Functional SARS-CoV-2 cross-reactive CD4+ T cells established in early childhood decline with age.

Pre-existing SARS-CoV-2-reactive T cells have been identified in SARS-CoV-2-unexposed individuals, potentially modulating COVID-19 and vaccination outcomes. Here, we provide evidence that functional cross-reactive memory CD4+ T cell immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is established in early childhood, mirroring early seroconversion with seasonal human coronavirus OC43. Humoral and cellular immune responses against OC43 and SARS-CoV-2 were assessed in SARS-CoV-2-unexposed children (paired samples at age two and six) and adults (age 26 to 83). Pre-existing SARS-CoV-2-reactive CD4+ T cell responses targeting spike, nucleocapsid, and membrane were closely linked to the frequency of OC43-specific memory CD4+ T cells in childhood. The functional quality of the cross-reactive memory CD4+ T cell responses targeting SARS-CoV-2 spike, but not nucleocapsid, paralleled OC43-specific T cell responses. OC43-specific antibodies were prevalent already at age two. However, they did not increase further with age, contrasting with the antibody magnitudes against HKU1 (β-coronavirus), 229E and NL63 (α-coronaviruses), rhinovirus, Epstein-Barr virus (EBV), and influenza virus, which increased after age two. The quality of the memory CD4+ T cell responses peaked at age six and subsequently declined with age, with diminished expression of interferon (IFN)-γ, interleukin (IL)-2, tumor necrosis factor (TNF), and CD38 in late adulthood. Age-dependent qualitative differences in the pre-existing SARS-CoV-2-reactive T cell responses may reflect the ability of the host to control coronavirus infections and respond to vaccination

Robust T cell immunity in convalescent individuals with asymptomatic or mild COVID-19

SARS-CoV-2-specific memory T cells will likely prove critical for long-term immune protection against COVID-19. Here, we systematically mapped the functional and phenotypic landscape of SARS-CoV-2-specific T cell responses in unexposed individuals, exposed family members, and individuals with acute or convalescent COVID-19. Acute-phase SARS-CoV-2-specific T cells displayed a highly activated cytotoxic phenotype that correlated with various clinical markers of disease severity, whereas convalescent-phase SARS-CoV-2-specific T cells were polyfunctional and displayed a stem-like memory phenotype. Importantly, SARS-CoV-2-specific T cells were detectable in antibody-seronegative exposed family members and convalescent individuals with a history of asymptomatic and mild COVID-19. Our collective dataset shows that SARS-CoV-2 elicits broadly directed and functionally replete memory T cell responses, suggesting that natural exposure or infection may prevent recurrent episodes of severe COVID-19

Immune maturation in early childhood and the influence of herpesvirus infections

The quality of immune responses develops from birth into adulthood and in the context of the host microbial environment. The aim of this work was to study immune maturation during childhood, and how this process can be affected by the common herpesviruses; Epstein-Barr virus (EBV) and cytomegalovirus (CMV). In paper I we studied monocytes, an important cell type for immunity in the newborn. We showed that the neonatal monocyte subsets exist in similar frequencies as adult subsets, and have a potent capacity for pro-inflammatory cytokine production. In paper II, III and IV we studied the effects of EBV and CMV infections on immune cell function in children. In paper II we found that monocyte-induced NK-cell production of IFN-γ, and plasma IFN-γ levels, were decreased in 2-year old EBV- and/or CMV-seropositive children and mostly so in co-infected children. In paper III we found that in 5-year old children, EBV and CMV co-infection was associated with the highest levels of differentiated NKG2C+ NK cells. CMV+ children had higher plasma IFN-γ and IL-15 levels and higher NK-cell cytotoxic capacity. In vitro PBMC systems showed elevated frequencies of NKG2C+ NK cells in the presence of EBV-infected cells. In paper IV we showed that a child’s age and subsequent capacity for anti-viral cytokine production affects in vitro EBV infection in terms of B-cell proliferation and B-cell acquisition of memory phenotype. PBMC from CMV+ children had lower EBV-induced accumulation of switched memory B cells, which was connected to high prevalence of CD57+CD8+ T cells and IFN-γ production. Taken together, this thesis work shows that monocyte subsets at birth can give potent functional responses and that latency with EBV and CMV has a significant effect on the differentiation process and functional capacity of anti-viral effector cells during childhood. This in turn could affect responses to related or unrelated infections or even to non-invasive antigens such as allergens.At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.</p

Deciphering natural killer cell homeostasis

Natural killer (NK) cells have a central role within the innate immune system, eliminating virally infected, foreign and transformed cells through their natural cytotoxic capacity. Release of their cytotoxic granules is tightly controlled through the balance of a large repertoire of inhibitory and activating receptors, and it is the unique combination of these receptors expressed by individual cells that confers immense diversity both in phenotype and functionality. The diverse, yet unique, NK cell repertoire within an individual is surprisingly stable over time considering the constant renewal of these cells at steady state. Here we give an overview of NK cell differentiation and discuss metabolic requirements, intra-lineage plasticity and transcriptional reprogramming during IL-15-driven homeostatic proliferation. New insights into the regulation of NK cell differentiation and homeostasis could pave the way for the successful implementation of NK cell-based immunotherapy against cancer

Herpesvirus seropositivity in childhood associates with decreased monocyte-induced NK-cell IFN-γ production

EBV infection is inversely associated with IgE sensitization in children, and this association is further enhanced by CMV coinfection. In mice, herpesvirus latency causes systemic innate activation and protection from bacterial coinfection, implying the importance of herpesviruses in skewing immune responses during latent infection. Early control of viral infections depends on IFN- release by NK cells, which generally requires the presence of accessory cells. We investigated IFN- production by NK cells in PBMCs from children seropositive (SP) for EBV alone, for both EBV and CMV, or seronegative for both viruses. The ability of classical (CD14++CD16–) and proinflammatory (CD14+CD16+) monocytes to induce autologous NK cell IFN- was studied by coculture experiments with enriched CD3–CD56+ cells. Transwell experiments were used to evaluate how monocytes interact with NK cells to induce IFN- synthesis. SP children had a significantly reduced proportion of IFN-+ NK cells and cognate intracellular IFN- levels, which was more pronounced in CMV-coinfected subjects. Also, resting PBMCs of SP children displayed lower proportions of proinflammatory monocytes. IFN- production by NK cells was dependent on interactions with monocytes, with the proinflammatory subset inducing the highest IFN-. Finally, SP children had markedly lower levels of plasma IFN-, concurrent with in vitro findings. Herpesvirus infections could be one contributing factor for maturation toward balanced Th1-Th2 responses. Our data indicate that early infection by herpesviruses may affect NK cell and monocyte interactions and thereby also influence the development of allergies

Intra-lineage plasticity and functional reprogramming maintain natural killer cell repertoire diversity

Natural killer (NK) cell repertoires are made up of phenotypically distinct subsets with different functional properties. The molecular programs involved in maintaining NK cell repertoire diversity under homeostatic conditions remain elusive. Here, we show that subset-specific NK cell proliferation kinetics correlate with mTOR activation, and global repertoire diversity is maintained through a high degree of intra-lineage subset plasticity during interleukin (IL)-15-driven homeostatic proliferation in vitro. Slowly cycling sorted KIR+CD56dim NK cells with an induced CD57 phenotype display increased functional potential associated with increased transcription of genes involved in adhesion and immune synapse formation. Rapidly cycling cells upregulate NKG2A, display a general loss of functionality, and a transcriptional signature associated with increased apoptosis/cellular stress, actin-remodeling, and nuclear factor κB (NF-κB) activation. These results shed light on the role of intra-lineage plasticity during NK cell homeostasis and suggest that the functional fate of the cell is tightly linked to the acquired phenotype and transcriptional reprogramming

Perturbed NK Cell Homeostasis Associated with Disease Severity in Chronic Neutropenia

Abstract Neutrophils have been thought to play a critical role in terminal differentiation of NK cells. Whether this effect is direct or a consequence of global immune changes with effects on NK-cell homeostasis remains unknown. In this study, we used high-resolution flow and mass cytometry to examine NK-cell repertoires in 64 patients with neutropenia and 27 healthy age- and sex-matched donors. A subgroup of patients with chronic neutropenia showed severely disrupted NK-cell homeostasis manifesting as increased frequencies of CD56bright NK cells and a lack of mature CD56dim NK cells. These immature NK-cell repertoires were characterized by expression of the proliferation/exhaustion markers Ki-67, Tim-3, and TIGIT and displayed blunted tumor target cell responses. Systems-level immune mapping revealed that the changes in immunophenotypes were confined to NK cells, leaving T-cell differentiation intact. RNA sequencing of NK cells from these patients showed upregulation of a network of genes, including TNFSF9, CENPF, MKI67, and TOP2A, associated with apoptosis and the cell cycle, but different from the conventional CD56bright signatures. Profiling of 249 plasma proteins showed a coordinated enrichment of pathways related to apoptosis and cell turnover, which correlated with immature NK-cell repertoires. Notably, most of these patients exhibited severe-grade neutropenia, suggesting that the profoundly altered NK-cell homeostasis was connected to the severity of their underlying etiology. Hence, although our data suggest that neutrophils are dispensable for NK-cell development and differentiation, some patients displayed a specific gap in the NK repertoire, associated with poor cytotoxic function and more severe disease manifestations