BNT162b2-boosted immune responses six months after heterologous or homologous ChAdOx1nCoV-19/BNT162b2 vaccination against COVID-19.

Heterologous prime/boost vaccination with a vector-based approach (ChAdOx-1nCov-19, ChAd) followed by an mRNA vaccine (e.g. BNT162b2, BNT) has been reported to be superior in inducing protective immunity compared to repeated application of the same vaccine. However, data comparing immunity decline after homologous and heterologous vaccination as well as effects of a third vaccine application after heterologous ChAd/BNT vaccination are lacking. Here we show longitudinal monitoring of ChAd/ChAd (n = 41) and ChAd/BNT (n = 88) vaccinated individuals and the impact of a third vaccination with BNT. The third vaccination greatly augments waning anti-spike IgG but results in only moderate increase in spike-specific CD4 + and CD8 + T cell numbers in both groups, compared to cell frequencies already present after the second vaccination in the ChAd/BNT group. More importantly, the third vaccination efficiently restores neutralizing antibody responses against the Alpha, Beta, Gamma, and Delta variants of the virus, but neutralizing activity against the B.1.1.529 (Omicron) variant remains severely impaired. In summary, inferior SARS-CoV-2 specific immune responses following homologous ChAd/ChAd vaccination can be compensated by heterologous BNT vaccination, which might influence the choice of vaccine type for subsequent vaccination boosts.

γδ T cells license immature B cells to produce a broad range of polyreactive antibodies.

Immature autoreactive B cells are present in all healthy individuals, but it is unclear which signals are required for their maturation into antibody-producing cells. Inducible depletion of γδ T cells show that direct interaction between γδ T cells and immature B cells in the spleen support an "innate" transition to mature B cells with a broad range of antigen specificities. IL-4 production of γδ T cells and cell-to-cell contact via CD30L support B cell maturation and induce genes of the unfolded protein response and mTORC1 signaling. Eight days after in vivo depletion of γδ T cells, increased numbers of B cells are already stuck in the transitional phase and express increased levels of IgD and CD21. Absence of γδ T cells leads also to reduced levels of serum anti-nuclear autoantibodies, making γδ T cells an attractive target to treat autoimmunity.

Intranasal Delivery of MVA Vector Vaccine Induces Effective Pulmonary Immunity Against SARS-CoV-2 in Rodents.

Antigen-specific tissue-resident memory T cells (Trms) and neutralizing IgA antibodies provide the most effective protection of the lungs from viral infections. To induce those essential components of lung immunity against SARS-CoV-2, we tested various immunization protocols involving intranasal delivery of a novel Modified Vaccinia virus Ankara (MVA)-SARS-2-spike vaccine candidate. We show that a single intranasal MVA-SARS-CoV-2-S application in mice strongly induced pulmonary spike-specific CD8+ T cells, albeit restricted production of neutralizing antibodies. In prime-boost protocols, intranasal booster vaccine delivery proved to be crucial for a massive expansion of systemic and lung tissue-resident spike-specific CD8+ T cells and the development of Th1 - but not Th2 - CD4+ T cells. Likewise, very high titers of IgG and IgA anti-spike antibodies were present in serum and broncho-alveolar lavages that possessed high virus neutralization capacities to all current SARS-CoV-2 variants of concern. Importantly, the MVA-SARS-2-spike vaccine applied in intramuscular priming and intranasal boosting treatment regimen completely protected hamsters from developing SARS-CoV-2 lung infection and pathology. Together, these results identify intramuscular priming followed by respiratory tract boosting with MVA-SARS-2-S as a promising approach for the induction of local, respiratory as well as systemic immune responses suited to protect from SARS-CoV-2 infections.

IL-4-Producing Vγ1+/Vδ6+ γδ T Cells Sustain Germinal Center Reactions in Peyer's Patches of Mice.

The mucosal immune system is the first line of defense against pathogens. Germinal centers (GCs) in the Peyer's patches (PPs) of the small intestine are constantly generated through stimulation of the microbiota. In this study, we investigated the role of γδ T cells in the GC reactions in PPs. Most γδ T cells in PPs localized in the GCs and expressed a TCR composed of Vγ1 and Vδ6 chains. By using mice with partial and total γδ T cell deficiencies, we found that Vγ1+/Vδ6+ T cells can produce high amounts of IL-4, which drives the proliferation of GC B cells as well as the switch of GC B cells towards IgA. Therefore, we conclude that γδ T cells play a role in sustaining gut homeostasis and symbiosis via supporting the GC reactions in PPs.

αß T cells replacing dermal and epidermal γδ T cells in Tcrd-/- mice express an MHC-independent TCR repertoire.

The epidermis of mouse skin is usually populated by dendritic epidermal T cells (γδDETC) expressing an invariant Vγ5Vδ1+ TCR. In Tcrd-/- mice, skin-resident γδDETC are replaced by αßDETC carrying polyclonal αß TCRs. Although they exhibit a dendritic morphology, αßDETC were reported to be less functional than genuine γδDETC, likely because their TCR is unable to interact with the original TCR ligands of γδDETC. However, the TCR repertoire of those replacement DETC in Tcrd-/- mice might provide clues for understanding the development and selection of canonical γδDETC. Here, we compare the phenotype and TCR repertoires of wild-type and Tcrd-/- mouse skin T cells. Our data reveal that αßDETC are CD4/CD8 double negative and express an MHC-independent TCR repertoire. Furthermore, we identify a second MHC-independent population of CD103hi CD4/ CD8 double-negative αß T cells in the dermis of Tcrd-/- mice.

Immune responses against SARS-CoV-2 variants after heterologous and homologous ChAdOx1 nCoV-19/BNT162b2 vaccination.

Currently approved viral vector-based and mRNA-based vaccine approaches against coronavirus disease 2019 (COVID-19) consider only homologous prime-boost vaccination. After reports of thromboembolic events, several European governments recommended using AstraZeneca's ChAdOx1-nCov-19 (ChAd) only in individuals older than 60 years, leaving millions of already ChAd-primed individuals with the decision to receive either a second shot of ChAd or a heterologous boost with mRNA-based vaccines. However, such combinations have not been tested so far. We used Hannover Medical School's COVID-19 Contact Study cohort of healthcare professionals to monitor ChAd-primed immune responses before and 3 weeks after booster with ChAd (n = 32) or BioNTech/Pfizer's BNT162b2 (n = 55). Although both vaccines boosted prime-induced immunity, BNT162b2 induced significantly higher frequencies of spike-specific CD4+ and CD8+ T cells and, in particular, high titers of neutralizing antibodies against the B.1.1.7, B.1.351 and P.1 variants of concern of severe acute respiratory syndrome coronavirus 2.

Expression of ACKR4 demarcates the "peri-marginal sinus," a specialized vascular compartment of the splenic red pulp.

The spleen comprises defined microanatomical compartments that uniquely contribute to its diverse host defense functions. Here, we identify a vascular compartment within the red pulp of the spleen delineated by expression of the atypical chemokine receptor 4 (ACKR4) in endothelial cells. ACKR4-positive vessels form a three-dimensional sinusoidal network that connects via shunts to the marginal sinus and tightly surrounds the outer perimeter of the marginal zone. Endothelial cells lining this vascular compartment express ACKR4 as part of a distinct gene expression profile. We show that T cells enter the spleen largely through this peri-marginal sinus and initially localize extravascularly around these vessels. In the absence of ACKR4, homing of T cells into the spleen and subsequent migration into T cell areas is impaired, and organization of the marginal zone is severely affected. Our data delineate the splenic peri-marginal sinus as a compartment that supports spleen homing of T cells.

A fetal wave of human type 3 effector γδ cells with restricted TCR diversity persists into adulthood.

Accumulating evidence suggests that the mouse embryonic thymus produces distinct waves of innate effector γδ T cells. However, it is unclear whether this process occurs similarly in humans and whether it comprises a dedicated subset of innate-like type 3 effector γδ T cells. Here, we present a protocol for high-throughput sequencing of TRG and TRD pairs that comprise the clonal γδTCR. In combination with single-cell RNA sequencing, multiparameter flow cytometry, and TCR sequencing, we reveal a high heterogeneity of γδ T cells sorted from neonatal and adult blood that correlated with TCR usage. Immature γδ T cell clusters displayed mixed and diverse TCRs, but effector cell types segregated according to the expression of either highly expanded individual Vδ1+ TCRs or moderately expanded semi-invariant Vγ9Vδ2+ TCRs. The Vγ9Vδ2+ T cells shared expression of genes that mark innate-like T cells, including ZBTB16 (encoding PLZF), KLRB1, and KLRC1, but consisted of distinct clusters with unrelated Vγ9Vδ2+ TCR clones characterized either by TBX21, FCGR3A, and cytotoxicity-associated gene expression (type 1) or by CCR6, RORC, IL23R, and DPP4 expression (type 3). Effector γδ T cells with type 1 and type 3 innate T cell signatures were detected in a public dataset of early embryonic thymus organogenesis. Together, this study suggests that functionally distinct waves of human innate-like effector γδ T cells with semi-invariant Vγ9Vδ2+ TCR develop in the early fetal thymus and persist into adulthood.

Microbial exposure drives polyclonal expansion of innate γδ T cells immediately after birth.

Starting at birth, the immune system of newborns and children encounters and is influenced by environmental challenges. It is still not completely understood how γδ T cells emerge and adapt during early life. Studying the composition of T cell receptors (TCRs) using next-generation sequencing (NGS) in neonates, infants, and children can provide valuable insights into the adaptation of T cell subsets. To investigate how neonatal γδ T cell repertoires are shaped by microbial exposure after birth, we monitored the γ-chain (TRG) and δ-chain (TRD) repertoires of peripheral blood T cells in newborns, infants, and young children from Europe and sub-Saharan Africa. We identified a set of TRG and TRD sequences that were shared by all children from Europe and Africa. These were primarily public clones, characterized by simple rearrangements of Vγ9 and Vδ2 chains with low junctional diversity and usage of non-TRDJ1 gene segments, reminiscent of early ontogenetic subsets of γδ T cells. Further profiling revealed that these innate, public Vγ9Vδ2+ T cells underwent an immediate TCR-driven polyclonal proliferation within the first 4 wk of life. In contrast, γδ T cells using Vδ1+ and Vδ3+TRD rearrangements did not significantly expand after birth. However, different environmental cues may lead to the observed increase of Vδ1+ and Vδ3+TRD sequences in the majority of African children. In summary, we show how dynamic γδ TCR repertoires develop directly after birth and present important differences among γδ T cell subsets.

TCR repertoire analysis reveals phosphoantigen-induced polyclonal proliferation of Vγ9Vδ2 T cells in neonates and adults.

The Vγ9Vδ2 T cell subset is the major γδ T cell subset in human peripheral blood and has the unique ability to contribute to immune surveillance by detecting pyrophosphorylated metabolites of isoprenoid synthesis, termed phosphoantigens (pAgs). Vγ9Vδ2 T cells are first detected at midgestation and show postnatal expansion. Interestingly, neonatal Vγ9Vδ2 T cells display a higher TCR repertoire diversity with more public clonotypes and lower pAg responsiveness than in adults. Notably, it is not known whether postnatal changes occur by TCR-dependent reactivity to pAg exposure. Here, we applied next-generation sequencing of γδ TCR repertoires to understand potential differences in the pAg-mediated response of neonatal and adult Vγ9Vδ2 T cells at the level of the expressed γδ TCR. We observed a polyclonal pAg-induced response of neonatal and adult Vγ9Vδ2 T cells, albeit neonatal γδ T cells showed less in vitro pAg responsiveness. Neonatal Vγ9Vδ2 T cells displayed a less pronounced bias for Jδ1 usage and a more frequent use of Jδ2 or Jδ3 that remained stable after pAg exposure. In addition, public and private Vδ2 TRD clones took part in the polyclonal pAg-induced response in neonates and adults. In conclusion, adult and neonatal Vγ9Vδ2 T cells both undergo polyclonal pAg-induced proliferation, whereas especially adult Vγ9Vδ2 T cells display a high stability at the level of the expressed TCR repertoire.

IL-1ß Promotes Staphylococcus aureus Biofilms on Implants in vivo.

Implant associated infections represent a serious health burden in clinics since some microorganisms are able to colonize biological surfaces or surfaces of indwelling medical devices and form biofilms. Biofilms represent communities of microorganisms attached to hydrated surfaces and enclosed in self-produced extracellular matrix. This renders them resistant to exogenous assaults like antibiotics or immune effector mechanisms. Little is known regarding the role of the immune system in the formation of biofilms during implant associated infections, largely due to the lack of suitable mouse models. Here we use colonized osmotic pumps in mice to study the interaction of an activated immune system with biofilm-forming Staphylococcus aureus encoding Gaussia luciferase. This approach permits biofilm formation on the osmotic pumps in living animals. It also allows the continuous supply of soluble immune cell activating agents, such as cytokines to study their effect on biofilm formation in vivo. Using non-invasive imaging of the bioluminescent signal emitted by the lux expressing bacteria for quantification of bacterial load in conjunction with light and electron microscopy, we observed that pump-supplied pro-inflammatory cytokine IL-1ß strongly increased biofilm formation along with a massive influx of neutrophils adjacent to the biofilm-coated pumps. Thus, our data demonstrate that immune defense mechanisms can augment biofilm formation.

The chemokine receptor CCR7 is a promising target for rheumatoid arthritis therapy.

The chemokine receptor CCR7 and its ligands CCL19 and CCL21 guide the homing and positioning of dendritic and T cells in lymphoid organs, thereby contributing to several aspects of adaptive immunity and immune tolerance. In the present study, we investigated the role of CCR7 in the pathogenesis of collagen-induced arthritis (CIA). By using a novel anti-human CCR7 antibody and humanized CCR7 mice, we evaluated CCR7 as a target in this autoimmune model of rheumatoid arthritis (RA). Ccr7-deficient mice were completely resistant to CIA and presented severely impaired antibody responses to collagen II (CII). Selective CCR7 expression on dendritic cells restored arthritis severity and anti-CII antibody titers. Prophylactic and therapeutic treatment of humanized CCR7 mice with anti-human CCR7 mAb 8H3-16A12 led to complete resistance to CIA and halted CIA progression, respectively. Our data demonstrate that CCR7 signaling is essential for the induction of CIA and identify CCR7 as a potential therapeutic target in RA.

CRISPR/Cas9 Immunoengineering of Hoxb8-Immortalized Progenitor Cells for Revealing CCR7-Mediated Dendritic Cell Signaling and Migration Mechanisms in vivo.

To present antigens to cognate T cells, dendritic cells (DCs) exploit the chemokine receptor CCR7 to travel from peripheral tissue via afferent lymphatic vessels to directly enter draining lymph nodes through the floor of the subcapsular sinus. Here, we combined unlimited proliferative capacity of conditionally Hoxb8-immortalized hematopoietic progenitor cells with CRISPR/Cas9 technology to create a powerful experimental system to investigate DC migration and function. Hematopoietic progenitor cells from the bone marrow of Cas9-transgenic mice were conditionally immortalized by lentiviral transduction introducing a doxycycline-regulated form of the transcription factor Hoxb8 (Cas9-Hoxb8 cells). These cells could be stably cultured for weeks in the presence of doxycycline and puromycin, allowing us to introduce additional genetic modifications applying CRISPR/Cas9 technology. Importantly, modified Cas9-Hoxb8 cells retained their potential to differentiate in vitro into myeloid cells, and GM-CSF-differentiated Cas9-Hoxb8 cells showed the classical phenotype of GM-CSF-differentiated bone marrow-derived dendritic cells. Following intralymphatic delivery Cas9-Hoxb8 DCs entered the lymph node in a CCR7-dependent manner. Finally, we used two-photon microscopy and imaged Cas9-Hoxb8 DCs that expressed the genetic Ca2+ sensor GCaMP6S to visualize in real-time chemokine-induced Ca2+ signaling of lymph-derived DCs entering the LN parenchyma. Altogether, our study not only allows mechanistic insights in DC migration in vivo, but also provides a platform for the immunoengineering of DCs that, in combination with two-photon imaging, can be exploited to further dissect DC dynamics in vivo.

Manifold Roles of CCR7 and Its Ligands in the Induction and Maintenance of Bronchus-Associated Lymphoid Tissue.

The processes underlying the development and maintenance of tertiary lymphoid organs are incompletely understood. Using a Ccr7 knockout/knockin approach, we show that spontaneous bronchus-associated lymphoid tissue (BALT) formation can be caused by CCR7-mediated migration defects of dendritic cells (DCs) in the lung. Plt/plt mice that lack the CCR7 ligands CCL19 and CCL21-serine do not form BALT spontaneously because lung-expressed CCL21-leucine presumably suffices to maintain steady-state DC egress. However, plt/plt mice are highly susceptible to modified vaccinia virus infection, showing enhanced recruitment of immune cells as well as alterations in CCR7-ligand-mediated lymphocyte egress from the lungs, leading to dramatically enhanced BALT. Furthermore, we identify two independent BALT homing routes for blood-derived lymphocytes. One is HEV mediated and depends on CCR7 and L-selectin, while the second route is via the lung parenchyma and is independent of these molecules. Together, these data provide insights into CCR7/CCR7-ligand-orchestrated aspects in BALT formation.

T cell specific Cxcr5 deficiency prevents rheumatoid arthritis.

The chemokine receptor CXCR5 is primarily expressed on B cells and Tfh cells and facilitates their migration towards B cell follicles. In the present study we investigated the role of the CXCL13/CXCR5 axis in the pathogenesis of rheumatoid arthritis (RA) and specifically addressed the impact of CXCR5-mediated T and B cell migration in this disease. Employing collagen-induced arthritis (CIA) we identify CXCR5 as an absolutely essential factor for the induction of inflammatory autoimmune arthritis. Cxcr5-deficient mice and mice selectively lacking Cxcr5 on T cells were completely resistant to CIA, showed impaired germinal center responses and failed to mount an IgG1 antibody response to collagen II. Selective ablation of CXCR5 expression in B cells also led to suppression of CIA owing to diminished GC responses in secondary lymphoid organs (SLO) and impaired anti-collagen II antibody production. Chimeric mice harboring Cxcr5-proficient and Cxcr5-deficient immune cells revealed SLO and not the synovial tissue as the compartment where CXCR5-mediated cell migration induces autoimmune inflammation in arthritis. Thus our data demonstrate that CXCR5-mediated co-localization of Tfh cells and B cells in SLOs is absolutely essential for the induction of RA and identify CXCR5 and Tfh cells as promising therapeutic targets for the treatment of RA.

Shift of graft-versus-host-disease target organ tropism by dietary vitamin A.

Gut-homing of donor T cells is causative for the development of intestinal GvHD in recipients of allogeneic hematopoietic stem cell transplantation (HSCT). Expression of the gut-specific homing receptors integrin-α4ß7 and chemokine receptor CCR9 on T cells is imprinted in gut-associated lymphoid tissues (GALT) under the influence of the vitamin A metabolite retinoic acid. Here we addressed the role of vitamin A deficiency in HSCT-recipients for donor T cell migration in the course of experimental GvHD. Vitamin A-deficient (VAD) mice were prepared by feeding them a vitamin A-depleted diet. Experiments were performed in a C57BL/6 into BALB/c model of acute GvHD. We found that expression of integrin-α4ß7 and CCR9 in GALT was reduced in VAD recipients after HSCT. Competitive in vivo homing assays showed that allogeneic T cells primed in VAD mice did not home as efficiently to the intestine as T cells primed in mice fed with standard diet (STD). The course of GvHD was ameliorated in VAD HSCT-recipients and, consequently, their survival was prolonged compared to recipients receiving STD. However, VAD-recipients were not protected and died of clinical GvHD. We found reduced numbers of donor T cells in the intestine but increased cell counts and tissue damage in other organs of VAD-recipients. Furthermore, we observed high IFN-γ(+)CD4(+) and low FoxP3(+)CD4(+) frequencies of total donor CD4(+) T cells in VAD as compared to STD recipients. Taken together, these results indicate that dietary vitamin A deficiency in HSCT-recipients changed target organ tropism in GvHD but also resulted in fatal inflammation after HSCT.

Deficient CCR7 signaling promotes TH2 polarization and B-cell activation in vivo.

The chemokine receptor CCR7 has a central role in regulating homing and positioning of T cells and DCs to lymph nodes (LNs) and participates in T-cell development and activation. In this study, we addressed the role of CCR7 signaling in T(H) 2 polarization and B-cell activation. We provide evidence that the lack of CCR7 drives the capacity of naïve CD4(+) T cells to polarize toward T(H) 2 cells. This propensity contributes to a lymph node environment in CCR7-deficent mice characterized by increased expression of IL-4 and increased frequency of T(H) 2 cells. We show that elevated IL-4 levels lead to B-cell activation characterized by up-regulated expression of MHC class II, CD23 and CD86. Activated B cells are in turn highly efficient in presenting antigen to CD4(+) T cells and thus potentially contribute to the T(H) 2 microenvironment. Taken together, our results support the idea of a CCR7-dependent patterning of T(H) 2 responses, with absent CCR7 signaling favoring T(H) 2 polarization, dislocation of T helper cells into the B-cell follicles and, as a consequence, B-cell activation.

Alloantigen-specific de novo-induced Foxp3+ Treg revert in vivo and do not protect from experimental GVHD.

Induced antigen-specific Foxp3(+) T cells (iTreg) are being discussed as a promising alternative to polyclonal natural Foxp3(+) T cells (nTreg) for cell-based therapies, particularly to achieve transplantation tolerance. Using Foxp3eGFP-reporter mice, we here establish an efficient protocol to induce and expand alloantigen-specific iTreg from Foxp3(-)CD4(+) T cells with cluster-disrupted DC. These iTreg were mainly CD62L(+) and showed efficient suppressive activity in vitro. However, in contrast to nTreg, adoptively transferred iTreg entirely failed to prevent lethal graft versus host disease (GVHD). Within irradiated recipients, the majority of adoptively transferred Foxp3(+) iTreg, but not Foxp3(+) nTreg quickly reverted to Foxp3(-)CD4(+) T cells. We therefore suggest that therapeutic approaches to treat GVHD should rely on nTreg, whereas the use of de novo alloantigen-induced iTreg should be handled with caution since the stability of the regulatory phenotype of the iTreg could be of major concern.