CD4+CD8αlow T cells in rheumatoid arthritis are clonally expanded and dependent on co-stimulation.

OBJECTIVES: CD4+CD8+ T cells are increased in patients with rheumatoid arthritis (RA). They are not only associated with joint erosions in established disease, but are also present in the pre-clinical stages of RA. This study aims to further investigate their expansion in the context of T cell clonality in patients with RA, as well as their responsiveness to T cell targeted treatment. METHODS: Single-cell-(sc)RNA- and scTCR-sequencing data were used to determine co-receptor expression and T cell receptor sequences to assess clonality of CD4+CD8+ T cells in RA (n=3) patients and healthy controls (n=2). Peripheral CD4+CD8+ T cells and their subpopulations were measured in patients with RA (n=53), PsA (n=52) and healthy donors (n=50) using flow cytometry. In addition, changes in CD4+CD8+ T cell frequency were prospectively followed in RA patients receiving therapy with abatacept for 12 weeks. RESULTS: We observed an increase of CD4+ T cells expressing CD8α in RA patients, both in comparison to PsA patients and to healthy controls. Clonality analysis revealed, that these CD4+CD8αlow T cells are part of large T cell clones, which cluster separately from CD4+CD8- T cell clones in the scRNA-seq gene expression analysis. Treatment with abatacept significantly reduced the frequency of peripheral CD4+CD8αlow T cells, and this was linked to reduction in disease activity. CONCLUSION: In RA, clonal expansion of CD4+ T cell clones culminates in the emergence of peripheral CD4+CD8αlow T cells, which are associated with disease activity and diminished upon abatacept treatment, and which could contribute to disease pathogenesis.

Comparable CD8+ T-cell responses to SARS-CoV-2 vaccination in single-cell transcriptomics of recently allogeneic transplanted patients and healthy individuals.

Despite extensive research on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination responses in healthy individuals, there is comparatively little known beyond antibody titers and T-cell responses in the vulnerable cohort of patients after allogeneic hematopoietic stem cell transplantation (ASCT). In this study, we assessed the serological response and performed longitudinal multimodal analyses including T-cell functionality and single-cell RNA sequencing combined with T cell receptor (TCR)/B cell receptor (BCR) profiling in the context of BNT162b2 vaccination in ASCT patients. In addition, these data were compared to publicly available data sets of healthy vaccinees. Protective antibody titers were achieved in 40% of patients. We identified a distorted B- and T-cell distribution, a reduced TCR diversity, and increased levels of exhaustion marker expression as possible causes for the poorer vaccine response rates in ASCT patients. Immunoglobulin heavy chain gene rearrangement after vaccination proved to be highly variable in ASCT patients. Changes in TCRα and TCRß gene rearrangement after vaccination differed from patterns observed in healthy vaccinees. Crucially, ASCT patients elicited comparable proportions of SARS-CoV-2 vaccine-induced (VI) CD8+ T-cells, characterized by a distinct gene expression pattern that is associated with SARS-CoV-2 specificity in healthy individuals. Our study underlines the impaired immune system and thus the lower vaccine response rates in ASCT patients. However, since protective vaccine responses and VI CD8+ T-cells can be induced in part of ASCT patients, our data advocate early posttransplant vaccination due to the high risk of infection in this vulnerable group.

Associations of myeloid cells with cellular and humoral responses following vaccinations in patients with neuroimmunological diseases.

Disease-modifying therapies (DMTs) are widely used in neuroimmunological diseases such as multiple sclerosis (MS) and neuromyelitis optica spectrum disorder (NMOSD). Although these treatments are known to predispose patients to infections and affect their responses to vaccination, little is known about the impact of DMTs on the myeloid cell compartment. In this study, we use mass cytometry to examine DMT-associated changes in the innate immune system in untreated and treated patients with MS (n = 39) or NMOSD (n = 23). We also investigated the association between changes in myeloid cell phenotypes and longitudinal responsiveness to homologous primary, secondary, and tertiary SARS-CoV-2 mRNA vaccinations. Multiple DMT-associated myeloid cell clusters, in particular CD64+HLADRlow granulocytes, showed significant correlations with B and T cell responses induced by vaccination. Our findings suggest the potential role of myeloid cells in cellular and humoral responses following vaccination in DMT-treated patients with neuroimmunological diseases.

Single-cell clonal tracking of persistent T-cells in allogeneic hematopoietic stem cell transplantation.

The critical balance between intended and adverse effects in allogeneic hematopoietic stem cell transplantation (alloHSCT) depends on the fate of individual donor T-cells. To this end, we tracked αßT-cell clonotypes during stem cell mobilization treatment with granulocyte-colony stimulating factor (G-CSF) in healthy donors and for six months during immune reconstitution after transfer to transplant recipients. More than 250 αßT-cell clonotypes were tracked from donor to recipient. These clonotypes consisted almost exclusively of CD8+ effector memory T cells (CD8TEM), which exhibited a different transcriptional signature with enhanced effector and cytotoxic functions compared to other CD8TEM. Importantly, these distinct and persisting clonotypes could already be delineated in the donor. We confirmed these phenotypes on the protein level and their potential for selection from the graft. Thus, we identified a transcriptional signature associated with persistence and expansion of donor T-cell clonotypes after alloHSCT that may be exploited for personalized graft manipulation strategies in future studies.

Primary ChAdOx1 vaccination does not reactivate pre-existing, cross-reactive immunity.

Currently available COVID-19 vaccines include inactivated virus, live attenuated virus, mRNA-based, viral vectored and adjuvanted protein-subunit-based vaccines. All of them contain the spike glycoprotein as the main immunogen and result in reduced disease severity upon SARS-CoV-2 infection. While we and others have shown that mRNA-based vaccination reactivates pre-existing, cross-reactive immunity, the effect of vector vaccines in this regard is unknown. Here, we studied cellular and humoral responses in heterologous adenovirus-vector-based ChAdOx1 nCOV-19 (AZ; Vaxzeria, AstraZeneca) and mRNA-based BNT162b2 (BNT; Comirnaty, BioNTech/Pfizer) vaccination and compared it to a homologous BNT vaccination regimen. AZ primary vaccination did not lead to measurable reactivation of cross-reactive cellular and humoral immunity compared to BNT primary vaccination. Moreover, humoral immunity induced by primary vaccination with AZ displayed differences in linear spike peptide epitope coverage and a lack of anti-S2 IgG antibodies. Contrary to primary AZ vaccination, secondary vaccination with BNT reactivated pre-existing, cross-reactive immunity, comparable to homologous primary and secondary mRNA vaccination. While induced anti-S1 IgG antibody titers were higher after heterologous vaccination, induced CD4+ T cell responses were highest in homologous vaccinated. However, the overall TCR repertoire breadth was comparable between heterologous AZ-BNT-vaccinated and homologous BNT-BNT-vaccinated individuals, matching TCR repertoire breadths after SARS-CoV-2 infection, too. The reasons why AZ and BNT primary vaccination elicits different immune response patterns to essentially the same antigen, and the associated benefits and risks, need further investigation to inform vaccine and vaccination schedule development.

BCL11B depletion induces the development of highly cytotoxic innate T cells out of IL-15 stimulated peripheral blood αß CD8+ T cells.

BCL11B, an essential transcription factor for thymopoiesis, regulates also vital processes in post-thymic lymphocytes. Increased expression of BCL11B was recently correlated with the maturation of NK cells, whereas reduced BCL11B levels were observed in native and induced T cell subsets displaying NK cell features. We show that BCL11B-depleted CD8+ T cells stimulated with IL-15 acquired remarkable innate characteristics. These induced innate CD8+ (iiT8) cells expressed multiple innate receptors like NKp30, CD161, and CD16 as well as factors regulating migration and tissue homing while maintaining their T cell phenotype. The iiT8 cells effectively killed leukemic cells spontaneously and neuroblastoma spheroids in the presence of a tumor-specific monoclonal antibody mediated by CD16 receptor activation. These iiT8 cells integrate the innate natural killer cell activity with adaptive T cell longevity, promising an interesting therapeutic potential. Our study demonstrates that innate T cells, albeit of limited clinical applicability given their low frequency, can be efficiently generated from peripheral blood and applied for adoptive transfer, CAR therapy, or combined with therapeutic antibodies.

SARS-CoV-2 mRNA vaccinations fail to elicit humoral and cellular immune responses in patients with multiple sclerosis receiving fingolimod.

BACKGROUND: SARS-CoV-2 mRNA vaccination of healthy individuals is highly immunogenic and protective against severe COVID-19. However, there are limited data on how disease-modifying therapies (DMTs) alter SARS-CoV-2 mRNA vaccine immunogenicity in patients with autoimmune diseases. METHODS: As part of a prospective cohort study, we investigated the induction, stability and boosting of vaccine-specific antibodies, B cells and T cells in patients with multiple sclerosis (MS) on different DMTs after homologous primary, secondary and booster SARS-CoV-2 mRNA vaccinations. Of 126 patients with MS analysed, 105 received either anti-CD20-based B cell depletion (aCD20-BCD), fingolimod, interferon-ß, dimethyl fumarate, glatiramer acetate, teriflunomide or natalizumab, and 21 were untreated MS patients for comparison. RESULTS: In contrast to all other MS patients, and even after booster, most aCD20-BCD- and fingolimod-treated patients showed no to markedly reduced anti-S1 IgG, serum neutralising activity and a lack of receptor binding domain-specific and S2-specific B cells. Patients receiving fingolimod additionally lacked spike-reactive CD4+ T cell responses. The duration of fingolimod treatment, rather than peripheral blood B and T cell counts prior to vaccination, determined whether a humoral immune response was elicited. CONCLUSIONS: The lack of immunogenicity under long-term fingolimod treatment demonstrates that functional immune responses require not only immune cells themselves, but also access of these cells to the site of inoculation and their unimpeded movement. The absence of humoral and T cell responses suggests that fingolimod-treated patients with MS are at risk for severe SARS-CoV-2 infections despite booster vaccinations, which is highly relevant for clinical decision-making and adapted protective measures, particularly considering additional recently approved sphingosine-1-phosphate receptor antagonists for MS treatment.

Cutting Edge: Serum but Not Mucosal Antibody Responses Are Associated with Pre-Existing SARS-CoV-2 Spike Cross-Reactive CD4+ T Cells following BNT162b2 Vaccination in the Elderly.

Advanced age is a main risk factor for severe COVID-19. However, low vaccination efficacy and accelerated waning immunity have been reported in this age group. To elucidate age-related differences in immunogenicity, we analyzed human cellular, serological, and salivary SARS-CoV-2 spike glycoprotein-specific immune responses to the BNT162b2 COVID-19 vaccine in old (69-92 y) and middle-aged (24-57 y) vaccinees compared with natural infection (COVID-19 convalescents, 21-55 y of age). Serological humoral responses to vaccination excee-ded those of convalescents, but salivary anti-spike subunit 1 (S1) IgA and neutralizing capacity were less durable in vaccinees. In old vaccinees, we observed that pre-existing spike-specific CD4+ T cells are associated with efficient induction of anti-S1 IgG and neutralizing capacity in serum but not saliva. Our results suggest pre-existing SARS-CoV-2 cross-reactive CD4+ T cells as a predictor of an efficient COVID-19 vaccine-induced humoral immune response in old individuals.

Cross-reactive CD4+ T cells enhance SARS-CoV-2 immune responses upon infection and vaccination.

The functional relevance of preexisting cross-immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a subject of intense debate. Here, we show that human endemic coronavirus (HCoV)­reactive and SARS-CoV-2­cross-reactive CD4+ T cells are ubiquitous but decrease with age. We identified a universal immunodominant coronavirus-specific spike peptide (S816-830) and demonstrate that preexisting spike- and S816-830­reactive T cells were recruited into immune responses to SARS-CoV-2 infection and their frequency correlated with anti­SARS-CoV-2-S1-IgG antibodies. Spike­cross-reactive T cells were also activated after primary BNT162b2 COVID-19 messenger RNA vaccination and displayed kinetics similar to those of secondary immune responses. Our results highlight the functional contribution of preexisting spike­cross-reactive T cells in SARS-CoV-2 infection and vaccination. Cross-reactive immunity may account for the unexpectedly rapid induction of immunity after primary SARS-CoV-2 immunization and the high rate of asymptomatic or mild COVID-19 disease courses.

Rapid T-cell receptor interaction grouping with ting.

MOTIVATION: Clustering T-cell receptor repertoire (TCRR) sequences according to antigen specificity is challenging. The previously published tool GLIPH needs several days to weeks for clustering large repertoires, making its use impractical in larger studies. In addition, the methodology used in GLIPH suffers from shortcomings, including non-determinism, potential loss of significant antigen-specific sequences or inclusion of too many unspecific sequences. RESULTS: We present an algorithm for clustering TCRR sequences that scales efficiently to large repertoires. We clustered 36 real datasets with up to 62 000 unique CDR3ß sequences using both an implementation of our method called ting, GLIPH and its successor GLIPH2. While GLIPH required multiple weeks, ting only needed about one minute for the same task. GLIPH2 is comparably fast, but uses a different grouping paradigm. In addition, we found that in naïve repertoires, where no or very few antigen-specific CDR3 sequences or clusters should exist, our method indeed selects much fewer motifs and produces smaller clusters. AVAILABILITY AND IMPLEMENTATION: Our method has been implemented in Python as a tool called ting. It is available from GitHub (https://github.com/FelixMoelder/ting) or PyPI under the MIT license. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Analysis of peripheral inflammatory T cell subsets and their effector function in patients with Birdshot Retinochoroiditis.

Birdshot Retinochoroiditis (BSRC) is a progressive non-infectious intraocular inflammation that affects choroid and retina. Inflammatory processes have adverse effects on vision by affecting photoreceptor-bearing cells that do not regenerate. This study aimed at characterizing inflammatory CD4+ and CD8+ T cell subsets in the peripheral blood of active and inactive BSRCs. Furthermore, we correlated phenotypical and functional immunological analyses with clinical data. We observed a slight increase of terminally differentiated effector memory CD8+ T cells expressing CD45RA (TEMRA) in blood of inactive, compared to active BSRCs. Moreover, we identified a trend for a decreased population of TH2 cells and increased TH1 frequencies in active BSRCs, a typical sign of ongoing autoimmune processes. Functional assays demonstrated severe and overall impairment of effector function of both, CD4+ and CD8+ inflammatory T cells, which might reflect T cell exhaustion. Although the eye is the main site of inflammation in BSRC, we observed altered T cell subset compositions in the peripheral blood, dependent on the disease status. Our results indicate that T cells may play a major role in BSRC pathology, although our cohort size is too limited for definitve conclusions. Future studies with larger BSRCs have to be performed.

SLAMF7 and IL-6R define distinct cytotoxic versus helper memory CD8+ T cells.

The prevailing 'division of labor' concept in cellular immunity is that CD8+ T cells primarily utilize cytotoxic functions to kill target cells, while CD4+ T cells exert helper/inducer functions. Multiple subsets of CD4+ memory T cells have been characterized by distinct chemokine receptor expression. Here, we demonstrate that analogous CD8+ memory T-cell subsets exist, characterized by identical chemokine receptor expression signatures and controlled by similar generic programs. Among them, Tc2, Tc17 and Tc22 cells, in contrast to Tc1 and Tc17 + 1 cells, express IL-6R but not SLAMF7, completely lack cytotoxicity and instead display helper functions including CD40L expression. CD8+ helper T cells exhibit a unique TCR repertoire, express genes related to skin resident memory T cells (TRM) and are altered in the inflammatory skin disease psoriasis. Our findings reveal that the conventional view of CD4+ and CD8+ T cell capabilities and functions in human health and disease needs to be revised.

SARS-CoV-2-reactive T cells in healthy donors and patients with COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the rapidly unfolding coronavirus disease 2019 (COVID-19) pandemic1,2. Clinical manifestations of COVID-19 vary, ranging from asymptomatic infection to respiratory failure. The mechanisms that determine such variable outcomes remain unresolved. Here we investigated CD4+ T cells that are reactive against the spike glycoprotein of SARS-CoV-2 in the peripheral blood of patients with COVID-19 and SARS-CoV-2-unexposed healthy donors. We detected spike-reactive CD4+ T cells not only in 83% of patients with COVID-19 but also in 35% of healthy donors. Spike-reactive CD4+ T cells in healthy donors were primarily active against C-terminal epitopes in the spike protein, which show a higher homology to spike glycoproteins of human endemic coronaviruses, compared with N-terminal epitopes. Spike-protein-reactive T cell lines generated from SARS-CoV-2-naive healthy donors responded similarly to the C-terminal region of the spike proteins of the human endemic coronaviruses 229E and OC43, as well as that of SARS-CoV-2. This results indicate that spike-protein cross-reactive T cells are present, which were probably generated during previous encounters with endemic coronaviruses. The effect of pre-existing SARS-CoV-2 cross-reactive T cells on clinical outcomes remains to be determined in larger cohorts. However, the presence of spike-protein cross-reactive T cells in a considerable fraction of the general population may affect the dynamics of the current pandemic, and has important implications for the design and analysis of upcoming trials investigating COVID-19 vaccines.

T cell pathology in skin inflammation.

Forming the outer body barrier, our skin is permanently exposed to pathogens and environmental hazards. Therefore, skin diseases are among the most common disorders. In many of them, the immune system plays a crucial pathogenetic role. For didactic and therapeutic reasons, classification of such immune-mediated skin diseases according to the underlying dominant immune mechanism rather than to their clinical manifestation appears to be reasonable. Immune-mediated skin diseases may be mediated mainly by T cells, by the humoral immune system, or by uncontrolled unspecific inflammation. According to the involved T cell subpopulation, T cell-mediated diseases may be further subdivided into T1 cell-dominated (e.g., vitiligo), T2 cell-dominated (e.g., acute atopic dermatitis), T17/T22 cell-dominated (e.g., psoriasis), and Treg cell-dominated (e.g., melanoma) responses. Moreover, T cell-dependent and -independent responses may occur simultaneously in selected diseases (e.g., hidradenitis suppurativa). The effector mechanisms of the respective T cell subpopulations determine the molecular changes in the local tissue cells, leading to specific microscopic and macroscopic skin alterations. In this article, we show how the increasing knowledge of the T cell biology has been comprehensively translated into the pathogenetic understanding of respective model skin diseases and, based thereon, has revolutionized their daily clinical management.

CD40L expression by CD4+ but not CD8+ T cells regulates antiviral immune responses in acute LCMV infection in mice.

CD40-CD40 ligand (CD40L) signaling plays multiple indispensable roles in cellular and humoral immunity. Impaired memory T-cell responses in the absence of CD40L have been well documented, but the requirement of this interaction for efficient priming of CD8+ T cells especially under inflammatory conditions has been under debate. In contrast to previous publications, we report here that virus-specific CD8+ T-cell responses as well as viral clearance are affected not only in the memory but also in the effector phase in CD40L-/- mice infected with lymphocytic choriomeningitis virus (LCMV) Armstrong strain. Interestingly, a considerable part of the LCMV-specific effector and memory T cells consists of CD40L+ CD8+ T cells. However, deficiency of CD40L in CD8+ T cells did influence neither the quantity nor the quality of primary T-cell responses in LCMV infection. Virus-specific CD8+ T cells in conditional knockout mice, with a selective deletion of the CD40L in CD8+ T cells, were fully functional regarding cytokine production and efficient pathogen clearance. Thus, our results unambiguously demonstrate that while CD40L is critical to generate effective primary CD8+ T-cell responses also under inflammatory conditions, CD40L expression by CD8+ T cells themselves is dispensable in acute LCMV infection.