Intermittent calorie restriction alters T cell subsets and metabolic markers in people with multiple sclerosis

Background: Intermittent fasting or calorie restriction (CR) diets provide anti-inflammatory and neuroprotective advantages in models of multiple sclerosis (MS); data in humans are sparse. Methods: We conducted a randomized-controlled feeding study of different CR diets in 36 people with MS over 8 weeks. Patients were randomized to receive either: a daily CR diet (22% reduction in calories, 7 days/week), an intermittent CR diet (75% reduction, 2 days/week; 100%, 5 days/week), or a weight-stable diet (100%, 7 days/week). Untargeted metabolomics was performed on plasma samples at weeks 0, 4 and 8 at Metabolon Inc (Durham, NC). Flow cytometry of cryopreserved peripheral blood mononuclear cells at weeks 0 and 8 were used to identify CD4+ and CD8+ T cell subsets including effector memory, central memory, and naïve cells. Results: 31 (86%) completed the trial. Over time, individuals randomized to intermittent CR had significant reductions in CD4+CM -4.87%; 95%CI: -8.59%, -1.15%; p=0.01), CD4+EM (-3.82%; 95%CI: -7.44, -0.21; p=0.04), and CD8+EM (-6.96%; 95%CI: -11.96, -1.97; p=0.006) with proportional increases in naïve subsets (CD4+Naïve: 5.81%; 95%CI: -0.01, 11.63%; p=0.05; CD8+Naïve: 10.11%; 95%CI: 3.30, 16.92%; p=0.006). No changes were observed for daily CR or weight-stable diets. Larger within-person changes in lysophospholipid and lysoplasmalogen metabolites in intermittent CR were associated with larger reductions in memory T cell subsets and larger increases in naïve T cell subsets. Conclusions: In people with MS, an intermittent CR diet was associated with reduction in memory T cell subsets. The observed changes may be mediated by changes in specific classes of lipid metabolites. Trial Registration: This study is registered on Clinicaltrials.gov with identifier NCT02647502. Funding: National MS Society, NIH, Johns Hopkins Catalyst Award

Pre-Existing Hypertension Is Related with Disproportions in T-Lymphocytes in Older Age

Age-related immune deficiencies increase the risk of comorbidities and mortality. This study evaluated immunosenescence patterns by flow cytometry of naïve and memory T cell subpopulations and the immune risk profile (IRP), expressed as the CD4/CD8 ratio and IgG CMV related to comorbidities. The disproportions in naïve and memory T cells, as well as in the CD4/CD8 ratio, were analysed in 99 elderly individuals (71.9 ± 5.8 years) diagnosed with hypertension (n = 51) or without hypertension (n = 48), using an eight-parameter flow cytometer. The percentage of CD4+ T lymphocytes was significantly higher in hypertensive than other individuals independently from CMV infections, with approximately 34% having CD4/CD8 > 2.5, and only 4% of the elderly with hypertension having CD4/CD8 < 1. The elderly with a normal BMI demonstrated the CD4/CD8 ratio ≥ 1 or ≤ 2.5, while overweight and obese participants showed a tendency to an inverted CD4/CD8 ratio. CD4/CD8 ratio increased gradually with age and reached the highest values in participants aged >75 years. The decline in CD4+ naïve T lymphocytes was more prominent in IgG CMV+ men when compared to IgG CMV+ women. The changes in naïve and memory T lymphocyte population, CD4/CD8, and CMV seropositivity included in IRP are important markers of health status in the elderly that are dependent on hypertension.

Waning immune responses against SARS-CoV-2 among vaccinees in Hong Kong

Background: Nearly 4 billion doses of the BioNTech-mRNA and Sinovac-inactivated vaccines have been administrated globally, yet different vaccine-induced immunity against SARS-CoV-2 variants of concern (VOCs) remain incompletely investigated. Methods: We compare the immunogenicity and durability of these two vaccines among fully vaccinated Hong Kong people. Findings: Standard BioNTech and Sinovac vaccinations were tolerated and induced neutralizing antibody (NAb) (100% and 85.7%) and spike-specific CD4 T cell responses (96.7% and 82.1%), respectively. The geometric mean NAb IC 50 and median frequencies of reactive CD4 subsets were consistently lower among Sinovacvaccinees than BioNTech-vaccinees. Against VOCs, NAb response rate and geometric mean IC 50 against B1.351 and B.1.617.2 were significantly lower for Sinovac (14.3%, 15 and 50%, 23.2) than BioNTech (79.4%, 107 and 94.1%, 131). Three months after vaccinations, NAbs to VOCs dropped near to detection limit, along with waning memory T cell responses, mainly among Sinovac-vaccinees. Interpretation: Our results indicate that Sinovac-vaccinees may face higher risk to pandemic VOCs breakthrough infection.

Comparison of the Development of SARS-Coronavirus-2-Specific Cellular Immunity, and Central Memory CD4+ T-Cell Responses Following Infection versus Vaccination

Memory T-cell responses following infection with coronaviruses are reportedly long-lived and provide long-term protection against severe disease. Whether vaccination induces similar long-lived responses is not yet clear since, to date, there are limited data comparing memory CD4+ T-cell responses induced after SARS-CoV-2 infection versus following vaccination with BioNTech/Pfizer BNT162b2. We compared T-cell immune responses over time after infection or vaccination using ELISpot, and memory CD4+ T-cell responses three months after infection/vaccination using activation-induced marker flow cytometric assays. Levels of cytokine-producing T-cells were remarkably stable between three and twelve months after infection, and were comparable to IFNγ+ and IFNγ+IL-2+ T-cell responses but lower than IL-2+ T-cell responses at three months after vaccination. Consistent with this finding, vaccination and infection elicited comparable levels of SARS-CoV-2 specific CD4+ T-cells after three months in addition to comparable proportions of specific central memory CD4+ T-cells. By contrast, the proportions of specific effector memory CD4+ T-cells were significantly lower, whereas specific effector CD4+ T-cells were higher after infection than after vaccination. Our results suggest that T-cell responses—as measured by cytokine expression—and the frequencies of SARS-CoV-2-specific central memory CD4+T-cells—indicative of the formation of the long-lived memory T-cell compartment—are comparably induced after infection and vaccination.

T cell-intrinsic vitamin A metabolism and its signaling are targets for memory T cell-based cancer immunotherapy

Memory T cells play an essential role in infectious and tumor immunity. Vitamin A metabolites such as retinoic acid are immune modulators, but the role of vitamin A metabolism in memory T-cell differentiation is unclear. In this study, we identified retinol dehydrogenase 10 (Rdh10), which metabolizes vitamin A to retinal (RAL), as a key molecule for regulating T cell differentiation. T cell-specific Rdh10 deficiency enhanced memory T-cell formation through blocking RAL production in infection model. Epigenetic profiling revealed that retinoic acid receptor (RAR) signaling activated by vitamin A metabolites induced comprehensive epigenetic repression of memory T cell-associated genes, including TCF7, thereby promoting effector T-cell differentiation. Importantly, memory T cells generated by Rdh10 deficiency and blocking RAR signaling elicited potent anti-tumor responses in adoptive T-cell transfer setting. Thus, T cell differentiation is regulated by vitamin A metabolism and its signaling, which should be novel targets for memory T cell-based cancer immunotherapy.

Results of Hematopoietic Stem Cells Transplantation with Tcrαβ and CD19-Depletion from Matched Related Donors and Infusions of CD45RA Depleted Donor Lymphocytes in Pediatric Severe Aplastic Anemia

Introduction HSCT from matched family donors results in most favorable outcomes among children with severe aplastic anemia (SAA). Despite overall success, morbidity, associated with acute and chronic graft-versus-host disease (GVHD) is not completely prevented with current standard of pharmacologic prophylaxis. Depletion of ab T cells from the graft prevents GVHD, and improves outcome of hematopoietic stem cell transplantation from haploidentical donors, while infusions of donor memory lymphocytes (mDLI) (CD45RA-depleted) are able to transfer pathogen-specific immunity without the risk of GVHD. We evaluated the outcomes of ab T cell depletion and add-back of intermediate-dose mDLI among the pediatric SAA recipients of matched related grafts. Materials and methods A total of 16 children with SAA (8 female, 8 male, median age 10,9 y) underwent allogenic HSCT from matched family donors (MFD) between february 2015 and may 2021. For 15 (94%) pts it was the first allo HSCT, for 1 pts it was the second HSCT. TCR αβ+/CD19+ depletion of HSCT with CliniMACS technology was implemented in all cases. The median dose of CD34+ cells was 7,1 x10 6/kg (range 2,6-13), αβ T cells - 28x10 3/kg (range 5,6-184). All pts received an additional injection of memory T-cell (CD45RA-depleted) on day 0 at 1 million T cells per kg. All patients received cyclophosphamide at 100 mg/kg, fludarabine at 100 mg/m 2, rituximab 100mg/m 2 and serotherapy with either rabbit ATG at 5 mg/kg (n-2) or horse ATG at 100 mg/kg (n-14). Post-transplant GVHD prophylaxis included calcineurin (CNI)-based regimen and abatacept 10mg/kg on days -1, +7, +14 and +28. All pts received a graft from a 10/10 HLA-matched sibling. Median time of follow-up for survivors was 1,1 years (range: 0.14 - 6.38). Results Primary engraftment was achieved in all evaluable patients (100%) with full donor chimerism, and the median time to neutrophil and platelet recovery was 11 (10-20) and 14 (11-20) days, respectively. One patient had aGVHD grade I, there were no incidence of grade II-IV aGVHD and TRM. Event-free and overall survival were 100%. CMV viremia was detected among two patients after a median of 40 (35-73) days after HSCT. No cases of ADV and Epstein-Barr virus (EBV) viremia and EBV disease were recorded. The median recovery of T cells on day+60 was 0,26 (0,04-0,9). Conclusion ab T cell-depleted transplantation with intermediate dose memory T cell add-back definitively prevents GVHD and provides a platform for safe HSCT from matched family donors in patients with SAA. Disclosures Maschan: Miltenyi Biotec: Speakers Bureau.

Duvelisib Promotes Mitochondrial Fusion and Epigenetic Reprogramming to Drive Therapeutic T Cell Persistence and Function

Chronic lymphocytic leukemia (CLL), a cancer of B-lymphocytes, is the most common leukemia in adults. While current frontline therapies for CLL, such as ibrutinib or combination venetoclax and obintuzumab, have significantly improved clinical outcome for patients with treatment naïve CLL and relapsed and refractory CLL (RR-CLL), complete response (CR) rates for RR-CLL patients on ibrutinib remain between 5-14% and 42% for patients on venetoclax and obintuzumab (1, 2). With the advent of chimeric antigen receptor T cells (CART), CR for RR-CLL have increased to around 26-29%, yet this is in sharp contrast to the 70-93% CR achieved in acute lymphocytic leukemia (2). This discrepancy in response is in part due to the inherently immunosuppressive nature of CLL, such that CLL patients are significantly deficient in CD8 co-receptor expressing (CD8+) T cells, including stem cell-like memory T (Tscm) and central memory T (Tcm) cells (2). As the prevalence of Tscm and Tcm cell populations is directly correlated to the in vivo persistence and efficacy of CART, elucidating translatable mechanisms to selectively expand Tscm and Tcm from CLL patients is key to improving the efficacy CART cell therapy for CLL patients. Memory T cell activation, differentiation, and maintenance are processes that are tightly regulated by mitochondrial fusion, fatty acid oxidation, and oxidative phosphorylation (OXPHOS) (3). Moreover, enforcing T cell mitochondrial fusion improves CART cell efficacy against solid tumors (4). As metabolism plays an important role in memory T cell biology, identifying key metabolic pathways that can be targeted ex vivo during CART expansion is of particular interest. To that end, we have shown that dual inhibition of Phosphoinositide 3-Kinase (PI3K) δ/γ isoforms with IPI-145 (duvelisib) during ex vivo T cell manufacturing, preferentially expands CD8+ T cells, including Tscm and Tcm, as well as improves the in vivo persistence (Figure 1A) and cytotoxicity (Figure 1B) of CD19-targeted CART (CD19-CART) (5). To investigate the role of mitochondrial dynamics during ex vivo expansion of duvelisib treated T cell cultures, we stimulated CLL patient-derived T cells with anti-CD3/CD28 beads, re-stimulated T cells on day 9, and harvested T cell cultures on day 15. Immunoblot analysis of day 15 samples indicates that ex vivo duvelisib treatment of CLL patient T cells increases expression of key mitochondrial fusion proteins, mitofusins 1 and 2 (MFN1/2), and decreases serine 637 phosphorylation of mitochondrial fission protein, DRP1, without coincident upregulation of the master regulator of mitochondrial biogenesis, PPARG coactivator 1 alpha (Figure 2A). In addition, duvelisib increased the expression of sirtuins 1 and 3 (SIRT1/3), which have known roles in the post-translational activation of MFN1/2, as well as other epigenetic regulators of memory T cell development and persistence, including FOXO1, TCF1/7, and ID3 (Figure 2B). Taken together, these data suggest that duvelisib promotes mitochondrial fusion and epigenetic reprogramming of T cells during ex vivo expansion. To further interrogate the role of PI3K δ/γ inhibition in mitochondrial dynamics and metabolism, we analyzed T cell cultures following 15 days of duvelisib treatment using a series of extracellular flux and transmission electron microscopy (TEM) experiments. Duvelisib promotes an increase in the total mitochondrial cross-sectional area of both un-transduced and CD19-CAR transduced T cells (Figure 3) and maintains basal, coupled, and spare respiratory capacity of un-transduced T cells on day 15 of expansion (Figure 4). In summary, our data suggest that mitochondrial fusion through MFN1/2 and epigenetic reprogramming facilitate PI3K δ/γ inhibition-mediated ex vivo T cell expansion, where the SIRT1/3-MFN1/2 axis serves as a potential intersection between mitochondrial fusion and epigenetic reprogramming. Figure 1 Figure 1. Disclosures Waller: Verastem Oncology: Consultancy, Research Funding; Cambium Oncology: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company.

645 Single-cell transcriptional and clonal characterization of CD4+ T cells across tissues in long-term melanoma survivors

BackgroundMelanoma patients who develop immunotherapy-related adverse events often have durable responses to treatment. We previously identified that long-term melanoma survivors presenting with the autoimmune adverse event, vitiligo, developed long lived CD8+ resident memory T cell (TRM) responses in skin and tumor with circulating memory T cell (TCIRC) clonal counterparts in blood.1 Despite the focus on CD8+ T cells in prior studies, CD4+ T cell features remain largely in the background.MethodsUsing the same patient cohort, we performed parallel single-cell RNA sequencing (scRNAseq) and single-cell TCR sequencing (scTCRseq) on CD4+ T cells sorted from matched skin, tumor, and blood using the 10X Genomics platform. The UMI counts-based gene expression matrix was processed using the R package Seurat (v.3.0).ResultsEleven distinct CD4+ T cell clusters were identified. The FOXP3 expressing regulatory T cell (Treg) cluster was comprised of cells from skin, tumor, and blood, and could be further sub-clustered into 3 distinct populations with one having transcripts associated with Treg activation. Of the T helper-like clusters, we identified subsets with transcripts associated with cytotoxicity (GZMA, GNLY, CX3CR1; TCYTO), exhaustion (PDCD1, HAVCR2, TOX; TEX), and three clusters that were excluded from blood with clear resident memory characteristics (high CD69, low KLF2, S1PR1). These three clusters were differentiated by expression of IL2 (TRM-IL2); ID2 and CD40LG (TRM-ACTIVATED) and CD28 (TRM-CD28). Paired scTCRseq revealed a high level of clonal overlap between the TCYTO and the TRM-ACTIVATED clusters, with RNA velocity analysis supporting a potential differentiation trajectory from TRM-ACTIVATED to TCYTO. Integrating our previously published CD8+ TRM and TCIRC profiles, we identified a core TRM signature and core TCIRC signature from both the CD4+ and CD8+ TRM and TCIRC cells, respectively, in melanoma patients. The core TRM signature predicted better overall survival of advanced melanoma patients in TCGA, while the core TCIRC signature did not.ConclusionsThis study supports the crucial anti-tumor role of TRM in cancer patients and extends this important observation to CD4+ T cells.ReferenceHan JC, Zhao YD, Shirai K, Molodtsov A, Kolling FW, Fisher JL, Zhang PS, Yan SF, Searles TG, Bader JM, Gui J, Cheng C, Ernstoff MS, Turk MJ, Angeles CV. Resident and circulating memory T cells persist for years in melanoma patients with durable responses to immunotherapy. Nature Cancer 2021;2(3).Ethics ApprovalIRB-approved written informed consent was obtained from patients with advanced melanoma, to perform skin and tumor biopsies, draw blood, and to access historical banked tissue and blood samples for analysis. All human studies were performed in accordance with ethical regulation, and pre-approved by the Committee for the Protection of Human Subjects at Dartmouth-Hitchcock Medical Center IRB (#00029821).