Memory T Cells Research Articles

A Novel Autologous CAR-T Therapy, YTB323, with Preserved T-cell Stemness Shows Enhanced CAR T-cell Efficacy in Preclinical and Early Clinical Development.

Traditional CAR T-cell manufacturing requires extended ex vivo cell culture, reducing naive and stem cell memory T-cell populations and diminishing antitumor activity. YTB323, which expresses the same validated CAR as tisagenlecleucel, can be manufactured in <2 days while retaining T-cell stemness and enhancing clinical activity at a 25-fold lower dose. See related commentary by Wang, p. 1961. This article is featured in Selected Articles from This Issue, p. 1949.

Revealing viral and cellular dynamics of HIV-1 at the single-cell level during early treatment periods

While combination therapy completely suppresses HIV-1 replication in blood, functional virus persists in CD4+ Tcell subsets in non-peripheral compartments that are not easily accessible. To fill this gap, we investigated tissue-homing properties of cells that transiently appear in the circulating blood. Through cell separation and invitro stimulation, the HIV-1 "Gag and Envelope reactivation co-detection assay" (GERDA) enables sensitive detection of Gag+/Env+ protein-expressing cells down to about one cell per million using flow cytometry. By associating GERDA with proviral DNA and polyA-RNA transcripts, we corroborate the presence and functionality of HIV-1 in critical body compartments utilizing t-distributed stochastic neighbor embedding (tSNE) and density-based spatial clustering of applications with noise (DBSCAN) clustering with low viral activity in circulating cells early after diagnosis. We demonstrate transcriptional HIV-1 reactivation at any time, potentially giving rise to intact, infectious particles. With single-cell level resolution, GERDA attributes virus production to lymph-node-homing cells with central memory Tcells (TCMs) as main players, critical for HIV-1 reservoir eradication.

Assessing SARS-CoV-2-specific T-cell reactivity in late convalescents and vaccinees: Comparison and combination of QuantiFERON and activation-induced marker assays, and relation with antibody status.

Monitoring of SARS-CoV-2 spread and vaccination strategies have relied on antibody (Ab) status as a correlate of protection. We used QuantiFERON™ (QFN) and Activation-Induced Marker (AIM) assays to measure memory T-cell reactivity in unvaccinated individuals with prior documented symptomatic infection (late convalescents) and fully vaccinated asymptomatic donors (vaccinees). Twenty-two convalescents and 13 vaccinees were enrolled. Serum anti-SARS-CoV-2 S1 and N Abs were measured using chemiluminescent immunoassays. QFN was performed following instructions and interferon-gamma (IFN-γ) measured by ELISA. AIM was performed on aliquots of antigen-stimulated samples from QFN tubes. SARS-CoV-2-specific memory CD4+CD25+CD134+, CD4+CD69+CD137+ and CD8+CD69+CD137+ T-cell frequencies were measured by flow cytometry. In convalescents, substantial agreement was observed between QFN and AIM assays. IFN-γ concentrations and AIM+ (CD69+CD137+) CD4+ T-cell frequencies correlated with each other, with Ab levels and AIM+ CD8+ T-cell frequencies, whereas AIM+ (CD25+CD134+) CD4+ T-cell frequencies correlated with age. AIM+ CD4+ T-cell frequencies increased with time since infection, whereas AIM+ CD8+ T-cell expansion was greater after recent reinfection. QFN-reactivity and anti-S1 titers were lower, whereas anti-N titers were higher, and no statistical difference in AIM-reactivity and Ab positivity emerged compared to vaccinees. Albeit on a limited sample size, we confirm that coordinated, cellular and humoral responses are detectable in convalescents up to 2 years after prior infection. Combining QFN with AIM may enhance detection of naturally acquired memory responses and help stratify virus-exposed individuals in T helper 1-type (TH1)-reactive (QFNpos AIMpos Abshigh), non-TH1-reactive (QFNneg AIMpos Abshigh/low), and pauci-reactive (QFNneg AIMneg Abslow).

Tissue-Resident Memory T Cells Shape Tumor-Immune Evolution in Lung Cancer.

Tissue-resident memory T-cell immunosurveillance is associated with cigarette smoking before tumor onset.

The Sympathetic Nervous System Is Necessary for Development of CD4+ T-Cell Memory Following Staphylococcus aureus Infection.

Lymph nodes and spleens are innervated by sympathetic nerve fibers that enter alongside arteries. Despite discovery of these nerve fibers nearly 40 years ago, the role of these nerves during response to infection remains poorly defined. We have found that chemical depletion of sympathetic nerve fibers compromises the ability of mice to develop protective immune memory to a Staphylococcus aureus infection. Innate control of the primary infection was not impacted by sympathectomy. Germinal center formation is also compromised in nerve-depleted animals; however, protective antibody responses are still generated. Interestingly, protective CD4+ T-cell memory fails to form in the absence of sympathetic nerves after S aureus infection.

Activated tissue resident memory T-cells (CD8+CD103+CD39+) uniquely predict survival in left sided "immune-hot" colorectal cancers.

Characterization of the tumour immune infiltrate (notably CD8+ T-cells) has strong predictive survival value for cancer patients. Quantification of CD8 T-cells alone cannot determine antigenic experience, as not all infiltrating T-cells recognize tumour antigens. Activated tumour-specific tissue resident memory CD8 T-cells (TRM) can be defined by the co-express of CD103, CD39 and CD8. We investigated the hypothesis that the abundance and localization of TRM provides a higher-resolution route to patient stratification. A comprehensive series of 1000 colorectal cancer (CRC) were arrayed on a tissue microarray, with representative cores from three tumour locations and the adjacent normal mucosa. Using multiplex immunohistochemistry we quantified and determined the localization of TRM. Across all patients, activated TRM were an independent predictor of survival, and superior to CD8 alone. Patients with the best survival had immune-hot tumours heavily infiltrated throughout with activated TRM. Interestingly, differences between right- and left-sided tumours were apparent. In left-sided CRC, only the presence of activated TRM (and not CD8 alone) was prognostically significant. Patients with low numbers of activated TRM cells had a poor prognosis even with high CD8 T-cell infiltration. In contrast, in right-sided CRC, high CD8 T-cell infiltration with low numbers of activated TRM was a good prognosis. The presence of high intra-tumoural CD8 T-cells alone is not a predictor of survival in left-sided CRC and potentially risks under treatment of patients. Measuring both high tumour-associated TRM and total CD8 T-cells in left-sided disease has the potential to minimize current under-treatment of patients. The challenge will be to design immunotherapies, for left-sided CRC patients with high CD8 T-cells and low activate TRM,that result in effective immune responses and thereby improve patient survival.

GM-CSF and IL-7 fusion cytokine engineered tumor vaccine generates long-term Th-17 memory cells and increases overall survival in aged syngeneic mouse models of glioblastoma.

Age-related immune dysfunctions, such as decreased T-cell output, are closely related to pathologies like cancers and lack of vaccine efficacy among the elderly. Engineered fusokine, GIFT-7, a fusion of interleukin 7 (IL-7) and GM-CSF, can reverse aging-related lymphoid organ atrophy. We generated a GIFT-7 fusokine tumor vaccine and employed it in aged syngeneic mouse models of glioblastoma and found that peripheral vaccination with GIFT-7TVax resulted in thymic regeneration and generated durable long-term antitumor immunity specifically in aged mice. Global cytokine analysis showed increased pro-inflammatory cytokines including IL-1β in the vaccinated group that resulted in hyperactivation of dendritic cells. In addition, GIFT-7 vaccination resulted in increased T-cell trafficking to the brain and robust Th-17 long-term effector memory T-cell formation. TCR-seq analysis showed increased productive frequency among detected rearrangements within the vaccinated group. Overall, our data demonstrate that aging immune system can be therapeutically augmented to generate lasting antitumor immunity.

O123 Significance of hepatitis A virus cellular receptor-1 (HAVCR1) and its association with tumour immune microenvironment in breast cancer

Abstract Introduction Immunotherapy has an important role in the intervention of breast cancer, but single-agent efficacy of immunotherapy is poor, owing in part to influence of the surrounding tumour microenvironment. Hepatitis A virus cellular receptor-1 (HAVCR1), plays vital roles in immune microenvironment in pancreatic adenocarcinoma, but not clear in breast cancer. The present study aimed to characterize the role of HAVCR1 in immune microenvironment of breast cancer. Methods The Cancer Genome Atlas was used to evaluate functional role of HAVCR1. Kaplan-Meier plotter and Tumor Immune Estimation Resource were applied to illustrate correlation of HAVCR1 with the prognosis and immune infiltration of breast cancer. Results In Pietenpol subtype, high HAVCR1 levels indicated a favourable overall survival (OS) (HR=0.08, 95%CI:0.01-0.61, P=0.0024) in mesenchymal stem–like breast cancer. However, it was connected to worse OS (HR=2.29, 95%CI:1.04-5.04, P=0.035) in immunomodulatory subtype. Significant correlation was found between HAVCR1 expression level and immune infiltration in breast cancer, especially with B cells, CD8+ T cells, CD4+ T cells, macrophages, neutrophils and Dendritic cell (P&amp;lt;0.05), and other immune markers, such as PDCD1 and CTLA4 (P&amp;lt;0.05). Decreased of HAVCR1 in Mesenchymal stem cells, Natural killer T-cells and enriched of B cell, CD4+ memory T-cells, CD8+ T-cells, Type 1 T-helper cells, Type 2 T-helper cells significantly improved the prediction of OS (P&amp;lt;0.05). Decreased impression of HAVCR1 in B cells and CD8+ T-cells were correlated with worse OS (P&amp;lt;0.05). Conclusion HAVCR1 is closely correlated with immune microenvironment and may serve as a potential tumour immunotherapy target for breast cancer.

Six-month immune responses to mRNA-1273 vaccine in combination antiretroviral therapy treated late presenter people with HIV according to previous SARS-CoV-2 infection.

Immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mRNA vaccines in people with HIV (PWH) with a history of late presentation (LP) and their durability have not been fully characterized. In this prospective, longitudinal study, we sought to assess T-cell and humoral responses to SARS-CoV-2 mRNA vaccination up to 6 months in LP-PWH on effective combination antiretroviral therapy (cART) as compared to HIV-negative healthcare workers (HCWs), and to evaluate whether previous SARS-CoV-2 infection modulates immune responses to vaccine. SARS-CoV-2 spike (S)-specific T-cell responses were determined by two complementary flow cytometry methodologies, namely activation-induced marker (AIM) assay and intracellular cytokine staining (ICS), whereas humoral responses were measured by ELISA [anti-receptor binding domain (RBD) antibodies) and receptor-binding inhibition assay (spike-ACE2 binding inhibition activity), before vaccination (T0), 1 month (T1) and 5 months (T2) after the second dose. LP-PWH showed at T1 and T2 significant increase of: S-specific memory and circulating T follicular helper (cTfh) CD4 + T cells; polyfunctional Th1-cytokine (IFN-γ, TNF-α, IL-2)- and Th2-cytokine (IL-4)-producing S-specific CD4 + T cells; anti-RBD antibodies and spike-ACE2 binding inhibition activity. Immune responses to vaccine in LP-PWH were not inferior to HCWs overall, yet S-specific CD8 + T cells and spike-ACE2 binding inhibition activity correlated negatively with markers of immune recovery on cART. Interestingly, natural SARS-CoV-2 infection, while able to sustain S-specific antibody response, seems less efficacious in inducing a T-cell memory and in boosting immune responses to vaccine, possibly reflecting an enduring partial immunodeficiency. Altogether, these findings support the need for additional vaccine doses in PWH with a history of advanced immune depression and poor immune recovery on effective cART.

Recruiting T-Cells toward the Brain for Enhanced Glioblastoma Immunotherapeutic Efficacy by Co-Delivery of Cytokines and Immune Checkpoint Antibodies with Macrophage-Membrane-Camouflaged Nanovesicles.

Immunotherapy with immune checkpoint inhibitors (CPIs) shows promising prospects for glioblastoma multiforme (GBM) but with restricted results, mainly attributed to the immunosuppressive tumor microenvironment (TME) and the limited antibody permeability of the blood-tumor barrier (BTB) in GBM. Here, nanovesicles with amacrophage-mimicking membrane are described, that co-deliverchemotactic CXC chemokine ligand 10 (CXCL10), to pre-activate the immune microenvironment, and anti-programmed death ligand 1 antibody (aPD-L1), to interrupt the immune checkpoint, aiming to enhance the effect of GBM immunotherapy. Consequently, the tumor tropism of the macrophage membrane and the receptor-mediated transcytosis of the angiopep-2 peptide allowthe nanovesicle to effectively cross the BTB and target the GBM region, with 19.75-fold higher accumulation of antibodies compared to the free aPD-L1 group. The CPI therapeutic efficacy is greatly enhanced by CXCL10-induced T-cells recruitment with significant expansion of CD8+ T-cells and effector memory T-cells, leading to the elimination of tumor, prolonged survival time, and long-term immune memory in orthotopic GBM mice. The nanovesicles, that relieve the tumor immunosuppressive microenvironment by CXCL10 to enhance aPD-L1 efficacy, may present a promising strategy for brain-tumor immunotherapy.

Patients with Covid-19 induced atypical thyroiditis have thyroid-resident memory T-cells specific for SARS-CoV-2

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Monomethyl fumarate prevents alloimmune rejection in mouse heart transplantation by inducing tolerogenic dendritic cells.

Dendritic cells (DCs) are important targets for eliciting allograft rejection after transplantation. Previous studies have demonstrated that metabolic reprogramming of DCs can transform their immune functions and induce their differentiation into tolerogenic DCs. In this study, we aim to investigate the protective effects and mechanisms of monomethyl fumarate (MMF), a bioactive metabolite of fumaric acid esters, in a mouse model of allogeneic heart transplantation. Bone marrow-derived DCs are harvested and treated with MMF to determine the impact of MMF on the phenotype and immunosuppressive function of DCs by flow cytometry and T-cell proliferation assays. RNA sequencing and Seahorse analyses are performed for mature DCs and MMF-treated DCs (MMF-DCs) to investigate the underlying mechanism. Our results show that MMF prolongs the survival time of heart grafts and inhibits the activation of DCs in vivo. MMF-DCs exhibit a tolerogenic phenotype and function in vitro. RNA sequencing and Seahorse analyses reveal that MMF activates the Nrf2 pathway and mediates metabolic reprogramming. Additionally, MMF-DC infusion prolongs cardiac allograft survival, induces regulatory T cells, and inhibits T-cell activation. MMF prevents allograft rejection in mouse heart transplantation by inducing tolerogenic DCs.

Asymmetrical Expression of PD1 and CD25 in T-cells Post-Initial Activation

Abstract After TCR engagement, T cells induce expression of CD25, which forms the IL2 high affinity receptor complex, concurrently with PD1, which plays a down-regulatory role in activation. Cessation of TCR stimulation leads to eventual contraction of T cell responses. Chimeric antigen receptor (CAR) T cell therapy has seen increased use in the past decade. CAR T-cells are activated and transduced ex vivo and expanded with low amounts of cytokines prior to administration. The expression of CD25 and PD1 on CAR T cells at different stages of the process has been incompletely characterized. Purified T cells were activated in vitro for 3 days followed by expansion using low dose IL2. We demonstrate that both CD25 and PD-1 are strongly upregulated during initial activation, but by day 8, under expansion conditions, PD1 is downregulated to negligible levels. Despite absence of TCR signaling, CD25 expression remains moderate-to-high explaining the ability to maintain proliferation. This discrepancy was higher in transduced CAR T-cells compared to activated non-transduced (NT) T cells indicating the possibility of CAR tonic signaling. Furthermore, restimulation of both NT and CAR-T cells after this expansion phase resulted in further increased CD25 expression whereas PD-1 expression remained low. Our studies suggest that during initial activation, TCR engagement is necessary to maintain PD-1, but not CD25 expression. However, upon restimulation, TCR engagement is capable of further upregulating CD25, but not PD-1 expression. These results indicate that TCR signaling induces asymmetrical expression of PD-1 and CD25, a dynamic interplay that may regulate T-cell function and memory response and that PD1 may not be a limiting factor in the process.

Pim2 negatively regulates T-cell immune responses through modulating autophagy

Abstract Pim kinases affect cell survival, cell proliferation, transcriptional activation, and protein translation by phosphorylating various target substrates. We previously showed that Pim2 plays a distinct role from Pim1 and Pim3, and negatively regulates T-cell allogeneic response and anti-tumor immunity. Pim2 was reported to promote the induction of autophagy in cancer cells, a cellular process that critically impacts T-cell effector function, survival and memory formation. We hypothesize that Pim2 constrains T-cell immune responses through modulating autophagy. Using western blot and electron microscopy, we evaluated the effect of Pim2 on autophagic flux in resting and activated T cells. Pim2 deficiency in T cells attenuated LC3 lipidation, P62 degradation as well as autophagosome formation, suggesting that Pim2 facilitates autophagy in T cells. Mechanistically, Pim2 directly bound with P62 and loss of Pim2 led to accumulation of P62 which further blocked autophagic flux in T cells. Furthermore, augmentation of autophagy via overexpressing Atg5 or metformin treatment reduced effector cytokine production in Pim2-deficient T cells in vitroand reversed the heightened ability of Pim2-deficient T cells for inducing graft-versus-host disease and for controlling breast cancer growth in vivo. Taken together, Pim2 is a key promotor of autophagy in T cells and targeting Pim2 to suppress autophagy may represent a promising strategy to improve T-cell effector function in cancer immunotherapy. The work was supported by NIH grants: R01 CA258440 and R21 CA263140.

A newborn with multiple intestinal atresias found to have variants in tetratricopeptide repeat domain 7A gene

We report a male infant born with multiple intestinal atresias (MIA) prompting early genetic testing. Bi-allelic, compound heterozygous variants in tetratricopeptide repeat domain 7A (TTC7A) gene were identified. TTC7A is involved in transporting phosphatidylinositol 4-kinase IIIα into the plasma membrane (1). Mutations in this gene disrupt intestinal development and polarity, causing intestinal diseases such as atresia, enteropathy, and very early onset inflammatory bowel disease (VEO-IBD), as well as variable degrees of immunodeficiency (1).The identification of variants in TTC7A gene prompted an immune evaluation since as many as 75% of patients with TTC7A mutations reported in the literature have an underlying immunodeficiency (2). Flow cytometry was relatively unremarkable except for a slightly low memory T-cell count and percentage. NK-cell and B-cell numbers and percentages were appropriate for age. His phytohemagglutinin response was relatively low, but still significant, suggesting a possible component of combined immunodeficiency. To further characterize his T-cell function, we assessed his T-cell proliferation response to anti-CD3, which showed moderately decreased response of CD45+ lymphocytes and CD3+ T-cells to soluble anti-CD3 alone. However, co-stimulation with anti-CD3+anti-CD28 and anti-CD3+IL-2 normalized his T-cell proliferative response. Immunoglobulins were all low. Given his subtle T-cell defect and hypogammaglobulinemia, he was classified as having MIA with combined immunodeficiency (MIA-CID) phenotype of TTC7A.Cobalamin was tried in this patient as it has a low side effect profile and showed benefits in gut motility and intestinal tract luminal narrowing in a TTC7A zebrafish model (3). We considered investigational use of leflunomide, which showed the most promising results in the same zebrafish model (3). Bowel and hematopoietic stem-cell transplantation were considered too. Enterectomy was also offered, as this was shown to be effective in a case report of two patients with similar MIA-CID TTC7A phenotype (4). Unfortunately, the patient did not respond to steroids or cobalamin. His clinical condition worsened, and he passed away at four months of age.Mutations in the TTC7A gene result in various gastrointestinal pathologies with varying degrees of immunodeficiency in approximately 75% of patients (2). All patients with MIA or VEO-IBD should have an immune evaluation and genetic testing.

CD3ζ ITAM Diversity Determines Chimeric Antigen Receptor Signaling and Function

Abstract One of the major challenges facing CD28-based CAR-T immunotherapy is a lack of long-term T-cell persistence in patients.CD28 CARs exhibit strong signaling patterns that result in an acute tumor-killing response. However, based on studies with the conventional TCR, strong signaling favors terminal differentiation of T-cells that get exhausted faster while weaker CAR signaling favors more stem-like memory T-cell states with longer persistence. Hence attenuation of CAR signaling strength is a viable strategy for long term survival of CD28-CAR T-cells. Previous work has suggested that reducing CD3 ITAM diversity to single ITAM sequences while keeping the number of ITAMs same, attenuates TCR signaling strength to different extents. Based on this, we have designed modified ITAM diversity restricted CD28 based anti-CD19 CARs with the goals of optimizing CAR signalingand studying the non-redundant signaling properties of the individual ITAMs in the context of CARs. Our approach is different from reducing ITAM multiplicity as there is evidence suggesting that ITAM numbers might be important for CAR sensitivity. We hypothesize that the individual ITAM non-redundancy and the reduction in ITAM diversity will allow for optimization of CAR T cell signaling and improve T-cell persistence while maintaining sensitivity to low ligand densities.We have observed that our modified CARs can kill CD19+ leukemia cells in vitro. We observe functional differences in terms of cytokine production, proliferation, AICD and exhaustion. We have also observed differences in signal strength based on ITAM usage. These observations support the non-redundancy of ITAM signaling and open the possibility of optimizing CAR persistence and function in patients.

Abstract A007: Proteogenomic landscape of KRASG12C lung adenocarcinomas reveals new subtypes and potential combination therapies

Abstract Despite the presence of the KRASG12C oncogene, not all patients respond to KRASG12C inhibitors (e.g., sotorasib, adagrasib) and duration of response has been variable. We hypothesized that subtypes of KRASG12C lung cancers could be responsible for such differential effects. To define subtypes, we performed targeted exon sequencing, transcriptomics, proteomics, and phosphoproteomics on 46 frozen surgically resected KRASG12C lung adenocarcinomas. We employed unbiased consensus clustering methods, pathway analysis, and PTM-SEA to identify kinase signatures from phosphoproteomics data. Transcriptomics data revealed 4 clusters. Pathway enrichment showed that gene cluster (GC) 1 was enriched for immune pathways including interferon gamma response. Epithelial-mesenchymal transition (EMT), assessed by a TGF-β gene signature, was also enriched in GC1. 100% of GC1 overlapped the Proximal-Inflammatory subtype (Wilkerson et al.) and 53% with the mesenchymal subtype (Daemen et al.). GC2 was enriched for inflammatory response and TNF-alpha signaling. GC3 represented a metabolic phenotype with enriched xenobiotic metabolism and other metabolism pathways. 83% of GC3 overlapped with the Terminal Respiratory Unit subtype (Wilkerson et al.) and 56% with the proliferative subtype (Daemen et al.). GC4 only had 5 samples and did not have any significant enrichment. We next identified four proteomic clusters with distinct biology. Protein Cluster 1 (PC1) was enriched for immune pathways, PC2 was enriched for EMT, and PC3 was enriched for TNF/NF-κβ signaling. PC4 was enriched for xenobiotic and fatty acid metabolism and had significantly more KEAP1 mutations. We observed concordance of protein based clusters with mRNA based clusters, as PC1 largely overlapped with GC1 (70%) and PC4 largely overlapped with GC3 (86%). Cell type inference using xCell largely recapitulated the immune subtypes identified by transcriptomics and proteomics. GC1/PC1 had significantly more CD8+ effector memory T-cells, CD4+ Th2 T-cells, and M1 macrophages, while GC3/PC4 had significantly less of these same cell types. Collectively, GC1/PC1 represents 43% of the cohort and was defined by enrichment of immune pathways, elevated immune populations, and significantly higher PD-L1. Finally, we leveraged the phosphoproteomics data to determine whether signaling activity differs across subtypes. Using PTM-SEA, we identified phosphoproteomic signatures of activity for 50 kinases and pathways. We identified 26% of samples (N=12) with high EGFR activity, which is known to affect sensitivity of cells to KRASG12C inhibitors. The EGFR-high samples were observed across all genomic, transcriptomic, and proteomic subtypes. Our results suggest concordance of some transcriptomic and proteomic subtypes, including an immune subtype, while a metabolic protein subtype and EGFR high signaling subtype may provide additional subclasses with potential relevance for treatment responses. Additional phosphoproteomics signatures such as PLK1 and AKT1 may identify subtypes amenable to combination therapy approaches. Citation Format: Paul A. Stewart, Hitendra Solanki, Eric Welsh, Yaakov Stern, Denis Imbody, Yonghong Zhang, Bruna Pellini, Bin Fang, Sean Yoder, Steven Eschrich, Jamie Teer, John Koomen, Eric B. Haura. Proteogenomic landscape of KRASG12C lung adenocarcinomas reveals new subtypes and potential combination therapies [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr A007.

Severe Hematologic Abnormality Associated with an Inborn Error of Immunity (IEI)

Genetic sequencing improves our recognition of IEI's through genotype/phenotype correlations; however, variants of uncertain significance (VUSs) remain an obstacle. Functional assays provide avenues to identify variant pathogenicity but are infrequently commercially available, resulting in delayed diagnosis. We report the case of one such experience, and utilization of research-based functional testing to help characterize variant pathogenicity.A 20-year-old male patient with celiac disease came to medical attention after developing profound fatigue with pancytopenia: white blood cell count (4,400cells/mL), hemoglobin (8.4 g/dL), and platelets (18,000cells/mL). Evaluation included normal iron studies, folate, B12, copper levels, lactate dehydrogenase and haptoglobin, and low immunoglobulin levels (IgG 122 mg/dL; IgM 7 mg/dL; IgA 12 mg/dL). Infectious evaluation was negative including CMV, EBV, HHV-6, and Parvovirus. Bone marrow biopsy confirmed hypocellular marrow consistent with aplastic anemia. Hematologic testing was unremarkable (karyotype, myelodysplastic mutation sequencing, telomere length, leukemia flow cytometry immunophenotyping, and T-cell clonality).Lymphocyte flow cytometry showed severe lymphopenia: absolute lymphocyte count (268cells/mL), CD3+ (222cells/mL;83%), CD4+ (107cells/mL;40.1%), CD8+ (88cells/mL;32.9%), CD45RA+/CD4+/CD62L+ (15cells/mL;14.4%), and CD19+ (3cells/mL;1.3%). T-cell proliferation to phytohemagglutinin and tetanus was normal, and antibody titers were protective. Infection history was unremarkable. A primary immunodeficiency next generation sequencing panel uncovered a VUS in CTLA-4, c.385T> A (p.Cys129Ser) for the patient and his father (received biologic therapy for rheumatologic disease). Without commercially available transendocytosis assays, the patient and paternal samples were sent for research analysis. Testing demonstrated reduced CD4+ & CD8+ naïve T-cells with increased memory T-cells, and CD80-GFP transendocytosis assay showed reduced GFP uptake by C129S variant-expressing cells relative to wild-type CTLA-4. The clinical and immune phenotype were highly suggestive of CLTA-4 haploinsufficiency. Despite conventional immunomodulatory/immunosuppressive treatment with calcineurin inhibitors, corticosteroids and high-dose IVIG, only a partial response was achieved. Abatacept trial was considered, but haploidentical transplant was elected due to clinical severity, and he no longer requires repetitive transfusions. Our case outlines a novel CTLA-4 variant resulting in CLTA-4 haploinsufficiency presenting as aplastic anemia and hypogammaglobulinemia/CVID. The absence of easily accessible testing for CTLA-4 resulted in delayed functional evaluation, prolonging consideration for abatacept. Fortunately, the patient has benefited from hematopoietic stem cell transplant, which remains the mainstay curative therapy for severe disease. [Display omitted] [Display omitted]

Mucosal vaccination with an adenoviral vector vaccine protects against pulmonary breast cancer metastasis by robust induction of tissue-resident memory T-cells in the lung

Abstract The presence of tissue-resident memory T-cells (TRM) in solid tumors positively correlates with an improved overall prognosis and responsiveness to checkpoint inhibitor therapy. Rationally designed vaccine approaches capable of inducing antitumor TRM at cancer sites are needed to harness the full potential of localized immunity in tumor therapy. Our project aims to establish tumor antigen-specific lung TRM by mucosal vaccination with an adenoviral (Ad) vaccine to prevent and treat lung metastasis in the murine 4T1 breast cancer model. We employ Ad serotype 5 vectors encoding for 4T1 tumor-associated antigens from Mage-b and MuLV gp70 for intranasal vaccination, supplemented with an Interleukin-1β-encoding vector to specifically boost lung TRM induction. Indeed, particularly if Ad-IL-1β was co-delivered, mucosal vaccination established high numbers of tumor antigen-specific CD69 +CD103 +lung TRM as shown by phenotypic and functional analyses in combination with intravascular staining. Vaccine efficacy was evaluated in a 4T1 pseudometastasis model mimicking a prophylactic vaccination against lung metastasis in breast cancer patients. Local immunization led to a significant protection against pulmonary metastasis and improved survival compared to non-vaccinated and, notably, intramuscularly immunized mice. By using FTY720 to inhibit T-cell circulation upon tumor challenge, we confirmed that TRM are crucial contributors to the vaccine efficacy. In conclusion, induction of tumor-antigen specific lung TRM are a key point in local protection against pulmonary metastasis. Ongoing studies analyze the therapeutic use of this vaccination strategy as well as its potential in combination with immune checkpoint inhibition. Supported by grants from 'Anschubfinanzierung (ELAN) im IZKF' and 'Doktor Robert Pfleger-Stiftung'.

Age upon RSV infection and epitope-specificity influence the memory differentiation of antiviral T cells

Abstract Background Despite the high rate of respiratory syncytial virus (RSV) infection and severe symptoms in infants, subsequent infections occur, indicating impaired mucosal immune memory at early life. CD8 T cells are associated with reduced disease in human adult challenge trials and murine models, demonstrating their protective roles. However, since primary RSV infection occurs in infancy, a better understanding of the memory differentiation of RSV-specific respiratory CD8 T cells in neonates is required. Methods CB6F1/J mice were infected at 7, 14, 25 days of life or adulthood to characterize the RSV-specific memory T-cell development. Cells from mediastinal lymph nodes (MLN) and lungs were analyzed by flow cytometry at 7, 11, 14, and 40 days post RSV infection (dpi) after staining with fluorescent antibodies and tetramers specific for M and M2 epitopes in RSV. Results RSV-specific CD8 T cell responses were overall lower in neonates from peak to memory phase, but differ by epitope. At 40dpi, M-specific CD8 T cells preferentially differentiated to central-memory (CM) and resided in the MLN in both adult and neonatal mice, whereas M2-specific CD8 T cells preferentially differentiated to effector-memory (EM) and resided in the lungs in adult but not neonatal mice. Importantly, lung tissue resident memory cell differentiation was limited in early life and increased with age upon infection. Conclusion Our results indicate that the magnitude and memory development of RSV epitope-specific CD8 T cells differ by tissue and age at infection, suggesting that age-associated mucosal immune factors contribute to the development of RSV-specific memory T cells. This work was supported by funding through the NIAID R01AI154619 (A.M.W.M.). Supplemental support provided by USUHS Department of Pediatrics grant PED-86-3658 (A.M.W.M.).