Mitochondrial Dysfunction Associates With Acute T Lymphocytopenia and Impaired Functionality in COVID-19 Patients

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection results in rapid T lymphocytopenia and functional impairment of T cells. The underlying mechanism, however, remains incompletely understood. In this study, we focused on characterizing the phenotype and kinetics of T-cell subsets with mitochondrial dysfunction (MD) by multicolor flow cytometry and investigating the association between MD and T-cell functionality. While 73.9% of study subjects displayed clinical lymphocytopenia upon hospital admission, a significant reduction of CD4 or CD8 T-cell frequency was found in all asymptomatic, symptomatic, and convalescent cases. CD4 and CD8 T cells with increased MD were found in both asymptomatic and symptomatic patients within the first week of symptom onset. Lower proportion of memory CD8 T cell with MD was found in severe patients than in mild ones at the stage of disease progression. Critically, the frequency of T cells with MD in symptomatic patients was preferentially associated with CD4 T-cell loss and CD8 T-cell hyperactivation, respectively. Patients bearing effector memory CD4 and CD8 T cells with the phenotype of high MD exhibited poorer T-cell responses upon either phorbol 12-myristate-13-acetate (PMA)/ionomycin or SARS-CoV-2 peptide stimulation than those with low MD. Our findings demonstrated an MD-associated mechanism underlying SARS-CoV-2-induced T lymphocytopenia and functional impairment during the acute phase of infection.

High-Sensitive TRBC1-Based Flow Cytometric Assessment of T-Cell Clonality in Tαβ-Large Granular Lymphocytic Leukemia

Flow cytometric (FCM) analysis of the constant region 1 of the T-cell receptor β chain (TRBC1) expression for assessing Tαβ-cell clonality has been recently validated. However, its utility for the diagnosis of clonality of T-large granular lymphocytic leukemia (T-LGLL) needs to be confirmed, since more mature Tαβ cells (i.e., T-LGL normal-counterpart) show broader TRBC1+/TRBC1− ratios vs. total Tαβ cells. We compared the distribution and absolute counts of TRBC1+ and TRBC1− Tαβ-LGL in blood containing polyclonal (n = 25) vs. clonal (n = 29) LGL. Overall, polyclonal TRBC1+ or TRBC1− Tαβ-LGL ranged between 0.36 and 571 cells/μL (3.2–91% TRBC1+ cells), whereas the clonal LGL cases showed between 51 and 11,678 cells/μL (<0.9% or >96% TRBC1+ cells). Among the distinct TCRVβ families, the CD28− effector-memory and terminal-effector polyclonal Tαβ cells ranged between 0 and 25 TRBC1+ or TRBC1− cells/μL and between 0 and 100% TRBC1+ cells, while clonal LGL ranged between 32 and 5515 TRBC1+ or TRBC1− cells/μL, representing <1.6% or >98% TRBC1+ cells. Our data support the utility of the TRBC1-FCM assay for detecting T-cell clonality in expansions of Tαβ-LGL suspected of T-LGLL based on altered percentages of TRBC1+ Tαβ cells. However, in the absence of lymphocytosis or in the case of TαβCD4-LGL expansion, the detection of increased absolute cell counts by the TRBC1-FCM assay for more accurately defined subpopulations of Tαβ-LGL-expressing individual TCRVβ families, allows the detection of T-cell clonality, even in the absence of phenotypic aberrations.

Cattle T Cell Phenotyping by an 8-Colour, 10-Parameter Panel

This multiplex staining panel was developed to differentiate cattle T cells into conventional (CD4 and CD8) and unconventional (γδ-TCR) subsets as well as their stage of differentiation and activation. The combination of CD45RO and CD62L allows the identification of naïve (TNaïve), central memory (TCM), effector memory (TEM) and terminal effector (TTE) T cells. Activated cattle T cells (TAV) can be identified by the cell surface expression of CD25. This panel was developed using cryopreserved cattle peripheral blood mononuclear cells (PBMCs) and tested on fresh as well as stimulated PBMCs. Therefore, this 8-colour, 10-parameter flow cytometry panel simultaneously identifies cattle TNaïve, TAV, TCM, TEM, TTE and γδ-TCR cells. This panel will improve our ability to examine T cell response to pathogens and vaccines in cattle including the potential to identify previously undescribed subpopulations. Furthermore, this panel can be readily optimised for other bovid species as many of these reagents are likely to cross react.

Microglia and infiltrating T-cells adopt long-term, age-specific, transcriptional changes after traumatic brain injury

Introduction: Traumatic brain injury (TBI) afflicts over 3 million Americans every year. Patients over 65 years of age suffer increased mortality as well as greater long-term neurocognitive and neuropsychiatric morbidity compared to younger adults. Microglia, the resident macrophages of the brain, are complicit in both. Our published and preliminary data have demonstrated a significant age-effect in which aged microglia are more prone to adopt a constitutively activated state associated with worse neurocognitive and neuropsychiatric outcomes. Therefore, we hypothesized that aged microglia would fail to return to a homeostatic state after TBI but instead adopt a long-term injury-associated state within the brain of aged mice as compared to young-adult mice after TBI. Methods: Young-adult (14-weeks) and aged (80-weeks) C57BL/6 mice underwent TBI via controlled cortical impact vs. sham injury. We utilized single-cell RNA sequencing to examine age-associated cellular responses after TBI. Four months post-TBI or sham injury, brains were harvested, and CD45+ cells (N=4,000 cells) were isolated via florescence-activated cell sorting. cDNA libraries were prepared via the 10x Genomics Chromium Single Cell 3' Reagent Kit, followed by sequencing on a HiSeq 4000 instrument. The raw data were processed using the Cell Ranger pipeline mapped to the mm10 mouse reference genome and Seurat following standard workflow. Seurat and GOrilla were used for downstream clustering, differential gene expression, and pathway analysis. All cell types were annotated using canonical markers and top expressed genes. ProjecTILs was additionally used to interpret T cell states. Results: Microglia from young-adult and aged mice have distinct transcriptional profiles pre-injury and markedly different transcriptional responses post-injury compared to young-adult mice. Pre-injury, aged mice demonstrated a disproportionate immune cell infiltration, including T cells, as compared to young-adult mice (aged versus young: 45.5% vs. 14.5%). Post-injury, the disparity was amplified with a proportional decrease in homeostatic microglia and greater increased infiltrating T cells compared to young-adult mice (Microglia: 27.5% vs. 71%; T cell: 45.5% vs. 4.5%). Of note, aged mice post-injury had a subpopulation of unique, age-specific, immune-inflammatory microglia resembling gene profiles of neurodegenerative disease-associated microglia (DAM) with enriched pathways involved in leukocyte recruitment and Alzheimer’s disease pathogenesis (FDR < 0.05). Contrastingly, post-injury, aged mice demonstrate a heterogenous T-cell infiltration with gene profiles corresponding to CD8 effector memory, CD8 native-like, CD4, and double-negative T cells (75.9%, 2.5%, 12.9%, and 8.6%, respectively) and enriched pathways including tau protein binding, macromolecule synthesis, and cytokine-mediated signaling pathways (FDR < 0.05). Conclusion: We hypothesized that aged microglia would fail to return to a homeostatic state after TBI and adopt a long-term, injury-associated state within the brain of aged mice as compared to young-adult mice after TBI. In particular, our data suggest an age-dependent reduction of homeostatic microglia post-injury yet an upregulation in a unique microglial subpopulation with a distinct immuno-inflammatory profile. Furthermore, aged subjects demonstrated a markedly disproportionate inflammatory infiltrate after TBI predominated by the presence of CD8+ T cells. In addition, post-injury, brain trauma reorganized the T cell milieu, especially CD8 effector memory T cells, via upregulating genes associated with macromolecule biosynthesis process and negative regulation of neuronal death, possibly linking TBI with its long-term sequelae and complications. Taken together, our data showed that age-specific gene signature changes in the T-cell infiltrates and the microglial subpopulation contributes to increased vulnerability of the aged brain to TBI. Age should be an a priori consideration in future TBI clinical trials.

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

Nicotinamide Inhibits T Cell Exhaustion and Increases Differentiation of CD8 Effector T Cells

One of the limitations of immunotherapy is the development of a state referred to as T cell exhaustion (TEx) whereby T cells express inhibitory receptors (IRs) and lose production of effectors involved in killing of their targets. In the present studies we have used the repeated stimulation model with anti CD3 and anti CD28 to understand the factors involved in TEx development and treatments that may reduce changes of TEx. The results show that addition of nicotinamide (NAM) involved in energy supply to cells prevented the development of inhibitory receptors (IRs). This was particularly evident for the IRs CD39, TIM3, and to a lesser extent LAG3 and PD1 expression. NAM also prevented the inhibition of IL-2 and TNFα expression in TEx and induced differentiation of CD4+ and CD8 T cells to effector memory and terminal effector T cells. The present results showed that effects of NAM were linked to regulation of reactive oxygen species (ROS) consistent with previous studies implicating ROS in upregulation of TOX transcription factors that induce TEx. These effects of NAM in reducing changes of TEx and in increasing the differentiation of T cells to effector states appears to have important implications for the use of NAM supplements in immunotherapy against cancers and viral infections and require further exploration in vivo.

Persistence of immunity and impact of a third (booster) dose of an inactivated SARS-CoV-2 vaccine, BBV152; a phase 2, double-blind, randomised controlled trial

Background: Neutralising antibody responses to SARS-CoV-2 vaccines have been reported to decline within 6 months of vaccination, particularly against Variants of Concern (VOC). We assessed the immunogenicity and safety of a booster dose of BBV152 administered 6 months after the second of a two-dose primary vaccination series. Methods: In an ongoing phase 2 trial (ClinicalTrials.gov: NCT04471519) the protocol was amended after six months to re-consent and randomise 184 previously vaccinated participants to receive a third dose of vaccine or placebo on Day 215. The primary outcome was to measure neutralising antibody titres by plaque-reduction neutralisation test (PRNT50) four weeks after the booster; safety as serious adverse events (SAE) was the key secondary outcome. Findings: Four weeks after a second BBV152 vaccination geometric mean titres (GMTs) of neutralising antibodies were 197.0 PRNT50 (95% CI: 155.6,249.4); this level declined to 23.9 PRNT50 (14.0,40.6) six months later, with a seroconversion rate of 75.4% (95% CI: 68.4,81.6). Four weeks after booster vaccination the GMT increased on Day 243 to 746.6 PRNT50 (514.9,1081) compared with 100.7 PRNT50 (43.6,232.6) in the placebo group. Corresponding seroconversion rates were 98.7% (92.8,99.9) and 79.8% (69.6,87.8). Increased titres in the placebo group were attributed to natural infection as the study was conducted during the second wave of COVID-19 in India. PRNT50 titres against the SARS-CoV-2 variants increased Alpha (32.6 fold), Beta (161.0 fold), Delta (264.7 fold), and Delta plus (174.2 fold) after the booster vaccination. We found that vaccine induces both memory B and T cells with a distinct AIM+ specific CD4+T central and effector memory phenotype, including CD8+ TEMRA phenotype. Reactogenicity after vaccine and placebo was minimal and comparable, and no SAEs were reported. Interpretation: Six months after a two dose BBV152 vaccination series cell mediated immunity and neutralising antibodies to both homologous (D614G) and heterologous strains (Alpha, Beta, Delta and Delta plus) persisted above baseline, although the magnitude of the responses had declined. Neutralising antibodies against homologous and heterologous SARS-CoV-2 variants increased 19 to 97 fold after a third vaccination. Booster BBV152 vaccination is safe and may be necessary to ensure persistent immunity to prevent breakthrough infections.

Cellular and Humoral Responses to SARS-CoV-2 Vaccination in Immunosuppressed Patients

Objective: SARS-CoV-2 vaccinations have demonstrated vaccine-immunogenicity in healthy volunteers, however, efficacy in immunosuppressed patients is less well characterised. There is an urgent need to address the impact of immunosuppression on vaccine immunogenicity. Methods: Serological, T-cell ELISpot, cytokines and immunophenotyping assays were used to assess vaccine responses (either BNT162b2 mRNA or ChAdOx1 nCoV-19) in double-vaccinated patients receiving immunosuppression for renal transplants or haematological malignancies (n=13). Immunological responses in immunosuppressed patients (VACC-IS) were compared to immunocompetent vaccinated (VACC-IC, n=12), unvaccinated (UNVACC, n=11) and infection-naïve unvaccinated (HC, n=3) cohorts. All participants, except HC, had prior COVID-19 infection. Results: T-cell responses were identical between VACC-IS and VACC-IC (92%) to spike-peptide (S) stimulation. UNVACC had the highest T-cell non-responders (n=3), whereas VACC-IC and VACC-IS both had one T-cell non-responder. No significant differences in humoral responses were observed between VACC-IC and VACC-IS, with 92% (12/13) of VACC-IS patients demonstrating seropositivity. One VACC-IS failed to seroconvert, however had detectable T-cell responses. All VACC-IC participants were seropositive for anti-spike antibodies. VACC-IS and VACC-IC participants elicited strong Th1 cytokine response with immunodominance towards S-peptide. Differences in T-cell immunophenotyping were seen between VACC-IS and VACC-IC, with lower CD8+ activation and T-effector memory phenotype observed in VACC-IS. Conclusion: SARS-CoV-2 vaccines are immunogenic in patients receiving immunosuppressive therapy, with responses comparable to vaccinated immunocompetent participants. Lower humoral responses were seen in patients treated with B-cell depleting therapeutics, but with preserved T-cell responses. We suggest further work to correlate both protective immunity and longevity of these responses in both healthy and immunosuppressed patients.

Acazicolcept (ALPN-101), a dual ICOS/CD28 antagonist, demonstrates efficacy in systemic sclerosis preclinical mouse models

Background Uncontrolled immune response with T cell activation has a key role in the pathogenesis of systemic sclerosis (SSc), a disorder that is characterized by generalized fibrosis affecting particularly the lungs and skin. Costimulatory molecules are key players during immune activation, and recent evidence supports a role of CD28 and ICOS in the development of fibrosis. We herein investigated the efficacy of acazicolcept (ALPN-101), a dual ICOS/CD28 antagonist, in two complementary SSc-related mouse models recapitulating skin fibrosis, interstitial lung disease, and pulmonary hypertension. Methods Expression of circulating soluble ICOS and skin-expressed ICOS was investigated in SSc patients. Thereafter, acazicolcept was evaluated in the hypochlorous acid (HOCL)-induced dermal fibrosis mouse model and in the Fra-2 transgenic (Tg) mouse model. In each model, mice received 400 μg of acazicolcept or a molar-matched dose of an Fc control protein twice a week for 6 weeks. After 6 weeks, skin and lung were evaluated. Results ICOS was significantly increased in the sera from SSc patients and in SSc skin biopsies as compared to samples from healthy controls. Similar body weight changes were observed between Fc control and acazicolcept groups in both HOCL and Fra-2 Tg mice suggesting a good tolerance of acazicolcept treatment. In mice challenged with HOCL, acazicolcept induced a significant decrease in dermal thickness, collagen content, myofibroblast number, and inflammatory infiltrates characterized by B cells, T cells, neutrophils, and macrophages. In the Fra-2 Tg mouse model, acazicolcept treatment reduced lung collagen content, fibrillar collagen, histological fibrosis score, and right ventricular systolic pressure (RVSP). A reduction in frequency of CD4+ and T effector memory cells and an increase in the percentage of CD4+ T naïve cells in spleen and lung of acazicolcept-treated Fra-2 Tg mice was observed as compared to Fc control-treated Fra-2 Tg mice. Moreover, acazicolcept reduced CD69 and PD-1 expression on CD4+ T cells from the spleen and the lung. Target engagement by acazicolcept was demonstrated by blockade of CD28 and ICOS detection by flow cytometry in treated mice. Conclusions Our results confirm the importance of costimulatory molecules in inflammatory-driven fibrosis. Our data highlight a key role of ICOS and CD28 in SSc. Using complementary models, we demonstrated that dual ICOS/CD28 blockade by acazicolcept decreased dermal and pulmonary fibrosis and alleviated pulmonary hypertension. These results pave the way for subsequent research on ICOS/CD28-targeted therapies.

Vaccines Elicit Highly Cross-Reactive Cellular Immunity to the SARS-CoV-2 Omicron Variant

The highly mutated SARS-CoV-2 Omicron (B.1.1.529) variant has been shown to evade a substantial fraction of neutralizing antibody responses elicited by current vaccines that encode the WA1/2020 Spike immunogen, resulting in increased breakthrough infections and reduced vaccine efficacy. Cellular immune responses, particularly CD8+ T cell responses, are likely critical for protection against severe SARS-CoV-2 disease. Here we show that cellular immunity induced by current SARS-CoV-2 vaccines is highly cross-reactive against the SARS-CoV-2 Omicron variant. Individuals who received Ad26.COV2.S or BNT162b2 vaccines demonstrated durable CD8+ and CD4+ T cell responses that showed extensive cross-reactivity against both the Delta and Omicron variants, including in central and effector memory cellular subpopulations. Median Omicron-specific CD8+ T cell responses were 82-84% of WA1/2020-specific CD8+ T cell responses. These data suggest that current vaccines may provide considerable protection against severe disease with the SARS-CoV-2 Omicron variant despite the substantial reduction of neutralizing antibody responses.