KD tumors was performed by tail vein injection. Anti-PD1 and anti-IL-1a antibodies were injected IP. RNA-Seq analysis was performed on human PDAC cells (AsPC-1 ShCNT and M2KD) and validation of IL-1α and IL-1R expression was done in human and mouse pancreas control, KD, OE cancer cell lines, and PSCs by q-PCR. We exposed PSCs to conditioned media from cancer cells with and without MICAL2 and checked the expression of genes related to inflammation and fibrosis by qPCR. Results: Orthotopic injections of KPC-M2KD cells led to decreased tumor growth compared to ShCNT (0.26 gm vs. 1 gm, p = 0.008). Sc-RNA and immunohistochemistry revealed reduced PDPN+ and a-SMA+ CAFs in M2KD tumors. We also observed less collagen and fibronectin deposition in M2KD tumors resulting in reduced tissue stiffness as measured by atomic force microscopy. Flow cytometry and immunofluorescence showed increased intertumoral infiltration of cytotoxic and effector CD8+T cells in M2KD tumors. CD8+T cell depletion reversed growth inhibition in M2KD tumors. Adoptive transfer of CD8+T cells enriched from M2KD tumors impeded control tumor growth. RNA-seq revealed decreased IL1-α expression in M2KD cells (p&amp;lt;0.05). IL1R expression was reduced on PSCs when cocultured with M2KD vs. control cell media. PSCs co-cultured with M2KD cancer cells also showed significant downregulation of genes including IL-6, Cxcl1, and LIF changes that were rescued by adding recombinant IL-1α in M2KD cells. Western blot showed MICAL2 regulates the phosphorylation of p38 MAP kinase which in turn regulates the expression of IL1-α and STAT3 activation confirmed by treating dox-inducible ShCNT and ShM2KD cancer cells with p38 inhibitor. Treating M2KD tumors with immune checkpoint blockade PD-1 alone significantly reduced tumor size and 50% of M2KD tumor-bearing mice had complete tumor regression after treatment with PD-1 and neutralizing IL-1a antibodies. Conclusion: MICAL2 mediates tumor-stromal crosstalk through the IL1-a/p38/STAT3 axis and creates an immunosuppressive environment in PDAC. MICAL2 may be a potential immunomodulatory target for pancreatic cancer therapy. Citation Format: Bharti Garg, Evangeline Mose, Edgar Esparaza, Jay Patel, Rithika Medari, Alexei Martsinkovskiy, Carrie Bishop, Gissele Gonzalez, Adam Engler, Parag Katira, Herve Tiriac, Andrew M Lowy. Silencing MICAL2 Expression in Pancreatic Cancer Cells rewires the tumor microenvironment through the IL1-a/p38 MAP kinase/STAT-3 axis and Sensitizes Tumors to Immune Checkpoint Blockade therapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A052.

Invasive cancer and spontaneous regression two weeks after papillomavirus infection.

AbstractDevelopment of invasive cancer in mammals is thought to require months or years after initial events such as mutation or viral infection. Rarely, invasive cancers regress spontaneously. We show that cancers can develop and regress on a timescale of weeks, not months or years. Invasive squamous cell carcinomas developed in normal adult, immune-competent mice as soon as 2 weeks after infection with mouse papillomavirus MmuPV1. Tumor development, regression or persistence was tissue- and strain-dependent. Cancers in infected mice developed rapidly at sites also prone to papillomavirus-induced tumors and cancers in humans – the throat, anus, and skin – and their frequency was increased in mice constitutively expressing the papillomavirus E5 oncogene, which MmuPV1 lacks. Cancers and dysplasia in the throat and anus regressed completely within 4-8 weeks of infection; however, skin lesions in the ear persisted. T-cell depletion in the mouse showed that regression of throat and anal tumors requires T cells. We conclude that papillomavirus infection suffices for rapid onset of invasive cancer, and persistence of lesions depends on factors including tissue type and host immunity. The speed of these events should promote rapid progress in the study of viral cancer development, persistence, and regression.Summary Graphic