Chronic Antigenic Stimulation Research Articles

Are immunosenescent T cells really senescent?

Loss of proper T-cell functioning is a feature of aging that increases the risk of developing chronic diseases. In aged individuals, highly differentiated T cells arise with a reduced expression of CD28 and CD27 and an increased expression of KLRG-1 or CD57. These cells are often referred to as immunosenescent T cells but may still be highly active and contribute to autoimmunity. Another population of T cells known as exhausted T cells arises after chronic antigen stimulation and loses its effector functions, leading to a failure to combat malignancies and viral infections. A process called cellular senescence also increases during aging, and targeting this process has proven to be fruitful against a range of age-related pathologies in animal models. Cellular senescence occurs in cells that are irreparably damaged, limiting their proliferation and typically leading to chronic secretion of pro-inflammatory factors. To develop therapies against pathologies caused by defective T-cell function, it is important to understand the differences and similarities between immunosenescence and cellular senescence. Here, we review the hallmarks of cellular senescence versus senescent and exhausted T cells and provide considerations for the development of specific therapies against age-related diseases.

Two cases demonstrate an association between Tropheryma whipplei and pulmonary marginal zone lymphoma

BackgroundMarginal zone lymphomas of mucosa-associated lymphatic tissues (MZL of MALT) are a group of indolent B-cell neoplasms, which are thought to arise from chronic antigenic stimulation of B-cells either due to underlying chronic infection or autoimmune disease. Little is known about potential causative pathogens in pulmonary MZL (PMZL), although some data suggests a potential role of Achromobacter (A.) xylosoxidans.MethodsAn index case of chronic pulmonary colonisation with Tropheryma (T.) whipplei and subsequent development of PMZL was identified by T. whipplei specific PCR and metagenomic next genome sequencing (mNGS). This case prompted a retrospectively conducted analysis of T. whipplei-specific PCRs in lung tissue from PMZL patients (n = 22), other pulmonary lymphomas, and normal controls. Positive results were confirmed by mNGS. A systematic search for T. whipplei and A. xylosoxidans in our in-house mNGS dataset comprising autopsy lungs, lung biopsies and lung resection specimens (n = 181) was subsequently performed.ResultsA 69-year-old patient presented with weight loss and persistent pulmonary consolidation. Subsequent mNGS analysis detected T. whipplei in the resected lung specimen. An antibiotic regimen eventually eliminated the bacterium. However, the consolidation persisted, and the diagnosis of PMZL was made in a second lung resection specimen. A second case of T. whipplei-associated PMZL was subsequently detected in the retrospectively analysed PMZL cohort. Both cases showed comparatively few mutations and no mutations in genes encoding for NF-κB pathway components, suggesting that T. whipplei infection may substitute for mutations in these PMZL. None of the samples in our in-house dataset tested positive for T. whipplei. In contrast, A. xylosoxidans was frequently found in both autopsy lungs and lung biopsy / resection specimens that were not affected by PMZL (> 50%).ConclusionsOur data suggests that T. whipplei colonisation of lungs may trigger PMZL as a potential driver. Systematic analyses with larger cohorts should be conducted to further support this hypothesis. The frequent detection of A. xylosoxidans in lung tissue suggests that it is a common component of the pulmonary microbiome and therefore less likely to trigger lymphomas.

Ag-driven CD8+ T cell clonal expansion is a prominent feature of MASH in humans and mice.

Chronic liver disease due to metabolic dysfunction-associated steatohepatitis (MASH) is a rapidly increasing global epidemic. MASH progression is a consequence of the complex interplay between inflammatory insults and dysregulated hepatic immune responses. T lymphocytes have been shown to accumulate in the liver during MASH, but the cause and consequence of T cell accumulation in the liver remain unclear. Our study aimed to define the phenotype and T cell receptor diversity of T cells from human cirrhotic livers and an animal model of MASH to begin resolving their function in disease. In these studies, we evaluated differences in T cell phenotype in the context of liver disease. Accordingly, we isolated liver resident T cell populations from humans with cirrhosis and from mice with diet-induced MASH. Using both 5' single-cell sequencing and flow cytometry, we defined the phenotype and T cell receptor repertoire of liver resident T cells during health and disease. MASH-induced human cirrhosis and diet-induced MASH in mice resulted in the accumulation of activated and clonally expanded T cells in the liver. The clonally expanded T cells in the liver expressed markers of chronic antigenic stimulation, including PD1, TIGIT, and TOX. Overall, this study establishes for the first time that T cells undergo Ag-dependent clonal expansion and functional differentiation during the progression of MASH. These studies could lead to the identification of antigenic targets that drive T cell activation, clonal expansion, and recruitment to the liver during MASH.

The TOX–RAGE axis mediates inflammatory activation and lung injury in severe pulmonary infectious diseases

Thymocyte selection-associated high-mobility group box (TOX) is a transcription factor that is crucial for T cell exhaustion during chronic antigenic stimulation, but its role in inflammation is poorly understood. Here, we report that TOX extracellularly mediates drastic inflammation upon severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection by binding to the cell surface receptor for advanced glycation end-products (RAGE). In various diseases, including COVID-19, TOX release was highly detectable in association with disease severity, contributing to lung fibroproliferative acute respiratory distress syndrome (ARDS). Recombinant TOX-induced blood vessel rupture, similar to a clinical signature in patients experiencing a cytokine storm, further exacerbating respiratory function impairment. In contrast, disruption of TOX function by a neutralizing antibody and genetic removal of RAGE diminished TOX-mediated deleterious effects. Altogether, our results suggest an insight into TOX function as an inflammatory mediator and propose the TOX-RAGE axis as a potential target for treating severe patients with pulmonary infection and mitigating lung fibroproliferative ARDS.

Hyper-N-glycosylated SEL1L3 as auto-antigenic B-cell receptor target of primary vitreoretinal lymphomas

Primary vitreoretinal lymphoma (PVRL) is a rare subtype of DLBCL and can progress into primary central nervous system lymphoma (PCNSL). To investigate the role of chronic antigenic stimulation in PVRL, we cloned and expressed B-cell receptors (BCR) from PVRL patients and tested for binding against human auto-antigens. SEL1L3, a protein with multiple glycosylation sites, was identified as the BCR target in 3/20 PVRL cases. SEL1L3 induces proliferation and BCR pathway activation in aggressive lymphoma cell lines. Moreover, SEL1L3 conjugated to a toxin killed exclusively lymphoma cells with respective BCR-reactivity. Western Blot analysis indicates the occurrence of hyper-N-glycosylation of SEL1L3 at aa 527 in PVRL patients with SEL1L3-reactive BCRs. The BCR of a PVRL patient with serum antibodies against SEL1L3 was cloned from a vitreous body biopsy at diagnosis and of a systemic manifestation at relapse. VH4-04*07 was used in both lymphoma manifestations with highly conserved CDR3 regions. Both BCRs showed binding to SEL1L3, suggesting continued dependence of lymphoma cells on antigen stimulation. These results indicate an important role of antigenic stimulation by post-translationally modified auto-antigens in the genesis of PVRL. They also provide the basis for a new treatment approach targeting unique lymphoma BCRs with ultimate specificity.

TIM-3+ CD8 T cells with a terminally exhausted phenotype retain functional capacity in hematological malignancies.

Chronic antigen stimulation is thought to generate dysfunctional CD8 T cells. Here, we identify a CD8 T cell subset in the bone marrow tumor microenvironment that, despite an apparent terminally exhausted phenotype (TPHEX), expressed granzymes, perforin, and IFN-γ. Concurrent gene expression and DNA accessibility revealed that genes encoding these functional proteins correlated with BATF expression and motif accessibility. IFN-γ+ TPHEX effectively killed myeloma with comparable efficacy to transitory effectors, and disease progression correlated with numerical deficits in IFN-γ+ TPHEX. We also observed IFN-γ+ TPHEX within CD19-targeted chimeric antigen receptor T cells, which killed CD19+ leukemia cells. An IFN-γ+ TPHEX gene signature was recapitulated in TEX cells from human cancers, including myeloma and lymphoma. Here, we characterize a TEX subset in hematological malignancies that paradoxically retains function and is distinct from dysfunctional TEX found in chronic viral infections. Thus, IFN-γ+ TPHEX represent a potential target for immunotherapy of blood cancers.

Abstract LB070: In vitro modeling of antigen-driven exhaustion in human CAR T cells

Abstract A major hurdle for chimeric antigen receptor (CAR) T cells to function in solid tumors is chronic antigen exposure leading to terminal lymphocyte exhaustion and impaired anti-tumor activity. Therefore, it is necessary to establish improved in vitro models of antigen-driven CAR T cell exhaustion to evaluate new CAR design candidates prior to validation with costly in vivo studies. Using the human tumor antigen mesothelin (MSLN) and MSLN-targeting CAR T cells as a model, we developed two in vitro assays that drive an exhausted phenotype in human CAR T cells and can be used to predict anti-tumor responses in vivo: 1) a chronic stimulation assay with plate-bound recombinant antigen (MSLN), ICAM-1, and CD58 and 2) a high antigen-expressing 3D tumor spheroid co-culture assay. In the chronic antigen stimulation assay, repetitive exposure to plate-bound MSLN (alongside co-stimulation signals provided by recombinant ICAM-1 and CD58) reprogrammed the transcriptomic profile of MSLN CAR T cells leading to increased surface expression of inhibitory checkpoint molecules and reduced cytokine production. Moreover, chronically stimulated MSLN CAR T cells demonstrated a drastically impaired ability to kill MSLN+ tumor cells compared to their unstimulated or acutely stimulated counterparts. A similar exhausted phenotype was observed in the high-antigen tumor spheroid model: when MSLN CAR T cells were co-cultured with MSLN-high human NCI-H226 spheroids for 7 days in a 96-well plate, expression of inhibitory checkpoint molecules markedly increased on the surface of CAR T cells. This increased expression of exhaustion markers coincided with impairment of NCI-H226 spheroid clearance. By contrast, other tumor spheroid models expressing low to intermediate levels of MSLN (NCI-H1650, NCI-H2052, NCI-H292, and SKOV-3 tumor cells) did not substantially induce expression of inhibitory checkpoint molecules and the spheroids were successfully cleared by MSLN CAR T cells. Importantly, the ability (or inability) of a CAR construct to clear NCI-H226 high-antigen tumor spheroids in vitro accurately predicted CAR T cell performance in in vivo solid tumor studies: the CAR construct that showed effective killing in the in vitro spheroid model was the only condition to provide durable regression of NCI-H1650 tumors in a NSG xenograft mouse model. Additionally, the rank order of CAR efficacy for a series of MSLN CAR binders obtained using the tumor spheroid model mirrored in vivo efficacy. As the CAR T cell field develops novel strategies to combat antigen-driven exhaustion for use in solid tumors, these new antigen-dependent in vitro models of exhaustion can enable more cost-efficient and effective evaluation of those strategies than using in vivo models alone. Citation Format: Maria Steele, Tina Tan, Jason Yokoyama, Allan Wang, Ty Crowl, Jared Hammer, Jerry Chen, Kevin Haworth, Howell Moffett, Aaron Foster, Marc Lajoie, Scott Boyken, Rupesh Amin. In vitro modeling of antigen-driven exhaustion in human CAR T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB070.

Large Granular Lymphocytic Leukemia: Clinical Features, Molecular Pathogenesis, Diagnosis and Treatment.

Large granular lymphocytic (LGL) leukemia is a lymphoproliferative disorder characterized by persistent clonal expansion of mature T- or natural killer cells in the blood via chronic antigenic stimulation. LGL leukemia is associated with specific immunophenotypic and molecular features, particularly STAT3 and STAT5 mutations and activation of the JAK-STAT3, Fas/Fas-L and NF-κB signaling pathways. Disease-related deaths are mainly due to recurrent infections linked to severe neutropenia. The current treatment is based on immunosuppressive therapies, which frequently produce unsatisfactory long-term responses, and for this reason, personalized approaches and targeted therapies are needed. Here, we discuss molecular pathogenesis, clinical presentation, associated autoimmune disorders, and the available treatment options, including emerging therapies.

Abstract 7034: Pooled CRISPR screening coupled with single-cell sequencing identifies modifiers of CAR T cell state in the context of chronic antigen stimulation

Abstract T cell exhaustion resulting from chronic antigen stimulation and an immunosuppressive tumor microenvironment limits the efficacy of T cell therapies in the solid tumor setting. The onset of T cell exhaustion is associated with distinct epigenetic and transcriptional changes. We hypothesized that genetic perturbations which shift T cells away from exhaustion associated states could increase the potency of immunotherapies. To this end, we utilized pooled, in vitro CRISPR/Cas9-based screening paired with deep sequencing readouts to characterize perturbation dependent T cell states in the context of chronic antigen stimulation. In order to achieve this, we developed a lentivirus-based workflow to perform CRISPRko, CRISPRi and CRISPRa pooled screens in human CAR T-cells that allowed for assessment of T cell phenotypes mediated by knockout, knockdown or overexpression of a large pool of target genes with single cell transcriptome readout. Subjecting these engineered CAR T-cells to an antigen-specific, cell-based repetitive stimulation assay (RSA) led to the progressive loss of T cell proliferation and effector function enabling in vitro modeling of T cell exhaustion. Moreover, characterization of the CAR T-cells by single cell sequencing recapitulated key hallmarks of the transcriptional and epigenetic landscape of T cell exhaustion. We also discovered T cell intrinsic gene perturbations that govern T cell states in the context of chronic antigen stimulation. These results demonstrate the power of pooled CRISPR screening with single cell readouts to identify novel target genes to enhance CAR T-cell therapies. Citation Format: Sahil Joshi, Glenn Wozniak, John Gagnon, Kristina Vucci, Allyson Merrell, Mandi Simon, Catherine Oh, Andrew Cardozo, David DeTomaso, Julie Chow, Grace Zheng, Angela Boroughs, Keith Joho, Pratiksha Thakore, Soyoung Oh, Jake Freimer, Ashley Cass, Vibhavari Sail, Carla Tocchini, Marian Sandoval, Andrea Liu, Eric Cui, Matt Drever, Brendan Galvin, Jeff Milush, Levi Gray-Rupp, Emily Wheeler, Bob Chen, Jacob Levine, Celine Eidenschenk, Jill Schartner, Katie Geiger-Schuller, Jan-Christian Huetter, Sascha Rutz, Orit Rozenblatt-Rosen, Ira Mellman, W. Nicholas Haining. Pooled CRISPR screening coupled with single-cell sequencing identifies modifiers of CAR T cell state in the context of chronic antigen stimulation [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 7034.

Deep phenotyping of post-infectious myalgic encephalomyelitis/chronic fatigue syndrome

Post-infectious myalgic encephalomyelitis/chronic fatigue syndrome (PI-ME/CFS) is a disabling disorder, yet the clinical phenotype is poorly defined, the pathophysiology is unknown, and no disease-modifying treatments are available. We used rigorous criteria to recruit PI-ME/CFS participants with matched controls to conduct deep phenotyping. Among the many physical and cognitive complaints, one defining feature of PI-ME/CFS was an alteration of effort preference, rather than physical or central fatigue, due to dysfunction of integrative brain regions potentially associated with central catechol pathway dysregulation, with consequences on autonomic functioning and physical conditioning. Immune profiling suggested chronic antigenic stimulation with increase in naïve and decrease in switched memory B-cells. Alterations in gene expression profiles of peripheral blood mononuclear cells and metabolic pathways were consistent with cellular phenotypic studies and demonstrated differences according to sex. Together these clinical abnormalities and biomarker differences provide unique insight into the underlying pathophysiology of PI-ME/CFS, which may guide future intervention.

Imprinted immune abnormalities in liver transplant patients cured of hepatitis C with antiviral drugs.

Chronic HCV infection induces interferon and dysregulates immune responses through inflammation and chronic antigenic stimulation. Antiviral drugs can cure HCV, providing a unique opportunity to examine the immunological restoration that does and does not occur when a chronic viral infection is eradicated. We quantified blood cytokines levels and used mass cytometry to immunophenotype peripheral blood mononuclear cells before and after HCV cure in 2 groups of patients and controls. At baseline, serum interferon α and soluble CD163 (a macrophage product) were elevated in both liver transplant and nonliver transplant patients compared to controls; the frequencies of several peripheral blood mononuclear cell populations differed from controls; and programmed death protein 1-positivity was increased in nearly all T cell subsets. Many abnormalities persisted after HCV cure, including elevated programmed death protein 1 expression on CD4 naïve and central memory T cells, elevated soluble CD163, and expansion of the plasmablast/plasma cell compartment. Several myeloid-lineage subsets, including Ag-presenting dendritic cells, remained dysregulated. In mechanistic studies, interferon α treatment increased programmed death protein 1 on human T cells and increased T cell receptor signaling. The data identify immunological abnormalities that persist after curative HCV treatment. Before cure, high levels of interferon α may stimulate programmed death protein 1 expression on human T cells, causing persistent functional changes.

Lymphoma involvement of the eyelid and eye

Lymphomas of the eye and ocular adnexa are rare lymphoproliferative diseases of the ocular and ocular adnexal tissue. The incidence of these diseases has been rapidly increasing over the past few decades. The exact pathogenesis remains unknown, but it is postulated to be multifactorial and includes genetic aberrations, epigenetic and environmental factors, infectious agents, and chronic antigenic stimulation. The majority of ocular and ocular adnexal lymphomas are of B-cell origin, except for eyelid lymphomas, which are more often of T-cell type. Lymphoproliferative diseases of ocular and ocular adnexal structures are either primary, when they arise in the eye, orbit, lacrimal gland, eyelid, and/or conjunctiva, or secondary extranodal manifestation of systemic lymphoma. Diagnosis is challenging and requires a multidisciplinary approach involving ophthalmologists, dermatologists, oncologists, and radiation oncologists.

TCell Exhaustion.

Tcell responses must be balanced to ensure adequate protection against malignant transformation and an array of pathogens while also limiting damage to healthy cells and preventing autoimmunity. Tcell exhaustion serves as a regulatory mechanism to limit the activity and effector function of Tcells undergoing chronic antigen stimulation. Exhausted Tcells exhibit poor proliferative potential; high inhibitory receptor expression; altered transcriptome, epigenome, and metabolism; and, most importantly, reduced effector function. While exhaustion helps to restrain damage caused by aberrant Tcells in settings of autoimmune disease, it also limits the ability of cells to respond against persistent infection and cancer, leading to disease progression. Here we review the process of Tcell exhaustion, detailing the key characteristics and drivers as well as highlighting our current understanding of the underlying transcriptional and epigenetic programming. We also discuss how exhaustion can be targeted to enhance Tcell functionality in cancer. Expected final online publication date for the Annual Review of Immunology, Volume 42 is April 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Transcriptional and epigenetic regulators of human CD8+ T cell function identified through orthogonal CRISPR screens

Clinical response to adoptive T cell therapies is associated with the transcriptional and epigenetic state of the cell product. Thus, discovery of regulators of T cell gene networks and their corresponding phenotypes has potential to improve T cell therapies. Here we developed pooled, epigenetic CRISPR screening approaches to systematically profile the effects of activating or repressing 120 transcriptional and epigenetic regulators on human CD8+ T cell state. We found that BATF3 overexpression promoted specific features of memory T cells and attenuated gene programs associated with cytotoxicity, regulatory T cell function, and exhaustion. Upon chronic antigen stimulation, BATF3 overexpression countered phenotypic and epigenetic signatures of T cell exhaustion. Moreover, BATF3 enhanced the potency of CAR T cells in both in vitro and in vivo tumor models and programmed a transcriptional profile that correlates with positive clinical response to adoptive T cell therapy. Finally, we performed CRISPR knockout screens that defined cofactors and downstream mediators of the BATF3 gene network.

Bclxl Prevents Progressive Exhaustion in BCMA CAR T Cells with Maintenance of High TCF1 Expressing T Cells

Although BCMA targeted CAR T cell therapies are highly effective in the treatment of multiple myeloma (MM), patients with high disease burden have not fully benefited from this therapy with shorter disease remissions despite initial responses. It is postulated that high disease burden results in chronic antigenic stimulation of CAR T cells with the induction of exhaustion-associated gene signatures and CAR T cells contraction, limiting their in vivo potency. We have previously reported that BCL2L1 (gene encoding for BCLxL) armoring of BCMA CAR T cells enhanced CAR T cells functional fitness and improved proliferation and cytotoxicity after chronic antigenic stimulation in vitro, as well as superior tumor clearance in xenograft models of MM. In the current study we aimed to define the molecular mechanisms that mediates BCL2L1-armored CAR T cell and examine the effect of BCLxL variants on CAR T cells fitness. In in vivo studies, NSG mice were systemically injected with OPM2 cells (stably transduced with firefly luciferase) and led to develop large MM disease burden, treatment with BCLxL armored (BCMA_BCL2L1) vs unarmored CARs (BCMA_CAR) or control T cells completely eliminated the MM disease and significantly improved animal survival. In animals treated with armored CARs (BCMA_BCL2L1) no disease recurrence was noted 180 days post treatment (in contrast to unarmored CARs where disease recurrence was noted within 60 days). BCLxL also improved CARs persistence, with armored CAR T cells being detectable in the marrow of these mice 7 and 14 days post treatment. Of note, BCLxL armored CAR T cells did not demonstrate uncontrolled proliferation as they were no longer detectable on day 120 post-infusion. Furthermore, CITEseq profiling on harvested human T cells 7 days post CAR T cells infusion to diseased mice revealed a differential T cell repertoires and phenotypes between BCMA_CAR and BCMA_BCL2L1 CARs. While unarmored CARs were significantly enriched for exhausted T cells (CD8_Tex), BCMA_BCL2L1 CAR T were enriched with early activated non-exhausted CD8 T cells, precursor exhausted T cells (CD8_Tpex) and naïve CD4 and CD8 T cells. Importantly, single cell genes set enrichment analysis (ssGSEA) revealed a differential metabolic signature with higher oxidative phosphorylation in BCMA_BCL2L1 CAR T cells compared to unarmored CARs. These findings suggested that armoring CAR T cells with BCL2L1 maintain CAR T cells in an activated and precursor exhausted T cell states with improved mitochondrial and metabolic fitness, preventing terminal T cells exhaustion. To further evaluate the impact of BCLxL in suppressing BCMA CAR T cells' exhaustion, we engineered BCMA CAR T cells with armored expression of two BCLxL mutants (BCLxL Δ26-83 or BCLxL Δ46-83) harboring large or small deletions involving a BCLxL regulatory loop. These mutants were previously reported to enhance BCLxL function (Chang et al The EMBO Journal 1997,16:968-977). Under chronic antigenic stimulation (CAS) or hypoxic conditions, wild type or mutants BClxL overexpression in BCMA CAR T cells resulted in higher proliferation rates compared to unarmored CARs. Compared to wild type BClxL, BCLxL Δ26-83 and BCLxL Δ46-83 mutants demonstrated higher proliferation capacity post CARs or under hypoxic conditions. Intriguingly, majority of BCLxL-overexpressing BCMA CAR-T cells, particularly BCMA CAR-T BCLxL_Δ26-83 cells, maintained a central memory (T CM) phenotype following a long co-culture with MM tumor cells with lower levels of exhaustion markers. Importantly BCLxL-overexpressing BCMA CAR-T cells had substantially higher level of TCF1 with lower TOX/TOX2 expression compared to unarmored BCMA CAR T cells. TCF1 high population was also enriched in unstimulated BCLxL-overexpressing BCMA CAR T cells suggesting a positive regulatory impact of BCL2L1 on TCF1 expression, either through direct or indirect pathways. Expectedly, TCF1 high BCMA CAR-T cells overexpressing a BCLxL were mainly associated with the naive/T CM phenotype. In summary, we have identified a role for BCLxL and two mutant variants lacking a loop regulatory domain (BCLxL Δ26-83 or BCLxL Δ46-83), in maintenance of BCMA CAR T cells in central memory and precursor exhausted states with retained high TCF1 expression, even under hypoxic and chronic antigenic stimulation conditions. These studies further support the clinical development of a BCL2L1 armored CAR T cells for the treatment of MM.

CD19-CAR T Cytotoxicity Is Improved By AMPKγ2 Overexpression, Which Is Further Enhanced By Metformin Treatment

Background Acute lymphoblastic leukemia (ALL) is the most common leukemia found in children. Chimeric Antigen Receptor (CAR) T cells induce high response rates against relapsed/refractory B-cell ALL, but 40% of CAR-T recipients suffer disease relapse, many due to poor in vivo survival of the CAR-T cells. Metabolic exhaustion has been proposed as a major barrier to optimal CAR-T cell performance. Therefore, modulating T cell metabolism may represent a promising method to improve CAR T-cell therapy. AMP-activated protein kinase (AMPK) is a heterotrimeric signaling complex which serves as a cellular energy sensor, promoting mitochondrial health and oxidative metabolism under conditions of energetic stress. We have demonstrated overexpressing the regulatory subunit AMPKγ2 increases AMPK signaling in human T cells, and hypothesized that this overexpression would enhance CAR-T metabolic fitness and improve anti-leukemia activity. Results We used lentiviral transduction to introduce a CD19-reactive, CD28 CAR into human T cells, followed by transduction of either AMPKγ2 or an empty vector (EV) control. Dual transduced cells were isolated by flow sorting and cultured with human IL2. The metabolism and cytotoxicity of these CAR-T cells were first assessed in vitro. AMPK-CART cells showed higher oxidative metabolism, with a 29% increase in basal oxygen consumption rates (OCR) and a 45% increase in maximal OCR (p<0.001) following overnight stimulation by NALM6 cells. In addition, co-culture with Zs-Green+ NALM6 cells revealed greater cytotoxicity by AMPK-CART cells (p<0.001) (Fig. 1A), accompanied by a 35% increase in CD25 expression (p<0.05). We then pivoted to studying AMPK-CART cells in vivo utilizing a murine xenograft leukemia model. Interestingly, despite their in vitro advantages, AMPK-CART cells demonstrated equivalent anti-tumor capacity in vivo. We reasoned that AMPKγ2 overexpression may simply protect AMPK signaling which is already occurring. To test this idea, we coupled AMPKγ2 overexpression with transient metformin treatment, a process known to activate AMPK signaling through inhibition of mitochondrial complex I. Metformin treatment was followed by the functional analysis of CAR-T cells in a chronic stimulation protocol where we repetitively stimulate CAR-T cells with NALM6 targets to induce exhaustion. After 8 days of chronic stimulation, metformin treated AMPK-CART cells (7mM for 4 hours) demonstrated the highest cytotoxicity against NALM6 cells compared to either metformin treated EV-CART cells or AMPK-CART cells without metformin treatment (Fig. 1B). This cytotoxic advantage was accompanied by an 18% increase in CD25 expression (2052.67±104.74 vs. 1742.67±14.64, p<0.05) and a 56% elevation in the percentage of central memory cells (9.15±1.32 vs. 5.88±0.25, p<0.05) in metformin treated AMPK-CART cells. These changes were not seen in EV-CART cells with metformin treatment. Conclusions Here, we report that AMPKγ2 overexpression in CD19-CAR T cells enhances oxidative metabolism and improves in vitro cytotoxicity but does not significantly increase in vivo anti-leukemia function. Metformin pre-treatment elevates CAR-T cytotoxicity following chronic antigen stimulation, but only when coupled with AMPKγ2 overexpression. This combination is what we predict will improve the function of CD19-CAR T cells in our murine xenograft leukemia model and human samples.

DNMT3A-Mutated Stem and Progenitor Cells Contribute to Altered T Cell Activation in Clonal Hematopoiesis

Clonal hematopoiesis is characterized by an expansion of blood cells derived from a single mutated hematopoietic stem cell (HSC). When this expansion is driven by a leukemia-associated mutation that exceeds a variant allele frequency (VAF) of 2%, it is termed clonal hematopoiesis of indeterminate potential (CHIP). Approximately half of CHIP cases are driven by mutations in the DNA methyltransferase gene DNMT3A. In patients with CHIP, greater clonal expansion is associated with an increased risk of cardiovascular diseases and hematologic malignancies. While DNMT3A impairment has been shown to alter inflammatory signaling in HSCs and myeloid cells, less is known about the impact of DNMT3A-mutated CHIP on T cell biology. Here, we leverage a cohort of human CHIP samples to assess how the expansion of DNMT3A-mutated cells affects gene expression across blood cell types and generate a new mouse model to assess antigen-specific immune cell interactions. We find evidence that the accumulation of DNMT3A-mutated stem and progenitor cells can cause T cell dysfunction. We first identified CHIP in a cohort of 191 sternum bone marrow samples collected during heart surgery. We detected CHIP in 45 samples (23.6%), including 22 samples with mutated DNMT3A (48.9% of CHIP samples). To mitigate confounding factors that might be introduced by comparing CHIP to healthy individuals, we used ten DNMT3A-mutated CHIP samples for this study, splitting them into a low-VAF (<10%, three samples) and high-VAF (>10%, seven samples) cohorts. We performed single-cell RNA sequencing on these 10 samples, generating high-quality data for 95,245 cells. We clustered and annotated 33 cell types according to the expression of canonical marker genes, representing the full spectrum of hematopoietic progenitors to myeloid, erythroid and lymphoid lineages. To evaluate how greater DNMT3A clonal expansion affects immune cell states, we performed differential gene expression analysis in the high-VAF compared to the low-VAF cohort with DESeq2 using a pseudobulk approach followed by gene set enrichment analysis using Hallmark pathways. We found that TGF-beta signaling, known for its immunosuppressive role, is upregulated in hematopoietic stem and progenitor cells (HSPCs), myeloid and T cell populations in the high-VAF compared to the low-VAF cohort. Both interferon alpha and gamma signaling pathways are consistently downregulated in HSPCs and multiple myeloid cell types as well as T cells. TNF-alpha signaling is upregulated in HSPCs and myeloid cells, consistent with recent observations linking TNF-alpha to the survival and proliferation of HSPCs and myeloid cells. Overall, greater expansion of DNMT3A-mutated cells is associated with variable gene expression changes in progenitor and myeloid cell types, whereas gene expression changes in T cells are consistent with diminished activation (Figure 1). To evaluate mechanisms contributing to altered T cell activation in DNMT3A-mutated CHIP, we combined our Dnmt3a-fl-R878H/+ inducible mouse model with the ovalbumin (OVA) trackable antigen system. Endogenously expressed OVA is processed into a peptide that is loaded onto MHCI and recognized by transgenic CD8+ T cells from OT-I mice. We crossed the Dnmt3a-fl-R878H/+ mouse with a mouse expressing OVA under the actin promoter, resulting in Dnmt3a-mutated mice with constitutive OVA expression. To interrogate antigen-specific T cell responses, we co-cultured Dnmt3a-mutated OVA+ lineage negative (Lin-) stem and progenitor cells with CD8+ T cells from OT-I mice. By measuring T cell proliferation using CFSE dilution, we observed an increase in T cell proliferation when CD8+ T cells were co-cultured with Dnmt3a-mutated OVA+ Lin- cells compared to wildtype OVA+ Lin- cells (Figure 2). These results suggest that Lin- Dnmt3a-mutated cells have an increased capacity for antigen-specific T cell activation. In conclusion, our studies demonstrate that greater expansion of DNMT3A-mutated CHIP is associated with reduced expression of activation signatures in T cells. In apparent contrast, Dnmt3a-mutated mouse cells have an increased capacity for antigen-specific T cell stimulation. Since chronic antigenic stimulation causes T cell exhaustion, our findings are consistent with a model in which the expansion of DNMT3A-mutated progenitor and myeloid cells causes chronic stimulation and dysfunction of T cells.

Geographic Prevalence Patterns and Modifiable Risk Factors for Monoclonal Gammopathy of Undetermined Significance

Monoclonal gammopathy of undetermined significance (MGUS) is a pre-malignant plasma cell disorder with an etiology that is incompletely understood. Modifiable risk factors and genetic predispositions likely interact to increase MGUS risk in specific individuals and populations. Identifying geographic prevalence patterns and modifiable risk factors is critical for understanding the etiology of MGUS. The aim of this review was to outline original research on MGUS prevalence across geographic locations and modifiable risk factors. We conducted a systematic review of 39 eligible studies from PubMed®, Embase®, and Web of Science® written in English and published by February 2023. Our protocol was registered in accordance with PROSPERO guidelines. Studies were synthesized using Research Electronic Data Capture and appraised using the National Heart, Lung, and Blood Institute study quality assessment tools. The prevalence of MGUS ranged from 0.24% to 9% across geographic locations. Modifiable risk factors for MGUS include infections, autoimmune diseases, chronic inflammatory conditions, lifestyle factors, environmental exposures, and ionizing radiation. Therefore, the development of MGUS may be related to chronic antigenic stimulation and genetic aberrations that promote clonal proliferation of plasma cells. Prospective studies assessing gene–environment interactions are needed to further define risk factors for MGUS and inform screening and preventative strategies.

Overcoming immune checkpoint blockade resistance in solid tumors with intermittent ITK inhibition

Cytotoxic CD8 + T cell (CTL) exhaustion is driven by chronic antigen stimulation. Reversing CTL exhaustion with immune checkpoint blockade (ICB) has provided clinical benefits in different types of cancer. We, therefore, investigated whether modulating chronic antigen stimulation and T-cell receptor (TCR) signaling with an IL2-inducible T-cell kinase (ITK) inhibitor, could confer ICB responsiveness to ICB resistant solid tumors. In vivo intermittent treatment of 3 ICB-resistant solid tumor (melanoma, mesothelioma or pancreatic cancer) with ITK inhibitor significantly improved ICB therapy. ITK inhibition directly reinvigorate exhausted CTL in vitro as it enhanced cytokine production, decreased inhibitory receptor expression, and downregulated the transcription factor TOX. Our study demonstrates that intermittent ITK inhibition can be used to directly ameliorate CTL exhaustion and enhance immunotherapies even in solid tumors that are ICB resistant.

Lower PTEN may be associated with CD8+ T cell exhaustion in diffuse large B-cell lymphoma

Initially discovered in chronic viral infection and then extended to tumor, 'T-cell exhaustion' is a broad term describing the response of T cells to chronic antigen stimulation. By definition, whether T-cell exhaustion occurs in diffuse large B-cell lymphoma (DLBCL) remains largely unknown because little has been described. Here, the immune-suppressing checkpoint molecules involved in T-cell exhaustion, including PD-1, PD-L1, TIM-3 and TIGIT, whose expression levels were analyzed in DLBCL, were retrieved from the GEPIA database. Compared with the normal control, CD8A, TNFA, IFNG and GZMA were markedly elevated in DLBCL, indicating that infiltrated CD8+ T cells predominate in DLBCL. Meanwhile, inhibitory immune checkpoints, such as PD-1, PD-L1, TIGIT and TIM-3 were drastically higher in DLBCL. PTEN, WNT2 and DKK3 expression were also appraised. It was revealed that PTEN was lower in DLBCL, without being statistically significant. In contrast with PTEN, DKK3 and WNT2 were shown to be pronouncedly higher in DLBCL relative to the normal control. Prognostically, only TIGIT was found to be associated with overall survival in DLBCL. Collectively, all the data we curetted from the GEPIA and TIMER 2.0 databases explicitly indicate that CD8+ T cell exhaustion took place, which may be linked with lower PTEN in DLBCL. To the best of our knowledge, this is the first bioinformatic report explicitly proposing that CD8+ T cell exhaustion occurs in DLBCL, which may be associated with lower PTEN.